Post-class review: Glisovic et al. (2008) RNA-binding proteins and posttranscriptional gene regulation. FEBS letters 582:

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1 BMB 632 Probing structure and function of complex RNA-protein machines Instructor: Kristen W Lynch Co-Instructor: Jeremy Wilusz Tuesdays, 12-2 CRB 302 (and Anat-Chem 250/255) Summary: RNA-Protein complexes or RNPs can range from simple assemblies to megadalton enzymatic machines. The latter include two of the most abundant and essential enzymatic complexes for converting genes to functional protein the ribosome and the spliceosome. Understanding the molecular interactions that hold these RNPs together and how these complexes function has required the development of new techniques and pushed the boundaries of quantitative biochemistry. In this course we will take an in-depth look at general concepts common to many RNA binding proteins, the methods used to study protein-rna and RNA-RNA interactions, and how the complex nature of large RNPs uniquely allow them to achieve their precise functions. The course will be a combination of both lectures and student-lead discussion of recent literature. Students will be evaluated based on their presentations of primary literature and their participation in class discussion and a final oral exam. Syllabus: Sept 6 Overview of RNA-Protein Machines Post-class review: Glisovic et al. (2008) RNA-binding proteins and posttranscriptional gene regulation. FEBS letters 582: Sept : Penn RNP Discussion Group (in JF Library, Anat-Chem 250) 1-2: Discussion of Basic Methodology: primer extension, RT-PCR, RNA-Seq, CLIP- Seq (Anat-Chem 255) Sept 20 RNA-folding I - Self-splicing: footprinting and NIAM/NAIS José Almeida Cruz and Eric Westhof. The Dynamic Landscapes of RNA Architecture. Cell 136, February 20, 2009 Konforti et al (1998) A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme. EMBO J 17: Mariel

2 * Boudvillain and Pyle (1998) Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: NAIM analysis. EMBO J 17: Charlotte Boudvillain, et al. (2000) A tertiary interaction that links active-site domains to the 5' splice site of a group II intron. Nature 406(6793): (focus just on what NAIS added to story) Sept 27 RNA folding II: Riboswitches: in-line probing and SHAPE Watcher (2014) Gene Regulation by structures mrna elements. Trends Genet 30: Winkler WC, et. al. (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419: Caleb Rice et. al. (2014) SHAPE Analysis of small RNAs and Riboswitches. Methods Enzy 549: *Johnson JE et al. (2012) B12 cofactors directly stabilize an mrna regulatory switch. Nature 492: Tyrell J et al. (2013) The Cellular Environment Stabilizes Adenine Riboswitch RNA structure. Biochemistry 52: (focus on how SHAPE is used in vivo) 4 Kiara Oct 4 Structure and determinants of RNA-RBP interface: NMR and FRET Lunde et al. (2007) RNA-binding proteins: modular design for efficient function. Nat Rev Mol Cell Biol 8: Oberstrass (2005) Structure of PTB bound to RNA: Specific binding and implications for splicing regulation. Science 309:2054 (use as background) 5 Krystal *Lamichhane et al (2010) RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression. Proc Natl Acad Sci U S A. 107:

3 Mackereth CD, et al. (2011) Multi-domain conformational selection underlies premrna splicing regulation by U2AF. Nature 475: Agrawal et al (2016) An extended U2AF65-RNA binding domain recognizes the 3 splice site signal. Nat Comm doi (these present competing structures so focus on methods and similarities /differences) 6 Nikki Oct : Penn RNP Discussion Group (note: in JF Library, Anat-Chem 250) 1-2: RNA binding proteins: Identifying and determining binding specificity by UV crosslinking, RNA affinity, EMSA (1-2 Anat Chem 255) *Rothrock et al., (2005) HnRNP L represses exon splicing via a regulated exonic splicing silencer. EMBO J 24: Gabriel Motta-Mena et al., (2011) A Disease-associated polymorphism alters splicing of the human CD45 Phosphatase Gene by Disrupting Combinatorial Repression by hnrnps. J Biol Chem 286: *(focus on Figs 4-6) Oct 18 Ribosomes: Interplay of RNA folding and RBP binding Priya and Woodson (2009) S16 throws a conformational switch during assembly of the 30S 5 domain. Nat Struct Mol Biol 4: (use as background) 8 Laura *Soper et al (2013) In vivo x-ray footprinting of pre-30s ribosomes reveals chaperone-dependent remodeling of late assembly intermediates. Mol Cell 52: Bunner et al (2010) Kinetic cooperativity in E. coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape. PNAS 107: (use as background) *Sashital et al (2014) A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli. elife 3:e Christian Oct 25 Spliceosome: From Biochemistry (Native gels, RNAse H, psoralen) to EM Wahl et al (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136: (an excellent but mammoth review- Fig 2 is critical, the rest is helpful)

