4 The mechanism of splicing A 1212 A -OH 1 2 YYYYYYYYYNCAGGTRAGTACAGG 5’ splice site 3’ splice siteBranch point
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
6 Some types of alternative splicing Exon skipping Alternative Acceptor Alternative Donor Mutually exclusive Intron retention
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
34 Conservation of near introns (from VISTA genome browser, http://pipeline.lbl.gov)
35 Collection of exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743
36 Finding the mouse homolog AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.
37 Conservation in the intronic sequence near exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.
38 Flanking conserved introns Results Alternative exons Constitutive exons ~100 bp from each side of the exon
40 Alternative splicing regulatory sequences? n Could serve as binding sites for splicing regulatory proteins
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)
42 Functional elements in the human genome n 5% of the human genomic sequence is considered functional
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!
45 How can we break the regulatory code? n 1. Comparative genomics n 2. High throughput methods
46 CLIP-seq Licatalosi et al, Nature 2008: 412,686 sequences Ule et al, Science 2003: 340 sequences
47 Nova, a brain-specific splicing regulator Ule et al, Science 2003: 340 sequences
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).
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
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
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
75 And the obligatory next generation sequencing study… (Li, Levanon et al, Science 2009) Editing sites in non-repetitive regions
76 Connection between editing and splicing Negative feedback loop ADAR gene (editing enzyme)