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 2008

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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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Roles of alternative splicing

• Functional
• creating protein diversity
• 30.000 genes, gt100.000 proteins
• maintaining protein identity
• e.g. membrane (receptor) and secreted isoforms
• dominant negative isoforms
• combinatorial (transcription factors, signaling
  domains)
• regulatory
• E.g. via chanelling to NMD
• Evolutionary

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Plan

• Evolution of alternative exon-intron structure
• mammals
• human compared to mouse and dog
• mouse and rat compared to human and dog
• paralogs
• dipteran insects
• Drosophila melanogaster, D. pseudoobscura,
  Anopheles gambiae
• many drosophilas
• Evolutionary rates in constitutive and
  alternative regions
• human and mouse
• D. melanogaster and D. pseudoobscura
• many drosophilas
• human-chimpanzee vs. human SNPs
• Alternative splicing and protein domains
• Regulation of AS via conserved RNA structures

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Elementary alternatives
Cassette exon
Alternative donor site
Alternative acceptor site
Retained intron
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EDAS a database of alternative splicing

• Sources
• human and mouse genomes
• GenBank
• RefSeq
• consider cassette exons and alternative splicing
  sites
• functionality potentially translated vs.
  NMD-inducing elementary alternatives (in-frame
  stops, length non divisible by 3)

human mouse
genes 28957 31811
mRNA / cDNA 114624 215212
proteins 91844 126797
ESTs 4294590 3817531
all alternatives 51713 44030
elementary alternatives 31746 21329
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(No Transcript)
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Alternative exon-intron structure in the human,
mouse and dog genomes

• 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 account for 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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Gains/losses loss in mouse
Commonancestor
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Gains/losses gain in human (or noise)
Commonancestor
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Gains/losses loss in dog (or possible gain in
humanmouse)
Commonancestor
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Triple comparison
Human-specific alternatives noise?
Human-specific alternatives noise?
Lost in mouse
Lost in dog
Conserved alternatives
Conserved alternatives
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Translated and NMD-inducing cassette exons

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

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Mouserat vs human and dog a possibility to
distinguish between exon gain and noise
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The rate of exon gain decreases with the exon
inclusion rate increases with the sequence
evolutionary rate

• Caveat spurious exons still may seem to be
  conserved in the rodent lineage due to short time
• Solution estimate FDR by analysis of
  conservation of pseudoexons

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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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Source of innovation Model of random site
fixation

• Plots Fraction of exon-extending alternative
  sites as dependent on exon length
• Main site defined as the one in protein or in
  more ESTs
• Same trends for the acceptor (top) and donor
  (bottom) sites
• The distribution of alt. region lengths is
  consistent with fixation of random sites
• Extend short exons
• Shorten long exons

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Genetic diseases

• Mutations in splice sites yield exon skips or
  activation of cryptic sites
• Exon skip or activation of a cryptic site depends
  on
• Density of exonic splicing enhancers (lower in
  skipped exons)
• Presence of a strong cryptic nearby

Av. dist. to a stronger site Skipped exons Cryptic site exons Non-mutated exons
Donor sites 220 75 289
Acceptor sites 185 66 81
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One more source of innovation site creation

• MAGE-A family of human CT-antigens
• Retroposition of a spliced mRNA, then duplication
• Numerous new (alternative) exons in individual
  copies arising from point mutations
• Creation of donor sites

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Improvement of an acceptor site
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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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Evolution of (alternative) exon-intron structure
in nine Drosophila spp.
Dana
D. melanogasterD. sechelia D. yakuba D.
erecta D. ananassae D. pseudoobscura D.
mojavensis D. virilis D. grimshawi
D. Pollard, http//rana.lbl.gov/dan/trees.html
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Gain and loss of alternative segments and
constitutive exons
0 / 2 0 / 2
Dyak

• CaveatWe cannot observe exon gain outside and
  exon loss within the D.mel. lineage

7 / 7 1 / 1
1 / 7 19 / 23
Dmel
Dmoj
5 / 7 2 / 3
Dere
Dsec
Dana
3 / 10 10 / 12
2 / 12 0 / 1
Dvir
Dgri
20 / 32 2 / 4
2 / 16 5 / 13
1 / 5 9 / 12
3 / 5 8 / 21
Dpse
8 / 10 3 / 5
1 / 16 7 / 8
5 / 8 1 / 2
6 / 15 8 / 33
Notation Patterns with single events / Patterns
with multiple events (Dollo parsimony)
Sample size 397 / 452 18596 / 18874
9 / 21 7 / 12
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Evolutionary rate in constitutive and alternative
regions

