After undergoing massive RNA and protein rearrangements during assembly, the spliceosome undergoes a final, more subtle, ATPdependent rearrangement that is essential for catalysis. This rearrangement requires the DEAH-box protein Prp2p, an RNAdependent ATPase. Prp2p has been implicated in destabilizing interactions between the spliceosome and the protein complexes SF3 and RES, but a role for Prp2p in destabilizing RNA-RNA interactions has not been explored. Using directed molecular genetics in budding yeast, we have found that a cold-sensitive prp2 mutation is suppressed not only by mutations in SF3 and RES components but also by a range of mutations that disrupt the spliceosomal catalytic core element U2/U6 helix I, which is implicated in juxtaposing the 5 ′ splice site and branch site and in positioning metal ions for catalysis within the context of a putative catalytic triplex; indeed, mutations in this putative catalytic triplex also suppressed a prp2 mutation. Remarkably, we also found that prp2 mutations rescue lethal mutations in U2/U6 helix I. These data provide evidence that RNA elements that comprise the catalytic core are already formed at the Prp2p stage and that Prp2p destabilizes these elements, directly or indirectly, both to proofread spliceosome activation and to promote reconfiguration of the spliceosome to a fully competent, catalytic conformation.

Pre-mRNA splicing is a central step in the shaping of the eukaryotic transcriptome and in the regulation of gene expression. Yet, due to a focus on fully processed mRNA, common approaches for defining pre-mRNA splicing genome-wide are suboptimalespecially with respect to defining the branch point sequence, a key cis-element that initiates the chemistry of splicing. Here, we report a complementary intron-centered approach designed to more efficiently, simply, and directly define splicing events genome-wide. Specifically, we developed a method distinguished by deep sequencing of lariat intron termini (LIT-seq). In a test of LIT-seq using the budding yeast Saccharomyces cerevisiae, we not only successfully captured the majority of annotated, expressed splicing events but also uncovered 45 novel splicing events, establishing the sensitivity of LIT-seq. Moreover, our libraries were highly enriched with reads that reported on splice sites; by a simple and direct inspection of sequencing reads, we empirically defined both 5 ′ splice sites and branch sites, as well as their consensus sequences, with nucleotide resolution. Additionally, our study revealed that the 3 ′ termini of lariat introns are subject to nontemplated addition of adenosines, characteristic of signals sensed by 3 ′ to 5 ′ RNA turnover machinery. Collectively, this work defines a novel, genome-wide approach for analyzing splicing with unprecedented depth, specificity, and resolution.

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