Chanfreau, G. F. | Search bioRxiv

bioRxiv2023

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.

molecular biology↗

bioRxiv2022

Splicing inactivation generates hybrid mRNA-snoRNA transcripts targeted by cytoplasmic RNA decay

Many small nucleolar RNAs (snoRNA)s are processed from introns of host genes, but the importance of splicing for proper biogenesis and the fate of the snoRNAs is not well understood. Here we show that inactivation of splicing factors or mutation of splicing signals leads to the accumulation of partially processed hybrid mRNA-snoRNA transcripts (hmsnoRNA). HmsnoRNAs are processed to the mature 3'-ends of the snoRNAs by the nuclear exosome and bound by snoRNP proteins. HmsnoRNAs are unaffected by translation-coupled RNA quality control pathways, but they are degraded by the major cytoplasmic exonuclease Xrn1p due to their mRNA-like 5'-extensions. These results show that completion of splicing is required to promote complete and accurate processing of intron-encoded snoRNAs and that splicing defects lead to degradation of hybrid mRNA-snoRNA species by cytoplasmic decay, underscoring the importance of splicing for the biogenesis of intron encoded snoRNAs. Significance StatementSmall nucleolar RNAs mediate modifications of nucleosides within ribosomal RNAs, which are necessary for proper ribosomal function and translation. Many snoRNAs are encoded within introns of host genes and accurate biogenesis of these small RNAs is required to produce functional snoRNAs. The work presented here shows that when the splicing reactions are inactivated, snoRNAs undergo a distinct biogenesis pathway which leads to the production of aberrant hybrid RNAs that contain both mRNAs and small RNAs components of the host genes. While snoRNAs are primarily found in the nucleolus, these hybrid RNAs are degraded by the cytoplasmic mRNA degradation pathway. These results demonstrate the importance of splicing to promote accurate snoRNA processing and prevent the production of aberrant mRNA-snoRNA hybrids.

molecular biology↗