Brenton Graveley - University of Connecticut Health Center Mike Duff Li Yang

1. modENCORE: Discovering the Encyclopedia Of Drosophila RNA cis-regulatory splicing Elements Brenton Graveley - University of Connecticut Health Center Mike Duff Li Yang Steven Brenner - UC Berkeley Angela Brooks Sandrine Dudoit - UC Berkeley Kasper Hansen

2. The Organization of Eukaryotic Genes

3. Overview of Splicing Regulatory Mechanisms ESE - Exonic splicing enhancer ESS - Exonic splicing silencer ISE - Intronic splicing enhancer ISS - Intronic splicing silencer

4. Types of RNA Splicing

5. Combined Effort for a Large Scale Analysis of Splicing Regulation ~125 RNA Binding Proteins

6. RNA binding proteins to knockdown • Choose ~125 RNA binding proteins • RRM or KH domain • Choose cell line that expresses all proteins • Deplete proteins using RNAi • Double knockdown with Upf1 (NMD inhibition) Graveley

7. Working Ranked List of 222 RNA binding proteins to knockdown on wiki • Proteins with RRM and KH domains were identified through Pfam, SMART, and Prosite motif searches. • Explanation of rankings are on wiki

8. RNAi Library of RNA binding proteins • Library consists of over 400 proteins that may bind to RNA • Our final list of ~125 RNA binding proteins may not be in the RNAi library

9. Identify changes in splicing upon depletion of RNA binding protein • Three biological replicates hybridized to arrays • Junction net expression and isoform deconvolution algorithms • RT-PCR validation of 20 exons for every splicing factor Graveley, Affymetrix, Brenner, Dudoit

10. Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila • RNAi of four known splicing regulators: dASF/SF2, B52/SRp55, hrp48, and PSI • Assayed global effect on alternative splicing by designing a splice junction microarray Blanchette, Green, Brenner, Rio, (2005) Genes & Development, 19:1306-1314

11. Splice Junction Microarray Design • “a” probes - All alternatively spliced junctions • “c” probes - Up to two constitutive junctions probes per gene • “e” probes - For each gene, two exonic probes common to all isoforms were selected to detect total gene expression. • Agilent 44K Blanchette et al., Genes & Dev.(2005)

12. Identified junction probes with >2-fold change upon RNAi

13. Identified many splice junctions affected by each factor. Subset verified by RT-PCR

14. Deciding on method to detect changes in alternative splicing • Tiling arrays are insufficient to detect changes in alternative splicing. • Probe sequences need to be specific to alternative isoforms.

15. Tiling arrays will not detect changes in specific isoforms. % Rel. Abundance Tiled Probes Isoform 1 99% Control Isoform 2 1%

16. Tiling arrays will not detect changes in specific isoforms. % Rel. Abundance Tiled Probes Isoform 1 99% Control Isoform 2 1% Tiled Probes Isoform 1 90% RNAi Isoform 2 10%

17. Tiling arrays will not detect changes in specific isoforms. % Rel. Abundance Tiled Probes Isoform 1 99% Control Isoform 2 1% 10% decrease in abundance of Isoform 1 Tiled Probes Isoform 1 90% RNAi Isoform 2 10%

18. Tiling arrays will not detect changes in specific isoforms. % Rel. Abundance Tiled Probes Isoform 1 99% Control Isoform 2 1% 10% decrease in abundance of Isoform 1 10-fold increase in Isoform 2 Tiled Probes Isoform 1 90% RNAi Isoform 2 10%

19. Junction probes will detect changes in specific isoforms. % Rel. Abundance 1 2 Isoform 1 99% Control 3 Isoform 2 1% 1 2 Isoform 1 90% RNAi 3 Isoform 2 10%

20. Junction probes will detect changes in specific isoforms. % Rel. Abundance 1 2 Isoform 1 99% Control 3 Isoform 2 1% Probe 3 detects 10-fold increase 1 2 Isoform 1 90% RNAi 3 Isoform 2 10%

21. What type of alternative splicing is going on?

22. Deciding on method to detect changes in alternative splicing • Affy tiling array with junction probes • Agilent or Nimblegen arrays • Uses probe lengths >25nt • Sequencing technology • Sequence an enrichment of exon-exon junctions sequences

23. Affy array with junction probe design Tyson Clark, Affymetrix • Each exon-exon junction requires 6-8 probes to tile across the junction • Additional ~300,000-400,000 junction probes to existing tiling arrays • ~9,000 FlyBase annotated alt. junctions x 8 = 72,000 • EST, transcript predictions? • ~38,000 const. junctions x 8 = 304,000 • To detect unknown alternative splicing events and aberrant splicing events caused by RNAi knockdown

