A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5’ splice site identity

Abstract

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.

Author summary

Pre-messenger RNA splicing is an important regulator of eukaryotic gene expression, changing the content, frame, and functionality of both coding and non-coding transcripts. Our understanding of how the spliceosome chooses where to cut has focused on the initial identification of splice sites. However, our results suggest that the spliceosome also relies on other components in later steps to maintain the identity of the splice donor sites. We are currently in the midst of a “resolution revolution”, with ever-clearer cryo-EM snapshots of stalled complexes, allowing researchers to visualize moments in time in the splicing cycle. These models are illuminating, but do not always elucidate mechanistic functioning of a highly dynamic ribonucleoprotein complex. Therefore, our lab takes a complementary approach, using the power of genetics in a multicellular animal to gain functional insights into the spliceosome. Using a C. elegans genetic screen, we have found novel functional splicing roles for two proteins, KIN17 and PRCC. Mutations in PRCC in particular promote nearby alternative 5’ splice sites at native loci. This work improves our understanding of how the spliceosome maintains the identity of where to cut the pre-mRNA, and thus how genes are expressed and used in multicellular animals.