Composition and Function of Ccr4-Not Protein Complexes

Transcription

1 Composition and Function of Ccr4-Not Protein Complexes N.C. LAU

2 ISBN: Cover design by family Lau: Looking Proteins Printed by Gildeprint Drukkerijen - The Netherlands Financial support for the publication of this thesis by the J.E. Jurriaanse Stichting, The Netherlands, and Promega Benelux BV is gratefully acknowledged.

3 Composition and Function of Ccr4-Not Protein Complexes Samenstelling en functie van Ccr4-Not eiwitcomplexen (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 30 november 2010 des middags te uur door Nga Chi Lau geboren op 21 maart 1981 te Hong Kong, Hongkong

4 Promotor: Prof.dr. H.Th.M. Timmers Dit proefschrift werd mogelijk gemaakt met financiële steun van het Netherlands Proteomics Centre.

5 Research is learned by imitation, experience, and reflection - Personal quote (2010)

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7 CONTENTS Abbreviations 8 Chapter 1 General introduction 9 Chapter 2 Human Ccr4-Not complexes contain variable deadenylase subunits 43 Chapter 3 Subcellular localization of human Ccr4-Not subunits 69 Chapter 4 Phosphorylation of Not4p functions parallel to BUR2 to regulate resistance to cellular stresses in Saccharomyces cerevisiae 85 Chapter 5 Summarizing discussion 105 Nederlandse samenvatting 119 Acknowledgements 123 Curriculum vitae 124

8 ABBREVIATIONS Caf Ccr4p associated factor CALIF Caf1-like factor Ccr carbon catabolite repression CDK cyclin-dependent protein kinase CNOT Ccr4-Not subunit CTD C-terminal domain of RNA polymerase II EJC exon-junction complex GFP green fluorescent protein H3K4me3 histone H3 lysine-4 tri-methylation mrna messenger RNA mrnp messenger ribonucleoprotein complex MS/MS tandem mass spectrometry NES nuclear export signal NLS nuclear localization signal NMD nonsense-mediated mrna decay Not negative on TATA NPC nuclear pore complex NSAF normalized spectral abundance factor NUP nuclear pore complex protein PABP poly(a)-binding protein PABPC1 cytoplasmic poly(a)-binding protein 1 P bodies processing bodies Pol II RNA polymerase II Pop PGK-promoter directed overproduction TBP TATA-binding protein TREX transcription/export UTR untranslated region

9 General introduction 1

10 Regulation of mrna metabolism in eukaryotes The Central Dogma of molecular biology is that DNA is transcribed into RNA (transcription), and RNA is translated into protein (translation). These processes occur in separate compartments of the eukaryotic cell. mrna (messenger RNA) forms the central connection between the genetic information in the nucleus and active protein production in the cytoplasm. This mrna is subjected to multiple regulatory processes during its life from nucleus to cytoplasm in eukaryotic cells. Chapter 1 10

11 Chromatin and modifiers The genetic material in eukaryotes is packaged as condensed chromatin in the nucleus. Chromatin is composed of nucleosomes consisting of DNA wrapped around a core of eight histone proteins (two copies of H2A, H2B, H3 and H4), which is sealed by the linker histone H1 [1]. The condensed chromatin structure creates barriers against protein complexes to access the DNA template. Chromatin-modifying enzymes such as the ATP-dependent remodelling complexes and histone modifiers modulate the chromatin structure and facilitate the accessibility of DNA to its binding factors [2]. The ATP-dependent remodelling complexes use ATP hydrolysis to disassemble, displace and/ or slide nucleosomes along the DNA to expose or occlude DNA binding sequences. In contrast, histone modifying enzymes catalyze the covalent addition or removal of chemical groups on histone cores and on histone tails that protrude from the nucleosomal particle. Common histone modifications are methylation of arginine, methylation, acetylation, ubiquitination and SUMOylation of lysines, and phosphorylation of serine and threonine [2-6]. These modifications are performed at specific histone sites by specific enzymes. According to the histone-code hypothesis, certain combinations of histone modifications form a code for the binding of specific regulatory proteins that have consequences for DNA associated processes such as DNA replication, DNA repair and gene expression [4-9]. A conserved histone modification pattern of enriched H3K4me3 (histone H3 lysine-4 tri-methylation) and lysine-acetylated histone H3 at the 5 -end of genes is highly associated with chromatin accessibility and active transcription [5, 6, 9]. General introduction 11 mrna synthesis The eukaryotic transcription of protein-coding genes by Pol II (RNA polymerase II) (Figure 1) involves the assembly of the pre-initiation complex, promoter melting, initiation, promoter clearance, elongation, and termination [10]. Transcription of genetic material starts with the binding of transcriptional activators to regulatory elements in the promoter region of a gene. Recruitment of co-activators such as ATP-dependent chromatin remodellers and histone modifiers assist in the formation of the pre-initiation complex, which includes Pol II and the general transcription factors TFIID (contains TBP; TATAbinding protein), TFIIA, TFIIB, TFIIF, TFIIE and TFIIH that bind the promoter region in this particular order [11]. The largest subunit of Pol II contains a hepta-peptide (YSPTSPS) tandem repeat at its CTD (C-terminal domain), which is subjected to phosphorylation of its serine residues at position 2, 5 and 7 (Ser2, Ser5 and Ser7) at different stages of the transcription cycle [12]. Promoter melting occurs by unwinding the DNA around the transcription start site by TFIIH helicase, followed by transcription initiation by Pol II, which pauses 30 to 50 nucleotides downstream from the start upon binding by DSIF

12 and NELF. The CTD of paused Pol II is phosphorylated on Ser5 (and Ser7) by the kinase within TFIIH and enables promoter clearance of most of the general transcription factors. It is suggested that the paused Pol II resumes elongation upon phosphorylation of DSIF, NELF, and Ser2 of Pol II CTD [13]. Phosphorylation of Ser2 of Pol II CTD by P-TEFb kinase marks the transition from paused transcription to transcription elongation. As Pol II elongates further downstream along the gene sequence, levels of phosphorylated Ser5 (and Ser7) drop while Ser2 phosphorylation increases [12, 14, 15]. Various factors are required for transcription elongation including the classical elongation factor TFIIS, Spt4/5/6, FACT, Bur1/2, the PAF complex and Elongator, which associate with Pol II and/ or the nascent transcript [16-18]. The multiple termination signals at the 3 -end of a gene will stop transcription by Pol II and release it from the DNA template to make it available for another round of transcription [10]. Chapter 1 12 mrna processing Transcription and processing of pre-mrna transcripts are inter-connected and influence the efficiency and specificity of mature mrna formation [19-21]. Pol II plays a central role in mediating the recruitment of processing factors to nascent mrna throughout transcriptional elongation and termination. Four main processing events occur during the formation of a mature mrna transcript: 5 -end capping, splicing, 3 -end cleavage and polyadenylation (Figure 1). The first pre-mrna processing event occurs soon after Pol II initiates transcription. When the nascent RNA reaches about nucleotides, phosphorylation of Ser5 of Pol II CTD triggers the addition of a 7-methylguanosine cap to the 5 -end of the transcript by the mrna capping enzyme [12]. This cap is bound by the cap binding complex, which protects the nascent transcript against degradation by 5-3 exonucleases [22, 23]. The transcript also undergoes splicing to remove noncoding sequences (introns) and to join coding sequences (exons), resulting in a functional message from the DNA template. Splicing is catalyzed by the spliceosome, which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins [24]. The final pre-mrna processing events are 3 -end cleavage and polyadenylation, which are controlled by sequence elements in the pre-mrna as well as protein factors [25, 26]. Specific sequence elements in the 3 -UTR are recognized as the polyadenylation site and the newly synthesized RNA is cleaved at the 3 -end. Multiple adenosine residues (poly(a)- tail) are added to this end of the transcript by poly(a) polymerase and are subsequently bound by a PABP (poly(a)-binding protein), which in turn enhances the processivity of poly(a) polymerase [20]. These mrna processing events create properly processed mrnas, which trigger the recruitment of protein factors which are necessary for mrna export.

13 exon intron exon intron exon CAP exon exon exon 5 -capping splicing 3 -end cleavage and polyadenylation SYNTHESIS (DNA) intron exon (pre-mrna) PROCESSING exon AAAAAAAA (mrna) EXPORT nucleus cytoplasm DECAY A A A G C G T T A G C G C C A T A G General introduction TRANSLATION LOCALIZATION P bodies Transport granules Stress granules 13 CAP exon exon exon...exon AAAAAAAA PABPC1 80S ribosome Figure 1. Overview of mrna metabolism in eukaryotes In the nucleus, pre-mrna is synthesized by DNA transcription, followed by co-transcriptional processing including 5 -end capping, splicing, 3 -end cleavage and polyadenylation to generate mature mrna. During these processes, many RNA-binding proteins interact with the mrna and the resulting mrnp is exported. In the cytoplasm, the mrna transcript can be transported and stored in subcellular compartments, become degraded and/or translated into proteins.

14 mrnp assembly Pre-mRNAs and mrnas exist in cells as mrnps (messenger ribonucleoprotein complexes). During the course of mrna maturation, the transcript interacts with many proteins including processing factors, hnrnps (heterogeneous nuclear ribonucleoproteins) and export factors [27, 28]. Some of these associations are transient for processing events, whereas other RNA-binding proteins remain bound for nuclear export of mature mrna. The assembly of mrnp particles starts with the association of the cap binding complex to nascent mrna [27]. In addition, the EJC (exon-junction complex) binds transcripts at the exon-exon junctions in a splicing-dependent manner and functions in downstream events like mrna export and NMD (nonsense-mediated mrna decay) [29]. The splicing factor UAP56 (Sub2p in yeast) binds the EJC-component ALY (Yra1p in yeast), and together they associate with the multi-subunit THO complex to form the TREX (transcription/export) complex [30, 31]. Interestingly, this TREX complex is recruited to mrna by the splicing machinery in higher eukaryotes, whereas it is recruited cotranscriptional to the nascent transcript in yeast [32-35]. Chapter 1 14 Nuclear quality control The nuclear phases of mrnp assembly are monitored by nuclear surveillance machineries that can degrade RNA, retain mrnp or downregulate transcription when mrnp formation is impaired [36, 37]. The nuclear RNA degradation machinery, consisting largely of exonucleases, assists in the elimination of inefficiently processed pre-mrnas and malformed mrnas [36, 38]. Prior to nuclear mrna decay, the cap and/or poly(a)- tail of transcripts must be removed to gain access to the transcript body. In yeast, nuclear pre-mrnas with splicing defects are decapped by the Lsm2-8p complex, and degraded by the Rat1p 5-3 -exonuclease [39, 40]. Moreover, pre-mrnas containing defects in splicing, packaging and/or 3 -end processing are degraded in the 3-5 direction by the nuclear exosome-tramp complex [41-43]. Besides RNA decay, quality control can also lead to mrnp retention in the nucleus at the NPCs (nuclear pore complexes), thereby preventing its nuclear export [44-46]. The NPC components are also involved in transcriptional downregulation of genes from which impaired mrnas are being produced [46]. All these nuclear quality control systems make sure that only properly assembled mrnp particles are exported to the cytoplasm.

15 Nuclear pore complex Transport of small molecules and macromolecules between the nucleus and the cytoplasm occurs through the NPC in the double-membrane of the nuclear envelope. The NPC consists of roughly 30 different proteins called NUPs (nuclear pore complex proteins) that can be grouped in scaffold, transmembrane and peripheral NUPs, depending on their location and function within the NPC (Figure 2). The scaffold NUPs are stable and form the core of the NPC. The membrane-spanning portion of the NPC is an eight-fold symmetrical channel with extensions in the nucleoplasm (nuclear basket) and cytoplasm (cytoplasmic filaments) that are different in form and protein composition [47, 48]. Most peripheral NUPs are highly dynamic and have phenylalanine-glycine repeats with a crucial role in mediating selectivity and permeability of the NPC [49]. Nucleocytoplasmic transport of RNA- and protein-based cargo is mediated by at least three different classes of soluble transport receptors. The karyopherin family (CRM1 and importin) is involved in protein transport and certain RNAs, whereas TAP and Mex67p proteins function in bulk mrna/mrnp transport in metazoans and yeast, respectively, and NTF2 imports the small GTPase Ran into the nucleus [50-56]. All nuclear transport factors can bidirectionally traverse the NPC. mrnp export The ALY/Yra1p subunit within the TREX complex binds mrna and forms the bridge between the mrnp particle and the non-rna binding export receptors TAP/Mex67p (Figure 2) [57-59]. Heterodimerization of TAP with p15 or Mex67p with Mtr2p directs their interaction with NUPs for directional transport of mrnps to the cytoplasm [50, 60-62]. In addition, the function of Dbp5, Gle1 and soluble IP6 (inositol hexakisphosphate) at the cytoplasmic fibrils of the NPC are required for proper directional transport. It is thought that these factors trigger a conformational change of mrnp that results in the removal of a subset of proteins, including the export receptor, just prior to the release of mrnps into the cytoplasm [63-70]. In contrast, a subset of endogenous transcripts is exported via the karyopherin CRM1 using adaptor proteins that bind RNA (Figure 2). This alternative exit route is suggested for adaptor protein HuR that binds ARE (adenosine/uridine-rich element) in the 3 -UTR of mrnas derived from early response genes [54]. Nuclear export of these transcripts by CRM1 is regulated by the small GTPase protein Ran [71-75]. CRM1 binds its cargo (adaptor and its transcript) and RanGTP in the nucleus, and disassembles on the cytoplasmic side of the NPC upon RanGTP hydrolysis, resulting in directional transcript release into the cytoplasm. RanGDP is transported back to the nucleus by NTF2 to maintain the RanGTP gradient over the nuclear membrane for another round of CRM1-mediated export [71, 76, 77]. General introduction 15

16 Chapter 1 16 RanGTP RanGDP RanGDP peripheral NTF2 EXPORT cytoplasmic filaments } cytoplasm transmembrane nucleoplasm nuclear basket } scaffold IMPORT TAP p15 mrnp adaptor CRM1 RanGDP RanGTP ALY mrnp RanGTP Figure 2. Nucleocytoplasmic mrna transport through nuclear pore complex Transport of molecules over the nuclear envelope occurs through the nuclear pore complex, which consists of scaffold, transmembrane and peripheral NUPs. The bulk of mrnp particles is exported via the ALY/Yra1p adaptor (in TREX complex) and TAP-p15/Mex67p-Mtr2p heterodimer, whereas certain (ARE-containing) mrnps are transported to the cytoplasm by the CRM1-Ran complex. Ran is a small GTPase, which is GTP hydrolysed in the cytoplasm, delivered to the nucleus by NTF2, and guanine nucleotides are exchanged in the nucleus to provide a nucleocytoplasmic RanGTP gradient over the nuclear membrane. This figure is adapted from [48, 49, 74, 262, 263].

17 mrna localization in the cytoplasm Proper cytoplasmic localization of mrna molecules is critical for spatially regulated protein production during important processes such as cell fate determination, cell polarity, synaptic plasticity, cell motility, and embryonic axis formation [78, 79]. Selected mrna transcripts are packaged into translational inert RNA granules for transport along cytoskeletal elements to sites where their protein products are required and only then activated and translated by ribosomes [80-82]. Moreover, packaging of cytoplasmic mrnas into discrete RNA granules such as P (processing) bodies and stress granules also regulates gene expression by delaying the translation of their transcripts [83]. A working model for the metabolism of cytoplasmic mrna has been proposed (Figure 1) [83]. mrnas present in polysomes can undergo repeated rounds of translation initiation, elongation, and termination to produce proteins. In response to defects in translation or through specific recruitment, mrnas interact with components of the general mrna degradation and translation repression machineries and accumulate in P bodies [84, 85]. The transcripts within P bodies can be degraded or stored for return to translation. Furthermore, mrnas stalled in translation initiation associate with factors that promote their accumulation in stress granules, potentially enhancing their re-entry into translation when translation arrest is relieved [81]. General introduction Cytosolic quality control Like in the nucleus, the cytoplasm also harbours RNA quality control systems. Three main mechanisms NMD, nonstop mrna decay, and no-go mrna decay survey mrnas during translation and use the general mrna decay enzymes to degrade mrnas that direct aberrant protein synthesis [29]. The best characterized mrna surveillance system is NMD, in which of transcripts harbouring a premature translational termination codon are rapidly degraded [29, 86]. In mammals, the EJC at the exon-exon junctions triggers the NMD when translation terminates >50-55 nucleotides upstream of an EJC binding site [27]. mrna decay activities are recruited and additional rounds of degradation depend on the SMG5-6-7 proteins [29]. In yeast, mrnas are targeted for NMD if they harbour an error in their 3 -UTR or have an abnormal long distance between the termination site and the poly(a)-bound Pab1p. Recruitment and/or activation of mrna decay activities are regulated by Upf factors [29]. In contrast to NMD, nonstop mrna decay degrades transcripts that lack a stop codon [87]. The exosome is recruited to the empty A site of the ribosome by the exosome-associated factor Ski7p of the Ski-complex and degrades the mrna independently of deadenylation. Nonstop mrna decay targets are also thought to be degraded by decapping followed by Xrn1p-mediated degradation. The no-go mrna 17

18 decay occurs when mrna translation elongation stalls and involves endonucleolytic cleavage(s) near to where the translational active ribosome stalls, followed by exonuclease activities to degrade both decay products [29]. Chapter 1 18 Cytoplasmic mrna turnover Properly processed transcripts that pass the quality controls and are translated into (active) proteins are degraded at some point to downregulate gene expression. Their cap at the 5 -end and poly(a) tail at the 3 -end protect them from rapid and uncontrolled degradation (Figure 1). To initiate decay, the mrna body must become exposed by either removal of one of these two structures or by internal cleavage (Figure 3) [88]. In eukaryotes, deadenylation is often the rate-limiting step for mrna decay and is performed by several deadenylases (Figure 3A) [89]. The initial trimming of the poly(a)- tail to a length of nucleotides is performed by PAN2-PAN3, which is recruited to the tail by PABPC1 (cytoplasmic poly(a)-binding protein 1) [90-92]. The major process of deadenylation is performed by the exonucleases within the Ccr4-Not complex [91, 93]. The exposed mrna body is then attacked at the 3 -end by a large protein complex of 3-5 exonucleases and several accessory proteins known as the exosome-ski complex [94]. Following this decay, the cap becomes metabolized by the scavenger decapping enzyme DCPS [95]. Alternatively, the cap is removed by the heterodimer DCP1-DCP2, with DCP2 as the catalytic subunit, and several accessory factors [96]. Following decapping, the mrna body can be degraded in the 5-3 direction by the exoribonuclease XRN1. Moreover, endonucleases can also generate two mrna fragments by internal cleavage, which are efficiently degraded by XRN1 and the exosome-ski complex (Figure 3B) [96, 97]. Furthermore, mrna stability can be modulated by distinct RNA-binding proteins that are recruited to the 3 -UTR, 5 -UTR or coding region. They either inhibit or facilitate mrna decay by recruitment of the specific enzymes involved in mrna turnover [96]. Figure 3. General mrna degradation pathways in eukaryotes (A) Deadenylation-dependent decay. The bulk of mrnas undergoes decay by poly(a)-tail shortening by PAN2-PAN3 activity and deadenylation by the Ccr4-Not complex. This is followed by mrna body degradation via one of two pathways: a 5-3 pathway that proceeds by DCP1-DCP2-mediated mrna decapping followed by degradation by the exoribonuclease XRN1; or 3-5 degradation by the exosome complex followed by DCPS-mediated decapping. (B) Deadenylation-independent decay. Endonucleases expose the mrna body by internal cleavage, followed by degradation by the exosome and XRN1. Both figures are adapted from [89, 96].