24. Identify regulatory sequences near affected splicing events • Find overrepresented motifs in sequences near co-regulated splicing events • For splicing factors with known motifs, identify binding sites near affected splicing events Brenner

25. Blanchette et al. Analysis of Putative Binding Sites for B52

26. Development of Computational Methods to Discover Regulatory Sequences Near Co-affected Splicing Events • Currently analyzing data from Blanchette et al. (2005) • RNAi of dASF/SF2, B52/SRp55, hrp48, PSI

27. modENCORE: Methods to Identify Regulatory Elements • Graveley, Affymetrix, Brenner, & Dudoit • Identify cis-regulatory sequences near splicing events co-affected upon depletion of known and putative RNA binding proteins • Graveley • RNA secondary structures in conserved intronic sequences near alternatively spliced exons • Selection of splicing regulatory elements using in-vivo selection • Brenner • Strictly computational predictions

28. modENCORE group • Brenton Graveley - Component Leader • Mike Duff - Bioinformatics • Li Yang - Experimental • RNAi of RNA binding proteins, array design/analysis, • RNA secondary structure elements • Steven Brenner • Angela Brooks - Bioinformatics • Array design/analysis, motif discovery and analysis • Sandrine Dudoit • Kasper Hansen - Statistics • Array design/analysis

29. Open Questions • Which ~125 proteins to knockdown? • Which cell line to use for RNAi? • Which technology to use to detect changes in splicing? • Strandedness?

31. modENCORE: Discovering the Encyclopedia Of Drosophila RNA cis-regulatory splicing Elements Brenton Graveley - University of Connecticut Health Center Mike Duff Li Yang Steven Brenner - UC Berkeley Angela Brooks Sandrine Dudoit - UC Berkeley Kasper Hansen

32. The Organization of Eukaryotic Genes

33. Overview of Splicing Regulatory Mechanisms ESE - Exonic splicing enhancer ESS - Exonic splicing silencer ISE - Intronic splicing enhancer ISS - Intronic splicing silencer

34. Types of RNA Splicing

35. Most Important Questions in the Alternative Splicing Field 1. What are the mechanisms by which alternative splicing is regulated? 2. Who are the regulators? 3. What are the regulatory targets? 4. What is the biological relevance of alternative splicing?

36. Matthews et al., Dendrite self-avoidance is controlled by Dscam., Cell (2007) Alternative Splicing is Important in the Production of Multiple Gene Products Down syndrome cell adhesion molecule (Dscam)

37. Alternative Splicing is Important in Gene Regulation • Alternative splicing can be coupled with nonsense-mediated decay(NMD) Translated normally Gm AAAAAAAAA mRNA Degraded by NMD Gm AAAAAAAAA mRNA >50 nt

38. Alternative splicing can be unproductive

39. SC35 SC35 SC35 SC35 SC35 SC35 Human SC35 Auto-regulationAlternative splicing coupled with nonsense-mediated decay ORF SC35 pre-mRNA Gm AAAAA SC35 mRNA SC35 protein SC35 pre-mRNA SC35 mRNA (with premature termination codon) Gm Gm AAAAA AAAAA Sureau et al. (2001) EMBO J20:1785-96

40. The entire family of human SR genes has mRNAs with premature termination codons SRp55 SRp20 SRp75 SRp30c 9G8 SRp38 ASF/SF2 SRp40 SC35 SRp54 SRp46 Lareau, Inada, Green, Wengrod, Brenner, Nature (2007)

41. Alternatively spliced regions overlap highly conserved and ultraconserved elements SRp20 618nt SRp30c 42, 44nt 144nt 150nt SRp38 SRp40 SRp54 355, 234nt 9G8 228nt SRp55 322nt SRp75 114, 77, 119nt ASF/SF2 217 188nt SC35 118 132nt Conclusion: This suggests the alternative splicing is functionally important

42. Many Splicing Factors are Conserved Between Human and Fly * Barbosa-Morais et al. (2006) Genome Research

43. About 20% of fly genes have an annotated alternative splice form • This is a lower bound on the actual extent of alternative splicing • Previous estimates have suggested 40% of genes are alternatively spliced. Stolc et al. (2004) Science

44. Goal of modENCORE • Identify sequences that are involved in splicing regulation. • What proteins recognize the sequence. • Examine proteins involved in splicing regulation • Their gene targets

45. Graveley Group’s Previous Work

46. Identification of Proteins Required for Dscam Exon 4 Alternative Splicing 250 dsRNAs (70% of Drosophila RNA binding proteins) Drosophila S2 Cells Analyze Alternative Splicing of Endogenous Dscam Transcripts by RT-PCR

47. Proteins that Affected Dscam Splicing

48. Dscam - Requirement for a New Mechanism

49. Identification of a Highly Conserved Intronic Sequence Graveley, Cell (2005)

50. Examples of Additional Conserved Intronic Sequences