IPAM Workshop Jan 2006

1. Searching for human splice-regulatory motifs &II. Network analysis of synthetic-lethal interactions Fritz Roth Harvard Medical School Dept. of Biological Chemistry & Molecular Pharmacology IPAM Workshop Jan 2006

2. Outline • Human alternative-splicing motif search • Yeast synthetic-lethal network analysis

3. Outline • Human alternative-splicing motif search • Review mRNA splicing • Splice-junction expression data • Sequence neighborhoods • Clustering splice-junctions by usage • Results • Yeast synthetic-lethal network analysis

4. Brief review of canonical mRNA splicing Adapted from Molecular Cell Biology, Lodish et al.

5. Importance of alternative splicing ~100,000 genes ~70% of human multi-exon genes alternatively spliced 35,000 25,000 human genes 5% alt. spliced Antiquity 2004 2001

6. From correlation to regulatory mechanism Roth et al , 1998, Hughes et al, 2000 Tavazoie et al 1999, Slides adapted from S. Tavazoie

7. Sequence neighborhood of a splice junction

8. Conservation: constitutive vs. alternative 5’ donor neighborhood 3’ acceptor neighborhood (Sorek & Ast, Gen. Research, 2003)

9. Previous splicing motif searches • Canonical splicing enhancers/repressors not associated with specific tissues (e.g. Brudno et al., Burge et al, Chasin et al) • Based on small (curated literature) set of alternatively spliced genes (e.g. , Brudno et al, Stamm et al., Fedorov et al.) • Based on expressed sequence tag (EST) datasets (Xu et al), biased towards 3’ ends of genes, can contain artificial splice variants due to Unigene clustering (Modrek & Lee, 2002)

10. 18 nt + 18 nt Exon-exon splice junction expression data Pre-mRNA Cassette exon Alternative mature mRNAs • Every splice junction for ~11K reference mRNAs for ~10K genes • ~100K probe sequences on five arrays • 49 tissues & cell lines run in fluor-reversed pairs • full-length mRNA amplification • Johnson et al. Science. 2003 Dec 19;302(5653):2141-4. • Castle et al. Genome Biology. 2003;4(10):R66.

11. Tissue-specific probes & data

12. Flowchart splice junction usage data

13. probes 3’ 5’ tissues Tissue-specific splice junction expression original intensities NPTB

14. probes 3’ 5’ tissues Tissue-specific splice junction expression original intensities rescaled intensities NPTB

15. probes 3’ 5’ tissues Tissue-specific splice junction expression original intensities rescaled intensities relative splicing NPTB

16. Tissue-specific splice junction expression Synexin (ANXA7)

17. cassette exons alternative donors / acceptors cassette exons Probe set choice all skipped cassette exons alternative donors / acceptors Flowchart splice junction usage data Clustered splice junctions

18. Results: heart & muscle Cassette exons skipped in heart & skeletal muscle

19. alternative donors / acceptors cassette exons Probe set choice all Sequence neighborhood choice intronic all exonic proximal to acceptor proximal to donor sequence neighborhoods Flowchart splice junction usage data clustered splice junctions

20. Sequence neighborhood of a splice junction

21. Alternative splice junction neighborhoods 1 3 3 2 Immediate neighborhood Neighborhood of junctions with shared splice site Neighborhood of junctions of other competing junctions

22. alternative donors / acceptors cassette exons Probe set choice all Sequence neighborhood choice intronic all exonic proximal to acceptor proximal to donor sequence neighborhoods word- based motif- based Pattern finding enriched sequence patterns Flowchart splice junction usage data clustered splice junctions

23. Results: heart & muscle Cassette exons skipped in heart & skeletal muscle • ACTAAC @ end of intron • - 8 out of 39 probes • - 14-fold enrichment • strong position bias • (often nearby)

24. Results: brain Cassette exons skipped in brain 26% of probes (9-fold enrichment)

25. Results: ileum 65% (15-fold) 76% (3-fold)

26. Cluster Results: jejunum, liver, pancreas

27. HNRPL Cluster Results: jejunum, liver, pancreas

28. Results: jejunum, liver, pancreas HNRPL Cluster HNRPL RRM1 RRM2 RRM3 RRM4

29. HNRPL Cluster Results: jejunum, liver, pancreas PTB RRM1 RRM2 RRM3 RRM4 Protein interaction HNRPL RRM1 RRM2 RRM3 RRM4

30. HNRPL Cluster Results: jejunum, liver, pancreas PTB RRM1 RRM2 RRM3 RRM4 Protein interaction HNRPL RRM1 RRM2 RRM3 RRM4

31. validated cis-regulatory motifs candidate cis-regulatory secondary strcture Map to trans-acting splicing factors Summary, Part I tissue-specific alternative splicing candidate cis-regulatory motifs

32. Adnan Derti George Church & Lab Roth Lab Rosetta/Merck Jason Johnson John Castle Lee Lim Adrian Krainer Acknowledgments, Part I

33. Outline • Human alternative-splicing motif search • Yeast synthetic-lethal network analysis

34. Outline • Human alternative-splicing motif search • Yeast synthetic-lethal network analysis • Background • Overlap with other biological relationships • Network motifs • Predicting synthetic lethality • Role of transcription compensation in mutational robustness • SSL vs. protein interaction in predicting function

35. What is Synthetic Lethality? Gene X Gene Y Cells live Cells live Cells die Gene X Gene Y Gene X Gene Y

36. What is Synthetic Sickness/Lethality (SSL)? Gene X Gene Y Cells live Cells live Cells die or grow slowly Gene X Gene Y Gene X Gene Y

37. An engine can run without one cylinder (from http://www.cs.unc.edu/~geom/collide/videos.shtml)

38. 3 compensatory pathways, 2 required Partially redundant genes 2 partially redundant pathways Protein complex tolerating 1 but not 2 mutations A B H H B K A F A J F G E C G E C L B B C1 A C2 C D D M D E F D E I I SSL Scenarios resulting in synthetic genetic interaction

39. A known sub-network of SSL interactions • A Canadian consortium (Boone et al.) has made many double mutants • As of 2001: 8 query genes x 4500 nonessential “array” genes ≈ 36,000 tested pairs (Tong et al., Science, 2001)

40. The known sub-network circa 2001 (Tong et al., Science, 2001)

41. The known SSL sub-network circa 2004 • 160 query x 4500 nonessential ≈ 700,000 tested pairs (≈4% pairs) ~3800 interactions (Tong et al., Science, 2004)

42. Outline • Human alternative-splicing motif search • Yeast synthetic-lethal network analysis • Background • Overlap with other biological relationships • Network motifs • Predicting synthetic lethality • Role of transcription compensation in mutational robustness • SSL vs. protein interaction in predicting function

43. Overlap between synthetic lethality & other “interactions”

44. Overlap between synthetic lethality & other “interactions”

45. Overlap between synthetic lethality & other “interactions”

46. Overlap between synthetic lethality & other “interactions”

47. Overlap between synthetic lethality & other “interactions”

48. Overlap between synthetic lethality & other “interactions”

49. Overlap between synthetic lethality & other “interactions”

50. 3 partially redundant pathways, 2 required Partially redundant genes 2 partially redundant pathways Protein complex tolerating 1 but not 2 destabilizing mutations A B H H B K A F A J F G E C G E C L B B C1 A C2 C D D M D E F D E I I SSL Scenarios resulting in synthetic interaction < 2% < 4% *