Eukaryotic RNA processing and chromatin modification

描述（由申请人提供）：严格控制真核基因表达以维持适当的细胞功能。真核基因由RNA聚合酶II转录以产生完全加工的（加帽、聚腺苷酸化和剪接）成熟信使RNA分子。虽然进行这些反应（RNA合成和RNA加工）的细胞机制通常被研究为生物化学上不同的反应，但事实上，它们在时间和空间上被组织起来，以协调地协调完全加工的mRNA的正确生产。基因表达中任何严格控制的事件的错误调节都可能对细胞产生灾难性的后果，最终导致疾病。例如，错误调节（例如，不正确的剪接、转录下调等）编码肿瘤抑制因子的基因的突变能够并确实导致癌症和许多其他人类疾病。 由于共转录剪接发生在染色质模板的背景下，重要的是要了解剪接因子和染色质修饰酶之间的功能联系。使用的模式生物，酿酒酵母，这里描述的工作的特点出乎意料的组蛋白乙酰转移酶，GCN 5，在一个完整的佐贺复合物的背景下，和U2 snRNP组件MSL 1和LEA 1之间的遗传相互作用。此外，编码Gcn 5的基因，通过其相关的HAT活性，需要在体内共转录募集Msl 1和Lea 1的前mRNA。这些研究导致了Gcn 5通过其HAT介导的对转录的影响来协调前mRNA剪接与转录的假设。为此，将实现以下具体目标：1.表征Gcn 5介导的组蛋白乙酰化在剪接体组装中的作用。染色质免疫沉淀和生物化学测定将用于绘制Gcn 5与DBP 2和ECM 33的关联、其与U2 snRNP组分的相互作用、其在基因内的组蛋白的乙酰化（以及这种乙酰化的功能后果）以及其对Pol II转录的影响。2.剪接敏感的微阵列将被用来确定基因的剪接是受影响的GCN 5活动时，细胞生长在正常和压力条件下。将分析这些内含子以鉴定使其剪接Gcn 5依赖的特征。Gcn 5影响这些基因的共转录剪接的机制的特征在于，首先，映射Msl 1/Lea 1与基因的关联。然后，将使用目标1中描述的方法分析体内剪接。

DESCRIPTION (provided by applicant): Eukaryotic gene expression is tightly controlled to maintain proper cellular function. Eukaryotic genes are transcribed by RNA polymerase II to generate fully-processed (capped, polyadenylated, and spliced), mature messenger RNA molecules. Although the cellular machineries that carry out these reactions (RNA synthesis and RNA processing) have typically been studied as biochemically distinct reactions, they are, in fact, temporally and spatially organized to coordinately orchestrate the proper production of a fully-processed mRNA. Misregulation of any of the tightly controlled events in gene expression can have catastrophic consequences for the cell that, ultimately, lead to disease. For example, misregulation (e.g. incorrect splicing, transcriptional down-regulation, etc.) of genes encoding tumor suppressors can and does lead to cancer and a host of other human diseases. Since co-transcriptional splicing occurs in the context of a chromatin template, it is important to understand the functional links between splicing factors and chromatin-modifying enzymes. Using the model organism, Saccharomyces cerevisiae, work described here characterizes unexpected genetic interactions between the histone acetyltransferase, GCN5, within the context of an intact SAGA complex, and the U2 snRNP components MSL1 and LEA1. Furthermore, the gene encoding Gcn5, via its associated HAT activity, is required for cotranscriptional recruitment of Msl1 and Lea1 to pre-mRNAs in vivo. These studies have led to the hypothesis that Gcn5 coordinates pre-mRNA splicing with transcription through its HAT-mediated effects on transcription. To this end, the following specific aims will be undertaken: 1. Characterize the role of Gcn5 mediated histone acetylation in spliceosome assembly. Chromatin immunoprecipitation and biochemical assays will be utilized to map Gcn5 association with DBP2 and ECM33, its interactions with the U2 snRNP components, its acetylation of histones within the genes (and the functional consequence of this acetylation), and its effect on Pol II transcription. 2. Splicing sensitive microarrays will be used to identify genes whose splicing is affected by Gcn5 activity when cells are grown under normal and stress conditions. These introns will be analyzed to identify features that render their splicing Gcn5-dependent. The mechanism by which Gcn5 affects cotranscriptional splicing of these genes will be characterized by, first, mapping Msl1/Lea1 association with the genes. Then, in vivo splicing will be analyzed using the approaches described in Aim 1.