Eukaryotic RNA processing and chromatin modification

真核RNA加工和染色质修饰

• 批准号: 8813069
• 负责人: TRACY L JOHNSON
• 金额: $ 12.43万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8813069
• 关键词: Eukaryotic; RNA; processing; chromatin; modification

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.

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。真核基因由RNA聚合酶II转录，以产生完整加工（限制，聚腺苷酸化和剪接）成熟的Messenger RNA分子。尽管通常将执行这些反应（RNA合成和RNA加工）的细胞机制作为生化不同的反应进行了研究，但实际上，它们是在时间和空间上组织的，以协调正确地生产完整处理的mRNA。基因表达中任何严格控制事件的任何不正调可能会对细胞最终导致疾病产生灾难性后果。例如，编码抑制肿瘤的基因的错误（例如，不正确的剪接，转录下调等）可能会并且确实会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，因此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。使用模型生物体，在此描述的工作中，在完整的SAGA复合物和U2 SNRNP成分MSL1和LEA1的背景下，组蛋白乙酰转移酶，GCN5之间的意外遗传相互作用表征了意外的遗传相互作用。此外，编码GCN5的基因通过其相关的HAT活性是将MSL1和LEA1募集到体内MRNA的共转录募集所必需的。这些研究导致了以下假设：GCN5通过其HAT介导的转录作用来坐标与转录的剪接前MRNA剪接。为此，将实现以下特定目标：1。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测定将用于映射与DBP2和ECM33的GCN5关联，其与U2 SNRNP成分的相互作用，其基因内组蛋白的乙酰化（以及该乙酰化的功能性结果）及其对POL II转录的影响。 2。剪接敏感的微阵列将用于识别当细胞在正常和应力条件下生长时剪接受GCN5活性影响的基因。这些内含子将进行分析，以识别呈现其剪接GCN5依赖性的特征。 GCN5影响这些基因的共转录剪接的机制将以首先映射MSL1/LEA1与基因的关联。然后，将使用AIM 1中描述的方法分析体内剪接。