Alternative%20splicing:%20A%20playground%20of%20evolution

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Alternative splicing A playground of evolution

• Mikhail Gelfand
• Research and Training Center for Bioinformatics
• Institute for Information Transmission Problems
  RAS,
• Moscow, Russia

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of alternatively spliced human and mouse genes
by year of publication
Human (genome / random sample)
All genes
Human (individual chromosomes)
Only multiexon genes
Genes with high EST coverage
Mouse (genome / random sample)
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Plan

• Evolution of alternative exon-intron structure
• mammals human, mouse, dog
• dipteran insects Drosophila melanogaster, D.
  pseudoobscura, Anopheles gambiae
• Evolutionary rate in constitutive and alternative
  regions
• human / mouse
• D. melanogaster / D. pseudoobscura
• human-chimpanzee / human SNPs
• Functional consequences of alternative splicing
  what does it do with proteins

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Alternative exon-intron structure in fruit flies
and the malarial mosquito

• Same procedure (AS data from FlyBase)
• cassette exons, splicing sites
• also mutually exclusive exons, retained introns
• Follow the fate of D. melanogaster exons in the
  D. pseudoobscura and Anopheles genomes
• Technically more difficult
• incomplete genomes
• the quality of alignment with the Anopheles
  genome is lower
• frequent intron insertion/loss (4.7 introns per
  gene in Drosophila vs. 3.5 introns per gene in
  Anopheles)

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Conservation of coding segments
constitutive segments alternative segments
D. melanogaster D. pseudoobscura 97 75-80
D. melanogaster Anopheles gambiae 77 45
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Observations

• Alternative splicing is less conserved than
  constitutive one
• D.melanogaster - D.pseudoobscura
• retained introns are the least conserved (are all
  of them really functional?)
• mutually exclusive exons are as conserved as
  constitutive exons
• D.melanogaster Anopheles gambiae
• mutually exclusive exons are conserved exactly
  (no intron insertions would disrupt
  regulation?)
• cassette exons are the least conserved

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The MacDonald-Kreitman test evidence for
positive selection in (minor isoform) alternative
regions

• Human and chimpanzee genome mismatches vs human
  SNPs
• Exons conserved in mouse and/or dog
• Genes with at least 60 ESTs (median number)
• Fishers exact test for significance

Pn/Ps (SNPs) Dn/Ds (genomes) diff. Signif.
Const. 0.72 0.62 0.10 0
Major 0.78 0.65 0.13 0.5
Minor 1.41 1.89 0.48 0.1

• Minor isoform alternative regions
• More non-synonymous SNPs Pn(alt_minor).12 gtgt
  Pn(const).06
• More non-synonym. mismatches Dn(alt_minor).91
  gtgt Dn(const).37
• Positive selection (as opposed to lower
  stabilizing selection) a 1 (Pa/Ps) /
  (Da/Ds) 25 positions
• Similar results for all highly covered genes or
  all conserved exons

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Alternative splicing avoids disrupting domains
(and non-domain units)

• Data
• SwissProt
• PROFAM
• PROSITE
• Control
• fix the domain structure randomly place
  alternative regions

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Positive selection towards domain shuffling (not
simply avoidance of disrupting domains by
occurring between domains )
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Short (lt50 aa) alternative splicing events within
domains target protein functional sites
c)
FT
positions
affected
FT
positions
unaffected
Prosite
patterns
affected
Prosite
patterns
unaffected
Expected
Observed
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An attempt of integration

• AS is often genome-specific
• alternative exons and sites are less conserved
  (more often lost or gained) than constitutive
  ones
• but still functional
• Even NMD-inducing isoforms are conserved in at
  least one lineage
• especially those supported by multiple ESTs
• AS regions show evidence for decreased negative
  (stabilizing) selection
• excess non-synonymous codon substitutions
• AS regions show evidence for positive
  (diversifying) selection
• excess non-synonymous SNPs
• AS tends to shuffle domains and target functional
  sites in proteins
• Thus AS may serve as a testing ground for new
  functions without sacrificing old ones

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Acknowledgements Authors

• Discussions
• Vsevolod Makeev (GosNIIGenetika)
• Eugene Koonin (NCBI)
• Igor Rogozin (NCBI)
• Dmitry Petrov (Stanford)
• Dmitry Frishman (GSF, TUM)
• Shamil Sunyaev (Harvard University Medical
  School)
• Data
• King Jordan (NCBI)
• Support
• Howard Hughes Medical Institute
• INTAS
• Russian Academy of Sciences (program Molecular
  and Cellular Biology)
• Russian Fund of Basic Research

• Andrei Mironov (Moscow State University)
• Ramil Nurtdinov (Moscow State University)
  human/mouse/dog
• Dmitry Malko (GosNIIGenetika)
  drosophila/mosquito
• Ekaterina Ermakova (Moscow State University,
  IITP) Kn/Ks
• Vasily Ramensky (Institute of Molecular Biology)
  SNPs
• Irena Artamonova (GSF/MIPS) human/mouse,
  plots
• Alexei Neverov (GosNIIGenetika) functionality
  of isoforms