Transport of mrna through the nuclear pore in Plasmodium falciparum

Transport of mrna through the nuclear pore in Plasmodium falciparum Synonyms mrna Movement through nuclear pore in Plasmodium falciparum Definition The nucleus is the most important part of the cell and is vital for eukaryotic cell survival. It stores the necessary genetic information and separates and protects this from the rest of the cellular components. The nuclear envelope that surrounds the nucleus is a double membrane with multiple pores and is composed of an outer and an inner phospholipid bilayer. The nucleus communicates with the cell through nuclear pores, which are numerous tiny openings in the nuclear envelope, and exchanges the proteins and RNA for a range of the cellular processes. The nuclear pores allow the passage of ATP, ions, water, and other small molecules freely, but it regulates the passage of macromolecules. This phenomenon is commonly known as nucleocytoplasmic transport (NCT) (Fahrenkrog and Aebi 2003). Nuclear pore complexes (NPCs) are large macromolecular structures embedded in the nuclear envelope, which control the flow between the nucleus and the cytoplasm. NPCs fuse both outer and inner membrane to make aqueous translocation channels which control the NCT of small and large molecules into and out of the nucleus (Raices and D'Angelo 2012). NPCs are highly selective, bidirectional transporter for a variety of cargoes. NPCs are composed of multiple copies of nearly 30 different proteins, which are known as nucleoporins or Nups and form the largest multiprotein assemblies of the cell (Alber et al. 2007). Introduction The nuclear envelope of a vertebrate cell contains an average of 2,000 NPCs, but the number varies considerably depending on cell type and the stage in the life cycle. NPCs have essential functions in gene expression, cell cycle progression, and chromatin organization. NPCs are active complexes, which permit shuttling of proteins and ribonucleoprotein complexes (RNPs) and transport of cargoes through their central aqueous channel. The cargoes which go into the nucleus include inner nuclear membrane proteins and all the proteins in the nucleoplasm. The cargoes transported out of the nucleus are RNA-associated proteins that are assembled into ribosomal subunits or messenger ribonucleoproteins (mrnps). After the completion of the transport, the NPC ensures that these cargos are retained in their respective nuclear and cytoplasmic compartments. In an interesting study, it has been reported that the number and the distribution of NPCs change during the various stages of intraerythrocytic development cycle in Plasmodium falciparum (Weiner et al. 2011). There are only few NPCs during the ring stage and as the parasite develops into trophozoite stage the number of NPCs increases to ~60. But again the number of NPCs decreases during schizogony, and in late schizonts there are only few NPCs on each nucleus (Weiner et al. 2011). These observations suggest that the biogenesis of nuclear pores is halted at the trophozoite stage in order to allow the distribution of fixed number of pores among the daughter nuclei (Weiner et al. 2011). The size of NPCs ranges from 60 Mda in yeast to 125 Mda in vertebrates and Nups are essentially required for the function of NPC, and these can be categorized into "unstructured" or "structured" forms depending on the presence or absence of hydrophobic phenylalanine-glycine (FG) repeats in their sequence (Raices and D'Angelo 2012). In a recent study, an unusual nucleoporin of P. falciparum PfSec13, which shows unique structural similarities suggesting that it is a fusion between Sec13 and Nup145C of yeast, has been characterized (Dahan-Pasternak et al. 2013). Using super-resolution fluorescence microscopy and in vivo imaging, it has been shown that the dynamic localization of PfSec13 during parasites' intraerythrocytic development corresponds with that of the NPCs (Dahan-Pasternak et al. 2013). It was also reported that the proteins associated with PfSec13 suggest that this unusual Nup is involved in several cellular processes and, in addition to the NPCs, PfSec13 is found in the nucleoplasm where it is associated with chromatin (Dahan-Pasternak et al. 2013). The transport through NPC also involves the nuclear transport receptors (NTRs) commonly known as karyopherins or 1

