1. Alternative Splicing Genomic DNA Sequence GmGm AAAAA Exon Intron Exon GmGm AAAAA Transcription mRNA RNA Processing pre-mRNA

2. Alternative Splicing Data Sources are Large and Growing Microarray detection Direct or indirect alternative splicing detection Curated databases SWISS-PROT and RefSeq both support annotation of experimentally supported alternative splicing Hu et al. (2001)Genome Res 11:1237-45 Yeakley et al. (2002) Nat Biotech 20:353-9 cDNA Sequencing Projects RIKEN sequenced >21000 full length mouse cDNAs Many other projects underway (human, fly, plants,…) Shinagawa et al. (2001) Nature 409:685-90 Public EST data sources (dbEST) >4.5 million human EST sequences >12 million total EST sequences About 1000 new sequences per day Boguski et al. (1993) Nat Gen 4:332-3

3. GmGm Nonsense-Mediated mRNA Decay Termination codon is on the last exon (not premature) Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5 Nagy and Maquat (1998) TIBS 23:198-9 Le Hir et al. (2000) Genes & Dev 14:1098-1108 Lykke-Andersen et al. (2001) Science 293:1836-9 Kim et al. (2001) EMBO 20:2062-68 Ishigaki et al. (2001) Cell 106:607-17 Leeds et al. (1991) Genes Dev 5:2303-14 Genomic DNA pre-mRNA mRNA Exon junction complex AAAAAAAAA Exon Intron Exon Intron Exon

4. Nonsense-Mediated mRNA Decay Termination codon > 50nt before last exon junction (Premature Termination Codon) Decapping and degradation Mitchell and Tollervey (2001) Curr Opin Cell Biol 13:320-5 Nagy and Maquat (1998) TIBS 23:198-9 Le Hir et al. (2000) Genes & Dev 14:1098-1108 Lykke-Andersen et al. (2001) Science 293:1836-9 Kim et al. (2001) EMBO 20:2062-68 Ishigaki et al. (2001) Cell 106:607-17 Leeds et al. (1991) Genes Dev 5:2303-14 mRNA GmGm AAAAAAAAA Interaction between EJC and release factors triggers NMD

5. Nonsense-Mediated mRNA Decay GmGm GmGm Translated normally Degraded by NMD >50 nt mRNA AAAAAAAAA ORF

6. NMD is Pervasive 1498 of 1500 genes surveyed from fungi, plants, insects and vertebrates obey the PTC rule Nagy and Maquat (1998) TIBS 23:198-9 “NMD is a critical process in normal cellular developement” Wagner and Lykke-Andersen (2002) J Cell Sci 115:3033-8 Wang et al. (2002) J Biol Chem 277:18489-93 Renders recessive many otherwise dominant mutations Cali and Anderson (1998) Mol Gen Genet 260:176-84 V(D)J recombination 4.3% of reviewed RefSeqs have PTCs 34% have start codon after first exon

7. NMD is Pervasive Only 2 of 1500 genes surveyed from fungi, plants, insects and vertebrates are NMD candidates Nagy and Maquat (1998) TIBS 23:198-9 “NMD is a critical process in normal cellular developement” Wagner and Lykke-Andersen (2002) J Cell Sci 115:3033-8 Wang et al. (2002) J Biol Chem 277:18489-93 Renders recessive many otherwise dominant mutations Cali and Anderson (1998) Mol Gen Genet 260:176-84 V(D)J recombination

8. Transcriptional Regulation RUST Gene locus pre-mRNA productive mRNA Protein transcription productive splicing translation

9. Transcriptional Regulation RUST Transcriptional Regulation Gene locus productive splicing Gene locus pre-mRNA transcription pre-mRNA Productive mRNA Productive mRNA

10. Alternative Splicing Can Yield Isoforms Differentially Subjected to NMD Nucleus Premature termination codon NMD pre-mRNA DNA mRNA DNA pre-mRNA Nucleus

11. SC35 Auto-regulation SC35 Locus SC35 pre-mRNA Productive SC35 mRNA SC35 protein splicing transcription translation alternative splicing Sureau et al. (2001) EMBO J 20:1785-96

12. GmGm GmGm SC35 Auto-regulation Alternative splicing coupled with nonsense-mediated decay Sureau et al. (2001) EMBO J 20:1785-96 SC35 SC35 pre-mRNA SC35 mRNA SC35 protein SC35 pre-mRNA SC35 mRNA (with premature termination codon) SC35 Locus SC35 pre-mRNA Productive SC35 mRNA SC35 protein AAAAA ORF SC35

13. SC35 Auto-regulation Alternative splicing coupled with nonsense- mediated decay Sureau et al. (2001) EMBO J 20:1785-96 GmGm GmGm SC35

