Spliceosomal mutations decouple 3′ splice site fidelity from cellular fitness

The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3′-splice site (3′SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3′SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3′SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3′SS. We show that the role of Prp18p in 3′SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3′SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3′SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.