Alternative splicing: A playground of evolution

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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
• October 2006

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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

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Elementary alternatives
Cassette exon
Alternative donor site
Alternative acceptor site
Retained intron
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Alternative exon-intron structure in the human,
mouse and dog genomes

• EDAS a database of human alternative splicing
  (human genome GenBank EST data from RefSeq)
• consider casette exons and alternative splicing
  sites
• functionality potentially translated vs.
  NMD-inducing elementary alternatives
• Human-mouse-dog triples of orthologous genes
• We follow the fate of human alternative sites and
  exons in the mouse and dog genomes
• Each human AS isoform is spliced-aligned to the
  mouse and dog genome. Definition of conservation
• conservation of the corresponding region
  (homologous exon is actually present in the
  considered genome)
• conservation of splicing sites (GT and AG)

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Caveats

• we consider only possibility of AS in mouse and
  dog do not require actual existence of
  corresponding isoforms in known transcriptomes
• we do not consider situations when alternative
  human exon (or site) is constitutive in mouse or
  dog
• of course, functionality assignments (translated
  / NMD-inducing) are not very reliable

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Translated cassette exons
constitutive
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NMD-inducing cassette exons
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Observations

• Predominantly included exons are highly conserved
  irrespective of function
• Predominantly skipped translated exons are more
  conserved than NMD-inducing ones
• Numerous lineage-specific losses
• more in mouse than in dog
• Still, 40 of skipped (lt1 inclusion) exons are
  conserved in at least one lineage

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Alternative donor and acceptor sites same trends

• Higher conservation of uniformly used sites
• Internal sites are more conserved than external
  ones (as expected)

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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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Conservation of D.melanogaster elementary
alternatives in D. pseudoobscura genes

• blue exact
• green divided exons
• yellow joined exon
• orange mixed
• red non-conserved
• retained introns are the least conserved (are all
  of them really functional?)
• mutually exclusive exons are as conserved as
  constitutive exons

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Conservation of D.melanogaster elementary
alternatives in Anopheles gambiae genes

• blue exact
• green divided exons
• yellow joined exons
• orange mixed
• red non-conserved
• 30 joined, 10 divided exons (less introns in
  Aga)
• mutually exclusive exons are conserved exactly
• cassette exons are the least conserved

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CG1517 cassette exon in Drosophila, alternative
acceptor site in Anopheles
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CG31536 cassette exon in Drosophila, shorter
cassette exon and alternative donor site in
Anopheles
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Evolutionary rate in constitutive and alternative
regions

• Human and mouse orthologous genes
• Estimation of the dn/ds ratio higher fraction
  of non-synonymous (changing amino acid)
  substitutionsgt weaker stabilizing (or stronger
  positive) selection

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Concatenates of constitutive and alternative
regions in all genes different evolutionary rates

• Relatively more non-synonimous substitutions in
  alternative regions (higher dN/dS ratio)

• Less amino acid identity in alternative regions

• Columns (left-to-right) (1) constitutive
  regions
• (24) alternative regions N-end, internal, C-end

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Individual genes the rate of non-synonymous to
synonymous substitutions dn/ds tends to be larger
in alternative regions (vertical acis) than in
constitutive regions (horizontal acis)
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Non-symmetrical histogram of dn/ds(const)dn/ds(a
lt)
Black shadow of the left half.In a larger
fraction of genes dn/ds(const)ltdn/ds(alt),
especially for larger values
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The same effect is seen in N-terminal,
internal, C-terminal parts
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Drosophilas less selection in alternative
regions?
More mutations in alt. regions
Similar level of mutations
More mutations in const. regions
In a majority of genes, both synonymous and
non-synonymous mutation rates are higher in
alternative regions than in constitutive regions
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Different behavior of N-terminal, internal and
C-terminal alternatives
N-terminal alternatives most genes have higher
syn. substit. rate in alt. regions most genes
have higher stabilizing selection in alt. regions
Internal alternatives intermediate situation
C-terminal alternatives more non-synonymous
substitutions and less synonymous substitutions
gt lower stabilizing selection in alternative
regions
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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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An attempt of integration

• AS is often genome-specific
• young AS isoforms are often minor and
  tissue-specific
• but still functional
• although unique isoforms may result from aberrant
  splicing
• AS regions show evidence for decreased negative
  selection
• excess non-synonymous codon substitutions
• AS regions show evidence for positive 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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What next?

• Multiple genomes
• many Drosophila spp.
• ENCODE data for many mammals
• Estimate not only the rate of loss, but also the
  rate of gain (as opposed to aberrant splicing)
• Control for
• functionality translated / NMD-inducing
• exon inclusion (or site choice) level major /
  minor isoform
• tissue specificity pattern (?)
• type of alternative N-terminal / internal /
  C-terminal
• Evolution of regulation of AS
• Splicing errors and mutations retained introns,
  skipped exons, cryptic sites

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Acknowledgements

• 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

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Authors

• 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