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Alternative Splicing Alternative Splicing Multiple control Mechanisms & Involvement in human disease Anabella Srebrow Laboratorio de Fisiología y Biología Molecular IFIBYNE-CONICET Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires outside cytoplasm nucleus protein-coding genes Cleavage and Polyadenylation Capping Splicing Co-transcriptional processing post-transcriptional co-transcriptional pre-mRNA processing pre-mRNA processing Pol II Pol II Transcription Capping Transcription Splicing Polyadenylation Primary transcript CAP Capping Splicing Polyadenylation An CAP An CAP Co-transcriptional processing • requires coordination between molecular machineries acting at different steps • coordination is mainly achieved by the function of RNA polymerase II CTD (Carboxi Terminal Domain) multiple repeats of “YSPTSPS” heptad Serine phosphorylations are critical for transcription & association with processing factors mRNA factory governed by pol II CTD capping polyadenylation factors factors P P P P P P P Splicing factors CTD (YSPTSPS)52 Bentley lab: McCracken et al., Nature 1997 Figure 6-23 Molecular Biology of the Cell (© Garland Science 2008) Pol II CTD is crucial for pre-mRNA processing TRANSCRIPTION CAPPING SPLICING polyA Pol III Normal Inhibited Inhibited Inhibited Cleveland lab: Sisodia et al., MCB 1987 Normal Normal Normal Normal Normal* Inhibited Inhibited Inhibited Bentley lab: McCracken et al., Nature 1997 What is Splicing ? AUG STOP Lynch, Nature Reviews Immunol. 2004 Sequences required for splicing 30-50 nt exon exon a c c a c ...AGgu agu........................//.... x u a ...//…………..ccucuucucuucuccuxcagG..... g u u g u Branch site Polypyrimidine ’ tract 5 splice site ’ (5’ss) 3 splice site (3’ss) DONOR SITE ACCEPTOR SITE The Spliceosome snRNP snRNP snRNP U5 U4 U6 snRNP U1 snRNP U2AF65 U2AF35 U2 BBP 30-50 nt exon exon a c c a c ...AGgu agu........................//.... x u a ...//…………..ccucuucucuucuccuxcagG..... g u u g u Branch site Polypyrimidine ’ tract 5 splice site ’ (5’ss) 3 splice site (3’ss) DONOR SITE ACCEPTOR SITE Snurps or snRNPs • Small nuclear ribonucleoprotein particles (small nuclear RNA + proteins) • U1, U2, U4, U5 and U6 involved in nuclear RNA splicing • Snurps constitute components of the spliceosome and are key components of splicing process • Each snurp has a unique role is splicing and the sequence of the RNA component of the snurp determines the role played in splicing alowing specific snRNA-premRNA interactions Downloaded from cshperspectives.cshlp.org on October 5, 2011 - Published by Cold Spring Harbor Laboratory Press C.L. Will and R. Lu¨hrmann E A 1 p O A OH H + 5¢SS BP 3¢SS p E1 p A ppE2 A E2 E1 p E2 Step 1 Step 2 B Branch Poly Y 5¢ splice site 3¢ splice site site tract The spliceosome cycle Exon GURAGU YNCURAC Y(n) YAG Exon Metazoans Exon GUAUGU UACUAAC YAG Exon Yeast C 5¢ SS BP 3¢ SS GU A AG Pre-mRNA Intron mRNA U1 Prp43 - ATP Brr2 - ATP Snu114 - GTP U1 U2 Complex E U5 U6 U2 U2 Prp5 - ATP U5 U6 Sub2/UAP56 - ATP Prp22 - ATP U1 U2 U6 U2 Pre-spliceosome U6 U4 (complex A) U5 U5 Post-spliceosomal Prp28 - ATP U4/U6.U5 complex tri-snRNP U1 Prp22 - ATP Prp16 - ATP U6 U4 2nd step U1 U5 U2 U6 Pre-catalytic U4 U2 spliceosome U5 Catalytic step 1 (complex B) Brr2 - ATP spliceosome (complex C) Snu114 - GTP U6 U2 U6 U2 1st step U5 U5 Bact B* (activated) Prp2 - ATP (catalytically activated) D SR protein U2AF RS U2 U1 65 35 RRM Exon GU A YYYYYY AG ESE Exon GU Wahl, Will & Luhrmann, 2011 Luhrmann, & Will Wahl, Figure 1. Pre-mRNA splicing by theU2-typespliceosome. (A) Schematicrepresentation of thetwo-step mechanism of pre-mRNA splicing. Boxes and solid linesrepresent theexons(E1, E2) and theintron, respectively. Thebranch site adenosineisindicated by theletter A and thephosphategroups( p) at the5′ and 3′ splicesites, which ar econser ved in the splicing products, ar e also shown. (Seefacing pagefor legend.) 4 Cite as Cold Spring Harb Perspect Biol 2011;3:a003707 Dynamic composition of spliceosome Wahl, Will & Luhrmann, 2011 Other proteins (SR proteins) cooperate with snRNPs for spliceosome assembly Three types of spliceosome In mammalian cells Trypanosomes and nematodes More than 99% Less than 1% Figure 6-34a Molecular Biology of the Cell (© Garland Science 2008) What is Alternative Splicing ? splice site sequences may deviate from the consensus recognition sites for the spliceosome, creating “weak splice sites” (less efficiently recognized by spliceosome) COMPETITION between different donor or different acceptor sites Recognition of splice sites by the spliceosome can be impaired by pre-mRNA secondary structure or by RNA binding proteins (splicing regulatory factors) Differential removal of exonic sequences or inclusion of intronic ones Different alternative splicing patterns mRNA pre-mRNA