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Rinn & Chang Ann. Rev. Biochem 81:145, 2012

“The New RNA World”. “RNA molecules perform a stunningly diverse and elegantly complex set of functions”. 2012. ENCODE project: although only ~1.5% human genome is protein-coding, up to 75% is transcribed (much being cell-type specific) Nature 489:101, 2012.

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Rinn & Chang Ann. Rev. Biochem 81:145, 2012

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  1. “The New RNA World” “RNA molecules perform a stunningly diverse and elegantly complex set of functions” 2012 ENCODE project: although only ~1.5% human genome is protein-coding, up to 75% is transcribed (much being cell-type specific) Nature 489:101, 2012 Rinn & Chang Ann. Rev. Biochem 81:145, 2012

  2. RNA content in eukaryotic cells Total cellular RNA ~ 4% mRNA ~ 96% non-coding RNAs (mostly rRNA and tRNA) and small regulatory RNAs (eg. snRNA, snoRNA, miRNA, siRNA…) Northern blot analysis using RNA isolated from cytoplasm vs. nucleus Nucl Cyto Probe: gene X Adapted from Imam Nucl. Acids Res. 38:1559, 2009

  3. “Traditional” view of gene expression in eukaryotes Why is this picture out-of-date? Zhang Nature Reviews Genetics 3: 698, 2002

  4. CTD code and pre-mRNA processing PIC: pre-initiation complex CTD - 52 heptad repeats (of YSPTSPS) in humans, 26 in yeast dynamic changes in its phosphorylation profile “Cracking the CTD code” Montes et al. Gene 501:104, 2012

  5. Types of splicing 1. Nuclear pre-mRNA (spliceosomal) & lariat excised intron 2-step transesterification 2. Nuclear pre-tRNA endonuclease & ligase machinery 3. Group I intron (autocatalytic, ribozyme) - self-splicing - mobile genetic element 4. Group II intron - self-splicing (autocatalytic, ribozyme) - mobile genetic retroelement - two-step transesterification & lariat intron 5. Archaeal pre-tRNA, pre-rRNA Cis-elements in spliceosomal introns Wahl et al Cell 136: 701, 2009

  6. Splicing of pre-mRNAs via two transesterification reactions 2’OH 1st step OH 2nd step Same biochemical pathway in group II intron ribozymes Brown Fig.10.14

  7. U1 snRNA base pairs with 5’ splice site U2 snRNA base pairs with branchpoint site U2AF-65kD protein binds pyrimidine tract U2AF-35kD protein recognizes AG at 3’ splice site BBP (or SF1 in mammals) – branchpoint bridging protein SR proteins (“Ser-Arg” rich splicing activators) bind enhancers (ESE or ISE) & recruit other splicing factors… hnRNPs (repressors) bind silencers (ESS or ISS) Irimia & Blencowe Curr Opin Cell Biol 24:323, 2012 McManus & Gravely Curr.Opin.Gen.Dev 21:373, 2011

  8. How to determine that specific nucleotides at splice site are important? Site-directed mutation (at A of 3’splice site) and monitor effect using in vitro splicing assay Time course of splicing for labelled pre-mRNAs in HeLa cell nuclear extract Gaur Proc Natl Acad Sci 97:115 (2000)

  9. Step-wise assembly of spliceosomal machinery 3D structure by cryo-EM Azubel Mol Cell Sept 2004 How many proteins does the spliceosome contain? Montes et al. Gene 501:104, 2012

  10. U1 snRNP U1 snRNP Conformational rearrangements during spliceosomal assembly RRM: RNA recognition motif RS: Arg-Ser rich motif - base-pairing between branchpoint region and U2 snRNA is stabilized by Arg-Ser rich domain of U2AF65 and components of U2 snRNP Wahl et al Cell 136: 701, 2009

  11. One way to monitor specific RNA-protein interactions: “Gel mobility shift” or “gel retardation” assays Incubate protein extract 5’ 3’ * * * with labelled RNA 2 3 1 4 Native, non-denaturing gel electrophoresis & autoradiography * * * 1 = RNA, no incubation (control) 2 = RNA incubated with protein 3 = 2 + “cold” competitor RNA 4 = 2 + more competitor RNA Cho PNAS 108:8233, 2011 Unlabelled RNA competitor

