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. Liliana Florea, CS/SEAS Alternative Splicing In DT3 rat prostate cancer cells, the constitutive exon IIIc ( ) is repressed and the alternative exon IIIb ( ) is expressed in the mRNA transcript of FGF-R2 (*). 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%)

3. Liliana Florea, CS/SEAS 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 Genomic axis cDNA1 cDNA2 cDNA3 cDNA4 XSV 1 XSV 2 XSV 3 XSV 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 11 22 33 44 55 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. Liliana Florea, CS/SEAS 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 ) ((((HUM,CHP),(MUS,RAT))DOG)CHK) 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) 1. Evolutionary analysis of exon creation 2. Sequence conservation in introns AltI introns sequences are more frequently conserved than for AltD, nonAlt AltD introns are less frequently conserved 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. Liliana Florea, CS/SEAS 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 exonic motifs 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