The impact of Prp18p on splicing fidelity and efficiency in budding yeast

Fidelity of 3´-splice site (3´SS) selection by the spliceosome is critical for proper gene expression but is a daunting task considering the low complexity of the 3´SS consensus YAG. Here we show that loss of the splicing factor Prp18p in budding yeast activates a diverse array of alternative 3´SS at more than half of all introns. Some alternative sites highly diverge from the YAG consensus, demonstrating a critical role for Prp18p in promoting spliceosome fidelity. Usage of alternative 3´SS is determined by distance from the branchpoint, local RNA secondary structures, upstream poly(U) content, and adenosine enrichment in exons. The 3´SS fidelity function of Prp18p can be genetically uncoupled from its role in splicing efficiency and is promoted by interactions with Slu7p and Prp8p. Taken together, these results provide a comprehensive mechanism into how the spliceosome achieves specificity of 3´SS selection and how it prevents aberrant activation of non-canonical splice sites.剪接体（spliceosome）对3'剪接位点（3´SS）的选择保真度对于基因的正确表达至关重要，但鉴于3´SS共有序列YAG的低复杂度，这一过程极具挑战性。本研究发现，酿酒酵母中剪接因子Prp18p的缺失会在超过半数的内含子（introns）中激活多种可变3'剪接位点。部分可变位点与YAG共有序列差异极大，这证明Prp18p在维持剪接体保真度方面发挥关键作用。可变3'剪接位点的使用情况由其与分支点（branchpoint）的距离、局部RNA二级结构（RNA secondary structures）、上游聚U含量以及外显子（exons）中的腺苷富集程度共同决定。Prp18p的3'剪接位点保真功能可在遗传上与其剪接效率相关功能解偶联，并可通过与Slu7p和Prp8p的相互作用得以增强。综上，本研究结果为剪接体如何实现3'剪接位点选择的特异性，以及如何阻止非经典剪接位点的异常激活提供了全面的机制阐释。