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信使核糖核酸的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，特别是对于RNA剪接，仍然有待阐明。在这项建议的第一部分，我们研究了一种蛋白质，它可能持有这些连接的一个子集的关键，转录延伸因子CA150。应用的第二部分扩大了它的范围，建议建立体外和体内系统来研究伸长和剪接之间的功能联系。具体研究目标如下：1.鉴定和鉴定CA150作用的细胞靶点。RNAi介导的CA150敲除结合整体转录分析表明，多达3%的基因受CA150调控。我们建议找出被CA150抑制或激活的基因，以及响应CA150的顺式作用元件。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揭示CA150对细胞靶点的作用位置和受影响的转录事件。3.鉴定和鉴定新生转录本转录和剪接之间的功能联系。我们和其他人已经确定了CA150和剪接机制之间的联系。使用我们开发的将转录与剪接相结合的体外系统，我们将解决CA150和其他潜在连接物的功能。我们还建议揭开转录延伸对受调控的选择性剪接影响的机制。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集有更充分的理解。

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.