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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 but not KIN17 changed 5’ splice sites genome-wide, promoting usage of nearby non-consensus sites. We further found that mutations in KIN17 and PRCC changed dozens of 3’ splice sites, promoting non-consensus sites upstream of canonical splice sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.Author SummaryPre-mRNA splicing is an essential step of gene regulation, carried out by an unusual molecular machine, the spliceosome. Unlike other molecular machines, such as ribosomes, that simply assemble and catalyze chemical reactions, “the spliceosome” is a highly-dynamic cycle, carried out by 5 specialized small nuclear RNAs and over 100 proteins, which sequentially join, rearrange, and withdraw from the splicing assembly during each splicing cycle. These assemblies initially choose “splice sites” where the pre-mRNA will be cut, and then undergo multiple rearrangements to finally form the active site which catalyzes the splicing reactions which remove an intron from a pre-mRNA. 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, 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. Our results suggest that the spliceosome does not just rely on its initial identification of the splice site, but in a later step, re-identifies where to cut. We liken this two-stage identification to using a microscope by first using the coarse focus to find the area of interest, and then using the fine focus to adjust as needed. This work moves us closer to full mechanistic understanding of how the spliceosome chooses where to cut a pre-mRNA message.