REGULATING ALTERNATIVE PRE-MRNA SPLICING IN DROSOPHILA

调节果蝇中的选择性前 mRNA 剪接

• 批准号: 2685030
• 负责人: WILLIAM W MATTOX
• 金额: $ 11.11万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2685030
• 关键词: Drosophilidae; HeLa cells; RNA binding protein; RNA splicing; animal genetic material tag; cell free system; chemical binding; crosslink; developmental genetics; eukaryote; genetic regulation; genetic regulatory element; genetically modified animals; germ cells; introns; male; nucleic acid sequence; precursor mRNA; protein sequence; spliceosomes

Alternative pre-mRNA splicing is used widely among eucaryotes in the control of both gene expression and function. Regulation of splicing plays a critical role in a wide variety of processes needed for normal development. Despite its importance in gene regulation, remarkably little is known about the molecular mechanisms that result in alternative splicing. Particularly lacking is information on the biochemical activities of the trans-acting regulators that are thought to direct usage of alternative splicing pathways. Very few such regulators have yet been identified and, of those known, only a few have been studied in sufficient detail to yield significant information about their mechanisms of action. We propose to investigate the molecular mechanism by which transformer-2 (tra-2), an established splicing regulator from Drosophila melanogaster, functions to repress the splicing of an intron found within the tra-2 pre- mRNA itself. We will first investigate the physical interaction of tra-2 protein and pre-mRNA by using a UV crosslinking assay to identify both RNA and protein sequences needed for binding. The significance of the identified binding sites with respect to regulated splicing will be evaluated in the developmental context of the fly's male germline where autoregulation normally occurs. This will be accomplished in experiments using stable transgenic fly strains in which the binding site sequences have been altered. Additional studies will follow up on our preliminary finding that indicate that a general factor plays an important role in this binding interaction. We describe an approach for both identifying this factor and assessing its role in regulation of splicing. Finally, we will use a cell free in vitro splicing system to examine how tra-2 protein molecules interfere with the splicing of the M1 intron. The aim of these studies will be to identify the specific interactions between the splicing machinery and the pre-mRNA that are blocked in the presence of RNA bound tra-2 protein molecules. The results of these studies will provide substantial insights into the mechanism by which tra-2 protein molecules affect splicing. Such insights should prove helpful in understanding how splicing regulators function to control alternative splicing in other systems.

替代性前 mRNA 剪接在真核生物中广泛使用 基因表达和功能的控制。拼接玩法的监管 在正常运行所需的各种过程中发挥着关键作用 发展。尽管它在基因调控中很重要，但很少有 已知导致替代的分子机制 拼接。尤其缺乏有关生化的信息 被认为指导使用的交易监管机构的活动 的选择性剪接途径。目前此类监管机构还很少 已确定，并且在已知的那些中，只有少数已得到充分研究 详细信息以产生有关其作用机制的重要信息。 我们建议研究transformer-2的分子机制 (tra-2)，一种来自果蝇的已建立的剪接调节因子， 其功能是抑制 tra-2 前体中发现的内含子的剪接 mRNA本身。我们将首先研究 tra-2 的物理相互作用 通过使用 UV 交联测定来鉴定蛋白质和前 mRNA 以及结合所需的蛋白质序列。的意义 确定的与受调控剪接相关的结合位点将是 在果蝇雄性种系的发育背景下进行评估，其中 通常会发生自动调节。这将在实验中完成 使用稳定的转基因果蝇品系，其中结合位点序列 已被更改。进一步的研究将跟进我们的初步研究 研究结果表明，一个普遍因素在 这种结合相互作用。我们描述了一种方法来识别 这个因素并评估其在剪接调节中的作用。最后，我们 将使用无细胞体外剪接系统来检查 tra-2 蛋白如何 分子干扰 M1 内含子的剪接。这些的目的 研究将确定剪接之间的具体相互作用 机器和前 mRNA 在 RNA 结合存在时被阻断 tra-2 蛋白质分子。这些研究的结果将提供 对 tra-2 蛋白分子机制的深入见解 影响拼接。这些见解应该有助于理解如何 剪接调节器的功能是控制其他细胞中的选择性剪接 系统。