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Pre-Mrna Splicing 27 Pre-mRNA splicing 27. November 2018 Clemens Grimm Discovery of splicing Richard J. Roberts Phillip A. Sharp Nobel Prize in Physiology or Medicine 1993 "for their discoveries of split genes" mRNA splicing: the initial observation Genomic globin gene annealed to heterogenous nuclear RNA (hnRNA) containing globin pre- mRNA Genomic globin gene annealed to cytoplasmic RNA containing globin Tilghman SM et al. (1978) PNAS 75, 1309 mRNA Pre-mRNA splicing Intron number can vary greatly among genes Anzahl Introns Beispiel 1 meiste Gene in Hefe 50 Huhn Proa2 Kollagen 363 Humanes Titin Gen 0<5% Intron and exon length Exon Durchschnitt: 150 Nukleotide Intron bis zu 800.000 Nukleotide The primary transcript (pre-mRNA) can be very long Human Dystrophin-Gene: 79 exons 2.400.000 bases 260 kb intron 2.4 Mb > Needs approx. 16 h for transcription! Canonical splice sites Polypyrimidine-Tract + 3‘ splice site Little information in cis... • Problem: How to reliably locate and contract reactive sites? • Note: (Guide) RNAs are ideally suited to recognize short consensus elements in nucleic acids. Chemistry of splicing (step 1) Chemistry of splicing (step 2) The chemistry of splicing (overview) 5' Spleißstelle Verzweigungspunkt 3' Spleißstelle 5' AG GURAGU YNYURAC Yn NYAG G 3' Pre-mRNA EXON 1 EXON 2 2'-OH 5' p GU A AG p 3' 2‘-5‘ Phospho- Erster katalytischer Schritt G diesterbond U G p 5' OH A AG p 3' Intermediate Zweiter katalytischer Schritt U G p 5' EXON 1 p EXON 2 3' Spleißprodukte A AG-OH + Intron mRNA Back-splicing generates a new class of ncRNA rnapairing.png 976×240 Pixel 08.12.17, 08 36 The spliceosome catalyzes splicing pre-mRNA Exon1 Exon2 “Spliceosome” Exon1 Exon2 Mature mRNA Exon1 Exon2 A large number of trans-acting factors Yeast ~ 90 proteins 5 snRNAs Fabrizio P et al. (2009) Mol Cell 36, 593 Human ~ 170 proteins 5 snRNAs • Essentially all yeast proteins conserved in humans • Approx. 80 additional proteins in humans • Yeast: Conserved core. U-rich small nuclearThe ribonucleoproteins spliceosome ( U snRNPs) The spliceosome consists of snRNPs (small nuclear Ribonucleoprotein Particles): U1 U2 snRNA U1, U2, U4, U5, U6 U4 common snRNP proteins specific snRNP proteins U5 U6 Common features of U snRNPs m G m G GAUUUUUGG 3 AAUUGUGG 3 U1 RNA U2 RNA m G 3 AAUUUUUUG U4 RNA U5 RNA U6 RNA Sm and LSm proteins and structures The U snRNP Assembly Reaction Illustrated by existing Crystal Structures (Nagai Lab) (Dreyfuss Lab) Protein composition of U snRNPs General architecture of U snRNPs Aus: Stark et al., 2001, Nature 409, 539-542. Function of U snRNPs in splicing The splice cycle 1 5´Exon1 GUU1 U2 A AGExon 2 3´ The splice cycle 2 U4 U6 5´ Exon1 U1 U5 U2 Complex BA The splice cycle 3 mRNA 5´Exon 1 Exon 2 3´ U4 U6 5´ Exon1 U1 U5 U2 CatalyticallyComplex activeB spliceosome Intron Assembly and catalysis intermediates • The spliceosome assembles stepwise in nuclear extract. 0’ 2’ 7’ 20’ 60’ C B A H/E mRNP Native gel electrophoresis of splicing complexes A large number of trans-acting factors Yeast ~ 90 proteins 5 snRNAs Fabrizio P et al. (2009) Mol Cell 36, 593 Human ~ 170 proteins 5 snRNAs • Essentially all yeast proteins conserved in humans • Approx. 80 additional proteins in humans • Yeast: Conserved core. Repeated, massive compositional changes • Purification under native-like conditions • Double affinity purification • Identification by mass spectrometry Repeated, massive compositional changes Repeated, massive compositional changes Repeated, massive compositional changes U1 snRNP defines the 5‘ SS U1 snRNP A U1 RNA 70K700K SmSm 3‘ 5‘ 5‘ Exon Intron Exon C 5‘ SS recognition by U1 Branch point recognition Functional pairing of U1 and U2: B-complex formation U1 snRNP U2 snRNP 3‘ Spleißstelle 5‘ Spleißstelle U2AF Intron BBP Consolidation of weak RNA-RNA interactions by proteins Repeated recognition • Different factors recognize the same RNA sites multiple times during one splicing event. Formation of a catalytic RNA network 1: tri-snRNP U6 CH 3 m G 3 AGUUUUUAA U4 U4/U6 snRNP U4/U6 RNA U4/U6.U5 + tri-snRNP Formation of a catalytic RNA network 2: C-complex Komplex A Komplex B 1. Spleißschritt katalytisch aktives Spleißosom Formation of a catalytic RNA network 3: U2-U6 U6 snRNA U4 snRNA U6 snRNA U2 snRNA Pre-mRNA U1 snRNA U2 snRNA Catalytic RNA network – Branch point U6 snRNA U2 snRNA Pre-mRNA Pre-mRNA U1 snRNA A catalytic triplex Mg2+ coordinates donor and acceptor groups Group II introns and spliceosome active center Why proteins? Positioning and fixation of the catalytic center through Spp42/Prp8 Remodeling down to the snRNP level Prp24 assists U6/U2 U6/U4 re-pairing Prp24=SART3 RNA-binding protein Controlling and driving the splicing cycle DExD/H Helicases The main driving forces: DExD/H-box proteins We have two types of introns >99 % of all pre-mRNAs: U2-dependent „GU-AG“ (major) 5' Splice SiteBranch site 3' Splice Site 5'Exon 103-105 nts 20- 40 nts 3'Exon 5'- AG GURAGU YNYURAC (Yn) YAG G -3' <1 % of all pre-mRNAs: U12-dependent „AT-AC“ (minor) 103-105 nts 10-20 nts 5'- AUAUCCUUU UCCUUAAC YAC -3' Splicing of AT-AC introns requires a specialized spliceosome 5' Splice SiteBranch site 3' Splice Site 5' Exon 3 5 3' Exon 10 -1 0 nts 20-40 nts 5'- AG GURAGU YNYURAC (Yn) YAG G -3' U1 U2 +U4/6.U5 3 5 10 -1 0 nts 10-20 nts 5'- AUAUCCUUU UCCUUAAC YAC -3' U11 U12 +U4/6(AT-AC).U5 Summary The Spliceosome is built up by U snRNPs and an exchangeble set of associated proteins The splicing process is accompagnied by drastic reconfigutations of the splicing machine which is assembled de novo for each splicing event Key remodelling events are under control of DExD/H helicases The core of the catalytically activated spliceosome is reminiscent of group II introns The spliceosome is an RNA sculptor that can handle a set of highly divergent substrates with extreme precision.
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