Eukaryotic RNA processing and chromatin modification

描述（由申请人提供）：真核基因表达受到严格控制，以维持适当的细胞功能。真核生物基因通过RNA聚合酶II转录，生成完全加工（盖帽、聚腺苷化和剪接）的成熟信使RNA分子。尽管进行这些反应（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，它们是在时间和空间上组织起来协调协调地适当生产完全加工的mRNA。基因表达中任何严格控制的事件的错误调控都可能对细胞造成灾难性的后果，最终导致疾病。例如，编码肿瘤抑制因子的基因的失调（例如不正确的剪接、转录下调等）可以而且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，了解剪接因子和染色质修饰酶之间的功能联系是很重要的。使用模式生物酿酒酵母，本文描述的工作描述了在完整的SAGA复合体背景下，组蛋白乙酰转移酶GCN5与U2 snRNP组分MSL1和LEA1之间意想不到的遗传相互作用。此外，编码Gcn5的基因，通过其相关的HAT活性，是体内Msl1和Lea1向pre- mrna的共转录募集所必需的。这些研究导致假设Gcn5通过其hat介导的转录作用协调前mrna剪接与转录。为此目的，将采取下列具体目标：表征Gcn5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化分析将用于绘制Gcn5与DBP2和ECM33的关联，它与U2 snRNP组分的相互作用，基因内组蛋白的乙酰化（以及这种乙酰化的功能后果），以及它对Pol II转录的影响。2. 剪接敏感微阵列将用于鉴定细胞在正常和应激条件下生长时剪接受Gcn5活性影响的基因。将对这些内含子进行分析，以确定使其剪接依赖于gcn5的特征。Gcn5影响这些基因共转录剪接的机制将首先通过定位Msl1/Lea1与这些基因的关联来表征。然后，体内剪接将使用Aim 1中描述的方法进行分析。

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. PUBLIC HEALTH RELEVANCE: Eukaryotic gene expression is tightly controlled to maintain proper cellular function; misregulation of any of the reactions that allow proper gene expression (such as RNA synthesis and RNA processing) can have catastrophic consequences for the cell that, ultimately, lead to diseases that pose serious public health crises. Understanding the molecular details underlying proper gene expression will allow us to develop tools that may target specific mechanisms in disease progression. Hence, research that elucidates these basic mechanisms of gene expression are an important part of any public health strategy.