RNA caps and meiotic pre-mRNA splicing

描述(由申请人提供)： M7G帽的核功能是RNA聚合酶II转录本的标志性特征，由异源二聚体核帽结合复合体(CBC)介导。CBC参与新生转录本的m7G帽，并促进协同和转录后RNA处理，包括剪接。三甲基鸟苷(TMG)帽是RNA聚合酶II转录子集的特征，包括指导前mRNA剪接的U1、U2、U4和U5SnRNA。TMG是由酶Tgs1形成的，它催化m7G帽的氮原子连续两次甲基加成。虽然m7G帽对于真核细胞的生存是必不可少的，但TMG帽对于真核细胞的营养生长是必不可少的。支持当前建议的关键发现是我们的发现：(1)TMG封顶是酵母孢子形成所必需的，它通过TMG要求剪接特定的减数分裂mRNAs；(2)尽管减弱CBC-m7G帽子相互作用的影响(通过改变Cbc2亚单位的帽子结合口袋)在酵母有丝分裂生长的剪接途径中被其他参与者缓冲，但完整的帽子结合口袋对于减数分裂的发展是必不可少的。基于这些发现，以及其他人(和我们)关于依赖Mer1/Nam8的减数分裂剪接的工作，我们假设具有非共识剪接位点(和其他不寻常的RNA特征)的减数分裂前mRNAs对一般剪接因子和RNA帽的控制特别敏感。为了支持这一想法，我们描述了减数分裂剪接“调节子”--包括不同的前mRNA集，其剪接取决于营养上可选的剪接因子(例如，Nam8，剪接SPO22、MER2、MER3、AMA1和PCH2前-mRNAs所需)、SnRNA修饰(TMG帽；SAE3和PCH2剪接所需)或Cbc2与前mRNAm7G帽结合(MER3和SAE3剪接所需)。我们的长期目标是阐明减数分裂剪接控制的全谱及其涉及的生化机制。在这里，我们建议：(1)剖析剪接途径中对减数分裂控制敏感的步骤(特别是。(2)研究核CBC如何调控特定减数分裂前mRNAs的剪接；以及(3)询问其他减数分裂剪接控制的存在和靶谱，首先集中在Mud2和分支点结合蛋白Msl5上。我们希望对剪接位点的选择如何被基础剪接机制的组件调节以响应Pre-mRNA中的不同特征这一问题获得新的和一般的见解。

DESCRIPTION (provided by applicant): The nuclear functions of the m7G cap, a signature feature of RNA polymerase II transcripts, are mediated by a heterodimeric nuclear cap binding complex (CBC). CBC engages the m7G caps of nascent transcripts and facilitates co- and post-transcriptional RNA processing, including splicing. Trimethylguanosine (TMG) caps are characteristic of a subset of RNA polymerase II transcripts, including the U1, U2, U4 and U5 snRNAs that direct pre-mRNA splicing. TMG is formed by the enzyme Tgs1, which catalyzes two successive methyl additions to the N2 atom of the m7G cap. Whereas m7G caps are essential for viability of eukarya, TMG caps are dispensable for vegetative growth of eukaryal cells. The key discoveries underlying the current proposal are our findings that: (1) TMG capping is essential for yeast sporulation via a TMG requirement for splicing of specific meiotic mRNAs, and (2) whereas the effects of weakening CBC-m7G cap interactions (by altering the cap binding pocket of the Cbc2 subunit) are buffered by other actors in the splicing pathway during yeast mitotic growth, an intact cap binding pocket is essential for meiotic development. Based on these findings, and the work of others (and us) regarding Mer1/Nam8-dependent meiotic splicing, we hypothesize that meiotic pre- mRNAs with non-consensus splice sites (and other unusual RNA features) are especially sensitive to control by general splicing factors and RNA caps. In support of this idea, we've delineated meiotic splicing "regulons" - encompassing distinct sets of pre-mRNAs whose splicing is dependent on vegetatively optional splicing factors (e.g. Nam8, required for splicing of SPO22, MER2, MER3, AMA1 and PCH2 pre-mRNAs), snRNA modifications (TMG caps; needed for SAE3 and PCH2 splicing) or pre-mRNA m7G cap binding by Cbc2 (required for MER3 and SAE3 splicing). Our long-term goal is to elucidate the full spectrum of meiotic splicing controls and the biochemical mechanisms involved. Here, we propose to: (1) dissect the steps in the splicing pathway that are sensitive to meiotic controls (esp. the TMG caps, and Nam8/Mer1 proteins) using in vitro splicing assays and site-specific crosslinking approaches; (2) investigate how nuclear CBC governs splicing of specific meiotic pre-mRNAs; and (3) interrogate the existence and target spectra of additional meiotic splicing controls, focusing first on Mud2 and the branchpoint binding protein Msl5. We expect to gain new and general insights to the question of how splice site choice can be modulated by components of the basal splicing machinery in response to distinct features in the pre-mRNA.