19 A DEADENYLATION-DEPENDENT DECAY CAP exon exon exon exon AAAAAAAA CAP exon exon exon exon AAA PAN2-PAN3 poly(a) shortening CAP exon exon exon exon deadenylation decapping Ccr4-Not exon exon exon exon CAP exon exon exon DCP1-DCP2 5-3 decay scavenger decapping exon exon exon exon exon XRN1 DCPS 3-5 decay exosome General introduction 19 B DEADENYLATION-INDEPENDENT DECAY CAP exon exon exon exon AAAAAAAA internal cleavage endonuclease 3-5 decay CAP exon exon exosome 5-3 decay XRN1 exon exon AAAAAAAA

20 The Ccr4-Not complex Regulation of mrna metabolism involves an integrated series of events involving multiple proteins and large protein complexes including the Ccr4-Not complex. This protein complex is evolutionarily conserved and has important functions in mrna synthesis and degradation. Moreover, the Ccr4-Not complex is involved in histone methylation and regulates protein degradation via the ubiquitin-proteasome pathway. The emerging picture for the Ccr4-Not complex is that of a regulatory platform with distinct enzymatic activities in mrna metabolism that controls important cellular processes. Chapter 1 20

21 Yeast Ccr4-Not complex The Ccr4-Not complex was first identified in the budding yeast S. cerevisiae and is composed of at least nine core subunits: Not1p, Not2p, Not3p, Not4p, Not5p, Ccr4p, Caf1p, Caf40, Caf130 (Table 1). Except for the latter two Caf proteins, all subunits were revealed by genetic studies in yeast. The five NOT genes were identified by mutations, which increased the expression of the HIS3 gene in the presence of a crippled transcriptional activator from the TATA-less core promoter and not from the TATA core promoter [98-100], and are therefore named NOT (negative on TATA). Co-immunoprecipitation of Not5p with Not1p, Not3p and Not4p revealed that the Not proteins are associated with each other [100]. Not3p and Not5p are considered as paralogous proteins. The N-terminal region of Not3p is very similar to that of Not5p, and they show some functional redundancy [100]. Moreover, the CCR4 (carbon catabolite repression) gene was identified in a genetic selection for negative regulation of transcription factors of the glucose repressed ADH2 gene [101]. Co-immunoprecipitation with Ccr4p isolated Caf1p (Ccr4p associated factor 1) [102], which was identified in another genetic screen as the POP2 (PGK-promoter directed overproduction) gene [103]. Interestingly, mass spectrometry analysis identified Caf40p and Caf130p as part of the complex containing Ccr4p and Caf1p [104], and all Not proteins are part of this protein complex as well [ ]. Table 1. The core Ccr4-Not subunits in yeast and human General introduction Yeast* Residues Human* Residues Not1p (Cdc39p/Ros1p/Smd6p) 2108 CNOT Not2p (Cdc36p/Dna19p) 191 CNOT2 540 Not3p 836 CNOT3L 753 CNOT3S 609 Not5p 560 Not4p (Mot2p/Sig1p) 587 CNOT4L 639 CNOT4S 572 CNOT4N 433 Ccr4p (Fun27p/Nut21p) 837 CNOT6 (hccr4a) 557 CNOT6L (hccr4b) 555 Caf1p (Pop2p) 433 CNOT7 (hcaf1) 285 CNOT8 (hpop2/calif) 292 Caf40p 373 CNOT9 (Rcd1/Rqcd1/hCaf40) 299 Caf130p 1122 CNOT * Alternative names are given between brackets

22 In addition, the yeast Ccr4-Not complex exists in two forms, a core complex of 1.0 MDa and a larger complex of 1.9 MDa [105], whereby the latter may contain transient and/or sub-stoichiometrical associated factors. Not1p is the largest and the only essential subunit for yeast viability [98, 107]. All core subunits, except for Ccr4p, interact with Not1p, which is considered to be the scaffold of the yeast Ccr4-Not complex (Figure 4) [99, 100, 104]. ubiquitination Chapter 1 22 mrna degradation Caf1p Ccr4p Dhh1p Ccr4-Not Ubc4p Ubc5p Ccr4p Caf1p Caf40p Not1p Not4p Not2p Not5p Caf130p Not3p transcription TFIID Ada2p SAGA? Dbf2p? Srb10p Srb9p Srb11p protein modification Figure 4. Yeast Ccr4-Not complex and its activities Yeast Ccr4-Not subunits interact directly with the scaffold protein Not1p, except for Ccr4p, which associates with the complex via interaction with Caf1p. Many different proteins interact with the core components and contribute to the distinct functions of the Ccr4-Not complex such as mrna degradation, ubiquitination and transcription initiation. Moreover, protein kinases such as Dbf2p and Srb10p also interact with the Ccr4-Not complex, but no functional links (indicated by question marks) have been found. This figure is adapted from [104, 264].

23 Ccr4-Not complex in human and other eukaryotes Orthologues of yeast Ccr4-Not proteins have been identified in human and are renamed to CNOT (Ccr4-Not) proteins (Table 1) by the HUGO Gene Nomenclature Committee. CAF1/CNOT7 was the first human CNOT identified by homology to murine CAF1 [108], which was isolated from a two-hybrid screen using yeast Ccr4p as the bait [102]. A specific study, designed to isolate human Ccr4-Not orthologs, identified the cdnas of human CNOT2, CNOT3S(mall), CNOT4N(LS-containing) and CNOT8/CALIF (Caf1- like factor) by their homology to yeast CCR4-NOT genes [109]. A longer form of human CNOT3, CNOT3L(ong), was isolated from a yeast two-hybrid screen and contains an addition of 144 residues at the C-terminus of the earlier isolated CNOT3S [110]. Further analysis by genomic screens and/or biochemical isolation identified the human CNOT1, hccr4a/cnot6, hccr4b/cnot6l(ike), Rcd1/Rqcd1/hCaf40/CNOT9 and CNOT10 [91, ], but no human ortholog of yeast NOT5 could be found. These human Ccr4-Not subunits form protein complexes similar to yeast [109, 113, 115, 116] and biochemical fractionation revealed three distinct CNOT-containing complexes with estimated sizes of approximately 1.9 MDa, MDa and 650 kda [116]. Besides human and yeast, Ccr4-Not orthologues have been identified in many other eukaryotes including mouse, fly, worm, plant, trypanosome and other fungi [102, ] [102, ]. Subcellular localization The localization of Ccr4-Not components has been studied in yeast and metazoans (Figure 5), often by fusion to GFP (green fluorescent protein) or by specific antibodies against the (epitope-tagged) Ccr4-Not subunits. Eukaryotic cells have multiple subcellular structures, called bodies, in both their nuclear and cytoplasmic compartments. The Cajal bodies and nuclear speckles are structures in the nucleus. Spliceosome assembly/reassembly, snrna and snorna maturation, histone mrna 3 -end formation, and telomerase biogenesis occurs in the Cajal bodies [128], whereas nuclear speckles are enriched in small nuclear ribonucleoproteins and splicing factors [129]. P bodies are known cytoplasmic structures that contain translational repressed mrnps and factors involved in translational repression and mrna degradation [85]. The yeast Not1-4 proteins have been found in P bodies when decapping was blocked [130]. Indirect immunofluorescence showed that yeast Not1p, Not2p, Ccr4p and Caf1p/ Pop2p are cytoplasmic localized [93, 131], although fractionation of yeast cells indicated that endogenous Not1p and Not2p are nuclear proteins [98, 99]. Human CNOT2 and murine CNOT9 are also predominantly in the nucleus [132, 133], whereas fly dnot1-4, human CNOT6, human CNOT6L, fly dcaf1 and murine CNOT8 are mainly localized to the cytoplasmic fraction [91, 114, 120]. Many Ccr4-Not complex components (CNOT1, General introduction 23

24 CNOT3, CNOT6, CNOT7 and CNOT9) are also detected in the P bodies of mammalian cells [119, ]. The RNA-binding protein NANOS2 promotes the Ccr4-Not localization to these cytoplasmic foci in vivo [134]. Moreover, an unconventional human Ccr4-Caf1 complex containing hccr4d and hcaf1z is detected in nuclear Cajal bodies and in the cytoplasm [139]. In addition, CNOT7 has a cell cycle-dependent distribution in the nucleus and cytoplasm of mammalian cells [91, 129, 140]. CNOT7 is almost exclusively in the nucleus during G0- and G1-phases, and the majority of CNOT7 is cytoplasmic by the time cells enter S-phase [116]. Furthermore, blocking nuclear protein export by CRM1 inhibition resulted in the accumulation of mammalian CNOT6 and CNOT7 in the nucleus [91]. It seems that eukaryotic Ccr4-Not complex components localize to multiple subcellular compartments, which may reflect their multiple roles in cellular activities. YEAST METAZOAN Chapter 1 24 cytoplasm Not1p nucleus Not2p Not1-2p Ccr4p Caf1p P body Not1-4p nucleus CNOT2,6,9 CNOT7 dnot1-4 dcaf1 cytoplasm CNOT6 CNOT6L CNOT8 CNOT7 P body CNOT1,3,6,7,9 Figure 5. Subcellular localizations of Ccr4-Not complex components in yeast and metazoan Ccr4-Not subunits can reside in the nucleus, cytoplasm, or in specialized structures like P bodies. Human CNOT7 has a cell cycle-dependent localization in the nucleus and cytoplasm [116]. Cellular proliferation One of the important cellular activities of the Ccr4-Not complex is regulation of cell cycle control. Mutations in yeast NOT1 and NOT2 genes arrest cells in G1-phase [141]. In addition, many Ccr4-Not complex components associate or genetically interact with gene products with known cell cycle roles including Clb2p, Cdc73p, Dbf2, Paf1p, and Sic1

25 [115, ]. Moreover, human Ccr4-Not complex is important for cell cycle transition from G1-to-S-phase [ ], which is inhibited by members of the BTG/TOB family of anti-proliferative proteins that interact with the CNOT7 and CNOT8 deadenylase subunits [116, 140, ]. Human CNOT6L also influences cell proliferation by downregulation of p27/kip1 mrna levels, whereas CNOT6 does not influence cellular proliferation [114]. Furthermore, Ccr4-Not complex components are implicated in several cancers. CNOT9 is overexpressed in breast cancer cells and plays an important role in the growth of these cells [155]. The deadenylase activities of CNOT7 and CNOT8 control genes that are important for efficient proliferation of breast cancer cells [156], and elevated expression of CNOT8 has been found in both primary and metastatic human colorectal cancer cells [157]. Environmental stress response The maintenance of genome stability is essential for an organism, which can be restored through DNA repair during transcription or replication, by recombination, and/or DNA damage checkpoint functions [158, 159]. A functional relationship between the Ccr4- Not complex and DNA damage is supported by the fact that several ccr4-not mutants are sensitive to UV, ionizing radiation, replication inhibitor hydroxyurea, methylating agent methyl methane sulphonate and other DNA-damaging agents [118, 145, ]. Although the precise mechanisms of Ccr4-Not response to DNA damage are unknown, both transcriptional and deadenylation activities of the Ccr4-Not complex may play important functions in the DNA damage response [145, 146, , 167]. The Ccr4- Not and PAF complexes are involved in transcription elongation and DNA repair of active genes in a transcription-coupled repair process [160]. Yeast Ccr4p contributes to resistance to replication stress through its mrna deadenylase activity [145, 146, , 167], Ccr4p and Caf1p are involved in recombinational DNA repair [ ], and CNOT6 is important for proper cellular response to DNA damage in human cells [163]. Interestingly, the ubiquitination activity of Not4p is also important for DNA damage responses and sensitivity to heat [166]. Besides maintaining genome stability, eukaryotic cells must also rapidly adapt to environmental stresses such as changes in temperature, osmolarity or nutrient availability. In most cases this involves transcriptional activation of stress genes, which protects the cell against damage. The Ccr4-Not complex controls transcription initiation of these stress genes indirectly through the general stress factor Msn2p or specific stress factor Skn7p [ ]. The Ccr4-Not complex serves as a regulatory platform that senses and transmits extracellular signals to its downstream effectors to avoid DNA damage and survive environmental stresses. General introduction 25

26 Chapter 1 26 Normal development Components of the Ccr4-Not complex are also important for embryonic development. For instance, the C. elegans homolog of CNOT1, CeNOT1, is required for spindle positioning and regulation of microtubule length in embryos for proper cell division [173], CeCAF1 is essential for both somatic and germline development in C. elegans [121], and CeNot1, CeNot2 and CeCcr4 are required for normal development of excretory duct cell fate, which functions in osmoregulation and is essential for viability [174]. In Drosophila, oogenesis and early embryonic patterning requires dnot3/5 and dnot4 [175, 176], and the deadenylase activity of the Ccr4-like subunits Smaug and Twin [137, ]. Moreover, sexual development of S. pombe requires the function of Caf40p [180], and Not1p and Not2p are involved in the pheromone response pathway in S. cerevisiae [181]. In mammals, CNOT9 is highly expressed in actively differentiating tissues such as the testes, ovaries, thymus [180] and in virtually all tissues of the early embryo [182]. The isolation of murine CNOT9 as an erythropoietin-responsive gene and its interaction with c-myb suggests its role in hematopoietic cell development [133, 183]. In addition, CNOT9 is involved in branching morphogenesis during the embryonic development of the murine lung [182]. Interestingly, RNAi-silencing of dnot3 in Drosophila showed heart defects, which is also seen in heterozygous CNOT3 knockout mice. These heart defects could be reversed by HDAC inhibitors, suggesting that histone acetylation is involved. In humans, a common CNOT3 SNP correlates with altered cardiac rhythms, indicating that CNOT3 is essential for normal heart function throughout species [184]. In addition, mice lacking CNOT7 appear healthy, but display male infertility due to impaired spermatogenesis [ ]. CNOT7 also have an important role in bone morphogenesis [188, 189]. These studies indicate that the Ccr4-Not complex is conserved and required for multiple important cellular processes within eukaryotes, providing a multi-functional protein complex. Transcription regulation The Ccr4-Not subunits are important regulators in eukaryotic gene transcription [98-100, 105, 190]. Direct involvement of the Ccr4-Not complex in transcription regulation is indicated from their genetic and/or physical interactions with transcription factors (Figure 4) such as the TFIID complex [ ], SAGA-subunits Ada2p and Spt3p [196, 197], and Mediator Srb9-Srb10-Srb11p sub-complex in yeast [198], and the TBP-interacting protein TIP120B [110] and activation transcription factor ATF2 in mammalian cells [182]. Several reports also link the Ccr4-Not complex to members of the nuclear receptor family [113, 153, 182, 185]. CNOT1 showed a ligand-dependent physical interaction with the ERα (estrogen receptor α) and RXR (retinoid X receptor),

27 resulting in transcriptional repression of their responsive genes [113]. In contrast, CNOT6, CNOT7 and CNOT9 potentiate the ligand-dependent transcriptional activity of nuclear hormone receptors [116, 153, 182, 185], which may be mediated via association with NIF-1 [199]. Transcription regulation by the Ccr4-Not complex is also indicated by its role in appropriate promoter distribution of TFIID across the yeast genome [171] and by its association with promoters in yeast and metazoans [182, 193, 200, 201]. Yeast Not5p showed increased association with several promoters upon stress, which is dependent on TAF1 [193]. Several yeast Ccr4-Not complex components are recruited to the ARG1 promoter in a Gcn4p-dependent manner in vivo [200]. Mammalian CNOT9 is recruited to the c-jun promoter in the presence of retinoic acid [182], and CNOT3 associates with the consensus motif CGGCXGCG in promoter regions close to the transcription start sites, which are highly methylated (H3K4me3, H3K27me3, and bivalent methylations) in mice embryonic stem cells [201]. In addition, components of the Ccr4-Not complex have been studied for their function in the expression of yeast genome. One study indicated that the NOT genes affect many of the same genes and processes as the CCR4 and CAF1 genes [105], whereas other studies showed separate functional modules for NOT, CCR4- CAF1 and CAF130 genes under glucose conditions [106, 202]. Another study in yeast revealed that despite the association of all Ccr4-Not subunits within a complex, each subunit has its very specific function in gene expression [190]. General introduction Transcriptional repression and activation The Ccr4-Not complex can repress or activate transcription of genes, which is dependent on the core promoter structure and the (co-)activators/repressors present [105, 112, 200]. Early studies in yeast indicated a transcriptional repressive role for the Ccr4-Not complex [98, 99, 192, ]. Deletion of genes encoding Ccr4-Not subunits resulted in derepression of genes or increased TBP binding to promoters, which may sequester TFIID from active transcription sites [170, 171, 203]. Moreover, promoter recruitment of human CNOT1, CNOT2 or CNOT9 to a reporter gene resulted in transcriptional repression [112, 113]. The repressive function of human CNOT2 is identified in a specific protein motif at its C-termini called the Not-box, which is also present in CNOT3 [112]. The repression potential of both human CNOT1 and CNOT2 was sensitive to a histone deadenylase inhibitor, suggesting involvement of histone acetylation in transcriptional repression by the Ccr4-Not complex [112, 113, 207]. In contrast, a positive role in transcription has also been indicated for the Ccr4-Not complex. CCR4 was the first found to act as a positive regulator for the ADH2 gene in yeast [101]. Moreover, mutations in yeast Ccr4-Not complex components resulted in reduced levels of the transcriptional active histone marks H3K9 acetylation and/or H3K4 tri-methylation [ ], 27

28 and Ccr4-Not proteins are required for elevated gene expression levels from distinct promoters in yeast and mammalian cells [ , 105, 200, 211]. The Ccr4-Not subunits may enhance transcriptional activity by mediating the signals between DNA-binding transcriptional activators and the general transcription machinery [182]. Alternatively, genetic and physical interactions with components of the elongation machinery suggest that the Ccr4-Not complex contributes to transcription elongation and thereby elevation of mrna levels within cells [212]. Chapter 1 28 Ccr4-Not deadenylases The Ccr4-Not complex also regulates mrna decay to maintain proper levels of endogenous transcripts in the cell. Specific domains in its deadenylase subunits are responsible for mrna deadenylation activity. Yeast Ccr4p and its human orthologues CNOT6 and CNOT6L contain an EEP (exonuclease-endonuclease-phosphatase) domain that is required for the RNA nuclease activity [93, 111, 114, 213, 214], whereas yeast Caf1p/Pop2p and its human orthologues CNOT7 and CNOT8 contain RNA nuclease activities mediated via their DEDD domains [109, 111, ]. The Ccr4-Not complex may be involved in nuclear RNA degradation through its interaction with the nuclear exosome-tramp complex [43, 219], but the Ccr4-Not complex is well-known for its central role in cytoplasmic mrna decay. Both Ccr4p and Caf1p are required for normal mrna deadenylation in S. cerevisiae (Figure 4) [131, 215, 220], but Ccr4p is identified as the major cytoplasmic 3-5 poly(a)-specific RNase, whereas Caf1p is most likely facilitating Ccr4p activity [93, 131, 213, 215, 216, 221, 222]. Interestingly, Caf1p in A. nidulans is essential for degradation of specific transcripts and is functionally distinct from Ccr4p, which plays a major role in basal degradation [127]. However, combined depletion of CNOT7 and CNOT8 in human cells and T. brucei inhibited deadenylation of bulk mrna, whereas simultaneous depletion of CNOT6 and CNOT6L did not affect the abundance or degradation of mrna [124], suggesting a more important role for CNOT7 and CNOT8 in mrna deadenylation in humans and trypanosomes. In addition, the deadenylase subunits of the Ccr4-Not complex are also required for degradation of specific mrna molecules mediated by RISC (RNA-induced silencing complex) [ ], which consists at the minimum of an Argonaute protein that binds a small noncoding RNA called mirna (microrna), and a TNRC6 protein [227, 229]. Interestingly, this RISC-mediated mrna deadenylation and decay is independent of RNA base pairing by mirnas and has greater involvement of the CNOT7 and CNOT8 deadenylases than CNOT6, while CNOT7 and CNOT8 have no significant association with the RISC components Agonaute2 or TNRC6A (GW182) [227].

