Connections between mRNA elongation and splicing

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。虽然这些连接的存在被广泛接受，但它们的性质，特别是RNA剪接，仍有待阐明。在这个提议的第一部分，我们研究了一种蛋白质，它可能是这些连接的一个子集的关键，转录延伸因子CA150。申请的第二部分通过提出建立体外和体内系统来研究延伸和剪接之间的功能连接来扩大其范围。所提出的研究分为以下具体目标：1。鉴定和表征CA150作用的细胞靶点。RNAi介导的CA150敲除结合基因转录分析表明，高达3%的基因由CA150调节。我们建议，以确定基因的抑制或激活的CA 150和顺式作用元件响应CA 150。2.研究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阐明CA 150对细胞靶点的作用位点和受影响的转录事件。3.鉴定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和剪接机制之间的联系。使用我们开发的体外系统将转录与剪接偶联，我们将解决CA 150和其他潜在连接器的功能。我们还建议解开转录延伸影响调节可变剪接的机制。基因表达在健康和疾病中的重要性是显而易见的，我们希望完成这里提出的研究将导致对基因表达的这两个关键步骤及其交叉点的更全面的理解。

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.