Connections between mRNA elongation and splicing

mRNA 延伸和剪接之间的联系

• 批准号: 6780516
• 负责人: Mariano A. Garcia-Blanco
• 金额: $ 34.43万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780516
• 关键词: Drosophilidae; HeLa cells; RNA interference; RNA splicing; affinity chromatography; gene expression profiling; gene induction /repression; genetic regulatory element; green fluorescent proteins; messenger RNA; microarray technology; nuclear proteins; posttranscriptional RNA processing; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): The long term goal of the project is to understand the physical and functional connections between the mRNA elongation and processing. Although the existence of these connections is widely accepted, their nature, particularly for RNA splicing, remains to be elucidated. In the first part of this proposal we study a protein that may hold a key to a subset of these connections, the transcription elongation factor CA150. The second part of the application broadens its scope by proposing to establish in vitro and in vivo systems for the study of the functional connections between elongation and splicing. The studies proposed are divided into the following specific aims: 1. To identify and characterize the cellular targets of CA150 action. RNAi-mediated CA150 knockdowns coupled to en masse transcript analysis suggest that up to 3% of genes are regulated by CA150. We propose to identify genes that are repressed or activated by CA150 and the cis-acting elements responsive to CA150. 2. To characterize the mechanism of CA150 action. We will use both in vitro and in vivo approaches in mammalian and drosophila cells to unravel the site of CA150 action on cellular targets and the transcriptional events affected. 3. To identify and characterize functional connections between transcription and splicing of nascent transcripts. We and others have identified associations between CA 150 and the splicing machinery. Using an in vitro system we developed to couple transcription to splicing we will address the function of CA 150 and other potential connectors. We also propose to unravel the mechanism by which transcription elongation impacts on regulated alternative splicing. The importance of gene expression in health and disease is clear and we hope that completing the studies proposed here will lead to a fuller understanding of these two critical steps in gene expression and their intersection.

描述（由申请人提供）：该项目的长期目标是了解 mRNA 延伸和加工之间的物理和功能联系。尽管这些连接的存在已被广泛接受，但它们的性质，特别是 RNA 剪接的性质仍有待阐明。在该提案的第一部分中，我们研究了一种可能掌握这些连接子集的关键的蛋白质，即转录延伸因子 CA150。该申请的第二部分扩大了其范围，提出建立体外和体内系统来研究伸长和剪接之间的功能联系。拟议的研究分为以下具体目标： 1. 识别和表征 CA150 作用的细胞靶标。 RNAi 介导的 CA150 敲低与整体转录物分析相结合表明，高达 3% 的基因受 CA150 调节。我们建议鉴定被 CA150 抑制或激活的基因以及对 CA150 敏感的顺式作用元件。 2. 表征CA150的作用机制。我们将在哺乳动物和果蝇细胞中使用体外和体内方法来揭示 CA150 对细胞靶标的作用位点以及受影响的转录事件。 3. 识别和表征新生转录本的转录和剪接之间的功能联系。我们和其他人已经确定了 CA 150 和拼接机械之间的关联。使用我们开发的将转录与剪接耦合的体外系统，我们将解决 CA 150 和其他潜在连接器的功能。我们还建议揭示转录延伸对受调控的选择性剪接的影响机制。基因表达在健康和疾病中的重要性是显而易见的，我们希望完成此处提出的研究将有助于更全面地了解基因表达中的这两个关键步骤及其交叉点。