29 mrna deadenylation It is proposed that mrna deadenylation is coupled to translation termination in eukaryotes via PABPC1 [92, 230]. The erf1-erf3 complex binds PABPC1 during translation termination and is exchanged for the PAN2-PAN3 complex by competitive binding to PABPC1, thereby initiating deadenylation. After this first phase of deadenylation, the Ccr4-Not complex may be recruited to the shortened poly(a)-tail by specific RNA-binding proteins such as TOB, which has interaction capabilities with PABPC1 and CNOT7 [91, 92, 231]. Moreover, RNA-binding protein Nanos is a translational repressor and promotes deadenylation of poly(a)-tails [232], possibly by recruitment of the Ccr4-Not complex via direct interaction with CNOT4 and/or via interaction with PUM1 and CNOT8 (Mpt5p-Pop2p in yeast) [93, 221, ]. It is also suggested that NANOS2 recruits the Ccr4-Not complex to specific mrnas involved in meiosis for degradation [134]. In addition, members of the PUF family of 3 -UTR binding-proteins may recruit and retain Ccr4-Not deadenylases at the mrna [221, 234, 237]. The Puf5p-Pop2p interaction is conserved in S. cerevisiae, C. elegans, Drosophila and human [233, 234, 238], and other PUF proteins in yeast (Puf1p, Puf3p and Puf4p) also cause deadenylation and decay of their target mrnas [ ]. Furthermore, the Ccr4-Not complex forms a bridge between the removal of poly(a) tails and mrna decapping. Not2p, Not4p and Not5p of the yeast Ccr4-Not complex are required for EDC1 mrna decapping [130]. The Ccr4p and Caf1p deadenylases associate with decapping enzymes through the interaction with Dhh1p in yeast (Figure 4) [222, 242] and human Pat1b connects the Ccr4-Not complex with the DCP1-DCP2 decapping enzymes as also seen in Drosophila [243, 244]. General introduction 29 Protein ubiquitination The Ccr4-Not complex also harbours enzymatic activity that is required for posttranslational ubiquitination of proteins (Figure 6). Protein ubiquitination involves the addition of mono-ubiquitin or poly-ubiquitin to specific sites on protein substrates that determines the fate of the ubiquitinated protein. Substrates with mono-ubiquitination are involved in membrane trafficking, DNA repair and histone modification in transcriptional regulation, whereas poly-ubiquitination via ubiquitin lysine-63 chains direct substrates to functions as signalling, endocytosis, and ribosome functioning and genome stability [ ]. In contrast, poly-ubiquitin lysine-48 linkages destines substrates for degradation by the 26S proteasome [248]. The protein substrates are ubiquitinated by the subsequent activities of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3). E1 activates ubiquitin in an ATP-dependent manner and transfers it to E2, which dictates the type of inter-ubiquitin linkages. This is followed by

30 specific E2-E3 interaction, which mediates the transfer of activated ubiquitin to an E3- specific substrate [249]. E2s can interact with two different types of E3 enzymes (Figure 6). Interaction with HECT-type E3s results in transfer of ubiquitin from E2 via E3 to the substrate, whereas RING-type E3s catalyze direct transfer of ubiquitin from E2 to substrate [250]. Chapter 1 30 E3 ubiquitin ligase activity The Ccr4-Not subunits Not4p (in yeast) and CNOT4 (in human) contain a RING-finger domain at their N-terminus, which is required for ubiquitin E3 ligase activity [166, 251, 252]. Ubc4p and Ubc5p are the E2 partners of yeast Not4p (Figures 4 and 6), and human CNOT4 interacts with the E2 enzymes UbcH5B (UBE2D2), UbcH5C (UBE2D3), UbcH6 (UBE2E1), UbcH9 (UBE2E3) and Ubc13 (UBE2N) [166, ]. Yeast Not4p has shown in vitro ubiquitination activity in the context of the complete Ccr4- Not complex via its RING finger-mediated interaction with Ubc4p, which is required for stress responses in vivo [166, 255]. Human CNOT4 displayed in vitro ubiquitination activity that is dependent on its interaction with UbcH5B [252, 253]. Very few substrates for Not4p or CNOT4 have been identified so far (Figure 6). In yeast, the NAC or EGD (nascent polypeptide-associated complex) components Egd1p and Egd2p are substrates for Not4p activity [255]. Ubiquitination of these components is important for the stability of the NAC/EGD complex [256]. Interestingly, ubiquitinated Egd1p associates with the ribosome, while ubiquitinated Egd2p interacts with the proteasome [256], suggesting a role for yeast Not4p in the coordination of protein synthesis and degradation. Moreover, a recent study showed control of protein levels of the H3K4-specific demethylase Jhd2p by Not4p through a poly-ubiquitin-mediated degradation process. The human homolog of Jhd2p, JARID1C, was also poly-ubiquitinated by the human CNOT4 E3 ligase activity [257]. Furthermore, human CNOT4 may be a substrate of its own enzymatic activity as is suggested by auto-ubiquitination activity in vitro [253]. Post-translational regulation Post-translational modifications of Ccr4-Not complex subunits have been identified [ ], but in many cases the consequences have not been extensively studied. They may modulate enzymatic activity, protein binding or stability. Yeast Caf1p is phosphorylated on threonine-97 by Yak1p upon glucose deprivation and blocking this phosphorylation by T97A modification inhibits proper arrest at G1-phase when yeast cells are depleted from glucose. Moreover, Yak1p was shown to translocate from the cytoplasm to the nucleus after glucose deprivation, indicating that Caf1p phosphorylation likely occurs in the nucleus

31 For Ccr4-Not complex: Ub E1 Ub E1 ATP ADP + Pi E1 Ubiquitin-activating enzyme Ub E2 E3 HECT E3 Ub HECT substrate substrate Ub Ub E2 Ub E2 E3 RING substrate substrate Ub Ub Ub Ub E2 Ubiquitin-conjugating enzyme Y: Ubc4p, Ubc5p H: UbcH5B, UbcH5C, UbcH6, UbcH9, Ubc13 E3 Ubiquitin protein ligase Y: Not4p H: CNOT4 Substrates Y: Egd1p, Egd2p, Jhd2p H: JARID1C, CNOT4 Poly-ubiquitination Degradation by 26S proteasome General introduction Figure 6. Protein ubiquitination by Not4p/CNOT4 Protein substrates are ubiquitinated by the subsequent activities of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin protein ligase (E3). In general, poly-ubiquitination by the E3 RING-mediated protein ligase Not4p/CNOT4 labels protein substrates for degradation by the 26S proteasome. Y = yeast, H = human. 31 [261]. In addition, Not3p and Not5p are also phosphorylated under similar conditions as Caf1p, but seem to be degraded upon phosphorylation by PKA [170]. These data suggest that the Ccr4-Not complex is regulated by glucose-sensing pathways controlled in part by Yak1p and PKA protein kinases. Furthermore, the cell cycle-regulated kinase Dbf2p physically and functionally associates with the yeast Ccr4-Not complex (Figure 4), but no direct regulation of Ccr4-Not subunits was reported [142]. This is also the case for yeast Ccr4-Not and the mediator Srb9p-Srb10p-Srb11p sub-complex (Figure 4) [198], and for human CNOT2 interaction with CDK11 p46 (caspase-processed C-terminal kinase domain of cyclin-dependent kinase 11) [132].

32 Outline of this thesis Chapter 1 32 The evolutionarily conserved Ccr4-Not complex regulates various steps of mrna metabolism and is essential for important cellular processes such as cell proliferation, adaptations to environmental changes and normal embryogenesis (chapter 1). The aim of the studies described in this thesis is to gain more insight in the functions and regulation of the multi-subunit Ccr4-Not complex. This was performed by studying the composition and localization of human Ccr4-Not complexes, and the phosphorylation of the Ccr4-Not complex in budding yeast. The composition of the Ccr4-Not complex is well-established in yeast, but unknown in human cells with their multiple deadenylases. Chapter 2 describes the identification of the composition and protein interactors of human Ccr4- Not complexes using a combination of biochemical and in-depth proteomic approaches. At least four distinct Ccr4-Not complexes differing in their deadenylase subunits are identified in human cells and CNOT4 resides in a separate protein complex. Involvement of the Ccr4-Not complexes in splicing, transport and localization of RNA molecules is suggested by their associated protein interactors. In chapter 3, the spatial organisation of human GFP-fused Ccr4-Not subunits at near endogenous levels is visualized in stable cell lines by (real-time) fluorescence microscopy. The CNOT proteins localize predominantly to the cytoplasm, with the exception of a cell cycle-dependent nuclear-cytoplasmic distribution of CNOT7 proteins. Moreover, the spatial regulation of Ccr4-Not proteins was explored in a pilot study using leptomycin B. More CNOT7, CNOT8 and CNOT9 proteins were detected in the nucleus upon inhibition of NES-mediated nuclear protein export by this drug. Interestingly, these three Ccr4-Not subunits harbour a predicted NES. The work described in chapter 4 used a combination of molecular biology, biochemistry, mass spectrometry, phenotypical assays and genetics to investigate Not4p phosphorylation in relation to Bur2p of the Bur1/2p kinase complex in yeast. Bur2p and Not4p genetically interact and are required for global H3K4me3 marks, but disruption of all known Not4p phosphorylation sites maintains normal H3K4me3 levels. In addition, Not4p is confirmed as a phospho-protein whose phosphorylation sites are targets of kinases other than Bur1p, indicating that the Bur1/2p kinase complex is not directly involved in Not4p phosphorylation. The five phospho-sites on Not4p are important for resistance to cellular stresses in molecular pathways parallel to BUR2. Finally, chapter 5 summarizes, integrates and discusses the described findings in the current literature.

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42 255. Panasenko, O., Landrieux, E., Feuermann, M., Finka, A., Paquet, N. and Collart, M. A. (2006) The yeast Ccr4-Not complex controls ubiquitination of the nascent-associated polypeptide (NAC-EGD) complex. J Biol Chem 281, Panasenko, O. O., David, F. P. and Collart, M. A. (2009) Ribosome association and stability of the nascent polypeptide-associated complex is dependent upon its own ubiquitination. Genetics 181, Mersman, D. P., Du, H. N., Fingerman, I. M., South, P. F. and Briggs, S. D. (2009a) Polyubiquitination of the demethylase Jhd2 controls histone methylation and gene expression. Genes Dev 23, Albuquerque, C. P., Smolka, M. B., Payne, S. H., Bafna, V., Eng, J. and Zhou, H. (2008) A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics 7, Gnad, F., de Godoy, L. M., Cox, J., Neuhauser, N., Ren, S., Olsen, J. V. and Mann, M. (2009) High-accuracy identification and bioinformatic analysis of in vivo protein phosphorylation sites in yeast. Proteomics 9, Gruhler, A., Olsen, J. V., Mohammed, S., Mortensen, P., Faergeman, N. J., Mann, M. and Jensen, O. N. (2005) Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol Cell Proteomics 4, Moriya, H., Shimizu-Yoshida, Y., Omori, A., Iwashita, S., Katoh, M. and Sakai, A. (2001) Yak1p, a DYRK family kinase, translocates to the nucleus and phosphorylates yeast Pop2p in response to a glucose signal. Genes Dev 15, Xylourgidis, N. and Fornerod, M. (2009) Acting Out of Character: Regulatory Roles of Nuclear Pore Complex Proteins. Dev Cell 17, Cullen, B. R. (2003) Nuclear RNA export. J Cell Sci 116, Collart, M. A. (2003) Global control of gene expression in yeast by the Ccr4-Not complex. Gene 313, 1-16 Chapter 1 42

43 Human Ccr4-Not complexes contain variable deadenylase subunits 2 Adapted from: Biochemical Journal (2009) Volume 422(3): pages

44 Human Ccr4-Not complexes contain variable deadenylase subunits Nga-Chi LAU 1,2, Annemieke KOLKMAN 2,3, Frederik M.A. van SCHAIK 1,2, Klaas W. MULDER 4, W.W.M. Pim PIJNAPPEL 1,2, Albert J.R. HECK 2,3 and H.Th. Marc TIMMERS 1,2 1 Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands, 2 Netherlands Proteomics Centre, Utrecht, The Netherlands, 3 Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands, and 4 Epithelial Cell Biology Laboratory, Cancer Research UK Cambridge Research Institute, Li Ka-Shing Centre, Cambridge, U.K. ABSTRACT Chapter 2 44 The Ccr4-Not complex is evolutionarily conserved and important for regulation of mrna synthesis and decay. The composition of the yeast complex has been described well. Orthologues of the yeast Ccr4-Not components have been identified in human cells including multiple subunits with mrna deadenylase activity. In the present study, we examine the composition of the human Ccr4-Not complex in an in-depth proteomic approach using stable cell lines expressing tagged CNOT proteins. We find at least four different variants of the human complex, consisting of seven stable core proteins and mutually exclusive associated mrna deadenylase subunits. Interestingly, human CNOT4 is in a separate ~200 kda complex. Furthermore, analyses of associated proteins indicate involvement of Ccr4-Not complexes in splicing, transport and localization of RNA molecules. Taken together, human Ccr4-Not complexes are heterogeneous in composition owing to differences in their deadenylase subunits, which may reflect the multi-functionality of these complexes in cellular processes.

45 INTRODUCTION The evolutionarily conserved Ccr4-Not complex is important for multiple cellular functions. It is involved in several aspects of the mrna regulatory pathways (for reviews, see [1, 2]). Besides that, the E3 ligase activity of yeast Not4p and human CNOT4 subunits [3, 4] implicates the Ccr4-Not complex in the protein ubiquitination/degradation pathways [3, 5]. Moreover, members of the Ccr4-Not complex are associated with various functions in several species, both in the nucleus and in the cytoplasm. These include involvement in DNA repair and histone methylation in yeast [6, 7], spindle positioning and regulation of microtubule length in C. elegans [8] and spermatogenesis in mice [9]. The Ccr4-Not complex may influence the synthesis of mrna molecules in several ways. It has been reported that some yeast Ccr4-Not subunits interact both physically and genetically with TBP (TATA-binding protein), TAFs (TBP-associated factors), and other regulators of TBP binding, indicating direct regulation of gene expression at the core promoter through binding of these basal transcription factors (reviewed in [10]). The Ccr4-Not complex may also influence gene transcription by affecting chromatin structure [7, 11, 12] or by repressing the nuclear receptor-mediated transcription in a ligand-dependent manner via CNOT1 [13]. Two other human CNOT proteins, CNOT2 and CNOT9/Rcd1/Rqcd1/hCaf40, are also implicated in repression of promoter activity [11]. The major repression function of CNOT2 was found in a conserved protein motif called the Not-Box, which is also present at the C-terminus of CNOT3 [11]. Furthermore, the Ccr4-Not complex also plays a direct role in mrna degradation and aberrant mrna surveillance (reviewed in [14]). The deadenylase subunits in the complex are responsible for these events. Ccr4p is the major mrna deadenylase in yeast [15, 16], whereas mammalian cells require both CNOT6/hCcr4a and CNOT7/hCaf1 activities for constitutive deadenylation [17]. The composition of the conserved Ccr4-Not complex has been well studied in yeast. Initially, it was identified as physically and functionally separate subcomplexes of Ccr4- associated proteins and of Not proteins [18]. Not1p/Cdc39p, Not2p/Cdc36p, Not3p, Not4p/Mot2p and Not5p form the Not module, which regulates transcription initiation and elongation (reviewed in [1, 2]). They were first isolated as repressors of HIS3 gene transcription from a non-canonical TATA element in yeast (reviewed in [10]). The Ccr4 module encompasses the two deadenylases Ccr4p and Caf1p in association with Caf40p and Caf130p [19, 20], and is involved in mrna degradation. Surprisingly, biochemical analyses revealed that the Ccr4 module also binds Not proteins [19, 20], resulting in the Ccr4-Not protein complex. Interestingly, the Ccr4p subunit affects transcription both Composition of human Ccr4-Not complexes 45

46 Chapter 2 46 negatively and positively [20]. Orthologues for most of the yeast Ccr4-Not components have been identified in human cells [21, 22]. Not3p and Not5p are similar yeast proteins, and the human genome contains only one orthologous protein, CNOT3 [21]. In contrast, two human orthologues have been found for yeast Ccr4p (CNOT6/hCcr4a and CNOT6L/ hccr4b) [23] and for yeast Caf1p (CNOT7/hCaf1 and CNOT8/hPop2/CALIF) [21]. These four deadenylases have been identified as components of human Ccr4-Not complexes [23, 24], but the exact composition of the human complex is less well studied. In the present study, we determine the composition and the interactors of human Ccr4-Not complexes from stable HeLa cell derivatives, which express epitope-tagged Ccr4-Not subunits at near endogenous levels. We identified the interactors of the purified complexes from these cell lines by MS (mass spectrometry). Strikingly, the CNOT7 and CNOT8 proteins specify the distinct human Ccr4-Not complexes, whereby CNOT6 and CNOT6L associate more tightly with CNOT7 than with CNOT8. Besides the known Ccr4-Not proteins, we invariably and abundantly co-purified TAB182 and C2ORF29 proteins in human Ccr4-Not complexes, indicating that these subunits are novel core complex components. Moreover, our results imply that association of CNOT4 with the Ccr4-Not complex may be a regulated event. Furthermore, additional GO (Gene Ontology) analyses of interactors of the complex suggest direct involvement of CNOT7- containing complexes in mrna splicing, transport and localization, whereas CNOT8- containing complexes seem to play a role in splicing only. Taken together, the results of the present study support the model wherein Ccr4-Not complexes are involved from RNA synthesis in the nucleus to RNA decay in the cytoplasm. MATERIALS AND METHODS Antibodies CNOT1, CNOT2 and CNOT3 antibodies have been previously described [13]. Crude serum raised against CNOT8 (described in [21]) were affinity-purified via immunoblotting against a GST (glutathione transferase)-cnot8 fusion protein and elution from the membrane using 100 mm glycine-hcl (ph 2.5). Antibodies directed against CNOT7 (clone 2F6), HA (haemagglutinin; clone 3F10), α-tubulin (DM1A) and TAB182 were obtained from Abnova, Roche Applied Science, Calbiochem and Novus Biologicals respectively. The HA (12CA5) antibody was purified from the hybridoma cell line 12CA5 (A.T.T.C.).

47 Immunoblotting Protein complexes or extracts were separated using SDS-PAGE and were then transferred onto PVDF membranes. The membranes were blocked using 5% (w/v) non-fat dried skimmed milk, probed with primary antibodies overnight at 4 C and then with HRP (horseradish peroxidase)-conjugated secondary antibodies for 45 min at room temperature (21 C). Detection was performed by ECL (enhanced chemiluminiscence) according to the manufacturer s protocol (PerkinElmer Life and Analytical Sciences). cdna sequences of human CNOT genes Human cdnas of CNOT1 (partial), CNOT2, CNOT3S, CNOT3L, CNOT4N, CNOT4L, CNOT8 and CNOT9 have been previously described [11, 21]. cdna sequences of human CNOT6 ORF (open reading frame) was obtained by PCR using pgal4-hccr4 [25] as a template. Full-length cdnas of human CNOT7 and CNOT10 were purchased from RZPD (German Science Centre for Genome Research, Germany). All cdna constructs were verified by DNA sequencing. Generation of stable cell lines Human CNOT cdnas were introduced by GATEWAY cloning [26] into a retroviral destination plasmid derived from pbabe-puro carrying an N-terminal FLAGPreScissionHA-tag (for CNOT2, CNOT3L, CNOT4L, CNOT6, CNOT8, CNOT9 and CNOT10) or a C-terminal HAPreScissionFLAG-tag (for CNOT3S and CNOT7). Details of primer sequences and the cloning strategy are available upon request from the corresponding author. HeLa S3 cell clones expressing a tagged CNOT protein were obtained by retroviral transduction and puromycin selection. Positive clonal cell lines were identified by immunoblotting using antibodies directed against HA, CNOT2, CNOT3, CNOT7 or CNOT8. HeLa S3 cells with integrated empty pbabe-puro plasmid served as a control. Composition of human Ccr4-Not complexes 47 Overexpression in HEK (human embryonic kidney)-293t cells Human CNOT7 and CNOT8 cdnas were introduced by GATEWAY cloning into pmt2sm carrying an N-terminal HA-tag. HEK-293T cells were transfected with 3 µg of pmt2sm-ha-cnot7 or pmt2sm-ha-cnot8 using FuGENE TM 6 according to the manufacturer s instructions (Roche Applied Science). Cells were extracted 40 hrs post-transfection in lysis buffer (20 mm Hepes-NaOH (ph 8.0), 5% (v/v) glycerol, 150 mm NaCl, 0.5 mm EDTA, 5 mm MgCl₂, 0.5% (v/v) Nonidet P40, 0.5 mm PMSF, 0.5 mm DTT (dithiothreitol) and Complete TM protease inhibitor cocktail (Roche Applied Science)) and analysed by immunoblotting.

48 Cell culture conditions HeLa S3 stable cell lines and HEK-293T cells were grown in DMEM (Dulbecco s modified Eagle s medium; 1 g/l glucose) with 10% (v/v) FBS (fetal bovine serum), 2 mm L-glutamine and 100 units/ml penicillin/streptomycin (and 1 µg/ml puromycin for HeLa S3 cells). HeLa S3 cells were grown in bioreactors in MEM (modified Eagle s medium with Hank s balanced salt solution), 10% (v/v) FBS, 2 mm L-glutamine, 1% 100x nonessential amino acids, 100 units/ml penicillin/streptomycin and 1 µg/ml puromycin. All reagents were obtained from Cambrex, except for puromycin (Sigma). Chapter 2 48 Protein extraction, affinity purification and gel filtration HeLa cells were lysed by douncing in hypotonic lysis buffer (10 mm Hepes-NaOH (ph 8.0), 10 mm KCl, 1.5 mm MgCl₂, 0.5 mm DTT and 0.5 mm PMSF). Proteins were extracted in 1/3 volume of 4x extraction buffer (80 mm Hepes-NaOH (ph 8.0), 50% (v/ v) glycerol, 1.68 M NaCl, 0.8 mm EDTA, 3 mm MgCl₂, 1 mm DTT and 4% (v/v) protease inhibitor cocktail (Sigma)), bringing the ionic strength to 420 mm NaCl. Lysates were centrifuged at g for 45 min at 4 C. Supernatants were dialysed overnight against dialysis buffer (20 mm Hepes-NaOH (ph 8.0), 20% (v/v) glycerol, 100 mm KCl, 0.2 mm EDTA, 0.5 mm PMSF and 0.5 mm DTT). Dialysed extracts (10-20 mg/ml) were incubated for 5 hrs with 1/20 volume of M2 affinity resin (Sigma) pre-equilibrated in BC150 buffer (40 mm Tris-HCl (ph 7.9), 20% (v/v) glycerol, 150 mm KCl, 0.2 mm EDTA and 0.1% (v/v) Nonidet P40). The resin was subsequently washed with 60 column volumes of BC300 buffer (20 mm Tris-HCl (ph 7.9), 20% (v/v) glycerol, 300 mm KCl, 0.2 mm EDTA and 0.1% (v/v) Nonidet P40). Proteins were eluted twice with two column volumes of BC300 buffer containing 200 µg/ml FLAG peptide (Sigma). FLAG elution fractions were pooled and subsequently incubated overnight with HA affinity resin pre-equilibrated in BC300 buffer. HA affinity resin was prepared by covalently binding HA (12CA5) antibody to Dynabeads protein A (Dynal Biotech) using dimethyl pimelimidate dihydrochloride (Pierce) according to the manufacturer s instructions. After overnight incubation, resin was washed with 15 resin volumes of BC300 buffer. Bait and interacting proteins were eluted for 20 min at 30 C with 2 mg/ml HA peptide (Sigma-Genosys), followed by precipitation [27] and identification by MS, or proteins were separated on 4-12% Bis-Tris gel (NuPAGE; Invitrogen) for silverstain detection. For affinity purification at lower salt conditions, cells were lysed in hypotonic lysis buffer, dounced and proteins were extracted in 1/3 volume of 4x extraction buffer containing 600 mm NaCl, bringing the ionic strength to 150 mm NaCl, followed by overnight dialysis. Extracts were purified via the FLAG tag as described above, using buffers containing 150 mm NaCl. Purified complexes were subjected to immunoblotting.

