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 GenomeTranscriptomeProteome DNARNAProtein

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

5. pre-mRNA Alternative Splicing ExonIntronExon Intron GTAG GT 3’5’ ExonIntronExon Intron GTAG GT 3’5’ AG ExonIntronExon GTAG GTAG 3’5’ ExonIntronExon GTAGGT 3’5’ AG

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

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

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

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

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 transcripts Transcriptome Space Proteome Space mRNA Amino Acid Sequences Predicted Domains

21. 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 “Swapping” Exon

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

23. “Clipping” Lengthening/shortening of a domain 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