Alternative splicing: A playground of evolution
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Alternative splicing: A playground of evolution
Mikhail Gelfand
Research and Training Center for BioinformaticsInstitute 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)
Human (individual chromosomes)
Mouse (genome / random sample)
All genes
Only multiexon genes
Genes with high EST coverage
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Roles of alternative splicing
• Functional:– creating protein diversity
• ~30.000 genes, >100.000 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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• 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
Plan
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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 mousegenes 28957 31811mRNA / cDNA 114624 215212proteins 91844 126797ESTs 4294590 3817531all alternatives 51713 44030elementary alternatives 31746 21329
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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 human+mouse)
Commonancestor
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Human-specific alternatives: noise?
Conserved alternatives
Triple comparison
Human-specific alternatives: noise?
Conserved alternatives
Lost in dog
Lost in mouse
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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 (<1% inclusion) exons are conserved in at least one lineage
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Mouse+rat 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 – exactgreen – divided exonsyellow – joined exonorange – mixedred – non-conserved
• retained introns are the least conserved (are all of them really functional?)
• mutually exclusive exons are as conserved as constitutive exons
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
CONSTANTexon
Donor site Acceptor site Retained intron Cassette exon Exclusive exon
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Conservation of D.melanogaster elementary alternatives in Anopheles gambiae genes
blue – exactgreen – divided exonsyellow – joined exonsorange – mixedred – non-conserved
• ~30% joined, ~10% divided exons (less introns in Aga)
• mutually exclusive exons are conserved exactly
• cassette exons are the least conserved
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
CONSTANTexon
Donor site Acceptor site Retained intron Cassette exon Exclusive exon
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Evolution of (alternative) exon-intron structure in nine Drosophila spp.
Dana
Dmel
Dsec
Dyak
Dere
Dpse
Dmoj
DvirDgri
D. melanogasterD. secheliaD. yakubaD. erectaD. ananassaeD. pseudoobscuraD. mojavensisD. virilisD. grimshawi
D. Pollard, http://rana.lbl.gov/~dan/trees.html
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Gain and loss of alternative segments and constitutive exons
Dmel
Dsec
Dyak
DereDana
Dpse
Dmoj
DvirDgri
Caveat:We cannot observe exon gain outside and exon loss within the D.mel. lineage
1 / 719 / 23
20 / 322 / 4
2 / 165 / 13
1 / 167 / 8
Notation:Patterns with single events /Patterns with multiple events
(Dollo parsimony)9 / 217 / 12
Sample size397 / 452
18596 / 18874
5 / 81 / 2
3 / 58 / 21
1 / 59 / 12
6 / 158 / 33
5 / 72 / 3
3 / 1010 / 12
7 / 71 / 1
0 / 20 / 2
2 / 120 / 1
8 / 103 / 5
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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) => weaker stabilizing (or stronger positive) selection
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Human/mouse genes: non-symmetrical histogram of
dn/ds(const)–dn/ds(alt)
1 5
3
5
9 1 0
1 8
4 0
6 7
1 3 6
3 2 9
7 5 2 6 4 2
1 9 9
7 3
2 71 8
7 7
01 0 01
1 0
1 0 0
1 0 0 0
– 1 – 0 .9– – 0 .8 – 0 .7 – 0 .6 – 0 .5 – 0 .4 – 0 .3 – 0 .2 – 0 .1 0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .7 0 .8 0 .9 1
G en es
C– A
Black: shadow of the left half.In a larger fraction of genes dn/ds(alt) > dn/ds(const), especially for larger values
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Concatenated regions:Alternative regions evolve faster than constitutive ones
AП
0,1680,183
П
0,068
A
0,076
0,405 0,414П A
dN
dN/dS
dS
П A
0,790,80
A
0,220,25
0,28
0,31
П
dN/dS
dS
dN
1
0
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Weaker stabilizing selection (or positive selection) in alternative regions
(insignificant in Drosophila)
AП
0,1680,183
П
0,068
A
0,076
0,405 0,414П A
dN/dS
dN
dS
П A
0,790,80
A
0,220,25
0,28
0,31
П
dN/dS
dS
dN
1
0
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Different behavior of terminal alternatives
П A
AN
AI
AC
1,43
0,790,80
0,90
0,62
A
AN
AI
AC
0,22
0,250,23
0,33
0,25
0,28
0,31
0,37
0,23
0,28
П
AN
AI
A AN
П
0,1680,183 0,186
AI
0,169
AC
0,297
П
0,068
A
0,076
AN
0,076
AI
0,074
AC
0,132
0,405 0,414 0,4100,437П A AN
AI
0,445
AC
dN/dS
dS
dN
1,5
0
Mammals: Density of substitutions increases in the N-to-C direction
Drosophila: Synonymous substitutions prevalent in terminal alternative regions; non-synonymous substitutions,
in internal alternative regions
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Many drosophilas:dN 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) • Fisher’s 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% >> Pn(const)=.06%• More non-synonym. substitutions: Kn(alt_minor)=.91% >> Kn(const)=.37%• Positive selection (as opposed to lower stabilizing selection):
α = 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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a)
6%
10%
15%
37%
40%
34%
21%
19%
6%13%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Expected Observed
Non-domain functional units partially
Domains partially
No annotated unit affected
Non-domain functional units completely
Domains completely
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
0
1
Ra
tio
(ob
serv
ered
/ex
pec
ted
)
Mouse Human Mouse Human Mouse Human
nonAS_Exons AS_Exons AS
AS&Exon boundaries and SMART domains
inside domains
outside domains
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Positive selection towards domain shuffling (not simply avoidance of disrupting domains)
a)
6%
10%
15%
37%
40%
34%
21%
19%
6%13%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Expected Observed
Non-domain functional units partially
Domains partially
No annotated unit affected
Non-domain functional units completely
Domains completely
b)
Domains completely
Non-domain units
completely
No annotated
units affected
Expected Observed
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Short (<50 aa) alternative splicing events within domains target protein functional sites
a)
6%
10%
15%
37%
40%
34%
21%
19%
6%13%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Expected Observed
Non-domain functional units partially
Domains partially
No annotated unit affected
Non-domain functional units completely
Domains completely
c)
Prosite
patterns
unaffected
Prosite
patterns
affected
FT
positions
unaffected
FT
positions
affected
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/mouse+rat/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
• >95% 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 => 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 => 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)