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Alternative Splicing

Alternative Splicing. Splicing. Eukaryotic genes. Mature mRNA. The mechanism of RNA splicing. 5’ splice site. Branch point. 3’ splice site. 1. 2. CAG. GTRAGT. A. YYYYYYYYYNCAG. G. 1. 2. -OH. A. A. 1. 2. The mechanism of splicing. 1. 3. 4. 1. 2. 3. 4.

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Alternative Splicing

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  1. Alternative Splicing

  2. Splicing Eukaryotic genes Mature mRNA

  3. The mechanism of RNA splicing

  4. 5’ splice site Branch point 3’ splice site 1 2 CAG GTRAGT A YYYYYYYYYNCAG G 1 2 -OH A A 1 2 The mechanism of splicing

  5. 1 3 4 1 2 3 4 Alternative splicing 1 2 3 4 Can be specific to tissue, developmental-stage or condition (stress, cell-cycle). 50-70% of mammalian genes Alternative Splicing Mature splice variant I Mature splice variant II

  6. Some types of alternative splicing Exon skipping Alternative Acceptor Alternative Donor Mutually exclusive Intron retention

  7. Sex determination in fly

  8. Sex determination in fly

  9. Sex determination in fly

  10. Many variants in one gene

  11. DSCAM

  12. Antibody secretion

  13. Antibody secretion immunoglobulin μ heavy chain

  14. Tissue specific alternative splicing

  15. Detection of alternative splicing • By sequencing of RNA • Old methods (1995-2007) – ESTs • New methods: • Splicing-sensitive microarrays • RNA-seq

  16. AAAAAAAAA AAAAAAAAA AAA AAA AAA AAA TTTTTTTTTT Expressed Sequence Tags (ESTs) mRNA RT cDNA Cloning Vector

  17. 5’ EST 3’ EST Random-primed EST EST preparation Picking a clone Average size of EST ~450bp

  18. Alignment of ESTs to the genome DNA EST EST EST EST EST EST 8 million public human ESTs, collected over >10 years (NCBI)

  19. Splicing microarrays

  20. Massive sequencing of RNA (RNA-seq)

  21. RNA-seq on multiple tissues Wang et al Nature 2008

  22. Splicing regulation

  23. Tissue specific alternative splicing How is this process regulated?

  24. Regulation of alternative splicing • Splicing Enhancers/Silencers • Specifically bind SR proteins

  25. SR brain Y(n) AG Weak splice site Exon Exonic Splicing Enhancer (ESE) Model for ESE action

  26. SR proteins structure

  27. Discovery of ESEs Exon Silent mutations can cause exon skipping

  28. SR proteins (Splicing factors) Signal transduction ISE ISS ESE/ESS Regulators of splicing • Complex regulation usually exists • Hard to find intronic elements • For most alt exons – regulation unknown

  29. How can we break the regulatory code? • 1. Comparative genomics • 2. High throughput methods

  30. Comparative genomics: Use the mouse genome to find sequences that regulate alternative splicing

  31. Human-mouse comparisons

  32. The mouse genome • 100 million years of evolution • Average conservation in exons: 85% • Only 40% of intronic sequences is alignable • Average conservation in alignable intronic sequences: 69% • Average conservation in promoters: 77% • Function => evolutionary conservation

  33. Conservation of near introns (from VISTA genome browser, http://pipeline.lbl.gov)

  34. BE616884 AI972259 Collection of exons Human DNA AF010316 AF217965 AF217972 BE614743

  35. BE616884 AI972259 Finding the mouse homolog Mouse DNA Human DNA AF010316 AF217965 AF217972 BE614743 1753 Const. 243 Alt.

  36. BE616884 AI972259 Conservation in the intronic sequence near exons Mouse DNA Human DNA AF010316 AF217965 AF217972 BE614743 1753 Const. 243 Alt.

  37. Results Alternative exons Constitutive exons Flanking conserved introns ~100 bp from each side of the exon

  38. Conservation of introns

  39. Alternative splicing regulatory sequences? • Could serve as binding sites for splicing regulatory proteins

  40. Motif searching • Top scoring hexamer in conserved downstream regions: TGCATG (9-fold over expected) • Not over-represented downstream to constitutive exons. • Binding site for FOX1 (splicing regulatory protein)

  41. Functional elements in the human genome • 5% of the human genomic sequence is considered functional

  42. Functional elements in the human genome

  43. Impact of splicing regulatory elements • ~12,000 alt. spliced exons in the genome • 77% have conserved flanking intronic sequences • ~100bp conserved on each side • 12,000 exons * 100 bp * 2 introns * 0.77=2M bases • ==>At least2 Million bases in the human genome might be involved in alternative splicing regulation. • >1% of all functional DNA in the genome regulates alt splicing!

  44. How can we break the regulatory code? • 1. Comparative genomics • 2. High throughput methods

  45. CLIP-seq Ule et al, Science 2003: 340 sequences Licatalosi et al, Nature 2008: 412,686 sequences

  46. Nova, a brain-specific splicing regulator Ule et al, Science 2003: 340 sequences

  47. Ule et al, Science 2003: 340 sequences

  48. Extracting the regulatory motifs

  49. The power of deep sequencing (2008)

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