importins, which function by recognizing a nuclear localization signal (NLS) or a nuclear export signal (NES) on the cargo protein. Karyopherins are able to recognize the nucleotide motifs also in RNA cargoes and thus can export RNAs. The karyopherins that export cargo are known as exportins and the karyopherins that import cargo are called importins. The activity of these NTRs is regulated by the cofactor small GTPase Ran, which exists in GDP-bound state in the cytoplasm and in GTP-bound state in the nucleus. The gradient of RanGTP-RanGDP is produced by the action of two regulators RanGAP (Ran-GTPase-activating protein) in the cytoplasm and RanGEF (Ran-GDP-exchange factor) in the nucleus and is responsible for the directional NCT process (Raices and D'Angelo 2012). In the cytoplasm, importins bind cargo, and upon binding by RanGTP in the nucleus, the cargoes are released. But exportins bind nuclear cargo along with RanGTP, and this complex releases the cargo in the cytoplasm after its dissociation upon hydrolysis of RanGTP by RanGAP. The hydrolysis of GTP in the cytoplasmic NPC face causes the recycling of the export receptor and restoration of the cytoplasmic RanGDP pool. NPC can sort the transport of proteins, protein complexes, and RNPs accurately. It has been reported that the time frame for mrna transport through the pore is 180-500 ms (Mor et al. 2010). mrnp translocation through the NPC occurred 15-fold faster than diffusion through the nucleus (Mor et al. 2010). It has been shown that the bulk mrnp transport and export, from transcription to the NPC, occurred within a 5-40 min time frame and the mrnp export was rapid (about 0.5 s) and faster than nucleoplasmic diffusion (Mor et al. 2010). It is well established now that the export of microrna, small nuclear RNA, ribosomal RNA, and transfer RNA involves the exportins of karyopherin family and the Ran cycles. But the transport of mrna is different, which is not related to karyopherins and therefore is independent of RanGTP-RanGDP cycle also. Furthermore, some additional factors such as adaptors and release factors are required for mrna export. The large RNP complexes transport the mrna transcripts from the nucleus to the polysomes in the cytoplasm. The composition of mrnp changes continuously as these mrnps advance from synthesis to processing and finally to export. RNA helicases in general belong to an abundant protein family that is conserved from bacteria to humans, and these proteins participate in all aspects of RNA metabolism. DEAD-box proteins are essentially ATP-dependent RNA helicases, which are named so because of the presence of a conserved amino acid sequence "DEAD" (Asp-Glu-Ala-Asp) in their motif II. Mostly these RNA helicases contain dsrna unwinding activity, or they are involved in RNP (ribonucleoprotein) modeling (association/dissociation) (Linder 2006). In particular, although there is conservation of the helicase core, different helicases clearly function in discrete aspects of RNA metabolism, with their non-conserved intervening and flanking regions playing an important part in their targeting to the right substrates in vivo and regulation of their activities (Linder 2006). Moreover, many proteins that are associated with RNA helicases in vivo function as cofactors to enhance unwinding activity. This scenario is especially intriguing, as many putative RNA helicases lack efficient unwinding activity in vitro compared to the more processive DNA helicases. RNA helicase can displace proteins from RNA molecules without duplex unwinding, anneal RNA strands, act as RNA clamps or placeholders, and stabilize on-pathway folding intermediates (Tanner and Linder 2001). The flow of genetic information in eukaryotic cells occurs through the nucleocytoplasmic translocation of mrnas. There is limited information about mrna export in P. falciparum. In the present context and in this chapter, an overview of the mrna export pathway in P. falciparum will be provided. The information about mrna export has been mainly interpreted using yeast and other higher eukaryotes as the model systems. Using the tools of bioinformatics, the components of the mrna export pathway in P. falciparum have been identified and described. It is interesting to note that the components of the mrna export pathway are conserved to a great extent in P. falciparum, but there are some differences. mrna Export Machinery of P. falciparum The splicing factors SR (Ser-/Arg-rich) proteins function by recruiting the export factor TAP/NXF to mrna destined for export. SR proteins contain RNA recognition motifs (RRMs) and Ser-/Arg-rich domains. The eukaryotic proteins, which bind single-stranded RNA, contain one or more copies of a putative RNA-binding domain of about 90 amino acids that is commonly known as RRM. TREX (transcription/export complex), a multisubunit complex, is involved in splicing, export, and transcript elongation. TREX complex is composed of THO (suppressor of the transcriptional defect of Hpr1 by o verexpression) subcomplex, Tex1, adaptor ALY/REF (metazoa) or Yra1p (yeast), and UAP56 (metazoa) or Sub2 (yeast). TREX complex loads UAP56, which in turn recruits TAP/Mex67 by interaction with ALY/REF. P. falciparum database "PlasmoDB" ( http://www.plasmodb.org/ ) contains two proteins as homologues of Npl3 and Gbp2 (Table 1). These proteins are annotated in the PlasmoDB as putative pre-mrna splicing factor and protein with unknown function, respectively. These proteins are composed of 538 and 246 amino acids and share significant similarity to the yeast 2

proteins Npl3 and Gbp2, respectively. Npl3 and Gbp2 are expressed in all the developmental stages of the parasite as reported in PlasmoDB. The ScanProsite analysis reveals that PfNpl3 homologue is serine/lysine rich and contains several bipartite nuclear localization signals and two RRMs in the N-terminal region of the protein (Fig. 1a). The presence of nuclear localization signals in PfNpl3 homologue suggests that this protein is most likely localized in the nucleus. The homologue of Gbp2, PfGbp2, is a small protein, and it contains an arginine-rich region and two RRMs, one in the N-terminal and other in the C-terminal region of the protein (Fig. 1b). The presence of RRMs in PfGbp2 suggests that it is an RNA-binding protein. None of these proteins have been functionally characterized so far from P. falciparum. Table 1 mrna export factors from Plasmodium falciparum S. No. PlasmoDB number Homologue Putative function 1. PF10_0217/PF3D7_1022400 Npl3 Putative pre-mrna splicing factor 2. PF10_0068/PF3D7_1006800 Gbp2 RNA binding 3. PFL2390c/PF3D7_1249800 Tho2 Unknown 4. PFF0760w/PF3D7_0615700 REF RNA and export factor binding 5. PFB0445c/PF3D7_0209800 DDX39/UAP56/Sub2p RNA helicase involved in splicing 6. PF14_0303/PF14_0305/PF14_0306/PF14_0785/PF3D7_1432400 TAP mrna export factor 7. PF14_0563/PF3D7_1459000 DDX19/DBP5 Dual helicase involved in RNA export 8. PFF1110c/PF3D7_0623100 Nab2 mrna binding Fig. 1 Schematic representation of some of the factors involved in mrna export in malaria parasite. All the sequence data used in the analysis were downloaded from PlasmoDB ( www.plasmodb.org). The downloaded sequences were used as query to match with the human/yeast homologue using BLAST search ( www.ncbi.nlm.nih.gov). The domain analysis was done by using "ScanProsite" at http://expasy.org/tools/scanprosite/, and the results were further checked manually to confirm that all the domains are present and then used in the figures. ( a) PfNpl3. Blue boxes are RNA recognition motifs (RRM), and brown boxes are nuclear localization signals (NLS), serine- and lysine-rich regions, respectively. The numbers in blue are position of RRM, in red are NLS, and in black are lysine-/asparagine-rich regions, respectively, in the protein. ( b) PfGbp2. Blue boxes are RRMs, and brown box is arginine-rich region, respectively. The numbers in blue are position of RRM, and in black are arginine-rich region, respectively, in the protein. ( c) PfTAP. Blue boxes are leucine-rich repeat (LRR) motifs, and open brown boxes are asparagine-rich regions, respectively. The numbers in black are asparagine-rich regions in the protein. ( d) PfNab2. Green boxes are zinc finger motifs, and brown box is asparagine-rich region, respectively. The numbers in red are position of zinc finger motifs, and in black are asparagine-rich region, respectively, in the protein. This figure is not drawn to scale 3

The bioinformatics analysis revealed that TREX complex of P. falciparum contains Tho2, UAP56 (PfU52), and REF (RNA and export factor binding proteins). The homologue of Tho2 is present in the P. falciparum genome (Table 1). PfTho2 is unusually long and contains several asparagine- and lysine-rich regions and a bipartite nuclear localization signal also. The expression data in PlasmoDB report that this protein is expressed in all the developmental stages of the parasite and it is annotated in PlasmoDB as protein with unknown function. REF is also present in P. falciparum, and the PlasmoDB number is PF3D7_0615700 (old number PFF0760w) (Table 1). In Plasmodb, it has been designated as putative RNA and export factor binding protein. It is highly similar (~62 %) to its human homologue and contains one RRM in its sequence. It is noteworthy that the P. falciparum REF protein is smaller in size as compared to other eukaryotic