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
• RECOMB, 20 May 2008

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of alternatively spliced human and mouse genes,
by year of publication
100
2008
C.Burge
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
• human 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 (nonsense-mediated
  decay)
• Evolutionary

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Plan

• Evolution of alternative exon-intron structure
• Origin of new (alternative) exons and sites
• Evolutionary rates in constitutive and
  alternative regions

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Elementary alternatives
Cassette exon
Mutually exclusive exons
Alternative donor site
Alternative acceptor site
Retained intron
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Sources of data

• ESTs 1999 global 2002-3 comparative
• mapping exon-intron structure to genome
• global alignment of genomes
• identifying non-conserved exons and splice sites
• oligonucleotide arrays (chips)2001 global2004
  comparative
• qualitative analysis (inclusion values)
• genome-specific constitutive / alternative exons
• mRNA-seq (new generation high-throughput)2008
  globalexpected 2009-10 comparative

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Alternative exons are often genome-specific
(Modrek Lee, 2003)
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25 AS events in 50 genes are not conserved
Na/K-ATPase Fxyd2/FXYD2
p53
NurtdinovGelfand, 2003
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Alternative exon-intron structure in fruit flies
and 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 challenging
• incomplete genomes
• the quality of alignment with the Anopheles
  genome is lower, especially for terminal exons
• frequent intron insertion/loss (4.7 introns per
  gene in Drosophila vs. 3.5 introns per gene in
  Anopheles)

MalkoGelfand, 2006
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Conservation of coding segments
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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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Genome-specific AS real or noise?young or
deteriorating?

• minor isoforms, small inclusion rate
• often frameshifting and/or stop-containing gt NMD
• regulatory role?

Sorek, Shamir Ast, 2004
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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)

NurtdinovGelfand, 2007
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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
• 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)
  human exons are conserved in at least one lineage
  (mouse or dog)

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Mouserat vs human and dog a possibility to
distinguish between exon gain and noise
NurtdinovGelfand, 2009
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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

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Conserved rodent-specific exons and pseudoexons

• Estimation of FDR by analysis of conservation
  of pseudoexons
• intronic fragments with the same characteristics
  (length distribution etc.)
• apply standard rules to estimate conservation
• obtain the number (fraction) of rodent-specific
  exons that could be pseudoexons conserved by
  chance (brown)
• obtain the number (fraction) of real
  rodent-specific exons (dark green) 50, that
  is, 15 of mouse-specific exons (the rest is
  likely noise)

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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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Evolution of (alternative) exon-intron structure
in 11 Drosophila spp.
Dana
D. melanogasterD. sechelia D. yakuba D.
erecta D. ananassae D. pseudoobscura D.
mojavensis D. virilis D. grimshawi
D.persimilis
D.willistonii
D. Pollard, http//rana.lbl.gov/dan/trees.html
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Gain and loss of alternative segments and
constitutive exons
Unique events per 1000 substitutions. Caveat We
cannot observe exon gain outside and exon loss
within the D.mel. lineage
34. 0.9
Dyak
184. 1.1
37. 8.7
Dmel
Dmoj
143. 1.1
Dere
Dsec
Dana
57. 0.5
100. 6.6
Dvir
Dgri
13. 0.6
131. 0.4
14. 1.6
24. 1.2
Dpse
75. 7.2
85. 0.8
Dper
134. 1.1
40. 2.3
175. 20.2
5. 0.2
45. 0.9
16. 0.3
57. 1.0
Sample size 397 / 18596
Dwil
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Gain and loss of alternative segments and
constitutive exons
151. 3.6
Dyak
213. 1.3
164. 11.7
Non-unique events per 1000 substitutions (Dollo
parsimony)
Dmel
Dmoj
226. 2.7
Dere
Dsec
Dana
272. 1.0
330. 9.3
Dvir
Dgri
68. 1.4
188. 0.7
40. 2.1
33. 2.9
Dpse
238. 9.8
98. 1.3
Dper
233. 1.8
83. 4.2
408. 27.6
72. 0.4
120. 1.7
49. 1.1
Sample size 452 / 18874
81. 1.3
Dwil
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Conserved alternative splicing in nematodes

