Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how nonessential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a nonessential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5′ splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing nonconsensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the first-step catalytic site, promoting second-step catalysis, and permitting alternate 5′ SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

中文翻译：

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酿酒酵母 Ecm2 调节前 mRNA 剪接的催化步骤


遗传、生化和结构研究已经阐明了剪接体催化的分子基础。剪接是由 RNA 催化的，并且必需的 snRNA 和蛋白质因子得到了很好的保守。然而，人们对剪接体的非必需成分如何促进反应并调节基本核心机制的活动知之甚少。 Ecm2 是一种非必需的酵母剪接因子，是 Prp19 相关蛋白质复合物的成员。冷冻电镜 (cryo-EM) 结构显示 Ecm2 结合 U6 snRNA 并与 Cwc2 缠结，Cwc2 是先前发现的促进剪接体催化活性构象的因子。这些结构还表明 Ecm2 和 U2 snRNA 可能在 5' 剪接位点 (SS) 切割过程中形成短暂的相互作用。我们已经表征了ECM2和剪接因子等位基因之间的遗传相互作用，这些剪接因子改变了剪接的催化步骤。此外，我们还研究了ECM2的丢失如何影响含有非共有或竞争性 SS 的前 mRNA 的剪接。我们的结果表明ECM2在剪接的催化阶段发挥作用。我们的数据与 Ecm2 一致，促进第一步催化位点的形成和稳定，促进第二步催化，并允许交替使用 5'SS。我们认为 Cwc2 和 Ecm2 都可以以独特的方式微调剪接体活性位点。它们的相互作用网络可以充当管道，通过该管道可以调节某些前mRNA（例如含有弱或替代剪接位点的那些）的剪接。