49 For gel filtration, cell extracts of the CNOT4L-tagged HeLa S3 stable cell line were prepared as described with the following adaptations: hypotonic lysis buffer also contained 2% (v/v) protease inhibitor cocktail (Sigma), cells were lysed by syringe douncing and proteins were extracted by addition of 1 volume of extraction buffer (20 mm Hepes- NaOH (ph 8.0), 20% (v/v) glycerol, 300 mm NaCl, 0.4 mm EDTA, 1.5 mm MgCl₂, 0.2% (v/v) Nonidet P40 and 0.5 mm DTT). Extracts were analysed on a Superose 6 HR 10/30 column (GE Healthcare) in 20 mm Hepes-NaOH (ph 7.9 at 4 C), 150 mm NaCl, 10 µm ZnCl₂, 1 mm DTT, 0.5 mm PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin and 1 µg/ml pepstatin A, followed by immunoblot analyses of the 0.4 ml fractions. MS/MS Precipitated Ccr4-Not complexes were dissolved in 8 M urea in 50 mm ammonium bicarbonate (ph 8.0), and incubated with 1 µg of LysC (Roche Diagnostics) for 4 hrs at 37 C. Following reduction and alkylation using 2 mm DTT and 4 mm iodoacetamide respectively, the sample was diluted to 2 M urea with 50 mm ammonium bicarbonate (ph 8.0), and incubated overnight with 2 μg of trypsin at 37 C. Protein digests were desalted using a small plug of C₁₈ material (3M Empore C₁₈ extraction disk) packed into a GELoader Tip in a similar manner as described [28]. The eluate was dried completely by vacuum centrifugation and subsequently reconstituted in 20% (v/v) acetonitrile and 0.05% (v/v) formic acid. SCX (strong cation exchange) was performed using two Zorbax BioSCX-Series II columns (internal diameter, 0.8 mm; length, 50 mm; particle size, 3.5 µm), a Famos autosampler (LCpackings), a Shimadzu LC-9A binary pump and a SPD-6A UV-detector (Shimadzu). After injection of the peptides, the first 10 min were run isocratically at 100% solvent A (0.05% (v/v) formic acid in 8:2 (v/v) water-acetonitrile (ph 3.0)), followed by a linear gradient of 1.3% min -1 solvent B (500 mm NaCl in 0.05% (v/v) formic acid in 8:2 (v/v) water-acetonitrile (ph 3.0)). A total number of 24 SCX fractions (1 min each, i.e. a 50 µl elution volume) were collected manually and dried in a vacuum centrifuge. These residues were reconstituted in 20 µl of 0.1 M acetic acid and approximately half of this solution was analysed by on-line nanoflow liquid chromatography FTICR (Fouriertransform ion cyclotron resonance)-ms/ms (tandem MS) as previously described [29]. Protein identification was processed as described in [29] using IPI (International Protein Index) human (release 3.24, sequences entries) and Scaffold software package version 2.1 ( with confidence level of 95% for peptide identification and 99% for protein identification. Unique peptide counts of the identified Ccr4-Not components are given in Supplementary Table S1. The total number of spectral counts for each component was divided by its mass (in Daltons) and converted into NSAFs (normalized spectral abundance factors) as described in [30]. Composition of human Ccr4-Not complexes 49

50 The data associated with this manuscript may be downloaded from ProteomeCommons. org Tranche ( using the following Tranche hash: EKYhwbHIfbis7ocZqc+18hDwmT7WZvSLAWp1dVKjhBWF9W2Ubb8IEScvJq hwzmjqmybpapathb/yvedmnlpmenej10caaaaaaaadpw==. The data can also be downloaded from Chapter 2 50 Bioinformatics IPI numbers of identified proteins (by at least two unique peptides, no peptides in control purification, excluding keratins) were converted to gene symbols using PICR ( ebi.ac.uk/tools/picr/), DAVID ( and UniProt ( Some IPI numbers could not be mapped to a gene and were renamed nm_x (non-mapped_x). A list of genes for each purification was submitted to GeneTrail ([31]; using all human genes as a reference set. GO term over-representation was assessed using a hypergeometric test with FDR (false discovery rate) adjustment and a significance threshold of In addition, a minimum number of two genes per GO term were required for a GO term to be included in the analyses. All statistically significant over-represented GO terms were collected and a matrix was generated consisting of the GO terms and their P values for each of the purifications. P values were transformed to a log₂ scale to reduce the effects of abnormal data distribution during subsequent analyses. Missing values were converted into zero. Matrices containing log₂ transformed P values of over-represented GO terms were loaded into the clustering module (GenesisWeb) of CARMAweb ([32]; to generate hierarchically clustered heatmaps according to Pearson s correlation. The HomoMINT database ( uniroma2.it/homomint/welcome.do; download date 10 June 2009) was used to obtain interologues. Yeast two-hybrid analysis The psh18-34 plasmid, pjg4-5 and peg202-nls vectors, B42-CNOT1C, B42-CNOT2, B42-CNOT4N and B42-CNOT8 (B42-CALIF) were described previously [12, 21]. Other yeast plasmids were created using full-length cdna sequences of human CNOT proteins, except for B42-CNOT7. This latter construct was obtained by insertion of a murine CNOT7 fragment into pjg4-5, derived from pgal4-mcaf1 [33] containing an extended N-terminal part of 11 amino acids and a point mutation to resemble human protein sequences. Details of the cloning strategy are available upon request from the corresponding author. Yeast strain EGY48 (MATa his3 trp1 ura3 LEU2::pLexAop(x6)- LEU2) was used for the present assay [21]. Interactions were visualised on X-Gal (5- bromo-4-chloro-3-indoyl-β-d-galactopyranoside)-containing plates at 30 C.

51 RESULTS Stable HeLa S3 cell lines with FLAG/HA-tagged CNOT proteins under endogenous conditions The human Ccr4-Not complex is biochemically less well defined than its yeast counterpart [18, 23, 24]. To obtain more insight into the composition of the human complex, we generated HeLa cell lines that stably express FLAG/HA-tagged versions of human CNOTs (Ccr4-Not subunits). The expression levels of these tagged proteins within the distinct cell lines were similar with the exception of the more highly expressed CNOT9 (Figure 1A). Immunoblot analyses using specific antibodies against CNOT2, CNOT7 or CNOT8 showed near endogenous expression levels of the tagged protein (Figure 1B). Immunoprecipitation of tagged CNOT3 or CNOT9 resulted in co-purification of the known subunits of the Ccr4-Not complex, such as CNOT2, CNOT3 and CNOT7 (Figure 1C), indicating that the tagged proteins were integrated into larger protein complexes. Notably, similar amounts of CNOT2 and CNOT7 could be co-immunoprecipitated from cell extracts containing tagged CNOT3L or the higher expressed tagged CNOT9 (Figures 1A and 1C). This indicated that the higher expression levels of CNOT9 did not disrupt normal Ccr4-Not complex formation. In addition, silver-stain analysis of large-scale affinity purifications of CNOT3L, CNOT7 and CNOT9 cell lines revealed co-purification of other proteins, which were identified as Ccr4-Not complex subunits by MS (Figure 1D and Table 1). Thus the generated HeLa S3 cell lines expressed their epitope-tagged proteins at near endogenous levels and were integrated into endogenous Ccr4-Not complexes. Composition of human Ccr4-Not complexes 51 Human Ccr4-Not complexes differ in deadenylase subunits To analyse the composition of human Ccr4-Not complexes from different entry points, tagged HeLa S3 cells were grown in bioreactors for large-scale affinity purifications of Ccr4- Not complexes, which were subjected to in-solution tryptic digestion, SCX fractionation and nano-reversed phase liquid chromatography coupled to high resolution MS (LTQ (linear ion trap)-fticr) analyses. Table 1 summarizes the results in NSAFs to indicate the relative abundances of co-purified proteins among the individual CNOT purifications [30] (Supplementary Figure S1 is a coloured representation of Table 1). In the present study, we define a core subunit of the Ccr4-Not complex as being identified by MS with at least an NSAF value of 0.4 in all Ccr4-Not purifications and with no peptides in control purification. We define a subunit as variable when the NSAF value is at least 0.4, but not in all purifications. According to these criteria, the deadenylases CNOT6, CNOT6L, CNOT7 and CNOT8 are variable subunits within the human complex and CNOT4 E3 ligase is not an integral subunit of human Ccr4-Not (Table 1). CNOT1, CNOT2, CNOT3,

52 A [kda] * CNOT2 CNOT3L * * CNOT6 CNOT7 CNOT3S * CNOT9 CNOT10 Control CNOT4L CNOT8 * * α-ha B CNOT2 Control α-cnot2 CNOT7 Control α-cnot7 CNOT8 Control Tagged Endogenous α-cnot * * * α-α-tubulin C CNOT3L Input CNOT9 Control Bound CNOT3L CNOT9 Control α-cnot2 D [kda] 200 CNOT7 CNOT9 Control CNOT3L Chapter 2 52 E CNOT3L input FT bound input CNOT9 FT bound input Control FT bound α-cnot3 α-cnot7 α-tab * * * Figure 1. Integration of FLAG/HA-tagged CNOT proteins in endogenous Ccr4-Not complexes (A) Cell extracts from stable human cell lines expressing the indicated tagged CNOT protein were analysed by immunoblotting using anti-ha antibodies. Asterisks indicate epitope-tagged proteins. Detection of α-tubulin serves as a loading control. Control cells are HeLa S3 stably transfected with pbabe-puro. Molecular mass markers are indicated in kda on the left-hand side of the gel. (B) Some extracts from (A) were analysed by specific antibodies. Both endogenous and exogenous tagged proteins can be detected. (C) Immunoblot analyses of FLAG/HA purifications of CNOT3L, CNOT9 or control cells using specific antibodies. (D) Silver stain of purified Ccr4-Not complexes. Asterisks indicate epitope-tagged CNOT3L, CNOT7 and CNOT9 protein bands respectively from left to right. Molecular mass markers (kda) are given on the left-hand side of the gel. (E) Cell extracts of CNOT3L, CNOT9 and control cell lines were FLAG/HA-purified and analysed by immunoblotting using specific antibodies against TAB182. FT (flow through) is also shown.

53 CNOT9, CNOT10 and the new interactors TAB182 and C2ORF29 were identified as core subunits of the human complexes (Table 1). The C2ORF29 protein is predicted from the human genome sequence with no known function and TAB182 has been described as a binding protein for tankyrase 1, which plays a role in telomere length and mitotic spindle composition [34, 35]. Immunoblot analysis showed that a small but significant, proportion of cellular TAB182 co-purified with CNOT3L and CNOT9 complexes (Figure 1E), confirming that TAB182 is a stable interactor of the human Ccr4-Not complex. Unfortunately, no antibodies against C2ORF29 are available, precluding confirmation by Western blot analysis. The detailed MS results revealed several interesting properties of human Ccr4-Not complexes (Table 1). In particular, the variable deadenylase subunits showed interesting differences among the tagged CNOT6, CNOT7 and CNOT8 purifications (shown in bold in Table 1). No significant CNOT7 was detected in CNOT8 purifications and vice versa, suggesting that CNOT7 and CNOT8 are present in distinct Ccr4-Not complexes. CNOT6 and CNOT6L were identified in CNOT7 purifications, whereas they were absent from CNOT8 purifications, suggesting that CNOT6/6L proteins stably interact with CNOT7, but not with CNOT8. No CNOT6L could be identified in two CNOT6 purifications (Table 1 and the Tranche database), indicating mutual exclusiveness of these subunits as expected from their distinct functions; CNOT6L, but not CNOT6, impairs cell growth [23]. It is interesting to note that the deadenylases CNOT6, CNOT6L, CNOT7 and CNOT8 are relatively lower represented in the purifications than the other CNOT proteins, except when they are the bait of the purification. Moreover, the deadenylase subunits could not be detected in CNOT10-purified complexes. This could, however, be due to the lower amounts of recovered proteins in this purification. Importantly, Ccr4-Not complexes obtained via other tagged subunits inversely identified CNOT10. Furthermore, comparison of full-length CNOT3L (residues 1-753) to CNOT3S lacking the Not-Box (residues 1-609) showed that the smaller isoform did not interact with any Ccr4-Not subunit. This indicates that the Not-Box is required for the stable interaction of CNOT3 with the rest of the complex [11, 21, 36]. Finally, human CNOT4 could hardly be detected in purified Ccr4-Not complexes in contrast with yeast [18, 20], indicating that CNOT4 is not stably integrated within human complexes. In conclusion, purified Ccr4- Not complexes from human cells using seven different entry points indicate the existence of several Ccr4-Not complexes differing only in the deadenylase subunits CNOT6, CNOT6L, CNOT7 and CNOT8. Composition of human Ccr4-Not complexes 53

54 Chapter 2 54 Table 1. NSAFs of identified Ccr4-Not components FLAG/HA affinity-purified complexes of a tagged Ccr4-Not subunit were analysed by MS. The control is a HeLa S3 cell line stably transfected with pbabe-puro. Numbers represent 10-fold NSAFs (as described in [30]) for identified proteins. The molecular masses (kda) of these interactors are given in the right-hand column. The sum of unique peptides and sum of all identified peptides of identified proteins per purification are shown. Bold numbers indicate the NSAF values of deadenylase subunits in the purified Ccr4-Not complexes using a tagged deadenylase. The numbers in italics indicate the NSAF values of the tagged CNOT protein. NSAF Tagged protein Mol. mass Identified protein CNOT2 CNOT3L CNOT6 CNOT7 CNOT8 CNOT9 CNOT10 Control CNOT3S (kda) CNOT CNOT CNOT CNOT CNOT CNOT6L CNOT CNOT CNOT CNOT TAB C2ORF Sum of unique peptides Sum of all identified peptides

55 Analyses of substoichiometric interactors suggest Ccr4-Not functioning in RNA splicing and nuclear export Besides subunits of Ccr4-Not complexes, the MS results identified 498 proteins with at least two unique peptides and no peptides in control purification. These proteins have low NSAF values and are not present in all purifications. We propose that these 498 proteins represent substoichiometric interactors of tagged Ccr4-Not proteins (Supplementary Table S2). Approximately 47% of these associated proteins were shared among the deadenylase subunits and the other CNOT proteins, indicating that almost half of them bind both the deadenylases as well as transcriptional Ccr4-Not subunits. In addition, 70% of the interactors were co-purified by at least two CNOT proteins, 44% by at least three Ccr4- Not components, and 26% by at least four subunits. Importantly, these proteins were not identified in control purification, suggesting that these substoichiometric interactors are relevant for the function of the Ccr4-Not complex. We searched the HomoMINT database [37] for interologues to find seven interactors shared between our MS/MS-based human dataset and the yeast dataset. These shared interologues are rather diverse in function and we find only a few peptides of each interactor (Supplementary Table S3). To gain insight into the biological functions of human Ccr4-Not complexes, GO term over-representation analyses were performed with the CNOT subunits and the substoichiometric Ccr4-Not interactors. The results are given in Supplementary Figure S2 of which the interesting clusters are represented in Figure 2. Clusters containing gene transcription and RNA stability-related terms were identified (Figures 2A and 2B), indicating that expected functions of Ccr4-Not can be retrieved from these data analyses. Interestingly, many subunits gave association with mrna splicing (Figure 2C), and mrna transport and localization (Figure 2D), except for CNOT6 and CNOT8 respectively. Moreover, CNOT6, CNOT8 and CNOT10 have low probability to interact with the nuclear envelope (Figures 2E and 2F), suggesting that these proteins may not be involved directly in nuclear mrna export. Most Ccr4-Not subunits co-purified NUPs (nuclear pore complex proteins) located at the nuclear basket, the cytosolic filaments and inside the nuclear membrane (Supplementary Figure S3). The majority of the NUPs inside the nuclear membrane were co-purified. However, more peptides were retrieved from the identification of NUPs on the cytosolic or nuclear membrane side (Supplementary Figure S3). This suggests that the Ccr4-Not complex may interact with the intact nuclear pore complex. Thus GO term over-representation of substoichiometric interactors indicates physical interactions of human Ccr4-Not complexes with proteins involved in mrna processing and nuclear export, suggesting involvement of Ccr4-Not in these cellular processes. Composition of human Ccr4-Not complexes 55

56 Chapter 2 56 A B C D E F CNOT2 CNOT3L CNOT6 CNOT7 CNOT8 CNOT9 CNOT10 CNOT3S cellular biopolymer metabolic process gene expression nucleobase, nucleoside, nucleotide and nucleic acid metabolic process RNA metabolic process regulation of gene expression regulation of macromolecule metabolic process regulation of cellular metabolic process regulation of metabolic process regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process transcription regulation of RNA metabolic process RNA biosynthetic process transcription, DNA-dependent regulation of macromolecule biosynthetic process regulation of transcription regulation of transcription, DNA-dependent DNA binding DNA-directed RNA polymerase complex nuclear DNA-directed RNA polymerase complex RNA polymerase complex transporter activity RNA 3 -end processing mrna 3 -end processing mrna polyadenylation RNA polyadenylation low-density lipoprotein receptor binding single-stranded DNA binding cellular macromolecule catabolic process macromolecule catabolic process mrna stabilization RNA stabilization mrna binding regulation of mrna stability regulation of RNA stability regulation of transcription from RNA polymerase II promoter transcription factor binding transcription from RNA polymerase II promoter ribonucleoprotein complex mrna metabolic process mrna processing RNA processing RNA splicing nuclear mrna splicing, via spliceosome RNA splicing, via transesterification reactions RNA splicing, via transesterification reactions with bulged adenosine as nucleophile spliceosome mrna transport nucleobase, nucleoside, nucleotide and nucleic acid transport RNA localization establishment of RNA localization nuclear pore pore complex nuclear import protein import into nucleus nuclear envelope protein localization establishment of protein localization nuclear lumen intracellular organelle lumen membrane-enclosed lumen organelle lumen membrane part nucleoplasm part nucleoplasm intracellular organelle organelle nucleic acid binding biopolymer metabolic process intracellular membrane-bounded organelle membrane-bounded organelle nucleus metabolic process cellular metabolic process cellular macromolecule metabolic process

57 CNOT1 binds CNOT4, and CNOT6 binds CNOT8 in yeast two-hybrid assays To further explore the composition of human Ccr4-Not complexes, we investigated binary interactions between known components of the complex by yeast two-hybrid interaction experiments [38]. Instead of using full-length CNOT1 protein, this large protein (2376 residues) was split up into smaller fragments (Figure 3A). Positive binary interactions between a B42-fusion and a LexA-fusion CNOT protein in yeast are shown in Figure 3B. The majority of the subunits, except for CNOT3L and CNOT6, interacted with the C- terminal part of CNOT1 (CNOT1L: residues ). This is in concordance with earlier findings that a shorter C-terminal CNOT1 (CNOT1C: residues ) interacts with CNOT2, CNOT4N and CNOT8 [21]. In addition, CNOT2 and CNOT9 interacted with the N-terminus of CNOT1 (CNOT1N: residues 1-716). These observations indicate a scaffold function for CNOT1 as suggested previously for its yeast orthologue Not1p [18]. Moreover, none of the examined subunits interacted with themselves, suggesting that they occur as monomers. Interestingly, CNOT3L only interacted with CNOT2 (Figure 3B), suggesting that CNOT3 integrates within the Ccr4-Not complex by CNOT2 interaction. Furthermore, CNOT6 interacted with CNOT7 and vice versa. Also, CNOT6 showed a binary interaction with CNOT8 in this assay, whereby the reciprocal interaction could not be performed owing to self-activation properties of LexA-CNOT8. It is interesting to note that CNOT4N and CNOT1L also interacted in this assay. CNOT4 resides in a smaller complex outside the human Ccr4-Not complex To further investigate the association of CNOT4 with the Ccr4-Not complex, we have generated a cell line that stably expresses the largest isoform of CNOT4, CNOT4L [21]. Extracts of these cells were separated by gel filtration under physiological salt conditions and were subjected to immunoblot analyses (Figure 4). Tagged CNOT4L protein resided in a ~200 kda complex, whereas the subunits CNOT1, CNOT3, CNOT7 and CNOT8 coeluted in fractions corresponding to ~1.2 MDa. In addition, a small fraction of CNOT8 and of CNOT2 was seen at a single protein size (results not shown). It would be interesting to determine the composition of the ~200 kda complex. However, the low abundance of this complex precluded our first attempts in this direction (results not shown). Composition of human Ccr4-Not complexes 57 Figure 2. GO term over-representation clusters of Ccr4-Not interactors Interactors identified by at least two unique peptides and without peptides in control purification (keratins excluded) were analysed for GO term over-representation (see also Supplementary Figure S2 at Colours represent the log₂ P values of Pearson s correlation clustering multiplied by -1 on a scale of (A) Transcription cluster. (B) RNA stability cluster. (C) Splicing cluster. (D) RNA transport and localization cluster. (E) Nuclear envelope cluster. (F) Nuclear localization cluster.

