Genome Analysis Research Group

1. Genome Analysis Research Group to appear soon in Genome Biology 8 (2007) Karol Szafranski presented by Rileen Sinha Leibniz Institute for Age Research – Fritz Lipmann Institute Violating the splicing rules:TG dinucleotides function as 3' splice sites in U2-dependent introns

2. subtle alternative splicing overview Cartegni et al., Nat Rev Genet 3, 285 (2002)

3. subtle alternative splicing UGAGAA... ...UGGAGA gu.....uuacug cag CAG UGAGAA... ...UGGAGAgu.....uuacug Exceptional splice sites? GNAS adenylyl cyclase stimulatory G-protein GS Quan & Forte MCB 10,910 (1990) Pollard et al. JBC 277,15241 (2002)

4. subtle alternative splicing Exceptional splice sites? GU-AG rulefor U2-dependent introns Cartegni et al., Nat Rev Genet 3, 285 (2002)

5. subtle alternative splicing genotype ` Exceptional splice sites? A/A A/G G/G 1 3 2 number of protein isoforms Hiller et al., Am J Hum Genet 78, 291 (2006)

6. subtle alternative splicing UGAGAA... ...UGGAGA gu.....uuacug cag CAG UGAGAA... ...UGGAGAgu.....uuacug Exceptional splice sites? GNAS adenylyl cyclase stimulatory G-protein GS NAGNAG no no SNP! Quan & Forte MCB 10,910 (1990) Pollard et al. JBC 277,15241 (2002) unusual UGacceptor site!

7. subtle alternative splicing Exceptional splice sites? Identification and validation of 36 TG acceptors in the human genome

8. mining spliced alignments NNNNNN... ...NNNNNN nn.....nnnnnn nnn NNN NNNNNN... ...NNNNNN nn.....nnnnnn Approach neutral approach to search for 3' splice variants: splice variant pairs (SVP) spliced alignment of ESTs: 3’ splice site (“E”) 3’ splice site (“I”)

9. mining spliced alignments NNNNNN... ...NNNNNN nnn.....nnnnnnnug Approach II neutral approach to search for non-canonical splice sites based on RefSeq transcripts (independent of alternative splicing) spliced alignment of RefSeq transcripts: UG 3’ splice site

10. mining spliced alignments Pipeline systematic in silico screening for unusual alternative 3‘ splice sites of human introns human EST-to-genome alignment human RefSeq-to-genome alignment quality filtering BLAST-validation of EST-supported splice variants manual inspection

11. mining spliced alignments quality aspects of EST-based screen for splice variants filter for paralogous gene loci validate putative splice variants by explicit BLAST searches; choose only high-quality BLAST alignments around the exon-exon junction require at least two ESTs as evidence for a splice variant Pipeline

12. mining spliced alignments ESTs vs. RefSeqs EST-based RefSeq-based raw cases of unusual intron termini 44 39 artifacts 8 30 34 (77%) considered valid 9 (23%) 6 28 3 92% reproducible in RT-PCR non-canonical TG 3'ss in U2 introns

13. mining spliced alignments exon4 Experimental validation CNBP(cellular nucleic acid binding protein) exon3 AG-derived splice variants UG-derived splice variants UG-derived splice variants were experimentally validated!

14. functional relevance More than just noise? noise produced by error-prone spliceosomes? variants awaiting functionalization during evolution? functionally relevant alternative splicing? Indicators : flanking sequence conservation splice site conservation tissue-specific splicing pattern

15. functional relevance intron sequence conservation H.sapiens conservation offlanking intron sequence ? M.musculus exon intron

16. functional relevance intron sequence conservation efficient TG alternative splicing under purifying selection

17. functional relevance intron sequence conservation RYK intron 7

18. functional relevance tissue-dependent splice patterns BRUNOL4 CNBP

19. structural and mechanistical implications structural propertiesand mechanistic implications

20. structural and mechanistical implications structural pattern Significant structural constraint observed: TGs were exclusively found as alternative acceptors, part of TG-AG splice site tandems 6 AG-TG, up to  4 nt 31 TG-AG, up to  28 nt

21. functional relevance Mechanism of SS choice

22. structural and mechanistical implications Mechanism of SS choice AG-AG tandems may have separate branchpoints BP AG BP AG ... ... ... allowing large-distance splice sites. TG-AG tandems share the same branchpoint (BP) BP TG AG ... ... ... limiting the distance of splice sites.

23. functional relevance Mechanism of SS choice rare but high-confidence TG acceptors TG acceptors always alternative TG-AG acceptor distance constraint high conservation of alternative splicing at TG acceptors ... specific mechanism?

24. functional relevance Mechanism of SS choice splicing step I intron recognition 65 U2 snRNP U2AF 35 U2AF typically 15-40 nt ...nnCUaAcnnnnnnnnnnnUGnnnnnnnnnAGNNNNNNNNN... exon Lallena et al. 2002, Cell 109,285 SPF45 ...or another factor? splicing step II splice site selection AG is required for intron recognition during splicing step I TG splice site selection takes place in splicing step II

25. Acknowledgments Inst. Clin. Mol. Biol., Univ. Kiel Stefan Schreiber Philip Rosenstiel Bioinformatics, Univ. Freiburg Michael Hiller Rolf Backofen Genome Analysis, FLI Jena Stefanie Schindler Stefan Taudien Klaus Huse Niels Jahn Matthias Platzer