isoforms constitutive exon cassette A mutually exclusive exons A B B Alternative alternative 5’splice sites Splicing alternative 3’ splice sites A intron retention The relevance of alternative splicing It is more a rule than an exception. It is estimated to affect the expression of more than 65% of human genes. It explains how a vast protein diversity is achieved with a limited number of genes. Mutations that affect alternative splicing regulatory sequences are a widespread source of human disease. Examples of alternative splicing AUG Thyroid AATAA AATAA A A 2 3 3 4 5 6 AUG Neurons fruitless (sex-specific AS) Calcitonin/CGRP transcription factor - droshophila sex hormone determination epithelial tissue Embryo VASE 7 8 7 IIIb IIIc 10 Adult mesenchymal tissue NCAM (develpmentally regultated FGFR-2 AS) GF receptor (diff. binding properties) neural cell adhesion molecule Smith and Valcárcel, TIBS 2000 Splicing regulatory proteins Nilsen & Graveley, Nature 2010, vol 463 Key regulators of alternative splicing They bind to regulatory sequences (ENHANCERS or SILENCERS) within exons or introns, stimulating or inhibiting inclusion of alternative regions into mRNA SR PROTEINS (serine/arg rich) hnRNP PROTEINS (Heterogenous nuclear ribonucleoproteins) Lynch, Nature Reviews Immunol. 2004 The balance of positive- and negative-acting factors present in a given cell determine the extent of regulated exon inclusion ISS intronic splicing silencer; ISE intronic splicing enhancer ESS exonic splicing silencer; ESE exonic splicing enhancer Regulation of splicing factor activity by extracellular signals extracellular stimuli signalling pathways (ERK, Akt, JNK) Expression level Post-translational modification Subcelular-localization of splicing factors Regulation of pre-mRNA alternative splicing Mutations that affect alternative splicing regulatory sequences are a widespread source of human disease. 15-50% of the mutations that cause genetic disease affect the splicing process -thalassemia abnormal hemoglobin abnormal processsing of -globin due to point mutations Figure 6-35 Molecular Biology of the Cell (© Garland Science 2008) Examples of mutations in cis-acting sequences that are associated with malignant disease Gene Disease Mutated sequence Molecular Reference consequences LKB1 Peutz-Jeghers Disruption of minor (tumour suppressor) Syndrome IVS2+1A>G spliceosome Hasting et al., 2005 (increased cancer risk) 5’ splice site. Lower protein levels KIT Gastrointestinal Deletion of 9 Aberrant splicing Chen et al., 2005 (oncogene) stromal tumour nucleotides including Constitutive protein IVS10 3’ splice site activation LI-Cadherin Hepatocellular IVS6+35 A>G Generation of a ISS? Wang et al., 2005 carcinoma Exon 7 skipping LI-Cadherin Hepatocellular E6 codon 651 Generation of a ESS? Wang et al., 2005 carcinoma Exon 7 skipping KLF6 Prostate cancer IVSDA Generation of binding Narla et al., 2005 (tumour suppressor) site (ISE) for SRp40 Novel splicing variants act as dominant negatives HAS1 Multiple myeloma E3 C7760T Exon 4 skipping Adamia et al. 2005 (hyaluronan synthase) BRCA1 Breast and ovarian E18 G5199T [E1694X] ESE disruption Mazoyer et al. 1998 (tumour suppressor) cancer Exon 18 skipping S Table 1. Examples of mutations in splicing cis-acting sequences that are associatedSrebrow with malignant & Kornblihtt disease. 2006, JCS Mutations disrupting cis-acting splicing sequences cause splicing defects and disease SMN protein: important for snRNP biogenesis SMN deficiencies = Motor neuron degenerative disease Mutation creates a splicing silencer, leading to exon skipping and a partially functional dystrophin protein. Dystrophin communicates the extracellular matrix with the contractile apparatus in skeletal muscle cells Tau: microtubule associated protein important for neuronal processes (neurite outgrowth/axonal transport) ex 10 = 4th microtubule binding domain altered ratio, aggregation of Tau neurodegenerative disease Transmembrane Chloride Channel important for proper function of secretory epithelium (lung, intestine, testes) Cooper, Wan and Dreyfuss.Wan 2009, Cell136: 777 Cooper, Minor splicing, Disrupted 700-800 genes in the human genome contain U12-type introns Pessa & Frilander, 2011 Science Mutations in the snRNA component of the “minor U4” (U4atac) linked to human disease (developmental disorder MOPD I or TALS) interaction between human U6atac and U4atac snRNAs, mutations identified in U4atac mutations distort the conserved stem-loop structure, preventing the binding of spliceosomal protein Genetic alterations of the major splicing components linked to human pathogenesis Mutations in multiple components of the splicing machinery associated to myelodysplasia, would lead to impaired recognition of 3’ss and the production of aberrantly
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