Biosynthesis of RNAs

描述(由申请人提供)： 项目总结我们未来的总体目标保持不变：我们继续寻找mRNA处理的保真度和调节的分子机制，特别是在酵母中的mRNA剪接。这一建议的三个目的如下：1)在我们先前证明剪接体是一个高度动态的RNA-蛋白质机器的基础上，我们现在利用单分子FRET来关注对剪接体的催化激活至关重要的依赖于RNA的ATPase的动力学。Brr2是U4从U6解离所必需的，允许U6采用其活性构象。通过在U6 RNA中明智地放置荧光团，然后将其重组为SnRNP，我们将确定解离的方向性、步长和加工性，并询问U5 SnRNP蛋白Prp8对这些参数的影响，这是依赖Brr2的解离所必需的。此外，我们将测试Prp8的翻译后修改的角色，包括 泛素化和去泛素化的循环。2)我们使用高密度遗传相互作用图的分析揭示了剪接和转录之间广泛相互作用的证据，特别是指出了染色质结构和修饰的重要作用。我们的目标是通过利用我们的剪接敏感微阵列结合剪接因子和染色质标记芯片来分析全球剪接谱，通过使用这一大组双突变来解析共转录环境。我们还将利用触发器LOP(由Craig Kaplan产生)中的一系列等位基因突变，询问polII延长率在剪接效率中的作用，这些突变会在40倍的范围内改变转录速度。从后生动物选择性剪接模式变化的“动力学模型”推断，我们可以预测，放慢剪接速度将通过允许更多的时间进行SNRNPs的共转录加载来增强剪接。有趣的是，我们的初步数据显示了一个更细微的、特定于文字记录的画面。同时，我们将在S.pombe中提出类似的机制问题，其中许多基因的多内含子结构更像后生动物的结构；因此我们可以明确地询问polII是否真的像最近在萌芽酵母中所建议的那样暂停以确保共转录剪接。3)共转录偶联的一个基本原理是它能够协调 多个步骤来微调基因表达，以响应细胞的即时需求。在这个目标中，我们探索了两个新的共转录过程的例子，这两个过程似乎依赖于耦合来对细胞外线索做出适当的生物反应。在一种情况下，氨基酸饥饿导致核糖体蛋白基因剪接迅速而特异地下调；令人惊讶的是，这种反应取决于启动子的身份，而不是内含子。在第二种情况下，对mRNA输出至关重要的PAB1基因的有效表达需要该基因与Brr6之间的功能连接，Brr6是一种重要的核膜蛋白。瞬时靶向的动态变化产生了双模式的细胞群体，解释了brr6-1输出表型的不完全外显。

DESCRIPTION (provided by applicant): Project Summary Our broad goals going forward remain unchanged: we continue to seek molecular mechanisms for the fidelity and regulation of mRNA processing, with a particular focus on mRNA splicing in yeast. The three aims of this proposal are as follows: 1) Building on our previous demonstration that the spliceosome is a highly dynamic RNA-protein machine whose fidelity relies on RNA-dependent ATPases of the DEAD-box family, we are now employing single molecule FRET to focus on the dynamics of the ATP-dependent rearrangement most critical for the catalytic activation of the spliceosome. Brr2 is required for the unwinding of U4 from U6, allowing U6 to adopt its active conformation. By judicious placement of fluorophores in U6 RNA, which is then reconstituted into a snRNP, we will determine the directionality, step-size and processivity of unwinding and ask how these parameters are influenced by the U5 snRNP protein Prp8, which is required for Brr2-dependent unwinding. Additionally, we will test roles for post-translational modifications of Prp8, including cycles of ubiquitination and deubiquitination. 2) Our analyses using high-density genetic interaction maps have revealed evidence for an extensive interplay between splicing and transcription and, in particular, point to important roles for chromatin structure and modification We aim to parse the co-transcriptional environment by employing this large battery of double mutants in analysis of global splicing profiles by our splicing-sensitive microarrays in combination with ChIP of splicing factors and chromatin marks. We will also ask the role of polII elongation rate in splicing efficiency, exploiting an allelic series of mutations in the Trigger Lop (generated by Craig Kaplan) that alter transcription speeds over a 40-fold range. Extrapolating from the "kinetic model" for changes in alternative splicing patterns in metazoa, we would predict that slowing the rate would enhance splicing by allowing more time for co-transcriptional loading of snRNPs. Interestingly, our preliminary data suggest a more nuanced and transcript-specific picture. In parallel, we will ask similar mechanistic questions in S. pombe, where the multi- intronic structure of many genes is more like that of metazoa; thus we can explicitly ask whether polII actually pauses to ensure co-transcriptional splicing, as recently suggested in budding yeast. 3) One rationale for co- transcriptional coupling is that it enables coordination of multiple steps to fine-tune gene expression in response to the immediate needs of the cell. In this aim we explore two novel instances of co-transcriptional processes that appear to rely on coupling for appropriate biological responses to extracellular cues. In one case, amino acid starvation results in a rapid and specific down-regulation of ribosomal protein gene splicing; surprisingly, this response is dependent on the identity of the promoter, not the intron. In the second, efficient expression of the PAB1 gene, which is essential for mRNA export, requires a functional connection between the locus and Brr6, an essential nuclear envelope protein. Alterations in the dynamics of transient targeting generate a bi-modal population of cells, explaining the incomplete penetrance of the brr6-1 export phenotype.