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基因组的测序显示，很大比例的pre- mrna以多种模式拼接，产生编码不同蛋白质的mrna。虽然交替剪接很普遍，并且必须调节成千上万种基因产物的表达，但控制它的基本机制尚未得到很好的理解。RNA结合蛋白Tra2已被证明影响几种前mrna的可调节的选择性剪接。在人类中，这些包括疾病相关基因（如SMN、tau和CD44）的剪接位点选择。在果蝇中，Tra2是多种mrna交替加工所必需的，而这些mrna是调节和实现性别分化所必需的。这些包括双性、无果和繁殖基因以及tra2本身。虽然Tra2在双性RNA加工中的剪接激活功能已经得到了很好的研究，并且作为外显子剪接增强子发育控制的重要范例，但对于Tra2如何影响其他pre- mrna的剪接知之甚少。在本项目中，将研究Tra2抑制其自身前mrna中特定内含子剪接的机制，并将其与激活双性剪接的调节元件进行比较。通过体外剪接实验，重组Tra2特异性地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，并验证Tra2阻断内含子上游所需的外显子剪接增强子活性的假设。将在Tra2 pre-mRNA上形成的调节复合物与在dsx剪接增强子中与之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。从体外剪接研究中建立的机制模型的准确性将通过产生转基因蝇株进行测试，其中pre-mRNA和具有改变序列的调节因子在正常发生tra2依赖性抑制的组织和发育阶段表达。在进一步的研究中，将使用遗传学方法来确定SR蛋白和其他剪接因子在控制剪接位点识别中与Tra2合作的需求。这些研究将阐明单个剪接调节因子以不同方式改变不同pre- 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.