1. 1 Alternative Splicing

2. 2 Eukaryotic genes Splicing Mature mRNA

3. 3 The mechanism of RNA splicing

4. 4 The mechanism of splicing A 1212 A -OH 1 2 YYYYYYYYYNCAGGTRAGTACAGG 5’ splice site 3’ splice siteBranch point

5. 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. 6 Some types of alternative splicing Exon skipping Alternative Acceptor Alternative Donor Mutually exclusive Intron retention

7. 7 Sex determination in fly

8. 8

9. 9

10. 10 Many variants in one gene

11. 11 DSCAM

12. 12 Antibody secretion

13. 13 Antibody secretion immunoglobulin μ heavy chain

14. 14 Tissue specific alternative splicing

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

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

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

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

19. 19 Splicing microarrays

20. 20 Massive sequencing of RNA (RNA-seq)

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

22. 22 Splicing regulation

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

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

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

26. 26 SR proteins structure

27. 27

28. 28 Discovery of ESEs Exon Silent mutations can cause exon skipping

29. 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

30. 30 How can we break the regulatory code? n 1. Comparative genomics n 2. High throughput methods

31. 31 Comparative genomics: Use the mouse genome to find sequences that regulate alternative splicing

32. 32 Human-mouse comparisons

33. 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. 34 Conservation of near introns (from VISTA genome browser, http://pipeline.lbl.gov)

35. 35 Collection of exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743

36. 36 Finding the mouse homolog AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.

37. 37 Conservation in the intronic sequence near exons AF217972 Human DNA AF010316 AF217965 AI972259BE616884 BE614743 Mouse DNA 243 Alt. 1753 Const.

38. 38 Flanking conserved introns Results Alternative exons Constitutive exons ~100 bp from each side of the exon

39. 39 Conservation of introns

40. 40 Alternative splicing regulatory sequences? n Could serve as binding sites for splicing regulatory proteins

41. 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. 42 Functional elements in the human genome n 5% of the human genomic sequence is considered functional

43. 43 Functional elements in the human genome

44. 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. 45 How can we break the regulatory code? n 1. Comparative genomics n 2. High throughput methods

46. 46 CLIP-seq Licatalosi et al, Nature 2008: 412,686 sequences Ule et al, Science 2003: 340 sequences

47. 47 Nova, a brain-specific splicing regulator Ule et al, Science 2003: 340 sequences

48. 48 Ule et al, Science 2003: 340 sequences

49. 49 Extracting the regulatory motifs

50. 50 The power of deep sequencing (2008)

51. 51 Mutations causing aberrant splicing Exon ~15% of all point mutations linked to genetic disorders involve splicing alterations

52. 52 Mutations causing aberrant splicing: SMN

53. 53 Summary – alt splicing n Increases the coding capacity of genes n We have 25,000 genes but much more protein isoforms

54. 54 RNA EDITINA

55. 55 RNA EDITING

56. 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”.

57. 57 Mode of operation: A-to-I editing A-> G Editing performed by ADAR enzymes (dsRNA specific adenosine deaminases) Double strand RNA is required

58. 58 Mechanism of RNA-editing (A-to-I)

59. 59 Functions of RNA editing n Defense against dsRNA viruses n Also involved in endogenous regulation

60. 60

61. 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. 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. 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

64. 64 Intramolecular dsRNA Exon RNA Intron Levanon et al, Nature Biotech 2004

65. 65 ESTs to genome Levanon et al, Nature Biotech 2004

66. 66 dsRNA regions Levanon et al, Nature Biotech 2004

67. 67 dsRNA regions Masking EST’s ends Levanon et al, Nature Biotech 2004

68. 68 dsRNA regions Masking EST’s ends Masking poor sequence regions

69. 69 dsRNA regions Masking EST’s ends Masking poor sequence regions Removing known genomic SNPs Levanon et al, Nature Biotech 2004

70. 70 dsRNA regions Masking EST’s ends Masking poor sequence regions Removing SNPs Collecting candidates Levanon et al, Nature Biotech 2004

71. 71 Results DNA RNA (ESTs)

72. 72 Levanon et al, Nature Biotech 2004

73. 73

74. 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. 75 And the obligatory next generation sequencing study… (Li, Levanon et al, Science 2009) Editing sites in non-repetitive regions

76. 76 Connection between editing and splicing Negative feedback loop ADAR gene (editing enzyme)

77. 77 Evolution of a new exon

78. 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