Characterizing Alternative Splicing With Respect To Protein Domains

1. Characterizing Alternative Splicing With Respect To Protein Domains BME 220 Project Charlie Vaske

2. Overview • Background • Methods and Data • Preliminary Results • Preliminary Conclusions

3. Big Picture Genome Transcriptome Proteome DNA RNA Protein

4. Splicing in Higher Eukaryotes Genome: 5’ 3’ Intergenic Gene Intergenic Transcription of pre-mRNA 5’ 3’ Exon Intron Exon Intron Exon GT AG GT AG Donor Site Acceptor Site U2 U1 Splicing to mRNA Transcriptome: 5’ 3’ Exon Exon Exon

5. Exon Intron Exon Intron Exon GT GT AG AG GT GT AG AG 5’ 3’ Exon Intron Exon Intron Exon GT GT AG GT AG 5’ 3’ Exon Intron Exon AG 5’ 3’ Exon Intron Exon GT GT AG GT AG Alternative Splicing 5’ 3’ pre-mRNA GT AG

6. Microarrays For Alt. Splicing • Use short oligonucleotides • Get a guess at the rate of expression of the oligo Exon 1 Exon 2 Exon 4 Exon 5 Exon 3

7. Probe types Constitutive Junction Exon Unique (“Cassette”) AffymetrixMicroarrays For Alt. Splicing Exon 1 Exon 2 Exon 4 Exon 5 Exon 3 Isoform 1: Exon 1 Exon 2 Exon 4 Exon 5 Isoform 2: Exon 1 Exon 3 Exon 5

8. Probe types Constitutive Exon Junction Unique (“Cassette”) Ideal Microarray Readings Expression a b c d e Probe Isoform 1: a c Exon 1 Exon 2 Exon 4 Exon 5 b Isoform 2: a d Exon 1 Exon 3 Exon 5 e

9. Motivation • Why alternatively splice? • How does it affect the resulting proteins? • Look at domains: • High level summary of protein • ~80% of eukaryotic proteins are multi-domain • Domains are big relative to an exon

10. Some Previous Work • Signatures of domain shuffling in the human genome. Kaessmann, 2002. Intron phase symmetry around domain boundaries • The Effects of Alternative Splicing On Transmembrane Proteins in the Mouse Genome. Cline, 2004. Half of TM proteins studied affected by alt-splicing.

11. Method • Predict Alternative Splicing • Predict Protein Domains • Look for effects of Alt-Splicing on predicted domains • “Swapping” • “Knockout” • “Clipping”

12. Microarray Design • Genes based on mRNA and EST data in mouse • Mapped to Feb. 2002 mouse genome freeze • ~500,000 probes (~66,000 sets) • ~100,000 transcripts • ~13,000 gene models

13. Technical work Genome Space Overlap gene models Generated Data transcripts Overlap Provided data Overlap Probe to transcript mapping E@NM_021320 cc-chr10-000017.82.0 G6836022@J911445 cc-chr10-000017.91.1 G6807921@J911524_RC cc-chr10-000018.4.0 probes

14. Predicting Alternative Splicing • Using mouse alt-splicing microarrays • Data from Manny Ares • 8 tissues • 3 replicates of each tissue

15. Predicting Alternative Splicing • General Approach: Clustering, then Anti-Clustering 107 Clusters Detail View

16. Predicting Alternative Splicing • Cluster pairs have both anti-correlation and overlap

17. First Attempt at Predictions • Concerned with prediction quality • Only took clusters-pairs with anti-correlation less than -0.9

18. First Attempt at Predictions • Greater than -0.9 anti-correlation • 121 genes • ~60 named genes • Many of these have documented isoforms

19. Predicting Protein Domains • Used local install of InterPro • Only used pfam Based on sequence motifs • Liberal e-value cut-off: ~1e-10

20. Technical work Genome Space Transcriptome Space Proteome Space Amino Acid Sequences mRNA transcripts Predicted Domains Predicted Domains Predicted Domains

21. “Swapping” I define to be: • Genome base pair annotated with >1 domain • 2 isoforms share a domain, then each has a domain of different types on the same side Exon Exon Exon Exon Exon Exon Exon Exon

22. “Knockout” • Cassette exon indels a predicted domain Exon Exon Exon Exon Exon or Exon Exon Exon Exon Exon

23. “Clipping” • Lengthening/shortening of a domain Exon Exon Exon Exon Exon Exon Exon Exon Exon

24. Results: Selected Prediction • Rabggtb 3’ C 5’ N

25. Preliminary Conclusions • Only a few genes examined • Analysis pipeline in infancy • Not thoroughly tested • I do have alternative splicing events • Example and literature suggest that some effects will be found