14. EST-inferred human isoforms 0 2000400060008000 NMD Candidates Alternative isoforms All isoforms, including canonical 1989 (35 % of 5693) 5693 8820 10000

15. Inferred Isoforms 0100020003000400050006000 NMD Candidates Alternative isoforms All isoforms, including canonical 2078 (34 % of 5970) 5970 9282 700080009000

16. Canonical Splice Forms Genomic Contigs Coding Refseqs Association via LocusLink Refseq mRNAs Extract coding regions align w/ Spidey Refseq-Contig Pairs ≥98% id, no gaps Construct genes from aligned Refseq exons & intervening genomic introns (overlap  choose mRNA w/ largest number of exons) Refseq-coding genes Lander et al. (2001) Nature 409: 860-921 Wheelan et al. (2001) Gen Res 11:1952-7 Pruitt, K.D. et al (2001) NAR 29: 137-40 mRNA Exon 1Exon 3Exon 2Exon 4 Refseq-coding gene Genomic DNA Sequence

17. Cluster ESTs w/ WU-BLAST2 ≥92% id, allow gaps Refseq-coding genes Boguski et al., (1993) Nat Genet 4, 332-3. Gish,(2002)(Wash.Univ.) Align ESTs w/ sim4 Alternative Isoforms of Refseq-coding genes Kan, et al. (2001) Gen Res 11, 889-900. Florea, et al.,(1998) Gen Res 8, 967-74. Identification of Alternative Isoforms ESTs from dbEST Use TAP to infer alternative mRNAs > 92% identity, gaps allowed Aligned EST 5’ end does not indicate reading frame

18. Premature Stop Codons

19. Alternative Isoform Inference from Splice Pairs

20. Alternative Splice Pairs, by Mode

22. Splice Pairs Generating Premature Stops

23. For 76% of isoforms with premature stops: ESTs cover a PTC & splice junction downstream EST coverage and premature stops In 80% of these isoforms, there is a PTC in every reading frame: Alternative polyadenlyation signals are biased against recovery

24. For 76% of isoforms with premature stops: ESTs cover a PTC & splice junction downstream EST coverage and premature stops In 80% of these isoforms, there is a PTC in every reading frame: Alternative polyadenlyation signals are biased against recovery RefSeq mRNA Alternatively spliced EST, reading frame 0 Alternatively spliced EST, reading frame 2 Alternatively spliced EST, reading frame 1

25. Alternative Polyadenylation? Polyadq once every 2560 nucleotides Tabaska and Zhang. (1999) Gene 231: 77-86 once every 3115 nucleotides RefSeq mRNA NMD-candidate alternatively spliced EST

26. Alternative Splicing in Human 0 2000400060008000 NMD Candidates Alternative isoforms All isoforms, including canonical 2078 (35 % of 5970) 5970 9527 10000

27. Class Experimental EvidenceAmong Our Results Splicing Factors AUF1, SC35 SRP20, SRP30b (in C. elegans) Sureau et al. (2001) EMBO J 20:1785-96 Wilson et al. (1999) Mol Cell Bio 19:4056-64 Morrison et al. (1997) PNAS 94:9782-9785 AUF1, *10 new Ribosomal Proteins L3, L7a, L10a, L12 (in C. elegans) L30, S14B (in S. cerevisiae) Mitrovich & Anderson (2000) GenesDev 14:2173-84 L3, L7a, L10a, L12, *11 new Previous and new RUST targets

28. Alternative Splicing Genomic DNA Sequence GmGm AAAAA Exon Intron GmGm AAAAA Transcription mRNA RNA Processing pre-mRNA Exon Intron

29. Alternative Splicing Recruitment of Sequence. Deletion of Sequence. *Frameshift and Truncation. not integer # codons

30. Premature Stop Codons

31. EST Limitations Single pass sequencing errors Incompletely processed transcripts 3’ end bias Library contamination Thanaraj (1999) NAR 27:2627-37

32. Alternative Splicing EST Analysis From data in Brett et al. (2000) FEBS Lett 474:83-6

33. Alternative Isoform Inference from Splice Pairs

34. Alternative Splice Pairs, by Mode

36. Splice Pairs Generating Premature Stops

37. For 76% of isoforms with premature stops: ESTs cover a PTC & splice junction downstream EST coverage and premature stops In 80% of these isoforms, there is a PTC in every reading frame: Alternative polyadenlyation signals are biased against recovery RefSeq mRNA Alternatively spliced EST, reading frame 0 Alternatively spliced EST, reading frame 2 Alternatively spliced EST, reading frame 1