REGULATING ALTERNATIVE PRE-MRNA SPLICING IN DROSOPHILA

选择性前体mRNA剪接在真核生物中广泛使用， 控制基因表达和功能。剪接作用的调节 在正常生产所需的各种过程中发挥关键作用， 发展尽管它在基因调控中很重要， 是已知的分子机制，导致替代 拼接特别缺乏的是关于生物化学的信息， 被认为直接使用的交易行为监管机构的活动 选择性剪接途径。很少有这样的监管机构已经 在已知的研究中，只有少数几个被充分研究过。 详细信息，以获得有关其作用机制的重要信息。 我们建议研究Transformer-2 （tra-2），一种来自黑腹果蝇（Drosophila melanogaster）的已确立的剪接调节子， 其功能是抑制在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 蛋白质和前体mRNA通过使用UV交联测定来鉴定两种RNA 和结合所需的蛋白质序列。的意义 关于受调控剪接的鉴定的结合位点将被 在果蝇雄性生殖系的发育背景下进行评估， 自动调节正常发生。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 被篡改了进一步的研究将跟进我们的初步研究。 这一发现表明，一个普遍的因素起着重要作用， 这种相互作用。我们描述了一种方法， 这一因素，并评估其在剪接调节的作用。最后我们 将使用无细胞的体外剪接系统来检测tra-2蛋白 分子干扰M1内含子的剪接。这些目标 研究将是确定剪接之间的特定相互作用， 在RNA结合的存在下被阻断的前体mRNA和机器 tra-2蛋白分子。这些研究的结果将提供 对tra-2蛋白分子 影响拼接。这些见解应该有助于理解 剪接调节子的功能是控制其它细胞中的可变剪接， 系统.

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.