counterparts and PfREF also contains the RNA-binding domain. So far, there are no reports of functional characterization of these proteins from P. falciparum. In addition to Tho2 and REF, the P. falciparum genome contains a homologue of UAP56, which is a splicing factor responsible for the export of mrna transcripts. The Plasmodb number of UAP56 homologue is PF3D7_0209800 (old number PFB0445c), and it has been designated as ATP-dependent RNA helicase UAP56 (Table 1). This homologue known as PfU52 is a bona fide member of DEAD-box family of helicases and has been well characterized. It has been reported that PfU52 contains RNA-dependent ATPase and RNA helicase activities and it plays a crucial role in the splicing processes (Shankar et al. 2008). It was also shown that mutations in PfU52 abolish all the enzymatic activities (Shankar et al. 2008). Tap-p15 Pathway The general mrna export receptors, which transport mrnps through the NPCs are the Mex67-Mtr2 complex (yeast) and the TAP-p15 (also known as NXF1-NXT1) complex (metazoan). Tap/Mex67 bind poly(a) RNA and are the principal export factors involved in eukaryotes. Tap/Mex67 protein contains three distinct domains: a leucine-rich repeat domain (LRR), a NTF2-like middle domain, and a C-terminal UBA (ubiquitin-associated fold) (Suyama et al. 2000). It also shows significant similarity with its yeast counterpart the export factor Mex67. The ScanProsite analysis revealed that PfTAP contains several LRR domains distributed throughout its length but concentrated mainly in the N- and C-terminal regions and a few asparagine-rich regions also (Fig. 1c). The LRR domain is formed from tandem arrays (2-52 repeats) containing a leucine-rich consensus sequence, and these domains are mainly responsible for macromolecular interactions. It is noteworthy that the P. falciparum genome contains no detectable homologue for Nxt1/P15 because it is well known that Nxt1 homologues are present in higher eukaryotes but they are absent in protozoans. Helicase Dbp5 and Its Substrates It is well established that two DEAD-box helicases Dbp5 and UAP56 play important roles in the RNA export and are essentially required for this export (Köhler and Hurt 2007; Tran et al. 2007). RNA helicases are enzymes involved in almost all the steps of RNA metabolism. The overall directionality of the process of mrna export is determined by Dbp5. IP6 (inositol hexaphosphate) bound Gle1 at the cytoplasmic side of the NPC controls the activation of Dbp5 and provides the energy required for these transport cycles (Fig. 2). Gle1 and IP6 are responsible for the stimulation of the ATPase activity of Dbp5 (Köhler and Hurt 2007). The stimulation of Dbp5 helps in displacement of proteins from RNA-protein complexes and also in RNA unwinding. DEAD-box RNA helicase Dbp5 works in RNPs remodeling at the NPC by removing Mex67 and other hnrnp like proteins from the exported mrnp. The RNP bound protein Nab2p is one of the substrates for Dbp5p, and it is removed from the mrnp by Dbp5p at the cytoplasmic face of the nuclear pore (Köhler and Hurt 2007; Tran et al. 2007). 4

Fig. 2 Schematics of mrna transport through the nuclear pore. The mrna is assembled into a pre-messenger ribonucleoprotein by the THO/UAP56/ALY complex. After association with more pre-mrna factors, such as p15 and TAP, and dissociation of THO and UAP56, this complex passes through the nuclear pore. On the cytoplasmic side, the RNA helicase DBP5 and its activator Gle1 also join the complex, and the transport is complete In the genome-wide analysis of helicases from P. falciparum, it was reported that the gene with PlasmoDB number PF14_0563 is a homologue of Dbp5/DDX19 (Tuteja and Pradhan 2006; Tuteja 2010; Table 1) and is annotated as ATP-dependent RNA helicase and as the homologue of Dbp5 (yeast) and DDX19 (human). Dbp5 is a well-characterized member of DEAD-box family of helicases, and it has been biochemically characterized in detail (Mehta and Tuteja 2011a, b). It was demonstrated that this Dbp5/DDX19 homologue from P. falciparum requires Q motif for its activity and it is a novel dual helicase (Mehta and Tuteja 2011a, b). Its characteristics suggest that it is most likely involved in similar RNA transport processes. A Nab2 homologue was detected in PlasmoDB ( www.plasmodb.org) using the protein sequences of Nab2 (yeast) and the human homologue ( Zinc finger CCCH domain-containing protein 14/NY-REN-37) as query. The parasite's gene shows considerable homology with its yeast and human homologues (Table 1). This gene has been annotated putative coronin binding protein