  12. - if RNA & protein physically very close, can covalently link by UV treatment UV-crosslinking studies - RS (Arg-Ser rich domain) & RBD (RNA binding domain) of U2AF65 - pre-mRNA labelled with single radioactive phosphate at 5’ ss or BP or... - RNase to degrade any RNA not protected by protein - TEV linker cleaved with specific protease Shen & Green Mol Cell 16:363, 2004

  13. How to detect protein-protein interactions? 1. Affinity chromatography 2. Co-immunoprecipitation Lodish Fig.3-43 Alberts Fig. 8-50 - fusion protein (eg. GST-CTD) attached to resin - determine which proteins bind specifically to column

  14. Fluorescence resonance energy transfer (in vivo) FRET Protein-protein interactions RNA-RNA interactions Chudakov Trends Biotech 12:605, 2005 2 different fluorophores (or quencher)

  15. Dynamic network of RNA interactions (snRNA-snRNA, snRNA-intron...) in spliceosome Precatalytic spliceosome Catalytically activated spliceosome - U1 and U4 snRNPs leave spliceosome - U6 snRNA base-pairs with 5’ end of intron & restructuring of base-pairing between U6 and U2 snRNAs “only stem-loop 1 of U5 snRNA is shown” Will & Luhrmann Cold Spring Harbor Perspectives Biol 3:7, 2011

  16. One way of examining specific, short RNA-RNA interactions: Site-directed mutagenesis – loss of function followed by suppressor mutations – restoration of function (eg. in vitro splicing assay…) Assay to monitor effect on splicing wt Mutant U6 5’ …UGAUC…3’ | | | | | U2 3 ’…ACUAG…5’ Double mutant Smith Mol Cell 26:883, 2007

  17. Is the spliceosome a ribozyme (like group II introns)? .... or is splicing catalyzed by protein? Candidate protein: Prp8 (in U5 snRNP) Prp8 can be crosslinked to5’ SS, the branchpoint region, and to 3’ SS “Colored regions encompass residues that exhibit interactions with the factors indicated.” Wahl et al Cell 136: 701, 2009

  18. Trans-splicing of pre-mRNAs - found in certain protists (eg. trypanosomes) and “lower” animals (eg. nematodes) - separate SL “spliced leader” RNA provides the first (non-coding) exon - biochemical mechanism is fundamentally the same as cis-splicing - excised intron is Y-shaped In C. elegans, ~ 70% mRNAs are trans-spliced and in trypanosomes virtually all mRNAs are trans-spliced (so have the same 22 nt at end of 5’ UTRs for all mRNAs) Blumenthal WormBook. 2005 pp1-9.

  19. In trypanosomes & nematodes, some genes are organized in operons ! Landfear PNAS 100:7, 2003

  20. Singh & Cooper Trends Mol Med 18:472, 2012 2d Minor Class of Spliceosomal Introns “AT-AC introns: an “ATtACk on dogma” Mount, Science 271:1690, 1996 Figure 1. Spliceosome assembly and disease-associated mutations in spliceosome components. The broken lines and rectangles represent introns and exons, respectively. The left panel shows the assembly of the major (U2 type) spliceosome. U1 and U2 small nuclear ribonucleoproteins (snRNPs) are recruited to the consensus 50 splice site (50 SS) and branch point (A), respectively. The U2-auxiliary factor heterodimer (U2AF2/U2AF1) interacts with the polypyrimidine track (Y) and 30 splice site (30 SS), forming complex A. The U4/6 and U5 snRNPs join the assembling spliceosome followed by remodeling of the complex leading to removal of the U1 and U4 snRNP and formation of the catalytic complex (complex C). Two transesterification reactions join the exons and release an intron lariat that is subsequently degraded and the spliceosome components are recycled for subsequent rounds of splicing. The right panel shows the assembly of the minor (U12 type) spliceosome, in which U1, U2, U4, and U6 are replaced by homologous U11, U12, U4atac, and U6atac snRNPs, respectively. The red star indicates the components that are mutated in neoplasias. The black star indicates the components that are mutated in retinitis pigmentosa. The orange star indicates the mutation in U4atac that is associated with microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Abbreviations: ESEs, exonic splicing enhancers; ESSs, exonic splicing silencers

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