Early splicing functions of fission yeast Prp16 and its unexpected requirement for gene Silencing is governed by intronic features

Prp16 is a DEAH box pre-mRNA splicing factor that triggers a key spliceosome conformational switch to facilitate second step splicing in <i>Saccharomyces cerevisiae</i>. However, Prp16 functions are largely unexplored in <i>Schizosaccharomyces pombe</i>, an attractive model with exon-intron architecture more relevant to several other eukaryotes. Here, we generated mis-sense alleles in SpPrp16 whose consequences on genome-wide splicing uncover its nearly global splicing role with only a small subset of unaffected introns. Prp16 dependent and independent intron categories displayed a striking difference in the strength of intronic 5ʹ splice site (5’SS)-U6 snRNA and branch site (BS)-U2 snRNA interactions. Selective weakening of these interactions could convert a Prp16 dependent intron into an independent one. These results point to the role of SpPrp16 in destabilizing 5’SS-U6snRNA and BS-U2snRNA interactions which plausibly trigger structural alterations in the spliceosome to facilitate first step catalysis. Our data suggest that SpPrp16 interactions with early acting factors, its enzymatic activities and association with intronic elements collectively account for efficient and accurate first step catalysis. In addition to splicing derangements in the <i>spprp16F528S</i> mutant, we show that SpPrp16 influences cell cycle progression and centromeric heterochromatinization. We propose that strong 5’SS-U6 snRNA and BS-U2 snRNA complementarity of intron-like elements in non-coding RNAs which lead to complete splicing arrest and impaired Seb1 functions at the pericentromeric loci may cumulatively account for the heterochromatin defects in <i>spprp16F528S</i> cells. These findings suggest that the diverse Prp16 functions within a genome are likely governed by its intronic features that influence splice site–snRNA interaction strength.

Prp16是一种DEAH盒型前体mRNA剪接因子（DEAH box pre-mRNA splicing factor），在酿酒酵母（Saccharomyces cerevisiae）中可触发关键的剪接体构象转换，以促进剪接反应的第二步进行。然而，目前在粟酒裂殖酵母（Schizosaccharomyces pombe）中对Prp16的功能研究仍有待深入——该物种是一种理想的模式生物，其外显子-内含子结构与其他多种真核生物更为相似。本研究构建了粟酒裂殖酵母Prp16（SpPrp16）的错义等位基因，通过分析其对全基因组剪接的影响，揭示了其几乎全局性的剪接调控作用，仅存在少量不受其影响的内含子。Prp16依赖型与非依赖型内含子类别，在内含子5'剪接位点（5' splice site, 5’SS）与U6小核RNA（U6 snRNA）、分支位点（branch site, BS）与U2小核RNA（U2 snRNA）的相互作用强度上呈现出显著差异。通过选择性减弱这些相互作用，可将Prp16依赖型内含子转化为非依赖型内含子。这些结果表明，SpPrp16可通过破坏5'SS-U6 snRNA与BS-U2 snRNA的相互作用，进而触发剪接体的结构改变，以促进剪接第一步的催化反应。我们的数据显示，SpPrp16与早期作用因子的相互作用、自身的酶活性以及与内含子元件的结合，共同保障了高效且精准的第一步催化反应。除了在spprp16F528S突变体中观察到剪接紊乱外，本研究还证实SpPrp16可影响细胞周期进程与着丝粒异染色质化。我们提出，非编码RNA中内含子样元件的5'SS-U6 snRNA与BS-U2 snRNA互补性过强，会导致完全的剪接停滞，并损害着丝粒旁区域Seb1蛋白的功能，这一累积效应或可解释spprp16F528S突变体细胞中的异染色质缺陷。上述研究结果表明，基因组内Prp16的多样功能可能由其内含子特征所调控，而这些特征会影响剪接位点-小核RNA的相互作用强度。