in the Plasmodb database. Furthermore, the ScanProsite analysis revealed that PfNab2 homologue contains an asparagine-rich region and two C3H1-type zinc finger motifs also (Fig. 1d). These zinc finger motifs are usually present in two copies in several eukaryotic proteins from yeast to mammals and have been shown to be responsible for interaction with mrnas. The export of mrna from the nucleus to the cytoplasm is a well-defined process and takes place through a well-organized pathway (Fig. 2). The assembly of export competent mrnp is linked to transcription, and this mrnp is composed of mrna and a number of other RNA export factors. The important factors mainly involved in export of mrna 5

are poly(a) binding protein (PABP), Dbp5, Mex67, and Npl3. The release of mrna from mrnp in the cytoplasm is mediated by Dbp5. The characterization of PABP and Dbp5 from malaria parasite P. falciparum has been reported (Tuteja 2009; Tuteja and Pradhan 2009; Mehta and Tuteja 2011a, b). Further detailed studies are essentially required in order to understand the important pathway of nuclear transport of mrna in the malaria parasite. Cross-References Helicases mrna Splicing and Alternative Splicing RNA References Alber F, Dokudovskaya S, Veenhoff LM, Zhang W, Kipper J, Devos D, Suprapto A, Karni-Schmidt O, Williams R, Chait BT, Sali A, Rout MP. The molecular architecture of the nuclear pore complex. Nature. 2007;450:695-701. Dahan-Pasternak N, Nasereddin A, Kolevzon N, Pe'er M, Wong W, Shinder V, Turnbull L, Whitchurch CB, Elbaum M, Timgilberger W, Yavin E, Baum J, Dzikowski R. PfSec13 is an unusual chromatin-associated nucleoporin of Plasmodium falciparum that is essential for parasite proliferation in human erythrocytes. J Cell Sci. 2013;126:3055-69. Fahrenkrog B, Aebi U. The nuclear pore complex: nucleocytoplasmic transport and beyond. Nat Rev Mol Cell Biol. 2003;4:757-66. Köhler A, Hurt E. Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol. 2007;8:761-73. Linder P. DEAD-box proteins: a family affair - active and passive players in RNP-remodeling. Nucleic Acids Res. 2006;34:4168-80. Mehta J, Tuteja R. A novel dual bipolar Dbp5/DDX19 homologue from Plasmodium falciparum requires Q motif for activity. Mol Biochem Parasitol. 2011a;176:58-63. Mehta J, Tuteja R. Inhibition of unwinding and ATPase activities of Plasmodium falciparum Dbp5/DDX19 homologue. Commun Integr Biol. 2011b;4(3):1-5. Mor A, Suliman S, Ben-Yishay R, Yunger S, Brody Y, Shav-Tal Y. Dynamics of single mrnp nucleocytoplasmic transport and export through the nuclear pore in living cells. Nat Cell Biol. 2010;12:543-52. Raices M, D'Angelo MA. Nuclear pore complex composition: a new regulator of tissue-specific and developmental functions. Nat Rev Mol Cell Biol. 2012;13:687-99. Shankar J, Pradhan A, Tuteja R. Isolation and characterization of Plasmodium falciparum UAP56 homologue: evidence for the coupling of RNA binding and splicing activity by site-directed mutations. Arch Biochem Biophys. 2008;478:143-53. Suyama M, Doerks T, Braun IC, Sattler M, Izaurralde E, Bork P. Prediction of structural domains of TAP reveals details of its interaction with p15 and nucleoporins. EMBO Rep. 2000;1:53-8. Tanner NK, Linder P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol Cell. 2001;8:251-62. Tran EJ, Zhou Y, Corbett AH, Wente SR. The DEAD box protein Dbp5 controls mrna export by triggering specific RNA: protein remodeling events. Mol Cell. 2007;28:850-9. Tuteja R. Identification and bioinformatics characterization of translation initiation complex eif4f components and poly(a)-binding protein from Plasmodium falciparum. Commun Integr Biol. 2009;2:1-16. Tuteja R. Genome wide identification of Plasmodium falciparum helicases: a comparison with human host. Cell Cycle. 2010;9:104-21. Tuteja R, Pradhan A. Unraveling the 'DEAD-box' helicases of Plasmodium falciparum. Gene. 2006;376:1-12. Tuteja R, Pradhan A. Isolation and functional characterization of eif4f components and poly(a)-binding protein from Plasmodium falciparum. Parasitol Int. 2009;58:481-5. Weiner A, Dahan-Pasternak N, Shimoni E, Shinder V, von Huth P, Elbaum M, Dzikowski R. 3D nuclear architecture reveals coupled cell cycle dynamics of chromatin and nuclear pores in the malaria parasite Plasmodium falciparum. Cell Microbiol. 2011;13:967-77. 6

Dr. Renu Tuteja Mammalian Biology: Malaria, International Centre for Genetic Engineering and Biotechnology, New Delhi, India DOI: URL: Part of: 10.1007/_372349 Encyclopedia of Malaria Editors: PDF created on: Prof. Marcel Hommel and Prof. Peter G. Kremsner June, 14, 2014 04:17 7