Using Isoform-Sensitive Microarrays to Study Different Modes of Alternative Splicing

1. Using Isoform-Sensitive Microarrays to Study Different Modes of Alternative Splicing Christina Zheng Ares Lab RNA Club September 14, 2006

2. Outline • Isoform-sensitive microarrays (splicing arrays) • introduction • challenges • Probe cross-hybridization • mapping of probes onto the genome • excluding potential cross-hybridizing probes • Analysis of different modes of alternative splicing • annotation of different modes • using splicing arrays to study different modes • Isoform Ratio (IR) • Isoform Expression (IE) • Future directions

3. Outline • Isoform-sensitive microarrays (splicing arrays) • introduction • challenges • Probe cross-hybridization • mapping of probes onto the genome • excluding potential cross-hybridizing probes • Analysis of different modes of alternative splicing • annotation of different modes • using splicing arrays to study different modes • Isoform Ratio (IR) • Isoform Expression (IE) • Future directions

4. Splicing Arrays • Used to assay and identify splicing changes associated with different biological conditions • muscle specific alternative splicing • alternative splicing associated with nonsense mediated decay • The first splicing array was made in yeast • Clark et al. Science 2002 • Mammalian splicing arrays • Johnson et. al. Science 2003 • Pan et. al. Mol. Cell 2004 • Li et. al. Cancer Research 2006 • Le et. al. Nucleic Acids Research 2004 • Sugnet et. al. PLoS 2006

5. Affymetrix Mouse Splicing Array • 5 X106 25mer probes • probes are grouped intro probesets (6-10 probes) • gene probesets - 8 – 10 probes placed in common regions • exon probesets • exon-exon junction probesets – 6 probesets across 30 nucleotides • 15,000+ genes • inflexible probe selection • greater chance of cross-hyb Sugnet et al. PLoS Comput. Bio. 2006

6. Splicing Arrays – AS events • All exon-exon junctions of human mRNA RefSeq • Johnson et. al. Science 2003 • Focused on simple cassette exon events • Pan et. al. Mol. Cell 2004 • Focused on simple AS events with two isoforms • Le et. al. Nucleic Acids Research 2004 • Ule et al. Nature Genetics 2005 • Sugnet et. al. PLoS 2006 • Li et. al. Cancer Research 2006 • Skip to include ratio • one measurement for each event • not applicable to more complicated modes of AS

7. Difficulties with Splicing Arrays • Greater potential of probe cross-hybridization • inflexibility in probe selection due to location of events • exon probes – restricted to the alternative exon • exon-exon junction probes – restricted to exon-exon junction • Alternative splicing (AS) events • identifying/annotating them • analyzing different modes of AS • more complex with a greater number of isoforms

8. Outline • Isoform-sensitive microarrays (splicing arrays) • introduction • challenges • Probe cross-hybridization • mapping of probes onto the genome • excluding potential cross-hybridizing probes • Analysis of different modes of alternative splicing • annotation of different modes • using splicing arrays to study different modes • Isoform Ratio (IR) • Isoform Expression (IE) • Future directions

9. Probe Remapping • Tools used to remap onto the May 2004 mouse assembly • GMAPWu et al. Bioinformatics 2005 • BLAT • home-made junction database • used GMAP to align all mRNA and EST from unigene • made a database of sequences and genomic coordinates of all exon-exon junctions • Remapped probes • uniquely mapped 25mer: 413502 • multiple hits: 25103 (cross-hyb to other genes) • not mapped 25mer: 62667 • missed exon-exon junction • SNPs • changed from old mouse assembly to new

10. Remapping Probes

11. Potential Cross-hybridization • Potential cross-hybridization • BLAST ~400,000 uniquely mapped probes • Cutoff for the level of similarity to other genes • how do different levels of similarity affect probe intensity? • took probes which only hit 2 genes • hit 25nt to one gene • hit at different level to another (24nt, 23nt, 22nt ….) • choose a cutoff based on the how the probe behavior in each class

12. Probe Analysis

13. Outline • Isoform-sensitive microarrays (splicing arrays) • introduction • challenges • Probe cross-hybridization • mapping of probes onto the genome • excluding potential cross-hybridizing probes • Analysis of different modes of alternative splicing • annotation of different modes • using splicing arrays to study different modes • Isoform Ratio (IR) • Isoform Expression (IE) • Future directions

14. Analysis of Affy Splicing Array • Previous work • Ule et al. Nature Genetics 2005 • Sugnet et al. PLoS 2006 • Focused on simple cassette exon events and or simple two isoform events • Using a variation of skip to include ratio • Array was designed with more complicated events

