REGULATING ALTERNATIVE PRE-MRNA SPLICING IN DROSOPHILA

选择性的前mRNA剪接在真核细胞中被广泛使用 对基因表达和功能的控制。对拼接游戏的监管 在正常运行所需的各种流程中扮演关键角色 发展。尽管它在基因调控中很重要，但令人惊讶的是 已知导致另一种选择的分子机制 拼接。尤其缺乏有关生物化学的信息。 被认为直接使用的反式作用调节器的活动 可供选择的剪接途径。到目前为止，很少有这样的监管机构 在已知的那些中，只有几个得到了充分的研究 详细信息，以提供有关其作用机制的重要信息。 我们建议研究Transform-2的分子机制 (tra-2)，一种已建立的来自黑腹果蝇的剪接调控因子， 功能，以抑制内含子剪接发现在tra-2前 信使核糖核酸。我们将首先研究tra-2的物理相互作用。 蛋白质和前-信使核糖核酸的UV交联法鉴定 和结合所需的蛋白质序列。这一事件的意义 已确定的与受调控剪接有关的结合位点将是 在果蝇雄性生殖系的发育背景下进行评估 自动调节通常会发生。这将在实验中完成。 使用稳定的转基因苍蝇品系，其中结合位点序列 已经被更改了。其他研究将在我们初步研究的基础上进行。 这一发现表明，一个一般因素在 这种结合的相互作用。我们描述了一种方法，用于识别 这一因素并评估其在剪接调控中的作用。最后，我们 将使用无细胞体外剪接系统来检测tra-2蛋白是如何 分子干扰M1内含子的剪接。这些活动的目的是 研究将确定剪接之间的具体相互作用 在RNA结合的存在下被阻断的机械和前-mRNA TrA-2蛋白分子。这些研究的结果将提供 对tra-2蛋白分子机制的实质性见解 影响拼接。这些洞察力应该有助于理解 剪接调节器的功能是控制其他 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.