58 A CNOT1 CNOT1N (1-716) CNOT1L ( ) CNOT1C ( ) B LexA CNOT1L CNOT1N CNOT3L CNOT6 CNOT7 Chapter 2 58 B42 CNOT1C CNOT2 CNOT3L CNOT4N CNOT6 CNOT7 CNOT8 CNOT9 Figure 3. Yeast two-hybrid interactions between CNOT proteins (A) A schematic representation of CNOT1 constructs is given. CNOT1N represents the N-terminal part (residues 1-716), CNOT1L contains the C-terminal part (residues ) and CNOT1C is a shorter C-terminal form (residues ). (B) Yeast strain EGY48 was transformed with the indicated LexA- and B42-fusion expression plasmids together with a LacZ reporter gene containing eight LexA operator sites. Self-activation of LexA-CNOT2, LexA-CNOT4N, LexA-CNOT8 and LexA-CNOT9 fusions (results not shown) prevented their inclusion as DNA-binding domain fusions. Transformants were spotted on X-Gal-containing plates and incubated for 2 days (LexA-CNOT1L and LexA-CNOT1N) or for 1 day (LexA-CNOT3L, LexA-CNOT6, LexA-CNOT7 and LexA) at 30 C. The different shades of grey in the figure indicate the blue colouring of yeast cells showing binary interactions between LexA- and B42-fusion proteins.

59 Taken together, gel-filtration analyses confirm the MS results that CNOT4 is not stably interacting with human Ccr4-Not components, but rather forms a distinct protein complex in human cells. The identity of CNOT4-associated subunits could reveal how interaction of CNOT4 with the Ccr4-Not complex would be regulated. input 2000 kda 450 kda 158 kda 67 kda 45 kda input α-ha-cnot4 α-cnot1 α-cnot3 α-cnot7 α-cnot8 Figure 4. CNOT4 resides in a smaller protein complex Cell extracts were subjected to gel filtration under conditions of 150 mm NaCl, and Ccr4-Not subunits were detected by specific antibodies. Input samples were loaded onto the far left-hand and right-hand lanes. Numbers across the top indicate elution fractions. Calibration proteins are given in kda with arrows: Blue Dextran (2000 kda), ferritin (450 kda), aldolase (158 kda), BSA (67 kda) and ovalbumin (45 kda). Composition of human Ccr4-Not complexes 59 CNOT6 interacts stronger with CNOT7 than with CNOT8 To further analyse the interactions among the four human deadenylases, we first determined the specificity of antibodies directed against CNOT7 or against CNOT8 as these deadenylases are 75% identical in their primary structure. Upon transient overexpression of CNOT7 and/or CNOT8, the CNOT7 antibodies specifically recognized CNOT7 (Figure 5A, first panel), whereas antibodies directed at CNOT8 (Figure 5A, second panel) detected both CNOT7 (upper band) and CNOT8 (lower band). The CNOT7 antibodies also specifically recognized endogenous CNOT7 in the stable cell lines (Figure 5A, fourth panel), whereas the CNOT8 antibodies detected endogenous CNOT8, but also displayed considerable background bands (Figure 5A, fifth panel). Thus the specificity of CNOT7 antibodies is exclusive for CNOT7 proteins, and antibodies directed against CNOT8 detect both CNOT7 and CNOT8 subunits, which can be distinguished by different mobilities in protein gels.

60 A Transient overexpression CNOT7 CNOT8 CNOT7+8 B Input CNOT6 CNOT7 CNOT8 CNOT2 Control Bound CNOT6 CNOT7 CNOT8 CNOT2 Control 7 α-cnot7 α-cnot α-cnot8 α-ha * * α-cnot8 α-cnot2 Stable cell lines 7-tag 7-endo CNOT7 CNOT8 Control α-cnot7 C Input Bound CNOT6 CNOT2 Control CNOT6 CNOT2 Control Chapter 2 8-tag 7-endo 8-endo 7-tag 8-tag α-cnot8 α-ha * * * * α-cnot7 α-cnot8 60 α-cnot3 α-ha Figure 5. CNOT6 interacts more strongly with CNOT7 than with CNOT8 (A) Cell extracts of HEK-293T cells with transiently overexpressed deadenylases (upper three panels) or stable HeLa cell line extracts (lower three panels) were immunoblotted using antibodies directed against CNOT7, CNOT8 or HA. Tag (tagged) or endo (endogenous) CNOT7 and CNOT8 proteins are indicated by arrows. (B) Extracts of cells containing epitope-tagged CNOT proteins or control cell extracts were affinity-purified and analysed by specific antibodies. Asterisks indicate the crossreactivity of the CNOT8 antibody with the CNOT7 protein. (C) Cell extracts of CNOT6, CNOT2 or control cells were FLAG-epitope affinity-purified under conditions of 150 mm NaCl (left of triangle) or 300 mm NaCl (right of triangle). Purified complexes were analysed by immunoblotting using specific antibodies. Asterisks indicate the cross-reactivity of the CNOT8 antibody with CNOT7 protein.

61 Immunoblot analyses using these antibodies showed the absence of CNOT7 proteins in CNOT8-purified complexes, and no CNOT8 proteins could be detected in CNOT6- and CNOT7-purified complexes (Figure 5B). As expected, both CNOT7 and CNOT8 were present in CNOT2-purified complexes (Figure 5B). These observations confirm the MS results (Table 1 and Supplementary Table S1) of the mutual exclusiveness of CNOT7 and CNOT8 within Ccr4-Not complexes. In addition, the interaction between CNOT6 and CNOT8 was further studied by immunoblot analyses using the described antibodies (Figure 5C). Yeast two-hybrid analysis showed CNOT6 binding to CNOT8, whereas MS results did not reveal CNOT6 in CNOT8 complexes and vice versa (Table 1 and Supplementary Table S1). One possibility is the loss of the CNOT6-CNOT8 interaction during the two-step purification prior to MS identification. To address this hypothesis, one-step FLAG purifications of Ccr4-Not complexes were performed under low- or high-salt concentrations (Figure 5C). The association of CNOT7 within CNOT6- or CNOT2-purified complexes was not sensitive to salt, indicating strong binding of CNOT7 to these complexes. Notably, CNOT8 could be detected just above background in CNOT6-purified complexes under low-salt conditions only, whereas CNOT8 was clearly detectable in CNOT2-purified complexes independent of the salt concentration. Taken together, this indicates that the interaction between CNOT6 and CNOT8 is sensitive to the salt concentration. Unfortunately, the lack of CNOT6 antibodies does not allow us to perform the converse experiment using CNOT7- and CNOT8-purified complexes. In conclusion, these experiments confirm that CNOT7 and CNOT8 deadenylases are mutually exclusive subunits of human Ccr4-Not complexes, whereby CNOT6 seems to interact less stably with CNOT8 than with CNOT7. Composition of human Ccr4-Not complexes 61 DISCUSSION In the present study, we have investigated the composition and the protein interactors of human Ccr4-Not complexes by an in-depth proteomic effort. We have defined the core proteins of human Ccr4-Not complexes including two new subunits, TAB182 and C2ORF29. These complexes are heterogeneous in composition by their associated mrna deadenylase subunits, which may reflect functional differences in coupling mrna synthesis and processing to nuclear export. Size-fractionation experiments have shown that the human CNOT4 E3 ligase resides in a separate ~200 kda protein complex. Interestingly, yeast two-hybrid assay displayed a CNOT4 interaction with the scaffold protein CNOT1. Moreover, CNOT3 requires its Not-Box to integrate into the human Ccr4-Not complex via CNOT2. Taken together, the results of the present study define

62 the composition of human Ccr4-Not complexes and have revealed the co-existence of multiple Ccr4-Not complexes differing in their deadenylase subunits. Defining the human Ccr4-Not complexes In the past, the components and the composition of the yeast Ccr4-Not complex were revealed by a variety of biochemical purifications and genetic experiments [18-20]. Gel filtration of the yeast complex revealed two peaks of 1.9 MDa and 1.0 MDa [18, 19]. In contrast, we have observed a rather broad peak for the human complex at ~1.2 MDa (Figure 4). Gavin et al. [39] purified the human Ccr4-Not complex via a TAP-tagged version of CNOT2, but they failed to identify CNOT3, CNOT4 and CNOT6L as Ccr4- Not components. Our MS results indicate a core of seven subunits for human Ccr4-Not complexes consisting of CNOT1, CNOT2, CNOT3, CNOT9, CNOT10, TAB182 and C2ORF29 with the associated deadenylases CNOT6, CNOT6L, CNOT7 and CNOT8 (Figure 6). The combined molecular mass of these components in the human complex is ~0.9 MDa, which corresponds with the gel-filtration results (Figure 4). Chapter 2 62 CNOT7-CNOT6 complex CNOT4 CNOT3 CNOT7 CNOT6 CNOT2 CNOT1 TAB182 CNOT9 CNOT10 C2ORF29 CNOT8-CNOT6 complex CNOT4 CNOT6 CNOT3 CNOT8 CNOT2 CNOT1 TAB182 CNOT9 CNOT10 C2ORF29 CNOT7-CNOT6L complex CNOT4 CNOT8-CNOT6L complex CNOT4 CNOT3 CNOT7 CNOT6L CNOT2 CNOT1 TAB182 CNOT9 CNOT10 C2ORF29 CNOT3 CNOT8 CNOT2 CNOT1 TAB182 CNOT9 CNOT10 C2ORF29 CNOT6L Figure 6. Model of human Ccr4-Not complexes differing in deadenylases Stable core subunits are shown in grey ovals and the regulated CNOT4 subunit is given in white. Variable deadenylases between complexes are indicated in black, and the arrow between CNOT6/ CNOT6L and CNOT8 indicate their less stable interaction.

63 The overall composition of human Ccr4-Not complexes is similar to the yeast complex [19]. We found that several CNOT proteins (CNOT2, CNOT7, CNOT8 and CNOT9) interact with the largest subunit of the complex, CNOT1 (Figure 3B), indicating that this protein acts as a scaffold similar to yeast Not1p. However, we have noted several differences between the human and yeast Ccr4-Not complexes. First, human cells contain multiple deadenylases, which have different binding properties. Our salt titration experiments indicate that CNOT6 interacted less stably with CNOT8 compared to CNOT7 (Figure 5C). Notably, CNOT6 only showed binary interactions with CNOT7 and CNOT8 in a yeast two-hybrid assay (Figure 3B), indicating that these two deadenylases link CNOT6 to the rest of the Ccr4-Not complex as proposed in [21, 40]. Secondly, CNOT3 did not bind directly to CNOT1 as reported for the yeast Ccr4-Not complex [18, 19], but it did bind CNOT2 (Figure 3B). Moreover, MS analyses showed that CNOT3 lacking the Not- Box (CNOT3S) could not interact with any Ccr4-Not subunits (Table 1). This suggests that CNOT3 integrates in the human complex via a Not-Box-mediated interaction with CNOT2. Finally, in contrast to yeast Not4p [18, 19], CNOT4 was not stably integrated in human Ccr4-Not complexes (Table 1 and Figure 4), but it could interact with the scaffold protein CNOT1 in a yeast two-hybrid assay (Figure 3B). In addition, human CNOT4 is a true orthologue of yeast NOT4 as it can complement not4δ not5δ lethality in yeast [21]. This suggests that the CNOT4 interaction with the Ccr4-Not complex may be a regulated event in human cells. Possibly, association of a core subunit precludes interaction of CNOT4 with CNOT1. It is also possible that interactors of CNOT4 or post-translational modifications are involved in restricting CNOT4 integration into the human complex. A model of the distinct Ccr4-Not complexes in human cells is depicted in Figure 6. Composition of human Ccr4-Not complexes 63 Multiple human Ccr4-Not complexes differing in mrna deadenylase subunits The yeast Ccr4-Not complex harbours two deadenylases, Ccr4p and Caf1p, of which Caf1p is dispensable for Ccr4p deadenylase activity [15, 16]. Human cells contain two orthologues of yeast Ccr4p, CNOT6/hCcr4a and the recently identified CNOT6L/hCcr4b, and two homologous Caf1p orthologues, CNOT7 and CNOT8. All of these Ccr4p/Caf1p orthologues display in vitro 3-5 poly(a) exoribonuclease activity [23, 41]. The results of the present study revealed that the highly homologous CNOT7 and CNOT8 proteins are in separate complexes (Table 1 and Figure 5B). Only a small portion of CNOT8- containing complexes harbour CNOT6, which seems to be less stable than the CNOT6 interaction in CNOT7-containing complexes (Figure 5C). It is plausible that CNOT7 and CNOT8 are competing for the same binding site on CNOT1 allowing association of either CNOT7 or CNOT8. In addition, CNOT6 and CNOT6L may also be mutually exclusive as no unique peptides of CNOT6L were found in two purifications of CNOT6- tagged complexes (Table 1 and the Tranche database). This is also supported by earlier

64 results showing that CNOT6L is functionally distinct from CNOT6 [23]. Furthermore, our results showed co-purification of CNOT6-CNOT7 and CNOT6L-CNOT7 (Table 1), which was previously reported in a transient overexpression approach [40]. Thus all of these findings support a model of distinct Ccr4-Not complexes allowing only a single Ccr4p and a single Caf1p orthologue (Figure 6). It is interesting to note that the substrate specificity for the enzymatic activity of CNOT7 and CNOT8 is different [41], which may allow differential regulation of mrna degradation. Chapter 2 64 Human Ccr4-Not complexes play various roles in mrna synthesis to degradation GO term over-representation analyses of associated Ccr4-Not proteins provided insight into the functional roles of human Ccr4-Not complexes such as in gene transcription and RNA stability (Figures 2A and 2B). A role in mrna transport and localization has also been indicated for CNOT7-, but not for CNOT8-, containing complexes (Figures 2D- 2F). Indeed, multiple NUPs interacted with all CNOT proteins tested, except for CNOT8 (Supplementary Figure S3). Interestingly, CNOT6 and CNOT7 proteins were reported to shuttle between the nucleus and cytoplasm [40]. In general, more peptides were retrieved from nuclear and cytosolic NUPs than for NUPs within the nuclear envelope (Supplementary Figure S3). This may indicate a shuttling function of the Ccr4-Not complex, which may link its nuclear and cytosolic functions. In addition, involvement in mrna splicing is also suggested by GO term over-representation analyses, which included the CNOT8-containing complexes (Figure 2C). Taken together, these findings support the view that human Ccr4-Not complexes can influence the mrna metabolism at multiple levels. In conclusion, the present study provides comprehensive composition analyses of Ccr4- Not complexes in human cells. We have identified multiple Ccr4-Not complexes differing in their deadenylase subunits and in complex stability. Our proteomic analyses provide a platform for further functional studies of human Ccr4-Not complexes defining the regulation of mrna metabolism by this evolutionarily conserved protein complex.

65 ACKNOWLEDGEMENTS We thank Dr. M.A. Collart (University of Geneva, Geneva, Switzerland) for the gift of the CNOT1 antibody, Dr. V.J. Bardwell (University of Minnesota, MN, U.S.A.) for the partial CNOT1 cdna constructs and Dr. T. Aoki (Chiba University, Chiba, Japan) for the gift of the CNOT3 antibody. We also thank Fred Koch for technical assistance in generating and purifying the HeLa S3 stable cell line expressing CNOT8, and Danny Navarro for the submission of the Scaffold data to the ProteomeCommons/Tranche database. We are grateful to Dr. P. Kemmeren (University Medical Center Utrecht, Utrecht, The Netherlands) for bioinformatic assistance. We thank members of the Timmers and Heck laboratories for discussions, and Dr. Nikolay Outchkourov and Loes van de Pasch for critical reading of this manuscript prior to submission. Composition of human Ccr4-Not complexes 65

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67 Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol 328, Wessel, D. and Flugge, U. I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138, Gobom, J., Nordhoff, E., Mirgorodskaya, E., Ekman, R. and Roepstorff, P. (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 34, Mousson, F., Kolkman, A., Pijnappel, W. W., Timmers, H. T. and Heck, A. J. (2008) Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes. Mol Cell Proteomics 7, Paoletti, A. C., Parmely, T. J., Tomomori-Sato, C., Sato, S., Zhu, D., Conaway, R. C., Conaway, J. W., Florens, L. and Washburn, M. P. (2006) Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc Natl Acad Sci U S A 103, Backes, C., Keller, A., Kuentzer, J., Kneissl, B., Comtesse, N., Elnakady, Y. A., Muller, R., Meese, E. and Lenhof, H. P. (2007) GeneTrail--advanced gene set enrichment analysis. Nucleic Acids Res 35, W Rainer, J., Sanchez-Cabo, F., Stocker, G., Sturn, A. and Trajanoski, Z. (2006) CARMAweb: comprehensive R- and bioconductorbased web service for microarray data analysis. Nucleic Acids Res 34, W Draper, M. P., Salvadore, C. and Denis, C. L. (1995) Identification of a mouse protein whose homolog in Saccharomyces cerevisiae is a component of the CCR4 transcriptional regulatory complex. Mol Cell Biol 15, Seimiya, H. and Smith, S. (2002) The telomeric poly(adp-ribose) polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor, 182-kDa tankyrase-binding protein (TAB182). J Biol Chem 277, Chang, P., Coughlin, M. and Mitchison, T. J. (2005) Tankyrase-1 polymerization of poly(adp-ribose) is required for spindle structure and function. Nat Cell Biol 7, Aoki, T., Okada, N., Wakamatsu, T. and Tamura, T. A. (2002) TBP-interacting protein 120B, which is induced in relation to myogenesis, binds to NOT3. Biochem Biophys Res Commun 296, Persico, M., Ceol, A., Gavrila, C., Hoffmann, R., Florio, A. and Cesareni, G. (2005) HomoMINT: an inferred human network based on orthology mapping of protein interactions discovered in model organisms. BMC Bioinformatics 6 Suppl 4, S21 Ito, T., Ota, K., Kubota, H., Yamaguchi, Y., Chiba, T., Sakuraba, K. and Yoshida, M. (2002) Roles for the two-hybrid system in exploration of the yeast protein interactome. Mol Cell Proteomics 1, Gavin, A. C., Bosche, M., Krause, R., Grandi, P., Marzioch, M., Bauer, A., Schultz, J., Rick, J. M., Michon, A. M., Cruciat, C. M., Remor, M., Hofert, C., Schelder, M., Brajenovic, M., Ruffner, H., Merino, A., Klein, K., Hudak, M., Dickson, D., Rudi, T., Gnau, V., Bauch, A., Bastuck, S., Huhse, B., Leutwein, C., Heurtier, M. A., Copley, R. R., Edelmann, A., Querfurth, E., Rybin, V., Drewes, G., Raida, M., Bouwmeester, T., Bork, P., Seraphin, B., Kuster, B., Neubauer, G. and Superti-Furga, G. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415, Yamashita, A., Chang, T. C., Yamashita, Y., Zhu, W., Zhong, Z., Chen, C. Y. and Shyu, A. B. (2005) Concerted action of poly(a) nucleases and decapping enzyme in mammalian mrna turnover. Nat Struct Mol Biol 12, Bianchin, C., Mauxion, F., Sentis, S., Seraphin, B. and Corbo, L. (2005) Conservation of the deadenylase activity of proteins of the Caf1 family in human. RNA 11, Composition of human Ccr4-Not complexes 67

68 Supplementary Figures and Tables See for online Supplementary Figures and Tables Figure S1. Heatmap of NSAFs of identified Ccr4-Not components Figure S2. Heatmap of GO term over-representation of Ccr4-Not interactors Figure S3. Heatmap of Ccr4-Not interacting components of the nuclear pore complex Table S2. Overview of all identified proteins in Ccr4-Not complex purifications Table S1. Unique peptide counts of identified Ccr4-Not components after MS analyses of purified Ccr4-Not complexes using distinct entry points Chapter 2 68 Unique peptide Tagged protein Mol. Identified protein CNOT2 CNOT3L CNOT6 CNOT7 CNOT8 CNOT9 CNOT10 Control CNOT3S mass (kda) CNOT CNOT CNOT CNOT CNOT CNOT6L CNOT CNOT CNOT CNOT TAB C2ORF Sum Table S3. Overlap of substoichiometric Ccr4-Not interactors with yeast interologues from the HomoMINT database Unique peptide counts of the overlapping Ccr4-Not interactors (identified protein given as gene name) after MS analyses of purified Ccr4-Not complexes using the indicated tagged protein are shown. Unique peptide Tagged protein Mol. Identified protein CNOT2 CNOT3L CNOT6 CNOT7 CNOT8 CNOT9 CNOT10 Control CNOT3S mass (kda) CHMP2A COPA AHSA GCN1L CCT6A NACA RPS27A

69 Subcellular localization of human Ccr4-Not subunits 3

70 Subcellular localization of human Ccr4-Not subunits Nga-Chi LAU, Frederic KOCH and H.Th. Marc TIMMERS Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands ABSTRACT Chapter 3 70 The evolutionarily conserved Ccr4-Not complex is involved in multiple steps of mrna metabolism in the nucleus and cytoplasm. To visualize the spatial regulation of Ccr4-Not subunits within living cells, stable U2OS cell lines have been generated that express human GFP-tagged CNOT proteins at near endogenous levels. It appears that the CNOT proteins localize predominantly to the cytoplasm, whereas CNOT7 are detected in the nucleus as well. Exposure of the generated U2OS cell lines to leptomycin B an inhibitor of the CRM1-mediated nuclear export increases the detection of CNOT7, CNOT8 and CNOT9 in the nucleus. These three Ccr4-Not components harbour a predicted nuclear export signal, suggesting a CRM1-regulated subcellular localization. Spatial regulation of the Ccr4-Not complex may be an important determinant of its cellular activities in gene expression.