15. Splicing Event Probe Groupings • Annotated AS events • exonwalk • identifies and annotates events, no matter how complicated the event • Mapped the probes onto annotated events • 3418 AS events: • 1 isoform: 2002 • 2 isoforms: 892 • 3 isoforms: 182 • 4 isoforms: 95 • 5 isoforms: 44 • 6 or more isoforms: 203

16. isoform1 isoform2 isoform3  isoform  isoform  isoform isoform i Isoform Ratio (IR) = isoform Isoform Ratio Isoform 3 Isoform 1 Isoform 2  isoform = isoform1+isoform2+isoform3

17. xt - xc # of false positives s+s0 # of significant isoforms Isoform Ratio • Significance Analysis of Microarrays (SAM) • identify statistically significant IRs • based on a modified t test - ‘relative difference’ • , s = standard deviation; s0 = small positive constant • q value - min false discovery rate (FDR) Storey J. Roy. Stat. Soc. Ser. B 2002 • (FDR) • the minimum FDR incurred for calling a specific isoform significant • analogous to p-value for false positive rate • can use a q-value as a specific cutoff much like a p-value

18. Isoform Ratio • Identifying muscle specific AS events • C2C12 myoblast differentiation system • Run samples on Affymetrix mouse splicing array C2C12 stem cells differentiate stem cells myo-tubule formation isolate control RNA isolate test RNA

19. Analysis Pipeline • Background correction, normalization, and probe summarization • RMA (Irizarry et al. Biostatistics 2003) • Grouping probesets into splicing events • mapping probesets onto annotated AS events • calculating IR • Grouping probesets into genes • average of all probesets within a gene • Use SAM (Tusher et al. PNAS 2001) to test significance differences between test and control • q-value (min false discovery rate) Storey J. Roy. Stat. Soc. Ser. B 2002 • Display results on dataviewer

20. Splicing Array Dataviewer

21. GeneViewer

22. Muscle Specific AS events DnaJ (Hsp40) homolog upregulated upregulated Coro6, actin binding protein

23. STOP STOP STOP STOP Stop codon is in last exon Premature stop codon (PTC) AAAAA AAAAA EJC EJC EJC EJC EJC >50nt Multiple rounds of normal translation NMD AAAAA AAAAA EJC Ribosome Ribosome Isoform Expression • Connection between AS and nonsense-mediated decay (NMD) • Block NMD and assay for changes in individual isoform changes Example: PTB include skip

24. Isoform Expression Isoform 3 Isoform 1 Isoform 2 log (isoform1) – log(gene) log (isoform2) – log(gene) log (isoform3) – log(gene) Isoform Expression (IE) = log (isoform i) – log (gene) gene =  probes in gene

25. Analysis Pipeline • Background correction, normalization, and probe summarization • RMA (Irizarry et al. Biostatistics 2003) • Grouping probesets into splicing events • mapping probesets onto predefined AS events • calculating IE • Grouping probesets into genes • average of all probesets within a gene • Use SAM (Tusher et al. PNAS 2001) to test the significance between test and control • q-value (min false discovery rate) • Display results on dataviewer

26. AS associated with NMD • SAT1 - spermidine/spermine N1-acetyl transferase 1 • down regulates polyamine levels in the cell • the inclusion of an alternative exon throws it out of frame NMD • block NMD under conditions which SAT1 is needed • polyamine and polyamine analog (BENSPM) • expect inclusion of the exon the be repressed • missed by previous analysis methods because this event is an example of having probes for only one of the isoforms

27. Outline • Isoform-sensitive microarrays (splicing arrays) • introduction • challenges • Probe cross-hybridization • mapping of probes onto the genome • excluding potential cross-hybridizing probes • Analysis of different modes of alternative splicing • annotation of different modes • using splicing arrays to study different modes • Isoform Ratio (IR) • Isoform Expression (IE) • Future directions

28. Future Directions • Probe cross-hybridization • 18bp cross-hyb level • behavior of exon probes vs exon-exon junction probes • Different modes of AS • better classification of the more complicated modes

29. Acknowledgements Ares Lab Manny Ares John-Paul Donohue Leslie Grate Roland Nagel Julie Ni Lily Shiue Charles Sugnet

30. Splice Junction (SJ) Index = log - log (SJmut) (EXmut) (SJwt) (EXwt) Normalize out gene expression Splicing Arrays • 40 nt probes • each intron-containing gene in yeast Clark et. al. Science 2002