Positive and Negative control of pre-mRNA processing

前 mRNA 加工的阳性和阴性对照

• 批准号: 6868356
• 负责人: WILLIAM W MATTOX
• 金额: $ 28万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6868356
• 关键词: Drosophilidae; RNA binding protein; RNA interference; RNA splicing; genetic enhancer element; genetic regulatory element; genetically modified animals; immunoprecipitation; messenger RNA; northern blottings; nucleic acid sequence; phosphorylation; polymerase chain reaction; posttranscriptional RNA processing; protein structure function; spliceosomes

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.

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表明，很大比例的前体 mRNA 以多种模式剪接，产生编码不同蛋白质的 mRNA。尽管交替剪接很普遍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尚不清楚。 RNA 结合蛋白 Tra2 已被证明可以影响几种前 mRNA 的受调控选择性剪接。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 是交替加工多种 mRNA 所必需的，而这些 mRNA 是调节和实现性分化所必需的。这些包括双性基因、无结果基因和 exuperantia 基因以及 tra2 本身的基因。虽然 Tra2 在双性 RNA 加工中的剪接激活功能已得到充分研究，并已成为外显子剪接增强子发育控制的重要范例，但人们对 Tra2 如何影响其他前 mRNA 的剪接知之甚少。在该项目中，将研究 Tra2 抑制其自身前 mRNA 中特定内含子剪接的机制，并将其与用于激活双性剪接的调节元件进行比较。使用重组 Tra2 特异性阻断果蝇核提取物对 M1 内含子的剪接的体外剪接测定，将鉴定 Tra2 抑制剪接的调节元件，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。 Tra2 前 mRNA 上形成的调节复合物将与其在 dsx 剪接增强子中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。体外剪接研究开发的机制模型的准确性将通过产生转基因果蝇菌株来测试，其中前体mRNA和序列改变的调节因子在通常发生Tra2依赖性抑制的组织和发育阶段中表达。在进一步的研究中，遗传方法将用于定义 SR 蛋白和其他与 Tra2 配合控制剪接位点识别的剪接因子的要求。这些研究将阐明个体剪接调节因子可以以不同方式改变不同前 mRNA 剪接的基本机制。