Positive and Negative control of pre-mRNA processing

描述(申请人提供)：对人类基因组和其他生物的基因组测序显示，很大比例的前-mRNAs以多种模式拼接，产生编码不同蛋白质的mRNAs。尽管交替剪接广泛存在，并且必须调节数千种基因产物的表达，但控制它的基本机制还没有被很好地理解。RNA结合蛋白Tra2已被证明影响几个前mRNAs的调节选择性剪接。在人类中，这些包括疾病相关基因的剪接位点选择，如SMN、tau和CD44。在果蝇中，Tra2是调节和实现性别分化所必需的多个mRNAs的交替处理所必需的。这些基因包括双性基因、无果基因和外膜基因，以及Tra2基因本身。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，并成为控制外显子剪接增强子发育的重要范例，但关于Tra2如何影响其他前-mRNAs的剪接却知之甚少。在这个项目中，我们将研究Tra2抑制其自身前-mRNA中特定内含子剪接的机制，并与激活双性剪接所使用的调节元件进行比较。利用重组Tra2特异性阻断果蝇核提取物对M1内含子剪接的体外剪接实验，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外显子剪接增强子活性的假设。在Tra2前-mRNA上形成的调控复合体将与它在DSX剪接增强子中相关的调控复合体进行比较，以确定决定剪接激活或抑制的关键元件。从体外剪接研究中发展出来的机制模型的准确性将通过产生转基因果蝇品系来测试，在转基因品系中，前mRNA和带有改变序列的调节因子在通常发生Tra2依赖抑制的组织和发育阶段表达。在进一步的研究中，将使用遗传方法来定义对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协同工作。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mRNAs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.