71 INTRODUCTION The Ccr4-Not complex is conserved from yeast to man and is important for multiple steps in mrna metabolism (reviewed in [1, 2]). The subunits of human Ccr4-Not complexes were first implicated in the regulation of gene transcription in the nucleus [3-5]. The Ccr4-Not complex also harbours multiple deadenylases, which are required for bulk removal of the poly(a) tail of mrna molecules in the cytoplasm [6-8]. In addition, indepth proteomic analyses of human Ccr4-Not complexes identified interactors, such as the hnrnps (heterogeneous nuclear ribonucleoproteins) and the nuclear pore complex, which further support their link to mrna processes in both compartments of the cell [9]. The hnrnps are believed to travel along with mrna molecules to regulate events in both nucleus as cytoplasm [10], whereas the nuclear pore complex is composed of nucleoporins that are implicated in protein and RNA transport over the nuclear membrane [11]. Selective nucleocytoplasmic transport through the nuclear pore complex is important for eukaryotes. Transport receptors are required to facilitate protein transport in a Randependent manner [12]. Importins facilitate nuclear import of proteins with a specific NLS (nuclear localization signal), whereas proteins containing a NES (nuclear export signal) are exported to the cytoplasm by exportins [12]. A specific class of exportins, called CRM1 (chromosome region maintenance 1) or exportin 1, transports leucine-rich NES-containing proteins. This CRM1-mediated protein translocation can be specifically inhibited by the drug leptomycin B [13]. In contrast, transport of mrnas over the nuclear membrane is not dependent on Ran, but requires the transporters TAP-p15 (NXF1- NXT1) and Dbp5 ATPase for proper mrna release into the cytoplasm [14]. The subcellular localization of human Ccr4-Not complex components have been studied sporadically, often as overexpressed epitope-tagged proteins. The CNOT2 subunit has been detected predominantly in the nucleus [15], whereas the deadenylases CNOT6, CNOT6L and CNOT7 are mainly localized to the cytoplasm [7, 16], of which a proportion of CNOT7 is also detected in the nucleus [16, 17]. Inhibition of CRM1- mediated nuclear protein export by leptomycin B also leads to more CNOT6 and CNOT7 in the nucleus [16]. Moreover, these deadenylases have also been detected in special cytosolic structures called P bodies [18-20]. Furthermore, endogenous CNOT7 showed a cell cycle-dependent localization, with almost exclusive nuclear staining during G0/G1- phase and being cytoplasmic in the S-phase [21]. Localization of human CNOT proteins 71

72 In the present study, we take the initial step to explore the spatial regulation of human Ccr4-Not complex components. Stable cell lines containing GFP(green fluorescent protein)-tagged CNOT proteins at near endogenous levels have been generated for this study. Most of the Ccr4-Not subunits (CNOT2, CNOT3L, CNOT6, CNOT7, CNOT8, CNOT9 and CNOT10) localize mainly to the cytoplasm, and CNOT7 is also detected in the nucleus in a cell cycle-dependent manner. Furthermore, cellular exposure to leptomycin B resulted in nuclear localization of CNOT7 in almost all cells, CNOT8 appearance in some nuclei and CNOT9 concentration in nuclear dot-like structures. Interestingly, a predicted NES in these three Ccr4-Not proteins suggests for a regulated nucleocytoplasmic translocation within cells. MATERIALS AND METHODS Chapter 3 72 Generation of stable U2OS cell lines Human CNOT cdnas (described in [9]) were introduced by GATEWAY cloning [22] into retroviral destination plasmid derived from pbabe-puro carrying an N- or C-terminal GFP-tag. U2OS cell clones expressing a GFP-tagged CNOT protein were obtained by retroviral transduction and puromycin selection. Positive clonal cell lines were identified by fluorescence microscopy and immunoblotting using antibodies against GFP. In addition, cell lines were enriched for GFP-containing cells using a FACSAria cell sorting system (BD Biosciences) compared to pbabe-puro control U2OS cells, which was followed by clonal cell line growth on plates. U2OS cells containing GFP-tagged TBP at near endogenous levels were obtained from Dr. Petra de Graaf. U2OS cells with GFP or GFP-tagged rat CLIP170 were provided by Dr. Marvin Tanenbaum. Cell culture conditions were as described in [9]. Immunoblotting and antibodies Stable U2OS cells expressing GFP-tagged proteins were lysed in 2x sample buffer (4% (w/v) SDS, 160 mm Tris-HCl (ph 6.8), 20% (v/v) glycerol, 4% (v/v) β-mercaptoethanol and 0.01% (w/v) bromophenol blue). DNA was sheared by several snap freeze and boil cycles or by sonication for 5 min in 5 cycles of 30 sec on a Bioruptor (Diagenode). Immunoblot analyses and antibodies used were as described in [9], with the exception that anti-cnot8 antibodies were not affinity-purified. Mouse antibodies against GFP were obtained from Roche Applied Science.

73 Fluorescence microscopy U2OS cells were cultured for two days on glass cover slips. Cells were fixed for 20 min in 4% (v/v) (para)formaldehyde, and DNA was stained by incubating cells for 5 min in 2 µg/ml DAPI. U2OS cells were analysed on an Axioskop 40 upright microscope (Zeiss) or on a LSM 510 META confocal laser scanning microscope (Zeiss). Time-lapse fluorescence microscopy GFP-CNOT7 cells were plated in 4-chamber glass-bottom dishes (Lab-Tek, Nunc, USA) and synchronized at the G1/S boundary by a double thymidine block. Cells were released from this block and followed by time-lapse imaging on an Axiovert 200M microscope (Zeiss) at 40x magnification. Images were captured for 80 msec at every 5 min (in total 11:40 hours) and processed using the Metamorph software (Universal Imaging). Leptomycin B treatment Cells were cultured on cover slips and synchronized at the G1/S boundary with a double thymidine block. First, cells were grown in medium containing 2.5 mm thymidine (Sigma) for 24 hours, followed by two washes in DPBS (Cambrex) and released in normal growth medium for 12 hours with a subsequent second thymidine block for 24 hours. Upon release of the second thymidine block, cells were cultured for 24 hours in medium containing 2 ng/ml leptomycin B (LC Laboratories) or ethanol (mock). Localization of human CNOT proteins FACS profiling Double thymidine blocked or asynchronous cells were fixed for at least 1 hour in 58% (v/v) ethanol at 4 C, followed by an incubation in a mixture containing 250 µg/ml RNase A (Roche) and 5 µg/ml propidium iodide (Sigma) for 30 min at 37 C. DNA profiling of the cells was performed on a FACSCalibur (BD Biosciences) using the Cellquest Pro software (BD Biosciences). 73 Bioinformatics Proteins were analysed for a NLS by the PredictNLS server ( edu/services/predictnls/; [23]) or for a NES by the NetNES 1.1 program ( cbs.dtu.dk/services/netnes/; [24]).

74 RESULTS Chapter 3 74 Stable expression of GFP-CNOT proteins under endogenous conditions The Ccr4-Not complex is regulating both the synthesis and bulk degradation of mrna molecules in eukaryotes, which are known to occur in the nucleus and cytoplasm, respectively. To understand its spatial regulation to fulfil these distinct cellular functions, a first step has been taken to investigate the subcellular localization of the Ccr4-Not complex components. The lack of appropriate antibodies prohibited the detection of endogenous CNOT proteins by immunofluorescence microscopy. Instead, chimeras of CNOT proteins and GFP were constructed to visualize the Ccr4-Not subunits within (living) cells. U2OS cell lines expressing each of these GFP-tagged CNOT proteins were generated by the pbabe-puro based retroviral system. This method has been used before and worked well to express epitope-tagged CNOT proteins at near endogenous levels [9]. Monoclonal cell lines were generated from U2OS transformants, but heterogeneous expression levels of the GFP-chimeras CNOT2, CNOT6, CNOT7, CNOT9 and CNOT10 were seen. FACS (fluorescence-activated cell sorting) was performed to enrich for GFPpositive cells in these populations (results not shown). In total eight stable U2OS cell lines expressing GFP-CNOT proteins were analysed further. The expression of GFP-proteins in the cell lysates of CNOT2, CNOT3S, CNOT3L, CNOT6, CNOT7, CNOT8, CNOT9 and CNOT10 cell lines were analysed by immunoblotting (Figures 1A and 1B). The CNOT3S lysate contains highly expressed proteins detectable by antibodies against GFP, of which one runs at similar height as GFP itself (Figure 1A), suggesting that free GFP exists in this cell line. Therefore, the CNOT3S cell line was excluded in subsequent experiments. Cell lysates of GFP-tagged transcription factor TBP (TATA-binding protein) and CLIP170 (cytoplasmic linker protein p170) were also analysed (Figure 1A) as they serve as controls in subsequent experiments. The GFP-TBP cell line was generated by the described retroviral system using human TBP, whereas the GFP-CLIP170 cell line was generated by stable transfection of GFPtagged rat CLIP170 constructs [25, 26]. The latter method may relate to the observed smaller GFP-products in the GFP-CLIP170 cell extract (Figure 1A). Nevertheless, the GFP-CLIP170 construct shows a normal localization pattern at the growing ends of microtubules in the cytoplasm [26, 27]. To compare the expression levels of GFP-tagged CNOT2, CNOT3L and CNOT7 to their endogenous counterparts, they were analysed by their specific antibodies (Figures 1C-1E). GFP-CNOT2 seemed to be lower expressed than its endogenous protein (Figure 1C), whereas GFP-tagged CNOT3 and CNOT7 were expressed at near endogenous protein levels (Figures 1D and 1E). Taken together, these results indicate that the generated U2OS cell lines stably express GFP-tagged CNOT proteins at comparable levels as the endogenous proteins.

75 A [kda] CNOT3S-N-GFP CNOT3L-N-GFP CNOT6-N-GFP CNOT7-N-GFP CNOT2-N-GFP * * * * * CNOT8-N-GFP * * * CNOT9-N-GFP CNOT10-C-GFP TBP-N-GFP * CLIP170-N-GFP * GFP B [kda] CNOT3S-N-GFP CNOT3L-N-GFP * * C [kda] CNOT2-N-GFP CNOT3S-N-GFP α-gfp D [kda] * CNOT3L-N-GFP CNOT3S-N-GFP E [kda] α-gfp CNOT7-N-GFP CNOT8-N-GFP Localization of human CNOT proteins * * * * 75 α-cnot2 α-cnot3 α-cnot7 Figure 1. Stable U2OS cell lines express different GFP-tagged subunits of the human Ccr4- Not complexes (A-B) Lysates from U2OS cells stably expressing the indicated GFP-tagged CNOT protein were analysed by immunoblotting using antibodies against GFP. CNOT3L was nearly detectable in (A) and was analysed again in (B). Controls are U2OS cell lines expressing GFP-tagged TBP or CLIP170. Lysate from U2OS cells with GFP serves as a control for immunoblotting. Molecular mass markers (kda) are indicated on the left-hand side of the gel. Asterisks indicate the GFP-tagged protein. (C-E) Some lysates from (A) were analysed by specific antibodies indicated below the panels. Both exogenous GFP-tagged (asterisk) and endogenous (arrow) proteins are detected. Molecular mass markers (kda) are indicated on the left-hand side of the gel.

76 Human Ccr4-Not subunits localize predominantly to the cytoplasm The GFP-CNOT cell lines were analysed for subcellular localization of its epitope-tagged CNOT protein by fluorescence microscopy (Figure 2). As expected, GFP-TBP was only seen in the nucleus [28] and GFP-CLIP170 was visible as comet-like staining at the ends of microtubules [26, 27]. The CNOT proteins appeared mainly in the cytoplasm, except for CNOT7 (Figure 2). This latter Ccr4-Not subunit localized both to the cytoplasm and nucleoplasm as reported earlier [17, 21, 29]. Interestingly, Morel and colleagues showed that endogenous CNOT7 in MRC5 cells was almost exclusively nuclear in the G0/G1- phases and became more cytoplasmic when entering the S-phase [21]. This cell-phase dependent localization of CNOT7 was studied in our GFP-CNOT7 cells beyond the S- phase. The cells were synchronized at the G1/S boundary by a double thymidine block and followed by time-lapse microscopy upon release. It showed that GFP-CNOT7 localized predominantly to the cytoplasm during S-phase, as reported for endogenous CNOT7, and gradually became more visible in the nucleus when the cell moved towards M-phase (Figure 3). Taken together, the majority of GFP-CNOT proteins display a cytoplasmic localization, suggesting that human Ccr4-Not complexes are predominantly located to the cytoplasm. Chapter 3 GFP CNOT2 CNOT3L CNOT6 CNOT7 CNOT8 76 DAPI CNOT9 CNOT10 TBP CLIP170 GFP DAPI Figure 2. Cellular localizations of GFP-tagged CNOT proteins Asynchronous GFP-tagged U2OS cells were fixed and analysed by fluorescence microscopy in the FITC channel (GFP) and in the DAPI channel.

77 00:05 01:30 03:00 04:30 06:00 07:00 08:00 09:00 09:15 09:55 10:00 11:30 Figure 3. Localization of GFP-CNOT7 proteins from S-phase to M-phase Synchronized U2OS GFP-CNOT7 cells at the G1/S boundary were released for time-lapse imaging. The numbers represent the time in hh:mm upon release. The depicted GFP-CNOT7 cell is representative for the whole population, and went from S-phase through M-phase and cytokinesis. Nuclear localization of CNOT7, CNOT8 and CNOT9 proteins Sequences of all Ccr4-Not subunits were analysed in silico for the presence of a NLS or NES. The PredictNLS server [23] did not identify a peptide signal for nuclear import for any of the Ccr4-Not subunits. Surprisingly, the NetNES 1.1 tool [24] predicted leucine-rich NES sites for CNOT7 (residues 74-81: IIQLGLTF), CNOT8 (residues 74-81: IIQLGLTF) and CNOT9 (residues : IPLCLRI). The potential nuclear export of these three CNOT proteins was studied using leptomycin B. This drug is known to inhibit leucine-rich NES-mediated protein export by CRM1 [13], and can also cause cell cycle arrest in G1-phase and partially in G2-phase in mammalian cells [30]. To circumstance these cell cycle side-effects of leptomycin B, GFP-CNOT cells were synchronized at the G1/S boundary using a double thymidine block and upon release exposed to leptomycin B for 24 hours, followed by confocal fluorescence microscopy analyses. The efficiency of the thymidine blocks was determined by FACS profiling (Figure 4A and Table 1), except for CNOT3L and CNOT9 cells due to their incomplete FACS profiles (Figure 4A). For the other cell lines, an accumulation of 70-86% of the synchronized cells in G1-phase was seen compared to 39-52% when untreated (Table 1). Moreover, microscopy analyses showed upon release and leptomycin B treatment nuclear localization of the three CNOT proteins with a predicted NES (Figure 4A). The GFP-CNOT7 cells showed an enhanced nuclear staining, GFP-CNOT8 became diffusely distributed in some nuclei and GFP- CNOT9 appeared in nuclear dot-like structures (Figures 4A and 4B). Taken together, these results indicate that besides CNOT7, both CNOT8 and CNOT9 can be detectable in the nucleus. It is possible that they may translocate to the cytoplasm via their predicted NES by CRM1 mediation. Localization of human CNOT proteins 77

78 A FACS profiles + Leptomycin B Double thymidine block Asynchronous counts FL3-A FL3-A FL3-A * counts counts FL3-A FL3-A FL3-A 150 counts ** FL3-A FL3-A FL3-A counts 250 counts counts counts counts 200 Chapter CNOT10 0 counts counts CNOT FL3-A FL3-A 150 CNOT FL3-A ** 0 CNOT7 DAPI counts CNOT6 GFP counts counts 0 CNOT3L DAPI CNOT2 GFP + Ethanol (mock) 0 B CNOT9 cells + Leptomycin B FL3-A 0 0 GFP FL3-A DAPI Merge

79 Table 1. Distribution of cells in G1-, S- and G2/M-phases given in percentages Double thymidine blocked cells Asynchronous cells Cell line G1-phase S-phase G2/M-phase G1-phase S-phase G2/M-phase CNOT CNOT3L CNOT CNOT CNOT CNOT CNOT DISCUSSION In the present study, the subcellular localization of all detectable human Ccr4-Not complex components have been investigated at near endogenous expression levels (Figure 1), whereas other studies often use overexpressed proteins [7, 15, 16, 19-21]. We found that the Ccr4-Not subunits are predominantly present in the cytoplasm (Figure 2), whereas the majority of CNOT7 is cytoplasmic during S-phase and becomes more visible in the nucleoplasm when the cell approaches towards mitosis (Figure 3). A nucleocytoplasmic distribution throughout the cell cycle has not been described for other CNOT proteins and can be examined using the established cell lines. Moreover, it would be interesting to explore what signals and proteins are required for the observed cell cycle-dependent localization of CNOT7. It is possible that this protein translocates to the nucleus independently from the complex as the other Ccr4-Not subunits are predominant cytoplasmic located (Figure 2). Interestingly, leptomycin B leads to nuclear localization of CNOT8 and CNOT9 besides CNOT7 (Figure 4). Localization of human CNOT proteins 79 Figure 4. Cellular localizations of GFP-tagged CNOT proteins upon exposure to leptomycin B (A) Cells were synchronized at the G1/S boundary using a double thymidine block. Synchronized and asynchronous cells were analysed by FACS profiling after propidium iodide staining (FACS profiles; left-hand panels). The cellular distribution of the cell lines in the different phases of the cell cycle are given as percentages in Table 1. Upon cell cycle release, leptomycin B (2 ng/ml) or ethanol (mock) was added to the cells for 24 hours. Cells were fixed and observed by confocal fluorescence microscopy (right-hand panels) using the 488 nm (GFP) or the 405 nm laser (DAPI). (B) The leptomycin B- treated GFP-CNOT9 cell from panel (A) is magnified to show the nuclear dot-like structures. The merged picture is a combined observation using both the 488 nm and the 405 nm laser of the confocal fluorescence microscopy. *FACS profile is not aligned properly along the x-axis. **Not a proper FACS profile.

