Download Evolution of alternative splicing

Alternative splicing:
A playground of evolution
Mikhail Gelfand
Research and Training Center for Bioinformatics
Institute for Information Transmission Problems RAS,
Moscow, Russia
% of alternatively spliced human and mouse genes
by year of publication
Human (genome / random sample)
All genes
Human (individual chromosomes)
Only multiexon genes
Mouse (genome / random sample)
Genes with high EST coverage
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
• Functional consequences of alternative
splicing: what does it do with proteins
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)
Conservation of coding segments
constitutive
segments
alternative
segments
D. melanogaster –
D. pseudoobscura
97%
75-80%
D. melanogaster –
Anopheles gambiae
77%
~45%
Observations
• Alternative splicing is
less conserved than
constitutive one
• D.melanogaster D.pseudoobscura
– retained introns are the
least conserved (are all of
them really functional?)
– mutually exclusive exons
are as conserved as
constitutive exons
• D.melanogaster –
Anopheles gambiae
– mutually exclusive exons
are conserved exactly (no
intron insertions – would
disrupt regulation?)
– cassette exons are the least
conserved
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
CONSTANT
exon
Donor site
Acceptor site Retained intron Cassette exon Exclusive exon
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
CONSTANT
exon
Donor site
Acceptor site Retained intron Cassette exon Exclusive exon
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)
Fisher’s exact test for significance
Pn/Ps (SNPs) Dn/Ds (genomes)
Const.
0.72
0.62
Major
0.78
0.65
diff.
– 0.10
– 0.13
Signif.
0
0.5%
Minor
+ 0.48
0.1%
1.41
1.89
Minor isoform alternative regions:
• More non-synonymous SNPs: Pn(alt_minor)=.12% >> Pn(const)=.06%
• More non-synonym. mismatches: Dn(alt_minor)=.91% >> Dn(const)=.37%
• Positive selection (as opposed to lower stabilizing selection):
α = 1 – (Pa/Ps) / (Da/Ds) ~ 25% positions
• Similar results for all highly covered genes or all conserved exons
Alternative splicing avoids disrupting domains
(and non-domain units)
a)
100%
90%
13%
21%
80%
70%
6%
Non-domain functional
units partially
Domains partially
Data:
• SwissProt
• PROFAM
34%
• PROSITE
60%
40%
50%
No annotated unit
affected
40%
37%
30%
15%
Non-domain functional
units completely
20%
10%
0%
10%
19%
6%
Expected
Observed
Domains completely
Control:
fix the domain
structure;
randomly place
alternative regions
Positive selection towards domain shuffling
(not simply avoidance of disrupting domains by
occurring between domains )
b)
No
annotated
units
affected
Nondomain
units
completely
Domains
completely
a)
100%
90%
13%
21%
80%
70%
6%
Non-domain functional
units partially
Domains partially
34%
60%
40%
50%
No annotated unit
affected
40%
37%
30%
15%
Non-domain functional
units completely
19%
Domains completely
20%
10%
0%
10%
6%
Expected
Observed
Expected
Observed
Short (<50 aa) alternative splicing events within
domains target protein functional sites
c)
FT
positions
affected
FT
positions
unaffected
Prosite
patterns
affected
a)
100%
90%
13%
21%
80%
70%
6%
Non-domain functional
units partially
Domains partially
Prosite
patterns
unaffected
34%
60%
40%
50%
No annotated unit
affected
40%
37%
30%
15%
Non-domain functional
units completely
19%
Domains completely
20%
10%
0%
10%
6%
Expected
Observed
Expected
Observed
An attempt of integration
• AS is often genome-specific
– alternative exons and sites are less conserved (more often lost or
gained) than constitutive ones
• … but still functional
– Even NMD-inducing isoforms are conserved in at least one lineage
– … especially those supported by multiple ESTs
• AS regions show evidence for decreased negative
(stabilizing) selection
– excess non-synonymous codon substitutions
• AS regions show evidence for positive (diversifying)
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
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
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
•
•
– King Jordan (NCBI)
• Support
– Howard Hughes Medical
Institute
– INTAS
– Russian Academy of Sciences
(program “Molecular and
Cellular Biology”)
– Russian Fund of Basic
Research
•
•
•
Irena Artamonova (GSF/MIPS)
– human/mouse, plots
Alexei Neverov (GosNIIGenetika)
– functionality of isoforms