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Alternative Splicing

Alternative Splicing. Genomic DNA Sequence. Transcription. pre-mRNA. Intron. Intron. Intron. Exon. Exon. Exon. Exon. Exon. RNA Processing. G m. AAAAA. G m. AAAAA. mRNA. mRNA. Alternative Splicing Data Sources are Large and Growing. Curated databases

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Alternative Splicing

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

  2. Alternative Splicing Data Sources are Large and Growing Curated databases SWISS-PROT and RefSeq both support annotation of experimentally supported alternative splicing cDNA Sequencing Projects RIKEN sequenced >21000 full length mouse cDNAs Many other projects underway (human, fly, plants,…) Shinagawa et al. (2001) Nature409:685-90 Microarray detection Direct or indirect alternative splicing detection Hu et al. (2001)Genome Res11:1237-45 Yeakley et al. (2002) Nat Biotech20:353-9 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 Gen4:332-3

  3. Nonsense-Mediated mRNA Decay Genomic DNA pre-mRNA Exon Intron Exon Exon Intron Exon junction complex mRNA Gm AAAAAAAAA Termination codon is on the last exon (not premature) Leeds et al. (1991) Genes Dev5:2303-14 Nagy and Maquat (1998) TIBS23:198-9 Le Hir et al. (2000) Genes & Dev14:1098-1108 Mitchell and Tollervey (2001) Curr Opin Cell Biol13:320-5 Ishigaki et al. (2001) Cell106:607-17 Lykke-Andersen et al. (2001) Science293:1836-9 Kim et al. (2001) EMBO 20:2062-68

  4. Nonsense-Mediated mRNA Decay Interaction between EJC and release factors triggers NMD Decapping and degradation Gm mRNA AAAAAAAAA Termination codon > 50nt before last exon junction (Premature Termination Codon) Leeds et al. (1991) Genes Dev5:2303-14 Nagy and Maquat (1998) TIBS23:198-9 Le Hir et al. (2000) Genes & Dev14:1098-1108 Mitchell and Tollervey (2001) Curr Opin Cell Biol13:320-5 Ishigaki et al. (2001) Cell106:607-17 Lykke-Andersen et al. (2001) Science293:1836-9 Kim et al. (2001) EMBO 20:2062-68

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

  6. 1498 of 1500 genes surveyed from fungi, plants, insects and vertebrates obey the PTC rule 4.3% of reviewed RefSeqs have PTCs 34% have start codon after first exon Nagy and Maquat (1998) TIBS23:198-9 NMD is Pervasive “NMD is a critical process in normal cellular developement” Wagner and Lykke-Andersen (2002) J Cell Sci115:3033-8 V(D)J recombination Wang et al. (2002) J Biol Chem277:18489-93 Renders recessive many otherwise dominant mutations Cali and Anderson (1998) Mol Gen Genet260:176-84

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

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

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

  10. SC35 Auto-regulation SC35 Locus transcription SC35 pre-mRNA alternative splicing splicing Productive SC35 mRNA translation SC35 protein Sureau et al. (2001) EMBO J20:1785-96

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

  12. EST-inferred human isoforms 0 10000 2000 4000 6000 8000 NMD Candidates 1989 (35 % of 5693) 5693 Alternative isoforms All isoforms, including canonical 8820

  13. Genomic DNA Sequence Pruitt, K.D. et al (2001) NAR29: 137-40 Refseq mRNAs Extract coding regions Lander et al. (2001) Nature409: 860-921 Genomic Contigs Coding Refseqs Association via LocusLink Refseq-Contig Pairs align w/ Spidey ≥98% id, no gaps Wheelan et al. (2001) Gen Res11:1952-7 Construct genes from aligned Refseq exons & intervening genomic introns (overlap choose mRNA w/ largest number of exons) Refseq-coding genes Canonical Splice Forms Exon 1 Exon 2 Exon 3 Exon 4 Refseq-codinggene mRNA

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

  15. Previous and new RUST targets

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

  17. Premature Stop Codons

  18. EST Limitations Single pass sequencing errors Incompletely processed transcripts 3’ end bias Library contamination Thanaraj (1999) NAR27:2627-37

  19. Alternative Splicing EST Analysis From data in Brett et al. (2000) FEBS Lett474:83-6

  20. Alternative Isoform Inference from Splice Pairs

  21. Alternative Splice Pairs, by Mode

  22. Alternative Splice Pairs, by Mode

  23. Splice Pairs Generating Premature Stops

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

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