4 Konarska and Sharp (1987) Interactions between small nuclear ribonucleoprotein particles in formation of spliceosome. Cell 49: *Wasserman and Steitz (1992) Interactions of small nuclear RNAs with precursor messenger RNA during in vitro splicing. Science 257: Ashley Sartoris Rauhut et al (2016) Molecular architecture of the Saccharomyces cerevisiae activated spliceosome. Science epub Galej et al (2016) Cryo-EM structure of the spliceosome immediately after branching. Nature epub *we could spend a entire semester on these papers. Just focus on the highlights and differences between the structures. See Kosmyna and Query (Nature N&V) for help. Other reviews are likely to come out before Oct 4. This is Nobel material. 11 Leann Nov 1 Regulation of Splicing: Site-specific labeling, psoralen, MS2 purification Fu and Ares (2014) Context-dependent control of alternative splicing by RNAbinding proteins. Nat Rev Genetics 15: *Chiou et al. (2013) HnRNP L and hnrnp A1 induce extended U1 snrna interactions with an exon to repress spliceosome assembly. Mol Cell 49: Jacob Sharma et al (2011) U1 snrna directly interacts with the polypyrimidine tractbinding protein during splicing repression. Mol Cell 41: (use as background) *Sharma et al (2014) Stem-loop 4 of U1 snrna is essential for splicing and interacts with the U2 snrnp-specific SF3A1 protein during spliceosome assembly. Genes Dev 28: Richard Nov : Penn RNP Discussion Group (note: in JF Library, Anat-Chem 250) 1-2: Nucleotide modifications in and by RNP complexes (Anat-Chem 255) McMahon et al (2015) Small RNAs with big implications: New insights into H/ACA snorna function and their role in human disease. WIRES RNA 6:

5 *Jack K, et al. (2011) rrna pseudouridylation defects affect ribosomal ligand binding and translational fidelity from yeast to human cells. Mol Cell 44: Zhao et al (2016) Psuedouridylation of 7SK snrna promotes 7SK snrnp formation to suppress HIV-1 transcription and escape from latency. EMBO Rep epub (this has lots of data, but just use as another example of H/ACA impact - and mention function of 7SK snrnp) 14 Ashley Viera Nov 15 Telomerase and SRP: Applying concepts to additional RNP machines Blackburn EH and Collins K (2011) Telomerase: an RNP enzyme synthesizes DNA. Cold Spring Harb Perspect Biol pii: a Mitchell et al. (2010) Structural basis for telomerase catalytic subunit TERT binding to RNA template and telomeric DNA. Nat Struct Mol Biol 17: Jiang, et al. (2013) The architecture of Tetrahymena telomerase holoenzyme. Nature 496: Will Akopian et al (2013). Signal recognition particle: an essential protein-targeting machine. Annu Rev Biochem 82: (long! just skim) Maity et al. (2006) Compartmentalization directs assembly of the Signal Recognition Particle. Biochemistry 45: (just for background) 16 *Chartron et al (2016) Cotranslational signal-independent SRP preloading during membrane targeting. Nature in press (there is also back-to-back paper that looks at the same thing for those interested) Nov 22 Helicases and understanding the dynamic nature of RNP machines: FRET and CoSMoS Semlow et al (2016) Splicoeosomal DEAH-box ATPases Remodel pre-mrna to Activate Alternative Splice Sites. Cell 164: Diane Hoskins et al (2011) Ordered and Dynamic Assembly of Single Spliceosomes Science 331: (just read for basic method) Hoskins AA et al (2016) Single molecule analysis reveals reversible and irreversible steps during spliceosome activation. Elife 5 pii: e Saurabh

6 Nov 29 RISC and CRISPR: How understanding RNP Machines can inspire useful tools Nakanishi (2013) Anatomy of RISC:how do small RNAs and chaperones activate Argonaute proteins. WIREs RNA 7: Hammond et al (2001) Argonaute 2, a link between genetic and biochemical analyses of RNAi. Science 293: (use for background) 19 Matt *Salomon et al (2012) Single-Molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides. Cell 162: Koonin and Makarova (2013) CRISPR-Cas. RNA Biol 10: Deltcheva et al (2011) CRISPR RNA matureation by trans-encoded small RNA and host factor RNAse III. Nature 471: (use for background) 20 Evan *Jinek et al (2012) A Programmable dual-rna guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337: Dec 6 Gideon Dreyfuss: The RNP world Dec 13 Penn RNP Discussion Group (note: in JF Library, Anat-Chem 250) and Exam (Final Date TBD)