• 92 of cassette exons from Caenorhabditis elegans
  are conserved in Caenorhabditis briggsae and/or
  Caenorhabditis remanei (EST-genome comparisons)
• in minor isoforms as well
• especially for complex events
• there is less difference between levels of AS
  (exon inclusion) in natural C.elegans isolates
  than in mutation accumulation lines (microarray
  analysis) gt positive selection on the level of
  AS.

IrimiaRoy, 2007 Barberan-Sohler Zaler, 2008
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Plants little conservation of alternative
splicing

• Arabidopsis thaliana Oriza sativa (rice)
• Oriza sativa (rice) Zea mays (maize)
• Few AS events are conserved (5 of genes compared
  to 50 of genes with AS)
• the level of conservation is the same for
  translated and NDM isoforms

Severingvan Hamm, 2009
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Constitutive exons becoming alternative

• human-mouse comparison, EST data gt 612 exons
  constitutively spliced in one species and
  alternatively in the other
• all are major isoform (predominantly included)
• analysis of other species (selected cases)
  ancestral exons have been constitutive
• characteristics of such exons (molecular
  evolution Kn/Ks, conservation of intron flanks
  etc) are similar to those of constitutive exons

Lev-MaorAst, 2007
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Changes in inclusion rate

• orthologous alternatively spliced (cassette)
  exons of human and chimpanzee
• quantitative microarray profiling
• estimate the inclusion rate by comparison of exon
  and exon-junction probes
• gt 6-8 of altertnative exons have significantly
  different inclusion levels

CalarcoBlencowe, 2007
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Sources of new exons

• exon shuffling and duplications
• mutually exlusive exons
• exonisation new exons, new sites
• in repeats
• constitutive exons becoming alternative

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Alternative splice sites 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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A natural model 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

Kurmangaliev Gelfand, 2008
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Creation of sites
Vorechovsky, 2006 BurattiVorechovsky, 2007
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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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Exonisation of repeats

• early studies 61 alternatively spliced
  translated exon with hits to Alu (no constitutive
  exons)
• 84 frame-shiting or stop-containing
• exonisation by point mutations in cryptic sites
  in the Alu consensus
• studied in experiment
• both donor and acceptor sites
• recent studiy 1824 human exons, 506 mouse exons
• Alu, L1, LTR may generate completely new exons

Sorek, Ast, Graur, 2002 Lev-MaorAst, 2003
SorekAst, 2004 SelaAst, 2007
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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. regions)dn/ds(alt. regions)
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() in some other
studies dN(alt)ltdN(const) less synonymous
substitutions in alternaitve regions
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 drosophilas, different alternatives
dN in mutually exclusive exons same as in
constitutive exons
dS lower in almost all alternatives regulation?
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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

• 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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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?

• Changes in inclusion rates (mRNA-seq)
• revisit constitutive-becoming-alternative exons
• Other taxonomical groups
• Evolution of regulation
• donor and acceptor splicing sites
• splicing enhabcers and silencers
• cellular context (SR-proteins etc.)
• 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 exons, alternative sites, etc.

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Acknowledgements

• Discussions
• Eugene Koonin (NCBI)
• Igor Rogozin (NCBI)
• Vsevolod Makeev (GosNIIGenetika)
• Dmitry Petrov (Stanford)
• Dmitry Frishman (GSF, TUM)
• Sergei Nuzhdin (USC)
• Support
• Howard Hughes Medical Institute
• 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 (IITP) Kn/Ks
• Vasily Ramensky (Institute of Molecular Biology,
  Moscow) SNPs, MacDonald-Kreitman test
• Irena Artamonova (Inst. of General Genetics and
  IITP, Moscow) human/mouse, plots, MAGE-A

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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)