Comparative Genomics Methods for Alternative Splicing of Eukaryotic Genes

1. Comparative Genomics Methods for Alternative Splicing of Eukaryotic Genes Liliana Florea Department of Computer Science Department of Biochemistry GWU florea@gwu.edu 202-994-1057 Jan 24th, 2007

2. Alternative Splicing • Alternative splicing = ability of a gene to produce different mRNAs and proteins under different developmental, tissue and disease-vs-normal conditions, by using distinct combinations of the gene’s exons. • Alternative splicing is important and interesting to characterize: • Possible mechanism to increase protein diversity during species evolution • Aberrant splicing is often associated with disease, in particular cancers (BRCA1, FGFR-2) • Potentially combinatorial numbers of splice variants per gene (DSCAM – 38,000) • Most human genes are alternatively spliced (~70%) In DT3 rat prostate cancer cells, the constitutive exon IIIc ( ) is repressed and the alternativeexon IIIb ( ) is expressed in the mRNA transcript of FGF-R2(*).

3. cDNA1 cDNA2 cDNA3 cDNA4 Genomic axis XSV 1 XSV 2 XSV 3 XSV 4 1 2 3 5 4 MappingPotential Coverage Fragmentation Longest IntronOri cumScore XSV 1 1.0 1.0 1.0 1.0 1.0 XSV 2 1.0 1.0 1/2 = 0.5 4/5 = 0.8 1.0 XSV 3 1.0 2/4 = 0.5 1/1 = 1.0 2/4 = 0.5 1.0 XSV 4 1.0 2/4 = 0.5 1/1 = 1.0 2/4 = 0.5 1.0 1.0 0.81 0.75 0.75 Alternative Splicing Annotation with AIR • The AIR pipeline annotates genes and splice variants in eukaryotic genomes based on mRNA, EST and protein evidence; used to annotate the Celera rat genome Map evidence to the genome: Splice graph = ‘gene’ Enumerate variants Score, rank and select variants (Florea et al., Genome Res. 2005; Florea et al., CSHL 2004; DiFrancesco et al., CSHL 2004)

4. ((((HUM,CHP),(MUS,RAT))DOG)CHK) Alternative Splicing and Evolution • Classes of exons: constitutive (nonAlt), alternative major-form (AltD), alternative minor-form (AltI) • Conservation: establish the presence (P)/ absence (A) of human exons in each of the other species (>50% presence in ‘multiz’ alignments; http://genome.ucsc.edu ) 1. Evolutionary analysis of exon creation • AltI exons are more frequently associated with exon creation (insertion)than the other categories • AltI exons have resulted mostly by recent insertions (~15% occurred before the chicken split, compared to ~80% for AltD and ~75% for nonAlt) • 2. Sequence conservation in introns • AltI introns sequences are more frequently conserved than for AltD, nonAlt • AltD introns are less frequentlyconserved than for nonAlt and AltI • These tendencies become stronger as the evolutionary distance increases • 3. Sequence variation in exons • AltI exons show increased I and V rates compared to nonAlt and AltD exons at all 3 codon positions, which may indicate positive selection (MUS, DOG, CHP comparisons) • AltD exons show decreased I and V rates compared to nonAlt and AltI exons at all 3 codon positions, which may indicate effects of purifying selection (Florea and Zhao, CFG 2005)

5. Prediction of Splicing Regulatory Elements • Exon selection during splicing is controlled by splicing regulatory elements within the exon (exonic) or in its vicinity (intronic) • Regulatory elements may act to promote the inclusion of the exon (enhancers) or to inhibit it (silencers) • Current work: • Identify exonicmotifs over-represented in alternative (Alt) versus constitutive (nonAlt) exons • Identify intronic motifs over-represented in the vicinity of alternative (Alt) versus constitutive (nonAlt) exons • Validate the motifs by comparing the sequence conservation within the motif regions versus within the entire gene in multiple species (human, mouse, rat, dog, chimp, cow, chicken) w/ Erhan Guven