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

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Human/mouse genes non-symmetrical histogram of
dn/ds(const)dn/ds(alt)
Black shadow of the left half.In a larger
fraction of genes dn/ds(alt) gt dn/ds(const),
especially for larger values
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Concatenated regionsAlternative regions evolve
faster than constitutive ones
1
dN/dS
dS
dS
dN/dS
dN
dN
0
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Weaker stabilizing selection (or positive
selection) in alternative regions (insignificant
in Drosophila)
1
dN/dS
dS
dS
dN/dS
dN
dN
0
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Different behavior of terminal alternatives
1,5
Drosophila Synonymous substitutions prevalent
in terminal alternative regions non-synonymous
substitutions, in internal alternative regions
dN/dS
Mammals Density of substitutions increases in
the N-to-C direction
dS
dN
0
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Many drosophilasdN in mut. exclusive exons
same as in constitutive exonsdS lower in almost
all alternatives regulation?
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Many drosophilas relaxed (positive?) 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 substitutions 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) Kn/Ks (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. substitutions
  Kn(alt_minor).91 gtgt Kn(const).37
• Positive selection (as opposed to lower
  stabilizing selection) a 1 (Pa/Ps) /
  (Ka/Ks) 25 positions
• Similar results for all highly covered genes or
  all conserved exons

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What does alternative splicingdo to proteins?

• SwissProt proteins
• PFAM domains
• SwissProt feature tables

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Alternative splicing avoids disrupting domains
(and non-domain units)
Control fix the domain structure randomly place
alternative regions
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and this is not simply a consequence of the
(disputed) exon-domain correlation
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Positive selection towards domain shuffling (not
simply avoidance of disrupting 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 species-specific
• young AS isoforms are often minor and
  tissue-specific
• but still functional
• although species-specific 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 fixation of non-synonymous substitutions
  (compared to 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?

• AS in one species, constitutive splicing, in
  another (data from microarrays)
• Changes in inclusion rates
• Evolution of regulation of AS
• Control for
• functionality translated / NMD-inducing
  (frameshifts, stop codons)
• exon inclusion (or site choice) level major /
  minor isoform
• tissue specificity pattern (?)
• type of alternative 1 N-terminal / internal /
  C-terminal
• type of alternative 2 cassette and mutually
  exclusive exon, alternative site

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Acknowledgements

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

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Authors

• Andrei Mironov (Moscow State University)
• Ramil Nurtdinov (Moscow State University)
  human/mouserat/dog
• Dmitry Malko (GosNIIGenetika, Moscow)
  drosophila/mosquito
• Ekaterina Ermakova (Moscow State University,
  IITP) Kn/Ks
• Vasily Ramensky (Institute of Molecular Biology,
  Moscow) SNPs, MacDonald-Kreitman test
• Evgenia Kriventseva (now at U. of Geneva) and
  Shamil Sunyaev (now at Harvard U. Medical
  School)
• protein structure
• Irena Artamonova (Inst. of General Genetics,
  Moscow) human/mouse, plots, MAGE-A
• Alexei Neverov (GosNIIGenetika, Moscow)
  functionality of isoforms

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Bonus track conserved secondary structures
regulating (alternative) splicing in the
Drosophila spp.

• 50 000 introns
• 17 alternative, 2 with alt. polyA signals
• gt95 of D.melanogaster introns mapped to at least
  7 of 12 other Drosophila genomes
• Search for conserved complementary words at
  intron termini (within 150 nt. of intron
  boundaries), then align
• Restrictive search gt 200 candidates
• 6 tested in experiment (3 const., 3 alt.). All 3
  alt. ones confirmed

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CG33298 (phopspholipid translocating ATPase)
alternative donor sites
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Atrophin (histone deacetylase) alternative
acceptor sites
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Nmnat (nicotinamide mononucleotide
adenylytransferase) alternative splicing and
polyadenylation
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Less restrictive search gt many more candidates
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Properties of regulated introns

• Often alternative
• Longer than usual
• Overrepresented in genes linked to development

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Authors

• Andrei Mironov (idea)
• Dmitry Pervouchine (bioinformatics)
• Veronica Raker, Center for Genome Regulation,
  Barcelona (experiment)
• Juan Valcarcel, Center for Genome Regulation,
  Barcelona (advice)
• Mikhail Gelfand (general pessimism)