1. A Non-EST-Based Method for Exon-Skipping Prediction Rotem Sorek, Ronen Shemesh, Yuval Cohen, Ortal Basechess, Gil Ast and Ron Shamir Genome Research August 2004 楊佳熒

2. Homologous human and mouse exon are, on the average, 85% identical in their sequences, but introns are more pooly conserved. (Waterston et al. Nature,2002) Segments and blocks >300kb in size with conserved in human are superimposed on the mouse genome

3. Reference Sorek, R. et al. Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse. Genome Research, 2003. Sorek, R. et al. How prevalent is functional alternative splicing in the human genome. TRENDS in Genetics, 2004. Sorek, R. et al. A Non-EST-Based Method for Exon-Skipping Prediction. Genome Research, 2004.

4. What is Exon-Skipping ? dbESTs exon1exon2exon3exon4exon5exon6gene est2 est3 est4 est1

5. Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse Rotem Sorek and Gil Ast Genome Research July 2003

6. Objective and Result 1. Alternatively spliced conserved exons 2. Constitutively spliced conserved exons exon1exon2exon3 Human est1 Human est2 Alternatively spliced internal exons Mouse est1 Mouse est2 Alternatively spliced conserved exons 3583 243 Human gene Human est1 exon1exon2exon3 Human est2 Human est3 Human est4 Constitutively spliced internal exons 7557 Mouse est Constitutively spliced conserved exons 1966 Mouse gene exon1exon2exon3 Mouse gene exon1exon2exon3 A1B1 A2B2 D1C1 C2D2 223/243=92%199/243=82%188/243=77% 886/1966=45%691/1966=35%343/1966=17%

7. Per-position conservation near alternatively and constitutively spliced exons

8. Human KCND3 gene (exon 4~8) Refseq:NM_004980

9. KCDN3 gene exon information

10. KCDN3 gene exon 6 sequences (bold) (alternatively spliced exon)

11. Compare to chimpanzee genome (NM_004980)

12. Compare to chimpanzee genome (NM_172198)

13. Review : Finding exon-skipping events that are conserved between human and mouse 243 Conserved exon skipping events (25%) 737(980-243) Non-Conserved exon skipping events(75%)

14. How prevalent is functional alternative splicing in the human genome ? Rotem Sorek, Ron Shamir and Gil Ast TRENDS in Genetics Vo1.20 February 2004

15. Motivation 1.How many of there predicted splice variants are functional? 2.How many are the result of aberrant splicing (noise data)?

16. The influence of alternatively spliced exon on the protein- coding sequence. are peptide cassettess

17. Features differentiating between conserved alternatively spliced exons and non-conserved alternatively spliced exons FeaturesConserved alternatively spliced exons Non-conserved alternatively spliced exons Average size87116 Percentage of exon that a multiple of three 77%(147/191)40%(206/510) Percentage of exons that are “peptide cassettes” 73%(139/191)21%(109/510) Percentage of exon insertion that result in a longer protein by a nearby stop codon 61%(27/44)8%(25/304) Percentage of exon insertions that result in a protein <100 amino acids 9%(4/44)30%(91/304) Average supporting expressed sequences 92.2 30%62%

18. Conclusion 1.We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. 2.By contrast, most non-conserved cassette exons do not share these characteristics. 3.We conclude that a portion of skipping exon evidence in EST databases is not functional, and might result from aberrant rather than regulated splicing.

19. Review : Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse 1. Alternatively spliced conserved exons 2. Constitutively spliced conserved exons exon1exon2exon3 Human est1 Human est2 Alternatively spliced internal exons Mouse est1 Mouse est2 Alternatively spliced conserved exons 3583 243 Human gene Human est1 exon1exon2exon3 Human est2 Human est3 Human est4 Constitutively spliced internal exons 7557 Mouse est Constitutively spliced conserved exons 1966 Mouse gene exon1exon2exon3 Mouse gene exon1exon2exon3 A1B1 A2B2 D1C1 C2D2 223/243=92%199/243=82%188/243=77% 886/1966=45%691/1966=35%343/1966=17%

20. Review : Features Differentiating Between Alternatively Spliced and Constitutively Spliced Exons Alternatively spliced exons Constitutively spliced exons Average size87128 Percent exons whose length is a multiple of 3 73%(177/243)37%(642/1753) Percent exons with upstream intronic elements conserved in mouse 92%(223/243)45%(788/1753) Pervent exons with downstream intronic elements conserved in mouse 82%(199/243)35%(611/1753) Percent exons with both upstream and downstream intronic elements conserverd in mouse 77%(188/243)17%(292/1753)

21. A Non-EST-Based Method for Exon-Skipping Prediction Rotem Sorek, Ronen Shemesh, Yuval Cohen, Ortal Basechess, Gil Ast and Ron Shamir Genome Research August 2004

22. Objective 1.Our goal was to find a combination of features that would detect a substantial fraction of the alternative exons. 2.The features we have chosen are the following : 1)exon length 2)divisible / not divisible by 3 3)percent identity when aligned to the mouse 4)conservation in the upstream and downstream intronic sequences

23. Result 1.The best rule is : 1)at least 95% identity with mouse exon counterpart 2)exon size is a multiple of three 3)a best local alignment of at least 15 intronic nucleotides upstream of the exon with at least 85% identity 4)a perfect match of at least 12 intronic nucleotides downstream of the exon 2.The combination of features identified 76 exons, 31% of the 243 alternatively spliced exons in the training sets, whether non of 1753 constitutively spliced exons matched these features.

24. To test this classifier in a genome-wide manner (cont.) 1.For 453(48%) of the 952 candidate alternative exon there was such skipping evidence. 2.Only(17%) of the 453 exons that were classified by our rule had their exon-skipping supported by only one EST. 3. The rest were supported by two or more. 108,983 human exons for which a mouse counterpart could be identified using these rules 108,983 952 candidate exon, ~1%, were found.

25. To test this classifier in a genome-wide manner (cont.) 1.In comparison, skipping was supported by only a single EST in 46% of the total 7495 exons. 2.This suggests that our classification rule enriches for alternatively spliced exons with higher probability of being “real” relative to alternative exons merely supported by EST evidence. 108,983 human exons for which a mouse counterpart could be identified search ESTs and cDNA 108,983 7% (7495 exons) out of our entire set

26. To test this classifier in a genome-wide manner 1.The remaining 499 candidate alternative exons (952-453) for which no EST/cDNA showing an exon skipping event was found. 2.Using the UCSC genome browser to check, we found that for 190 additional exons there was a human expressed sequence showing patterns of alternative splicing other than exon skipping cases. 1)Alternative donor/acceptor  22% 2)Intron retention  17% 3)Mutually exclusive exon  7% 3.Thus, for 643(453+190 ; 68%) of the 952 candidate alternative exons identified by this method, there was independent evidence for alternative splicing in dbEST.

27. Conclusion 1.We show that a substantial fraction of the splice variants in the human genome could not be identified through current human EST or cDNA data. 2.In the future, we hope it could develop into a more general alternative splicing predictor that would identify other types of alternative splicing.

28. Classification of alternative splicing 1.Skipped Exons 2. Multiple Skipped Exons 3. Alternative Donor / Acceptors 4. Retained Introns