80 Chapter 3 80 It was surprising to detect human Ccr4-Not subunits predominantly in the cytoplasm (Figure 2). This spatial organization is in concordance with the deadenylase function, but not with the nuclear functions of transcription regulation and mrna processing. It is tempting to speculate that human Ccr4-Not components form a protein complex that is located in the cytoplasm during steady-state and upon signalling may become imported to the nucleus to exert nuclear function. This event may be undetectable by microscopy when they are low in abundance and/or are dispersedly distributed in the nucleoplasm. The steady-state cytoplasmic localization may be a result of fast export of leucine-rich NES-containing CNOT proteins via protein-protein interactions with CRM1. This is suggested for the CNOT7, CNOT8 and CNOT9 proteins as they show increased nuclear staining upon inhibition of this export pathway (Figure 4A). Alternatively, Ccr4-Not components may be exported via the TAP-p15 complexes along with mrna molecules. This pathway is not affected by leptomycin B and may explain why the majority of Ccr4- Not proteins were not retained in the nucleoplasm upon exposure to this drug. Taken together, important questions remain to understand the spatial regulation of the Ccr4- Not complex in relation to its regulatory functions in mrna metabolism. The precise mechanism of nuclear export and import of CNOT7, CNOT8 and CNOT9 has not been established. A predicted leucine-rich NES suggests that CRM1 is required for their export. Localization studies of NES mutants will provide more insight in this. However, no canonical NLS was found in the three CNOTs that could explain their mechanism of nuclear import. Alternatively, these proteins may contain a NLS motif that is not recognized by the PredictNLS server. Another possibility is that a NLS-containing adaptor protein is responsible for their nuclear import. Identification of interactor proteins with a NLS may lead to insights to their regulation of nuclear and cytoplasmic localization. Another question that arises is why CNOT7 is predominantly located in the cytoplasm during S-phase (Figure 3), when DNA replication takes place. The orthologue of CNOT7 in S. pombe, SpCaf1, may provide an answer. Activation of the RNR (ribonucleotide reductase) complex is essential for DNA replication and repair. In S. pombe, the RNR regulatory subunit Suc22 is retained in the nucleoplasm by binding to Spd1. In the presence of DNA-replication stress, SpCaf1 interacts with Suc22 in the nucleoplasm to facilitate the degradation of Spd1 and the translocation of Suc22 to the cytoplasm to form an active RNR complex with Cdc22 [31]. In mammalian cells, the RNR regulatory subunit p53r2 is cytoplasmic and relocates to the nucleus in response to DNA damage [32]. It is possible that the cytoplasmic located CNOT7 translocates the mammalian regulatory subunit to the nucleoplasm in a similar manner as described for S. pombe during DNA replication stress [31], although it is in the opposite direction.

81 Another interesting observation is the localization of CNOT9 in nuclear dot-like structures (Figure 4). The cell nucleus consists of numerous subcellular compartments, collectively referred to as nuclear bodies [33]. According to the number and arrangement of the CNOT9 structures (Figure 4B), they may represent three possible nuclear bodies [34]. First, they may be PML (promyelocytic leukaemia) bodies, which are involved in transcriptional regulation, nuclear protein sequestration and/or nuclear proteasomal degradation of proteins [34, 35]. Secondly, it is possible that the dot-like structures are Cajal bodies, involved in snrnp and snornp biogenesis and post-transcriptional modification of newly assembled spliceosomal snrnas [34, 36]. Finally, they may represent nuclear speckles that are involved in storage, assembly, and/or modification of pre-mrna splicing factors [34, 37]. Interestingly, the functions of the Cajal bodies and nuclear speckles correspond well with the enhanced isolation of CNOT9-interactors functioning in RNA splicing [9], and other human Ccr4-Not subunits have been observed in these nuclear structures as well [17, 38]. Co-staining with specific markers for the nuclear bodies will help to identify the precise nature of the CNOT9-containing nuclear structures and will provide insight in CNOT9 functioning. ACKNOWLEDGEMENTS We thank Drs. Petra de Graaf and Marvin Tanenbaum for providing the GFP-TBP and GFP-CLIP170 cell lines, Livio Kleij and Dr. Markus Kleinschmidt for technical assistance in FACS profiling. We also thank members of the Timmers laboratory for discussions. Localization of human CNOT proteins 81

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83 , Rouault, J. P., Prevot, D., Berthet, C., Birot, A. M., Billaud, M., Magaud, J. P. and Corbo, L. (1998) Interaction of BTG1 and p53- regulated BTG2 gene products with mcaf1, the murine homolog of a component of the yeast CCR4 transcriptional regulatory complex. J Biol Chem 273, Yoshida, M., Nishikawa, M., Nishi, K., Abe, K., Horinouchi, S. and Beppu, T. (1990) Effects of leptomycin B on the cell cycle of fibroblasts and fission yeast cells. Exp Cell Res 187, Takahashi, S., Kontani, K., Araki, Y. and Katada, T. (2007) Caf1 regulates translocation of ribonucleotide reductase by releasing nucleoplasmic Spd1-Suc22 assembly. Nucleic Acids Res 35, Tanaka, H., Arakawa, H., Yamaguchi, T., Shiraishi, K., Fukuda, S., Matsui, K., Takei, Y. and Nakamura, Y. (2000) A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 404, 42-9 Matera, A. G., Izaguire-Sierra, M., Praveen, K. and Rajendra, T. K. (2009) Nuclear Bodies: Random Aggregates of Sticky Proteins or Crucibles of Macromolecular Assembly? Dev Cell 17, Spector, D. L. (2006) SnapShot: Cellular bodies. Cell 127, 1071 St-Germain, J. R., Chen, J. and Li, Q. (2008) Involvement of PML nuclear bodies in CBP degradation through the ubiquitinproteasome pathway. Epigenetics 3, Patel, S. B. and Bellini, M. (2008) The assembly of a spliceosomal small nuclear ribonucleoprotein particle. Nucleic Acids Res 36, Lamond, A. I. and Spector, D. L. (2003) Nuclear speckles: a model for nuclear organelles. Nat Rev Mol Cell Biol 4, Wagner, E., Clement, S. L. and Lykke-Andersen, J. (2007) An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies. Mol Cell Biol 27, Localization of human CNOT proteins 83

84 Chapter 3 84

85 Phosphorylation of Not4p functions parallel to BUR2 to regulate resistance to cellular stresses 4in Saccharomyces cerevisiae Adapted from: PLoS ONE (2010) Volume 5(4): pages e9864

86 Phosphorylation of Not4p functions parallel to BUR2 to regulate resistance to cellular stresses in Saccharomyces cerevisiae Nga-Chi LAU 1,2, *, Klaas W. MULDER 1, *, Arjan B. BRENKMAN 1, Shabaz MOHAMMED 2,3, Niels J. F. VAN DEN BROEK 1, Albert J. R. HECK 2,3 and H. Th. Marc TIMMERS 1,2 1 Department of Physiological Chemistry, University Medical Center Utrecht, Utrecht, The Netherlands, 2 Netherlands Proteomics Centre, Utrecht, The Netherlands, 3 Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands *Equally contributing authors ABSTRACT Chapter 4 86 The evolutionarily conserved Ccr4-Not and Bur1/2 kinase complexes are functionally related in S. cerevisiae. In this study, we explore the relationship further between the subunits Not4p and Bur2p. Firstly, we investigated the presence of post-translational modifications on the Ccr4-Not complex. Using mass spectrometry analyses we identified several SP/TP phosphorylation sites on its Not4p, Not1p and Caf1p subunits. Secondly, the influence of Not4p phosphorylation on global H3K4 tri-methylation status was examined by immunoblotting. This histone mark is severely diminished in the absence of Not4p or of Bur2p, but did not require the five identified Not4p phosphorylation sites. Thirdly, we found that Not4p phosphorylation is not affected by the kinase-defective bur1-23 mutant. Finally, phenotypic analyses of the Not4p phospho-mutant (not4s/t5a) and bur2δ strains showed overlapping sensitivities to drugs that abolish cellular stress responses. The double-mutant not4s/t5a and bur2δ strain even revealed enhanced phenotypes, indicating that phosphorylation of Not4p and BUR2 are active in parallel pathways for drug tolerance. Not4p is a phosphoprotein with five identified phosphorylation sites that are likely targets of a cyclindependent kinase(s) other than the Bur1/2p complex. Not4p phosphorylation on the five Not4 S/T sites is not required for global H3K4 tri-methylation. In contrast, Not4p phosphorylation is involved in tolerance to cellular stresses and acts in pathways parallel to BUR2 to affect stress responses in S. cerevisiae.

87 INTRODUCTION The evolutionarily conserved Ccr4-Not complex consists of nine core subunits in S. cerevisiae and regulates mrna biogenesis at multiple levels (reviewed in [1, 2]). The Ccr4-Not complex can regulate gene transcription both negatively and positively [3, 4], and its Ccr4p and Caf1p subunits initiate mrna degradation by their cytoplasmic deadenylase activity [5]. Beside this enzymatic activity, a protein ubiquitin ligase (E3) function has been described for the RING domain of Not4p [6]. The interaction of Not4p with ubiquitin-conjugating enzymes (E2s) Ubc4p and Ubc5p is required for a proper stress response to drugs like hydroxyurea and hygromycin B [6]. Moreover, Not1p, Not3p, Not5p and Caf1p are phospho-proteins that probably play a role in the signal transduction cascade in stress responses [1]. Synthetic lethal interactions of several CCR4-NOT genes with BUR1 and BUR2 have been observed [7]. The BUR genes have been identified in a genetic screen for mutations that increase transcription from the basal SUC2 promoter in yeast [8]. BUR2 encodes a cyclin for the essential CDK (cyclin-dependent protein kinase) Bur1p [9]. This Bur1/2p CDK/cyclin-pair is involved in transcription elongation [10-12] and activates polymerase II promoters by facilitating H3K4me3 (histone H3 lysine-4 tri-methylation) [13-15]. H3K4 tri-methylation is mediated by the Set1p-complex/COMPASS complex in yeast, which requires ubiquitination of histone H2B and Bur1/2p-facilitated PAF complex recruitment [13, 14]. Notably, Not4p and other Ccr4-Not subunits are also required for the H3K4me3 mark [7, 16, 17]. It has been suggested that the H3K4-specific demethylase Jhd2p is a direct substrate for the E3 ligase activity of Not4p [17], but E3 ligase-inactive Not4p mutants did not display reduced levels of H3K4 methylation [7]. It remains unclear how Ccr4-Not subunits function in relation to the Bur1/2p kinase complex. To investigate the functional relationship between the Ccr4-Not and Bur1/2 kinase complexes, we explored the phosphorylation status of Ccr4-Not components. We confirmed that Not4p is a phospho-protein in vivo. Mass spectrometry analyses identified several serine/threonine sites as phosphoacceptor-sites for Not4p. Substitution of these sites to alanine (Not4p-S/T5A) indicates that the mechanism by which the Ccr4-Not complex and the Bur1/2p complex regulate H3K4 tri-methylation is independent of Not4p phosphorylation. In addition, we found that a severe kinase-defective allele of BUR1 did not affect Not4p phosphorylation. Further analysis indicates that Not4p phosphorylation is functionally important for tolerance to drugs that induce replication stress and protein translation errors. Yeast strains containing the Not4p penta-phosphomutant in combination with a BUR2 deletion show a more severe phenotype than either single mutant, which argues against a linear pathway relationship between NOT4 and BUR2. Phosphorylation of yeast Not4p 87

88 Taken together, our data indicate that phosphorylation of Not4p is involved in tolerance of DNA replication stress and protein processing errors, likely in pathways parallel to BUR2. MATERIALS AND METHODS Chapter 4 88 Yeast strains, genetic manipulation and plasmids Yeast strains used in this study are listed in Table 1. TAP-tagged strains were constructed by PCR-mediated introduction of the tag to the 3 -end of the gene. The proper strains were identified by immunoblot and co-immunoprecipitation analyses. In addition, strains were tested for known phenotypes to exclude functional interference by the TAP-tag. YSB787 (bur1δ) contained the BUR1 allele on a prs316-ura3 marked plasmid [10]. The NOT4 gene in this strain was TAP-tagged, followed by a transformation with prs315-bur1-ha3 or prs315-bur1-23-ha3 [10], and subsequently selected on 0.1% 5-FOA (5-fluoroorotic acid) plates to remove prs316-bur1. The not4:kanmx6 and bur2:ura3 strains were generated using a PCR fragment from genomic DNA of strain KMY58 and KMY187, respectively. Genomic NOT4 or not4s/t5a mutants were obtained by integrating the prs305-not4 or prs305-not4s/t5a into the NOT4 locus in a not4δ background using the SmaI restriction site in the NOT4 promoter region (nt -226 relative to the ATG). Integrated mutants and gene disruption were verified by PCR and/or phenotypic rescue. For yeast strains and mrna analysis used in the Supplementary Figures S1 and S2, please see the Supplementary Materials and Methods, and Supplementary Table S1. Affinity purification and mass spectrometry TAP-tag mediated protein purifications were performed essentially as described [7]. The Ccr4-Not complex was isolated from a Caf40-TAP strain and a fraction of the purified proteins was precipitated as described [18] and resolved on a 4-12% SDS-PAGE gradient gel (NuPage, Invitrogen), stained with Biosafe (Bio-Rad) and processed for mass spectrometry analyses. In-gel proteolytic digestion of Coomassie-stained bands was performed essentially as described [19], using trypsin (Roche) or trypsin/v8 (Roche). Samples were subjected to nanoflow liquid chromatography (LC, Agilent 1100 series) and concentrated on a C₁₈ precolumn (100 µm ID, 2 cm). Peptides were separated on an analytical column (75 µm ID, 20 cm) at a flow rate of 200 nl/min and a 60 min linear acetonitrile gradient from 0 to 80%. The LC system was directly coupled to a QTOF Micro tandem mass spectrometer (Micromass Waters, UK). A survey scan was performed from amu s -1 and precursor ions were sequenced in MS/MS mode at a threshold

89 Table 1. Saccharomyces cerevisiae strains used in this study Strain Genotype Source BY4741 MATa his3 1 leu2 0 met15 0 ura3 0 EUROSCARF KMY58 Isogenic to BY4741 except not4:kanmx6 EUROSCARF KMY161 Isogenic to BY4741 except bur2:kanmx6 EUROSCARF DS1 MATa ade2 arg4(rv-) leu trp1-289 ura3-52 [24] KMY90 Isogenic to DS1 except set1:ura3 [24] KMY164 Isogenic to DS1 except CAF40-TAP:URA3 This work KMY86 Isogenic to BY4741 except NOT1-TAP:URA3 This work KMY87 Isogenic to BY4741 except NOT4-TAP:URA3 This work KMY88 Isogenic to BY4741 except CAF40-TAP:URA3 This work YSB787 MATa bur1:his3 ura3-52 leu2 1 trp1 63 his3 200 lys2 202 (prs316-bur1) [10] KMY143 Isogenic to YSB787 except NOT4-TAP:TRP1 (prs315-bur1 HA3) a This work KMY145 Isogenic to YSB787 except NOT4-TAP:TRP1 (prs315-bur1-23 HA3) a This work NCY1 Isogenic to KMY86 except not4:kanmx6 This work NCY2 Isogenic to NCY1 except NOT4:LEU2 This work NCY16 Isogenic to NCY1 except not4s/t5a:leu2 b This work NCY29 Isogenic to KMY58 except not4s/t5a:leu2 b This work NCY35 Isogenic to KMY58 except NOT4:LEU2 This work 2922 MATα mfa1 ::MFA1pr-HIS3 his3 1 ura3 0 lys2 0 can1 [28] KMY187 Isogenic to 2922 except bur2:ura3 This work NCY37 Isogenic to NCY29 except bur2:ura3 This work NCY43 Isogenic to NCY35 except bur2:ura3 This work NCY45 Isogenic to BY4741 except bur2:ura3 This work Phosphorylation of yeast Not4p 89 a subjected to 5-FOA selection b not4s/t5a = not4-s92a/s312a/t334a/s342a/t543a of 150 counts. Additional analyses were performed by nanolc-ltq-orbitrap-ms (Thermo). Data were processed and subjected to database searches using Proteinlynx Global Server version 2.1 (Micromass) or the MASCOT software (Matrixscience) against Swiss-Prot and the NCBI nonredundant database, with a 0.25 Da mass tolerance for both precursor ion and fragment ion. The identified phospho-peptides were confirmed by manual interpretation of the spectra. In vitro dephosphorylation assays Proteins of TAP-tagged Notp strains were captured on IgG beads and washed three times with E-buffer (20 mm HEPES-KOH (ph 8.0), 350 mm NaCl, 10% glycerol, 0.1% Tween-20). Immunoprecipitated material was (mock-) treated at 37 C for 45 min with

90 SAP (shrimp alkaline phosphatase) or SAP pre-incubated with 4 mm Na-Vanadate and 800 mm NaF. Alternatively, TAP-tagged purification of proteins from NOT4 or not4s/ T5A strains occurs via Not1-TAP. A fraction of these purified proteins were resolved on a 4-12% SDS-PAGE gradient gel and silver stained. Other fractions were incubated at 37 C for 45 min with or without SAP. All reactions were quenched by addition of 2x sample buffer and incubated at 95 C for 5 min. Proteins were subjected to immunoblot analyses. Immunoblotting and antibodies TAP-tagged proteins were detected using the PAP antibody (against protein A moiety of the TAP-tag; Sigma). Rabbit polyclonal antibody against Not1p and Not4p was generously provided by Dr. M.A. Collart. TBP antiserum was a kind gift from Dr. P.A. Weil. For detection of H3K4 methylation status, yeast were grown in YPD and extracts were prepared as described previously [20]. Proteins were separated by 15% SDS-PAGE gels and analyzed by immunoblotting. Antibodies against H3K4me3 (Ab8580), H3K4me2 (Ab7766), H3K4me1 (Ab8895) and H3 (Ab1791) were obtained from AbCam. Chapter 4 90 Drug sensitivity assay Ten-fold serial dilutions of the indicated strains were spotted on YPD plates without or with the indicated concentrations of hydroxyurea, hygromycin B or cycloheximide. The indicated strains were also 10-fold serial diluted and spotted on SC plates or SC-R plates containing the indicated concentration of canavanine. The plates were grown at 30 C (or 37 C for YPD) for 3 days. RESULTS Not4p is a phospho-protein in vivo The shared function of the Ccr4-Not and the Bur1/2 kinase complexes in H3K4 trimethylation [7] prompted us to test involvement of this kinase complex in posttranslational modification of the Ccr4-Not complex. To identify the phosphorylation sites on its subunits, mass spectrometry analyses were performed on the purified Ccr4-Not complex using Caf40-TAP as the bait (Figure 1A). Ccr4-Not components were in-gel digested with trypsin or trypsin/v8 and subjected to LC-MS/MS analyses. Unique phospho-peptides, corresponding to Not1p (T2102), Caf1p (S39) and Not4p (S92, S312, and S542 or T543), were identified (Figure 1A). These sites correspond to phospho-sites identified in large-scale phospho-proteome analyses [21-23]. Notably,

91 Not1p, Caf1p and Not4p were phosphorylated on SP and TP sites, which are often targets of CDK/cyclin-pair kinases. In addition, another Not4p peptide ( AQLHHDSHTNAGTPVLTPAPVPAGSNPWGVTQSATPVTSINLSK) was identified by mass spectrometry as a singly-phosphorylated peptide (data not shown), but the phospho-acceptor site could not be identified unequivocally. Manual inspection of the MS/MS spectrograms indicates that T334 and/or S342 of this Not4p peptide are the most likely phospho-acceptor sites. The phosphorylation status of several Ccr4-Not subunits was assessed by their electrophoretic mobility upon dephosphorylation (Figure 1B). TAPtagged proteins (Not1p, Not4p or Caf40p) were captured on IgG-Sepharose beads and subjected to treatment with SAP. As a control, SAP was inactivated using phosphatase inhibitors prior to the reaction. Interestingly, the phosphatase-activity dependent mobility was increased for Not4p, but not for Not1p or Caf40p. This confirms that Not4p is a phosphorylated protein. To further investigate the involvement of the Bur1/2p kinase complex in Not4p phosphorylation, electrophoretic migration of Not4p was assessed from Not4p-TAP yeast containing the wild-type BUR1 or the temperature-sensitive bur1-23 allele. The latter yeast strain possesses severely decreased kinase activity, even at permissive temperature [10]. Clearly, the electrophoretic mobility of Not4p was not affected in the bur1-23 yeast, whereas the level of H3K4 tri-methylation has significantly decreased as expected (Figure 1C). This suggests that efficient phosphorylation of Not4p is not dependent on Bur1p kinase activity. Phosphorylation of yeast Not4p The Not4p penta-phosphomutant displays wild-type levels of H3K4me3 To further investigate the effect of Not4p phosphorylation, phospho-not4p mutants were generated by substitution of the identified SP/TP phospho-sites and putative phosphosites on Not4p to alanine (not4s/t5a). To this end, wild-type NOT4 or the not4s/t5a mutant allele was chromosomally integrated into the NCY1 strain, which carries a TAP-tagged NOT1 allele and a NOT4 deletion (see also Table 1). Ccr4-Not complexes were isolated from these yeast strains using Not1-TAP as the bait. Mutation of Not4p phosphorylation-sites did not affect the assembly of the Ccr4-Not core complex (Figure 2A). Furthermore, purified Ccr4-Not complexes from both NOT4 as not4s/t5a strains were subjected to electrophoretic migration analyses. Notably, Not4p-S/T5A shows an increased migration compared to wild-type Not4p (Figure 2B, upper left panel), that did not change upon SAP treatment (Figure 2B, right upper panel). As expected, mobility of wild-type Not4p was increased by SAP activity (Figure 2B, middle upper panel). This confirms that the identified Not4p phosphorylation sites are responsible for the observed electrophoretic mobility change. 91

