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.

替代前MRNA剪接在桉树中广泛使用 控制基因表达和功能。剪接戏剧的调节 在正常所需的各种过程中的关键作用 发展。尽管它在基因调节中的重要性很少 已经知道导致替代的分子机制 剪接。尤其缺乏生化的信息 被认为可以指导使用的跨扮演监管机构的活动 替代剪接途径。很少有这样的监管机构 确定并在已知的人中只有少数人被足够研究 细节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），果蝇果蝇的固定调节器， 抑制在Tra-2 pre-中发现的内含子的剪接的功能 mRNA本身。我们将首先研究Tra-2的物理相互作用 蛋白质和前MRNA通过使用紫外线交联测定法鉴定两个RNA 和结合所需的蛋白质序列。意义的意义 相对于调节的剪接确定的结合位点将是 在苍蝇的男性种系的发育环境中进行评估 自动调节通常发生。这将在实验中完成 使用稳定的转基因蝇菌株，其中结合位点序列 已经改变了。其他研究将跟进我们的初步 发现表明一般因素在 这种结合相互作用。我们描述了一种识别的方法 该因素并评估其在调节剪接中的作用。最后，我们 将使用无细胞的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。这些目的 研究将是确定剪接之间的特定相互作用 在RNA结合的存在下阻塞的机械和前MRNA Tra-2蛋白分子。这些研究的结果将提供 对Tra-2蛋白分子的机制有实质性的见解 影响剪接。这样的见解应该证明有助于理解如何 剪接调节器的功能可以控制其他的替代剪接 系统。