Abstract
The properties of genotype–phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function—the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection.
Highlights
NN N N Effect in WT N nNAbsolute mutation effectWT 49−T 21−C 53−G 38−C 2−C 34−A 54−G 29−A 10−T6−T 12−T 20−A 57−A 6−C 53−T 36−G 11−A 37−G 16−G 54−T 51−G 37−A 34−C 31−C 19−T2−T 15−A 43−G 61−T 9−G 19−C 5−G 38−A 59−A 53−C 61−C 55−G59−C 60−C 45−G 57−T 52−A 50−G 24−A 55−C 42−G 56−C 26−G 17−C 26−C 35−C 35−A 62−A 43−A 32−A 47−A9−A 5−A 32−C 35−G 17−T 15−G 31−G 56−G 27−G 46−A 10−G 38−G 32−G 20−C 22−C 59−G 58−G 62−C BackgroundsFourth, the landscape suggests that selection may be acting on the robustness of splicing regulatory mechanisms as well as on levels of inclusion: the wt exon is more robust to the effects of mutations than its immediate neighbours in genotype space with the same level of inclusion
We generated a library of sequence variants designed to cover all possible single and double mutations in exon 6 of human FAS/CD95 using doped oligonucleotide synthesis[10]
The library was cloned into a minigene cassette spanning FAS exons 5–7, transfected into HEK293 cells under conditions that lead to approximately 50% exon inclusion, matching the levels of exon 6 inclusion in endogenous transcripts in this cell line, RNA was isolated and the inclusion score of each variant exon was quantified by reverse transcription, PCR amplification with minigene-specific primers and deep sequencing (Fig. 1a)
Summary
Single nucleotide changes at 58/63 positions (92%) affected splicing, demonstrating that splicing regulatory sequences are distributed across nearly every nucleotide in the exon (Fig. 2a), expanding previous estimates[3,11,12]. The distribution of mutation effects (Fig. 2c) is shifted towards reduced inclusion (one sample Wilcoxon test Po10 À 6), indicating selection for sequences that promote inclusion in the wt exon. Single nucleotide changes can quantitatively alter splicing over nearly the complete range of inclusion levels (Fig. 2a,c and Supplementary Fig. 3). This high mutation sensitivity means that the alternative splicing of this exon will evolve extremely rapidly unless constrained by purifying selection. Exon 6 Neighbouring exon Intron Vector Exon 6 sequence A Mutation pA Polyadenylation site
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