92 A Caf40-TAP [kda] Not1p Caf130p Not3p Ccr4p Not5p Not4p Caf1p Caf40p-CBP Not2p Chapter 4 92 Protein Peptide sequence a Phosphorylated residue Not1 RQTPLQSNA b T Caf1 QASEQHQQQNMGPQVYSPK b S39 83 Not4 YVTLSPEELK b S92 53 Not4 YVTLSPEELKMER c S92 52 Not4 SGIHNNISTSTAGSNTNLLSENFTGTPSPAAMR b S Not4 NFTGTPSPAAMR c S Not4 LQTVSQQIQPPLNVSTPPPGIFGPQHK c S542 or T Not4 LQTVSQQIQPPLNVSTPPPGIFGPQHK b S542 or T Mascot score B [kda] Input (15%) TAP IP SAP Inh. C Not4-TAP BUR1 bur Not1-TAP Not4-TAP Caf40-TAP h at 37 C Not4-TAP H3K4me3 TBP

93 Not4p and Bur2p were reported previously to be involved in the regulation of H3K4 tri-methylation [7, 13, 14, 16, 17]. We examined whether the phospho-sites of Not4p are required for this methylation event. Cellular extracts of yeast containing the pentaphosphomutant of Not4p (not4s/t5a) were analysed by immunoblot analysis (Figure 3). In agreement with previous observations [7, 13, 14, 16, 17], deletion of NOT4 or BUR2 resulted in severely decreased levels of H3K4me3 (Figure 3A). The SET1 deletion strain serves as a control for abolished tri-, di-, and mono-methylation levels of H3K4 (Figure 3) [24, 25]. In contrast, the not4s/t5a strain shows wild-type levels of H3K4me3 (Figure 3). No additional effects on tri-methylation levels by the not4s/t5a mutant are observed in strains deleted for BUR2 (Figure 3B). This suggests that the mechanism by which the Ccr4-Not and Bur1/2p complexes regulate H3K4 tri-methylation levels is independent of Not4p phosphorylation. Phosphorylation of Not4p is required for cellular stress tolerance The drug hydroxyurea introduces DNA replication stress [4] and yeast strains deleted for NOT4 or BUR2 display a similar sensitivity to hydroxyurea, which is supported by a reduced induction of RNR3 mrna upon hydroxyurea treatment (Supplementary Figure S1). Besides this drug, yeast NOT4 deletion mutants are also sensitive to high temperature and hygromycin B, which leads to errors during protein synthesis [6]. To explore the role of Not4p phosphorylation under these stress conditions, the not4s/t5a strain was subjected to 37 C, hydroxyurea or hygromycin B growth conditions. The not4s/t5a strain shows a temperature tolerance at 37 C unlike the NOT4 deletion strain (Figure 4A). Both not4δ and bur2δ mutants are highly sensitive to hydroxyurea and hygromycin B, Phosphorylation of yeast Not4p 93 Figure 1. Not4p is a phospho-protein (A) Phospho-proteomics on the Ccr4-Not complex. Ccr4-Not complexes were TAP-tagged purified from a strain expressing Caf40-TAP and visualized on gradient SDS-PAGE gel by Coomassie (upper left panel), marker proteins (kda) are indicated on the left. Tryptic digestion of Coomassie stained bands was followed by LC-MS/MS analyses, leading to the identification of the Ccr4-Not subunits and their phosphorylated peptides (inserted Table; a Phosphorylated amino acids are underlined; b Cleaved with trypsin and detected by ESI-QTOF mass spectrometry; c Cleaved with trypsin/v8 and detected by ESI-LTQ-Orbitrap mass spectrometry). A representative spectrum including peak assignment of Not4p phosphorylation on S92 is given (upper right panel; inset represents the b- and y-ion coverage of the phospho-peptide). (B) Not4p is phosphorylated in vivo. TAP-tagged versions of Not1p, Not4p or Caf40p were captured on IgG beads and subjected to treatment with SAP or SAP pre-incubated with Inh. (phosphatase inhibitors). Samples were resolved by SDS-PAGE and analyzed by immunoblotting using anti-pap antibodies that recognize the protein A moiety of the TAP-tag. Marker proteins (kda) are indicated on the left. (C) Bur1p kinase activity is not required for phosphorylation of Not4p. Strains expressing Not4-TAP and either the BUR1 or the bur1-23 allele were incubated at 37 C for the indicated h (hours). Samples were analyzed by immunoblotting with antibodies against PAP, H3K4me3 or TBP.

94 while the not4s/t5a yeast strain is weakly sensitive to hydroxyurea and mildly sensitive to hygromycin B (Figure 4A). To further examine the role of phosphorylated Not4p in the protein processing pathways, not4s/t5a mutants were tested for sensitivity to cycloheximide, an inhibitor for protein synthesis, and to canavanine, an arginine analogue that induces protein misfolding. The not4s/t5a strain displays a similar sensitivity to cycloheximide and canavanine as the bur2δ strain, while the growth of not4δ strains is severely reduced under these conditions (Figure 4B). Notably, different combinations of Not4p phospho-site mutations resulted in wild-type growth on the indicated drug plates (Supplementary Figure S2). These results indicate a redundancy among the five serine/threonine sites on Not4p. Moreover, phosphorylation of these sites is functionally important, but not essential, for resistance to replication stress and for proper processing of proteins in the cell. A Not1-TAP B Chapter 4 94 [kda] NOT4 not4s/t5a Not1p-CBP Caf130p Not3p Ccr4p Not5p Not4p [kda] 58 NOT4 not4s/t5a Not1-TAP NOT4 not4s/t5a - SAP + SAP - SAP + SAP Not4p Caf1p Caf40p Not2p 175 Not1p-CBP 14 Figure 2. The penta-phosphomutant of Not4p has increased electrophoretic mobility (A) The identified phospho-sites of Not4p are not required for Ccr4-Not complex assembly. Ccr4- Not complexes were TAP-tagged purified from a strain expressing Not1-TAP containing or lacking the Not4p penta-phosphomutant (not4s/t5a). Purified proteins were visualized by silver staining on a gradient SDS-PAGE gel. Marker proteins (kda) are indicated on the left. (B) Mutation of the five phospho-sites of Not4p results in increased gel migration. Purified proteins from panel (A) were subjected to (mock-) SAP treatment and analyzed by immunoblotting with antibodies against Not1p or Not4p. Marker proteins (kda) are indicated on the left.

95 A NOT4 not4 bur2 NOT1-TAP not4 NOT4 NOT1-TAP not4 not4s/t5a NOT1-TAP not4 set1 H3K4me3 H3K4me2 B NOT4 not4 NOT4 not4 not4s/t5a not4 NOT4 bur2 not4 not4s/t5a bur2 bur2 set1 H3K4me1 H3 Phosphorylation of yeast Not4p 95 H3K4me3 H3K4me2 H3K4me1 H3 Figure 3. Not4p phosphorylation has no influence on global H3K4 tri-methylation Extracts of strains indicated on the top were subjected to immunoblotting to detect the indicated proteins on the right. The SET1 deletion strain was used as an antibody specificity control. (A) Mutation of all five phospho-sites of Not4p does not affect H3K4 tri-methylation. (B) Addition of the Not4p pentaphosphomutant (not4s/t5a) into the bur2 strain does not enhance its H3K4 tri-methylation defect. Asterisks indicate aspecific bands.

96 A B NOT4 not4 bur2 NOT1-TAP not4 NOT4 NOT1-TAP not4 not4s/t5a NOT1-TAP not4 NOT4 not4 bur2 NOT1-TAP not4 NOT4 NOT1-TAP not4 not4s/t5a NOT1-TAP not4 YPD 30 C YPD Chapter 4 YPD 37 C YPD nm cycloheximide 96 YPD mm hydroxyurea SC YPD + 50 µm hygromycin B SC-R µm canavanine Figure 4. The not4s/t5a phenotypic analyses show overlapping drug sensitivity with the BUR2 deletion strain (A-B) Strains indicated on the top were spotted in 10-fold serial dilutions on the indicated plates and incubated at 30 C (or 37 C when indicated).

97 The drug sensitivity assays showed overlapping effects for phosphorylated Not4p and Bur2p (Figure 4). To explore the synthetic genetic relationship between BUR2 and phosphorylation of Not4p, the not4s/t5a and bur2δ double mutant was assayed for drug tolerance levels. Interestingly, this double mutant is more sensitive for hydroxyurea, cycloheximide and canavanine than its single mutants (Figure 5). Taken together, the observed additional effect of the combination of not4s/t5a and BUR2 deletion suggests that (phosphorylation of) Ccr4-Not and Bur1/2p complexes function in parallel molecular pathways to resist DNA replication stress and cellular stress upon misfolded and/or mistranslated proteins. NOT4 not4 bur2 not4 NOT4 not4 not4s/t5a not4 NOT4 bur2 not4 not4s/t5a bur2 bur2 YPD 30 C NOT4 not4 bur2 not4 NOT4 not4 not4s/t5a not4 NOT4 bur2 not4 not4s/t5a bur2 bur2 YPD nm cycloheximide Phosphorylation of yeast Not4p 97 YPD 37 C SC YPD + 10 mm hydroxyurea SC-R µm canavanine Figure 5. Yeast strains containing both not4s/t5a and BUR2 deletion show increased drug sensitivity Strains indicated on the top were spotted in 10-fold serial dilutions on the indicated plates and incubated at 30 C (or 37 C when indicated). Last three lanes of each plate show yeast strains with an integration of URA3 to inactivate the BUR2 locus.

98 DISCUSSION Chapter 4 98 In this study, we describe that Not4p is a phospho-protein in vivo (Figures 1A and 1B) and that this protein modification is not dependent on the kinase activity of Bur1p (Figure 1C). Our mass spectrometry analyses confirmed several phosphorylation sites on Not4p (S92, S312, S542/T543, putatively T334 and/or S342), Not1p (T2102) and Caf1p (S39) (Figure 1A). Absence of Not4p phosphorylation preserves the Ccr4-Not complex stoichiometry and H3K4 tri-methylation levels (Figures 2A and 3), but results in sensitivity to drugs that induce replication stress or aberrant protein synthesis (Figure 4). Moreover, the combination of a BUR2 deletion with the Not4p phospho-pentamutation leads to a more severe phenotype than single phospho-mutants (Figure 5), indicating that a synthetic genetic relationship between phosphorylated Not4p and BUR2 exist for various cellular stresses. The identification of Not4p phospho-acceptor sites on SP/TP positions (Figure 1A) suggests that Not4p is a substrate for CDK/cyclin kinase pairs. The replacement of BUR1 by the bur1-23 allele resulted in a severely reduced Bur1p kinase activity [10], but had no effect on the phosphorylation status of Not4p (Figure 1C). This suggests that Not4p is not a direct substrate for Bur1/2 kinase activity, and other CDK/cyclinkinase complexes may be required for Not4p phosphorylation. Ctk1p is, like Bur1p, a cyclin-dependent kinase that associates with the transcription elongation complex. It is suggested that Ctk1p and Bur1p are paralogues of the higher eukaryotic Cdk9 protein based on their sequence similarities [10]. Mutants deleted for CTK1 did not alter the electrophoretic mobility of Not4p (data not shown), indicating that this CDK is not the required kinase for Not4p. Interestingly, yeast mutants deleted for PHO85 showed an increased electrophoretic mobility of Not4p (data not shown), suggesting that Pho85p is involved in Not4p phosphorylation. Pho85p is a CDK that interacts with ten different cyclin partners to exert its diverse roles in the regulation of cellular responses to nutrient levels, environmental conditions and progression through the cell cycle [26]. One can speculate that Not4p is a direct substrate for Pho85p or alternatively be phosphorylated by one or multiple CDK/cyclin pairs that are targets of Pho85p. We observed that mutation of the identified phospho-acceptor sites of Not4p to alanine does not affect global H3K4 tri-methylation levels (Figure 3), indicating that phosphorylation of Not4p on S92, S312, T543, T334 and S342 does not contribute to the regulation of histone methylation. It is important to note that we achieved 87% coverage of Not4p peptides in our mass spectrometry analyses. Conceivably, Not4p could be phosphorylated at other sites not included in our analyses, but the unchanged electrophoretic mobility of Not4p penta-phosphomutant upon phosphatase treatment suggests that we covered the major phosphorylation sites on Not4p (Figure 2B). Moreover,

99 the penta-phosphomutant is less sensitive to certain drugs compared to a NOT4 deletion (Figure 4) and more sensitive than the different combinations of Not4p phospho-site mutants (Supplementary Figure S2), suggesting that abolishment of the majority of Not4p phospho-sites and not a particular phospho-site per se disrupts the function of Not4p. It is formally possible that the penta-phosphomutant is defective for reasons other than removal of phospho-sites such as misfolding, which may lead to aberrant or abolished protein interactions. However, the intact Ccr4-Not complex stoichiometry in the presence of the Not4p penta-phosphomutant (Figure 2A) indicates that the stability of the Ccr4-Not complex is preserved for Not4p penta-phosphomutant to function. Another interesting point is that the enzymatic E3 ligase function of Not4p, like Not4p phosphorylation, is important during cellular stress situations. The Ubc4/5p-interaction defective and ubiquitination-inactive Not4p-L35A mutant displayed slow growth on hydroxyurea, hygromycin B and cycloheximide plates ([6]; data not shown). Moreover, the E2 Ubc4p is important for proper Not4p functioning under these conditions, since absence of Ubc4p resulted in higher sensitivity to the same drugs as seen for the Not4p- L35A mutant ([6, 27]; data not shown). In addition, the Not4p-L35A mutant, like for Not4p penta-phosphomutant, has normal levels of H3K4me3 [7]. These observations raise the possibility that Not4p phosphorylation is involved in the E3 ligase function of Not4p. Given the reduced sensitivity of the Not4p penta-phosphomutant compared to the Not4p-L35A mutant, phosphorylation of Not4p would be modulating rather than being essential for its enzymatic function. Previous data indicated that Not4p is functionally related to Bur1/2p for global H3K4me3 in yeast, and that NOT4 did not influence the recruitment of Bur1p or Bur2p to genetic loci [7]. These and other observations suggest that Not4p functions downstream of the Bur1/2p complex in the histone tri-methylation pathway [13, 14]. Recently, it was suggested that loss of E3 ligase activity by deleting the RING of Not4p results in elevated levels Jhd2p, the H3K4-specific demethylase in yeast [17]. This would link degradation of Jhd2p to Not4p-mediated regulation of H3K4me3. However, the L35A mutant in the RING of Not4p abolishes its E3 ligase activity, but normal H3K4me3 levels are maintained in not4l35a cells [7]. Results reported in this study now indicate that Not4p and Bur1/2p act in parallel pathways. First, Not4p is not a direct substrate for the kinase activity of Bur1p (Figure 1C). Secondly, bur2δ strains displayed normal growth at 37 C unlike not4δ strains (Figures 4A and 5). Thirdly, bur2δ and not4δ strains displayed different sensitivities to inhibitors of protein synthesis/folding (cycloheximide, canavanine; Figures 4B and 5). Finally, the combination of the not4s/t5a and bur2δ alleles showed synthetic rather than epistatic growth phenotypes (Figure 5). These observations are consistent with a model wherein the Ccr4-Not and Bur1/2 kinase complexes act in parallel pathways to regulate cellular stress responses. Phosphorylation of yeast Not4p 99

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102 Supplementary Materials and Methods Yeast strains, genetic manipulation and plasmids Strains used in Supplementary Figures S1 and S2 are listed in Supplementary Table S1. The KMY86 strain was constructed by PCR-mediated attachment of the TAP-tag to the 3 -end of the NOT1 gene in the BY4741 background. This strain was checked by immunoblot and co-immunoprecipitation analyses. The NCY1 strain was generated via a PCR fragment from the NOT4 locus of strain KMY58 into the KMY86 strain. The NCY1 strain was checked for temperature sensitivity at 37 C. Genomic NOT4 or not4s/txa mutants (x = 1, 2, 3 or 4) were obtained by integrating the prs305-not4 or prs305-not4s/txa into the NOT4 locus of the NCY1 strain using the SmaI restriction site in the NOT4 promoter region (nt -226 relative to the ATG). Integrated mutants and gene disruption were verified by PCR or phenotypic analyses. Drug sensitivity assay Ten-fold serial dilutions of the indicated strains were spotted on YPD plates without or with the indicated concentrations of hydroxyurea, hygromycin B or cycloheximide. The indicated strains were also 10- fold serial diluted and spotted on SC plates or SC-R plates containing the indicated concentration of canavanine. All plates were grown at 30 C for 3 days. Chapter 4 Quantitative PCR analysis RNA extraction, reverse-transcription and quantitative PCR analysis were performed as described previously [1]. 102

103 Table S1. Saccharomyces cerevisiae strains used in Supplementary Figures Strain Genotype Source BY4741 MATa his3 1 leu2 0 met15 0 ura3 0 EUROSCARF KMY58 Isogenic to BY4741 except not4:kanmx6 EUROSCARF KMY161 Isogenic to BY4741 except bur2:kanmx6 EUROSCARF KMY86 Isogenic to BY4741 except NOT1-TAP:URA3 This work NCY1 Isogenic to KMY86 except not4:kanmx6 This work NCY3 Isogenic to NCY1 except NOT4:LEU2 This work NCY4 Isogenic to NCY1 except not4-s92a:leu2 This work NCY5 Isogenic to NCY1 except not4-s312a:leu2 This work NCY6 Isogenic to NCY1 except not4-t543a:leu2 This work NCY7 Isogenic to NCY1 except not4-s342a:leu2 This work NCY8 Isogenic to NCY1 except not4-t334a:leu2 This work NCY9 Isogenic to NCY1 except not4-s92a/t543a:leu2 This work NCY10 Isogenic to NCY1 except not4-s92a/s312a:leu2 This work NCY11 Isogenic to NCY1 except not4-s312a/t543a:leu2 This work NCY12 Isogenic to NCY1 except not4-s92a/s312a/t543a:leu2 This work NCY13 Isogenic to NCY1 except not4-s92a/s312a/s342a/t543a:leu2 This work NCY14 Isogenic to NCY1 except not4-s92a/s312a/t334a/t543a:leu2 This work A WT not4 bur2 WT not4 bur2 WT not4 0 mm 25 mm 50 mm bur2 HU B RNR3 not4 bur2 Phosphorylation of yeast Not4p 103 Figure S1. Deletion of NOT4 or BUR2 leads to similar hydroxyurea sensitivity (A) HU (hydroxyurea) sensitivity of cells lacking NOT4 or BUR2. BY4741, not4δ and bur2δ strains were spotted in 10-fold serial dilutions on YPD or YPD containing 25 mm or 50 mm HU. (B) HUinduced RNR3 transcription in cells lacking NOT4 or BUR2. Exponentially growing BY4741, not4δ and bur2δ strains were treated with 200 mm HU for 2 hours in YPD. RNA was extracted and subjected to quantitative reverse-transcriptase PCR. Standard deviations of four experiments are indicated as error bars.

104 not4-s92a/s312a/s342a/t543a not4-s92a/s312a/t334a/t543a not4-s92a/s312a/t543a not4-s312a/t543a not4-s92a/t543a not4-s92a/s312a not4-t543a not4-s342a not4-t334a not4-s312a not4-s92a NOT4 YPD YPD + 50 µm hygromycin B Chapter 4 YPD nm cycloheximide 104 SC SC-R µm canavanine Figure S2. All five phospho-sites on Not4p are required for drug tolerance Yeast strains of several combinations of Not4p phospho-site mutations were spotted in 10-fold serial dilutions on YPD or YPD containing the indicated concentrations of hygromycin B or cycloheximide. Strains were also spotted in 10-fold serial dilutions on SC or SC without R (arginine) containing the indicated concentration of canavanine. Lane 1-11 show the yeast strain NYC1 (NOT1-TAP not4δ) with an integration of not4s/txa (x = 1, 2, 3 or 4) at the NOT4 locus. Lane 12 shows yeast strain NYC1 with wild-type NOT4 at the NOT4 locus (see Table S1 for yeast strains).