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) Human (individual chromosomes) Mouse (genome / random sample) All genes Only multiexon genes Genes with high EST coverage

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 Plan

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

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)diff.Signif. Const – Major – % Minor % 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) Data: SwissProt PROFAM PROSITE Control: fix the domain structure; randomly place alternative regions

Positive selection towards domain shuffling (not simply avoidance of disrupting domains by occurring between domains )

Short (<50 aa) alternative splicing events within domains target protein functional sites c) Prosite patterns unaffected Prosite patterns affected FT positions unaffected FT positions affected ExpectedObserved

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 Authors Discussions –Vsevolod Makeev (GosNIIGenetika) –Eugene Koonin (NCBI) –Igor Rogozin (NCBI) –Dmitry Petrov (Stanford) –Dmitry Frishman (GSF, TUM) –Shamil Sunyaev (Harvard University Medical School) Data –King Jordan (NCBI) Support –Howard Hughes Medical Institute –INTAS –Russian Academy of Sciences (program “Molecular and Cellular Biology”) –Russian Fund of Basic Research 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 Irena Artamonova (GSF/MIPS) – human/mouse, plots Alexei Neverov (GosNIIGenetika) – functionality of isoforms