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1 Alternative Splicing
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2 Eukaryotic genes Splicing Mature mRNA
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3 The mechanism of RNA splicing
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4 The mechanism of splicing A 1212 A -OH 1 2 YYYYYYYYYNCAGGTRAGTACAGG 5’ splice site 3’ splice siteBranch point
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5 Alternative splicing 3214 3214 Splicing 314 Alternative Mature splice variant II Mature splice variant I Can be specific to tissue, developmental- stage or condition (stress, cell-cycle). 50-70% of mammalian genes
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6 Some types of alternative splicing Exon skipping Alternative Acceptor Alternative Donor Mutually exclusive Intron retention
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7 Sex determination in fly
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10 Many variants in one gene
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11 DSCAM
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12 Antibody secretion
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13 Antibody secretion immunoglobulin μ heavy chain
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14 Tissue specific alternative splicing
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15 Detection of alternative splicing n By sequencing of RNA n Old methods (1995-2007) – ESTs n New methods: –Splicing-sensitive microarrays –RNA-seq
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16 Expressed Sequence Tags (ESTs) AAAAAAAAA TTTTTTTTTT AAAAAAAAA RT Cloning AAA cDNA mRNA Vector
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17 EST preparation 5’ EST 3’ EST Random-primed EST Average size of EST ~450bp Picking a clone
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18 Alignment of ESTs to the genome EST DNA EST 8 million public human ESTs, collected over >10 years (NCBI)
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19 Splicing microarrays
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20 Massive sequencing of RNA (RNA-seq)
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21 Wang et al Nature 2008 RNA-seq on multiple tissues
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22 Splicing regulation
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23 Tissue specific alternative splicing How is this process regulated?
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24 Regulation of alternative splicing n Splicing Enhancers/Silencers n Specifically bind SR proteins
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25 Exon Weak splice site AGY(n) Model for ESE action SR brain Exonic Splicing Enhancer (ESE)
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26 SR proteins structure
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28 Discovery of ESEs Exon Silent mutations can cause exon skipping
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29 Regulators of splicing SR proteins (Splicing factors) ESE/ESS ISEISS Complex regulation usually exists Hard to find intronic elements For most alt exons – regulation unknown Signal transduction
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30 How can we break the regulatory code? n 1. Comparative genomics n 2. High throughput methods
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31 Comparative genomics: Use the mouse genome to find sequences that regulate alternative splicing
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32 Human-mouse comparisons
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33 The mouse genome n 100 million years of evolution n Average conservation in exons: 85% n Only 40% of intronic sequences is alignable n Average conservation in alignable intronic sequences: 69% n Average conservation in promoters: 77% n Function => evolutionary conservation
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34 Conservation of near introns (from VISTA genome browser, http://pipeline.lbl.gov)
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35 Collection of exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743
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36 Finding the mouse homolog AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.
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37 Conservation in the intronic sequence near exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.
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38 Flanking conserved introns Results Alternative exons Constitutive exons ~100 bp from each side of the exon
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39 Conservation of introns
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40 Alternative splicing regulatory sequences? n Could serve as binding sites for splicing regulatory proteins
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41 Motif searching n Top scoring hexamer in conserved downstream regions: TGCATG (9-fold over expected) n Not over-represented downstream to constitutive exons. n Binding site for FOX1 (splicing regulatory protein)
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42 Functional elements in the human genome n 5% of the human genomic sequence is considered functional
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43 Functional elements in the human genome
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44 Impact of splicing regulatory elements n ~12,000 alt. spliced exons in the genome n 77% have conserved flanking intronic sequences n ~100bp conserved on each side n 12,000 exons * 100 bp * 2 introns * 0.77= 2M bases n ==>At least 2 Million bases in the human genome might be involved in alternative splicing regulation. n >1% of all functional DNA in the genome regulates alt splicing!
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45 How can we break the regulatory code? n 1. Comparative genomics n 2. High throughput methods
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46 CLIP-seq Licatalosi et al, Nature 2008: 412,686 sequences Ule et al, Science 2003: 340 sequences
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47 Nova, a brain-specific splicing regulator Ule et al, Science 2003: 340 sequences
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48 Ule et al, Science 2003: 340 sequences
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49 Extracting the regulatory motifs
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50 The power of deep sequencing (2008)
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51 Mutations causing aberrant splicing Exon ~15% of all point mutations linked to genetic disorders involve splicing alterations
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52 Mutations causing aberrant splicing: SMN
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53 Summary – alt splicing n Increases the coding capacity of genes n We have 25,000 genes but much more protein isoforms
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54 RNA EDITINA
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55 RNA EDITING
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56 What is RNA editing? n Alters the RNA sequence encoded by DNA in a single-nucleotide, site-specific, manner n If splicing is “cut and paste” editing is the “spelling checker”.
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57 Mode of operation: A-to-I editing A-> G Editing performed by ADAR enzymes (dsRNA specific adenosine deaminases) Double strand RNA is required
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58 Mechanism of RNA-editing (A-to-I)
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59 Functions of RNA editing n Defense against dsRNA viruses n Also involved in endogenous regulation
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61 Functional consequences of RNA editing Protein change RNA stability Splicing n In human, RNA editing is particularly pronounced in brain tissues, due to excess of ADAR expression in brain n Neural disorders (glioblastoma, epilepsy, ALS) are linked to changes in RNA-editing patterns n Editing levels vary in other tissues (minimal editing in skeletal muscle, pancreas).
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62 Finding RNA-editing sites n Theoretically easy : find mismatch between genome to RNA n Huge number of sequencing errors n Mutations n Duplications n SNPs Signal drowns in noise
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63 Computational approach for identification of editing sites n Alignment of ESTs to genome n Find potential intramolecular dsRNA n Data cleaning Levanon et al, Nature Biotech 2004
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64 Intramolecular dsRNA Exon RNA Intron Levanon et al, Nature Biotech 2004
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65 ESTs to genome Levanon et al, Nature Biotech 2004
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66 dsRNA regions Levanon et al, Nature Biotech 2004
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67 dsRNA regions Masking EST’s ends Levanon et al, Nature Biotech 2004
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68 dsRNA regions Masking EST’s ends Masking poor sequence regions
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69 dsRNA regions Masking EST’s ends Masking poor sequence regions Removing known genomic SNPs Levanon et al, Nature Biotech 2004
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70 dsRNA regions Masking EST’s ends Masking poor sequence regions Removing SNPs Collecting candidates Levanon et al, Nature Biotech 2004
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71 Results DNA RNA (ESTs)
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72 Levanon et al, Nature Biotech 2004
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74 RNA-editing – a source for human transcripts diversity n >12,000 editing sites in >1,600 human genes n Vast majority of editing – in UTRs n Vast majority of editing – in Alu (repetitive) n A few editing sites in protein-coding regions Levanon et al, Nature Biotech 2004
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75 And the obligatory next generation sequencing study… (Li, Levanon et al, Science 2009) Editing sites in non-repetitive regions
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76 Connection between editing and splicing Negative feedback loop ADAR gene (editing enzyme)
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77 Evolution of a new exon
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78 Summary – alt splicing and RNA editing n Increases the coding capacity of genes n We have 25,000 genes but much more protein isoforms
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