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

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Alternative splicing of human(and mouse) genes
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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

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Data and Methods (routine)

• known alternative splicing
• HASDB (human, ESTsmRNAs)
• ASMamDB (mouse, mRNAsgenes)
• additional variants
• UniGene (human and mouse EST clusters)
• complete genes and genomic DNA
• GenBank (full-length mouse genes)
• human genome
• TBLASTN (initial identification of orthologs
  mRNAs against genomic DNA)
• BLASTN (human mRNAs against genome)
• Pro-EST (spliced alignment, ESTs and mRNA against
  genomic DNA)

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• Pro-Frame (spliced alignment of proteins against
  genomic DNA)
• confirmation of orthology
• same exon-intron structure for at least one
  isoform
• gt70 identity over the entire protein length
• analysis of conservation of human alternative
  splicing in the mouse genome align human protein
  to mouse genomic DNA the isoform is conserved if
• all exons or parts of exons are conserved
• all sites are conserved
• same procedure for mouse proteins and human DNA
• We do not require that the isoform is actually
  observed as mRNA or ESTs

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166 gene pairs
Known alternative splicing
human
mouse
42
84
40
126
124
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Elementary alternatives
Cassette exon
Alternative donor site
Alternative acceptor site
Retained intron
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Human genes
mRNA EST
cons. non-cons. cons. non-cons.
Cassette exons 56 25 74 26
Alt. donors 18 7 16 10
Alt. acceptors 13 5 19 15
Retained introns 4 3 5 0
Total 96 30 114 51
Total genes 45 28 41 44
Conserved elementary alternatives 69 (EST) -
76 (mRNA)Genes with all isoforms conserved 57
(45)
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Mouse genes
mRNA EST
cons. non-cons. cons. non-cons.
Cassette exons 70 5 39 9
Alt. donors 24 6 17 6
Alt. acceptors 15 6 16 9
Retained introns 8 7 10 4
Total 117 24 82 28
Total genes 68 22 30 26
Conserved elementary alternatives 75 (EST) -
83 (mRNA)Genes with all isoforms conserved 79
(64)
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Real or aberrant non-conserved AS?

• 24-31 human vs. 17-25 mouse elementary
  alternatives are not conserved
• 55 human vs 36 mouse genes have at least one
  non-conserved variant
• denser coverage of human genes by ESTs
• pick up rare (tissue- and stage-specific) gt
  younger variants
• pick up aberrant (non-functional) variants
• 17-24 mRNA-derived elementary alternatives are
  non-conserved (compared to 25-32 EST-derived
  ones)

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Comparison to other studies.Modrek and Lee,
2003 skipped exons

• inclusion level is a good predictor of
  conservation
• 98 constitutive exons are conserved
• 98 major form exons are conserved
• 28 minor form exons are conserved
• inclusion level of conserved exons in human and
  mouse is highly correlated
• Minor non-conserved form exons are errors? No
• minor form exons are supported by multiple ESTs
• 28 of minor form exons are upregulated in one
  specific tissue
• 70 of tissue-specific exons are not conserved
• splicing signals of conserved and non-conserved
  exons are similar

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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

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Fruit fly and mosquito

• Technically more difficult than human-mouse
• incomplete genomes
• difficulties in alignment, especially at gene
  termini
• changes in exon-intron structure irrespective of
  alternative splicing (4.7 introns per gene in
  Drosophila vs. 3.5 introns per gene in Anopheles)

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Methods

• Pro-Frame Align Dme protein isoforms to Dps and
  Aga genes
• coding segments regions in Dme genes between Dme
  intron shadows
• We follow the fate of Dme exons and coding
  segments in Dps and Aga genomes
• slices regions between all exon-exon junctions
  (intron shadows) from all three genomes (Dme,
  Dps, Aga) mapped to Dme isoforms
• slice is conserved if it aligns with ?35 identity

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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
• 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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CG1587 alternative acceptor site in Drosophila,
candidate retained intron in intronless gene of
Anopheles
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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

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Alternative splicing in a multigene family the
MAGEA family of cancer/testis specific antigens

• A locus at the X chromosome containing eleven
  recently duplicated genes two subfamilies of
  four genes each and three single genes
• Retrogene one protein-coding exon, multiple
  different 5-UTR exons
• Mutations create new splicing sites or disrupt
  existing sites

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Birth of donor sites (new GT in alternative
intial exon 5)
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Birth of an acceptor site (new AG and polyY
tract in MAGEA8-specific cassette exon 3)
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Birth of an alternative donor site (enhanced
match to the consensus (AG) in cassette exon 2)
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Birth of an alternative acceptor site (enhanced
polyY tract in cassette exon 4)
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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

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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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dn/ds (con) dn/ds (alt)
complete genes
N-terminal regions
internal regions
C-terminal regions
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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

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Na/Ns (alternative) gt Na/Ns (constitutive)for
all evidence levels
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• Evolution of alternative exon-intron structure
• human-mouse
• Drosophila and Anopheles
• Evolution of alternative splicing sites MAGE-A
  family of CT antigens
• Evolutionary rate in constitutive and alternative
  regions
• human-mouse
• human SNPs
• Alternative splicing and protein structure

34
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 young (as opposed to degenerating)
• young AS isoforms are often minor and
  tissue-specific
• but still functional
• although unique isoforms may be result of
  aberrant splicing
• AS often arises from duplication of exons
• or point mutations creating splicing sites
• or intron insertions
• AS regions show evidence for positive selection
• excess non-synonymous and damaging SNPs
• excess non-synonymous codon substitutions
• AS tends to shuffle exons 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

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

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Authors

• Andrei Mironov (Moscow State University)
  spliced alignment
• Ramil Nurtdinov (Moscow State University)
  human/mouse, data
• Irena Artamonova (GSF/MIPS) human/mouse,
  MAGE-A
• Dmitry Malko (GosNIIGenetika, Moscow)
  mosquito/drosophila
• Ekaterina Ermakova (Moscow State University)
  evolution of alternative/constitutive regions
• Vasily Ramensky (Institute of Molecular Biology,
  Moscow) SNPs
• Shamil Sunyaev (EMBL, now Harvard University
  Medical School) protein structure
• Eugenia Kriventseva (EBI, now EMBL) protein
  structure