Alternative splicing: A playground of evolution Mikhail Gelfand Research and Training Center for Bioinformatics Institute for Information Transmission Problems RAS, Moscow, Russia October 2006

% 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 Plan

Elementary alternatives Cassette exon Alternative donor site Alternative acceptor site Retained intron

Alternative exon-intron structure in the human, mouse and dog genomes EDAS: a database of human alternative splicing (human genome + GenBank + EST data from RefSeq) –consider casette exons and alternative splicing sites –functionality: potentially translated vs. NMD-inducing elementary alternatives 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)

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 consider situations when alternative human exon (or site) is constitutive in mouse or dog of course, functionality assignments (translated / NMD-inducing) are not very reliable

Translated cassette exons constitutive

NMD-inducing cassette exons

Observations Predominantly included exons are highly conserved irrespective of function Predominantly skipped translated exons are more conserved than NMD-inducing ones Numerous lineage-specific losses –more in mouse than in dog Still, ~40% of skipped (<1% inclusion) exons are conserved in at least one lineage

Alternative donor and acceptor sites: same trends Higher conservation of ~uniformly used sites Internal sites are more conserved than external ones (as expected)

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%

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

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

CG1517: cassette exon in Drosophila, alternative acceptor site in Anopheles Dme, Dps Aga a)

CG31536: cassette exon in Drosophila, shorter cassette exon and alternative donor site in Anopheles Dme, Dps Aga

Evolutionary rate in constitutive and alternative regions Human and mouse orthologous genes Estimation of the d n /d s ratio: higher fraction of non-synonymous (changing amino acid) substitutions => weaker stabilizing (or stronger positive) selection

Concatenates of constitutive and alternative regions in all genes: different evolutionary rates Columns (left-to-right) – (1) constitutive regions; (2–4) alternative regions: N-end, internal, C-end Relatively more non-synonimous substitutions in alternative regions (higher dN/dS ratio) Less amino acid identity in alternative regions

Individual genes: the rate of non-synonymous to synonymous substitutions d n /d s tends to be larger in alternative regions (vertical acis) than in constitutive regions (horizontal acis)

Non-symmetrical histogram of d n /d s (const)– d n /d s (alt) Black: shadow of the left half. In a larger fraction of genes d n /d s (const)< d n /d s (alt), especially for larger values

The same effect is seen in: N-terminal, internal, C-terminal parts

Drosophilas: less selection in alternative regions? More mutations in alt. regions Similar level of mutations More mutations in const. regions In a majority of genes, both synonymous and non- synonymous mutation rates are higher in alternative regions than in constitutive regions

Different behavior of N-terminal, internal and C-terminal alternatives N-terminal alternatives: most genes have higher syn. substit. rate in alt. regions; most genes have higher stabilizing selection in alt. regions Internal alternatives: intermediate situation C-terminal alternatives: more non-synonymous substitutions and less synonymous substitutions => lower stabilizing selection in alternative regions

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

An attempt of integration AS is often genome-specific young AS isoforms are often minor and tissue-specific … but still functional –although unique 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 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

What next? Multiple genomes –many Drosophila spp. –ENCODE data for many mammals Estimate not only the rate of loss, but also the rate of gain (as opposed to aberrant splicing) Control for: –functionality: translated / NMD-inducing –exon inclusion (or site choice) level: major / minor isoform –tissue specificity pattern (?) –type of alternative: N-terminal / internal / C-terminal Evolution of regulation of AS Splicing errors and mutations: retained introns, skipped exons, cryptic sites

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 –King Jordan (NCBI) Support –Howard Hughes Medical Institute –INTAS –Russian Academy of Sciences (program “Molecular and Cellular Biology”) –Russian Fund of Basic Research

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 Irena Artamonova (GSF/MIPS) – human/mouse, plots Alexei Neverov (GosNIIGenetika) – functionality of isoforms