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Comparative Genomics Methods for
Alternative Splicing of Eukaryotic Genes
Liliana Florea
Department of Computer Science
Department of Biochemistry
GWU
[email protected]
202-994-1057
Jan 24th, 2007
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)
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(*).
 Potentially combinatorial numbers of splice variants per gene (DSCAM – 38,000)
 Most human genes are alternatively spliced (~70%)
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
Map evidence
to the genome:
Splice graph
= ‘gene’
Enumerate
variants
Score, rank and
select variants
Liliana Florea, CS/SEAS
cDNA1
cDNA2
cDNA3
cDNA4
Genomic axis
XSV 1
XSV 2
XSV 3
XSV 4
XSV 1
XSV 2
XSV 3
XSV 4
1
2
3
5
4
MappingPotential Coverage Fragmentation Longest IntronOri
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1/2 = 0.5
4/5 = 0.8
1.0
1.0
2/4 = 0.5
1/1 = 1.0
2/4 = 0.5
1.0
1.0
2/4 = 0.5
1/1 = 1.0
2/4 = 0.5
1.0
cumScore
1.0
0.81
0.75
0.75
(Florea et al., Genome Res. 2005; Florea et al., CSHL 2004; DiFrancesco et al., CSHL 2004)
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
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
((((HUM,CHP),(MUS,RAT))DOG)CHK)
nonAlt
Alt
AltD
AltI
Error
Early inserts or ancestral (CF)
Dog/Rodent/Primate inserts (D)
Rodent/Primate inserts (R)
Primate inserts (C)
Human inserts (H)
• 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
Liliana Florea, CS/SEAS
(Florea and Zhao, CFG 2005)
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)
Liliana Florea, CS/SEAS
w/ Erhan Guven