Positive and Negative control of pre-mRNA processing

描述（由申请人提供）：人类基因组和其他生物体基因组的测序显示，大部分前mRNA以多种模式剪接，产生编码不同蛋白质的mRNA。虽然选择性剪接是广泛存在的，并且必须调节成千上万的基因产物的表达，但控制它的基本机制尚未得到很好的理解。RNA结合蛋白Tra2已显示影响几种前体mRNA的受调节的选择性剪接。在人类中，这些包括疾病相关基因如SMN、tau和CD44中的剪接位点选择。在果蝇中，Tra2是调节和实现性分化所必需的多种mRNA的交替加工所必需的。这些基因包括doubletex，fruitless和exuperantia基因以及tra2本身。虽然Tra2在双链RNA加工中的剪接激活功能已经得到了很好的研究，并且已经作为外显子剪接增强子的发育控制的重要范例，但关于Tra2如何影响其他前体mRNA的剪接知之甚少。在这个项目中，Tra2抑制剪接的一个特定的内含子在其自己的前mRNA的机制将进行调查，并与用于激活doubletex剪接的调控元件进行比较。使用体外剪接试验，其中重组Tra2特异性阻断果蝇核提取物对M1内含子的剪接，将鉴定Tra2抑制剪接的调控元件，并测试Tra2阻断内含子上游所需外显子剪接增强子活性的假设。将Tra2前mRNA上形成的调节复合物与dsx剪接增强子中与其相关的调节复合物进行比较，以确定决定剪接激活或抑制的关键元件。将通过产生转基因果蝇品系来测试从体外剪接研究开发的机制模型的准确性，其中具有改变的序列的前mRNA和调节剂在Tra2依赖性抑制通常发生的组织和发育阶段中表达。在进一步的研究中，遗传学方法将用于确定SR蛋白和其他剪接因子的要求，这些剪接因子与Tra2在控制剪接位点识别中合作。这些研究将阐明单个剪接调节因子以不同方式改变不同前体mRNA剪接的基本机制。

DESCRIPTION (provided by applicant): Sequencing of the human genome and those of other organisms has revealed that a large percentage of pre-mRNAs are spliced in multiple patterns to produce mRNAs encoding distinct proteins. Although alternate splicing is widespread and must regulate the expression of thousands of gene products, the fundamental mechanisms that control it are not yet well understood. The RNA binding protein Tra2 has been shown to affect the regulated alternative splicing of several pre-mRNAs. In humans these include splice site choices in disease-associated genes such as SMN, tau and CD44. In Drosophila, Tra2 is required for the alternate processing of multiple mRNAs that are necessary for the regulation and realization of sexual differentiation. These include those of the doublesex, fruitless and exuperantia genes as well as tra2 itself. While the splicing activation function of Tra2 in doublesex RNA processing has been well studied and has served as an important paradigm for developmental control of exonic splicing enhancers, little is known about how Tra2 affects splicing of other pre-mRNAs. In this project the mechanism by which Tra2 represses splicing of a specific intron in its own pre-mRNA will be investigated and compared with regulatory elements used in the activation of doublesex splicing. Using an in vitro splicing assay in which recombinant Tra2 specifically blocks splicing of the M1 intron by Drosophila nuclear extracts, the regulatory elements through which Tra2 represses splicing will be identified and the hypothesis that Tra2 blocks the activity of a required exonic splicing enhancer upstream of the intron will be tested. Regulatory complexes that form on the Tra2 pre-mRNA will be compared to those that it associates with in the dsx splicing enhancer to define key elements that determine activation or repression of splicing. The veracity of mechanistic models developed from in vitro splicing studies will be tested by generating transgenic fly strains in which pre-mRNA and regulators with altered sequences are expressed in the tissue and stage of development where Tra2-dependent repression normally occurs. In further studies genetic approaches will be used to define the requirements for SR proteins and other splicing factors that cooperate with Tra2 in the control splice site recognition. These studies will elucidate the fundamental mechanisms by which individual splicing regulators can alter splicing in different ways for different pre-mRNAs.