Defining Adaptors for mRNA Degradation in Bacteria

项目摘要/摘要 转录和RNA周转塑造了细胞中的转录库。对RNA降解的调节， 然而，与转录调控的可列举机制相比，仍然难以捉摸。在……里面 细菌，信使核糖核酸的衰退被认为是由少数具有混杂活性的内切核酸酶引起的， 然而，信使核糖核酸的半衰期变化很大。在这里，我建议在全球范围内定义内切核酸酶的适配子集合 这就像转录因子调节RNA聚合酶的活性一样，会导致信使核糖核酸的衰退。 在细菌中只发现了几个降解适配子，而且它们都是偶然发现的。 为了提供更全面的观点，我将开发几种通用的方法，这些方法将是 可翻译给其他生物体的。我的第一种方法将识别潜在目标的RNA类别 相同的适配器，使用它们对核酸酶水平的依赖作为分类器。自上而下的演示 方法利用革兰氏阳性菌枯草芽孢杆菌的核糖核酸酶Y。 完成这一目标的第一部分将是对枯草杆菌中RNA降解的第一个高分辨率研究 并展示了定义混杂中央核酸内切酶直接靶标的一般策略。 其次，为了补充系统范围的方法，我设计了遗传和生化两种方法 确定降解适配子分子组成的方法。新型适配器的发现 信使核糖核酸衰变将扩大我们对基因调控的理解，并为分子生物学提供变革性的工具 生物学。总而言之，这套自上而下和自下而上的方法将改变我们对 细菌核酸内切酶的特异性，为研究转录后调控提供了一般策略 关于基因表达的。 奖学金培训计划利用两位导师吉恩博士的互补专业知识和经验- 魏丽和艾伦·格罗斯曼博士。研究战略中的技术培训包括(1)量化 我的发起人李博士的建模和下一代测序技术以及(2)未来的方法学 来自我的共同赞助人格罗斯曼博士的遗传学分析和核酸生物化学。李博士是一位训练有素的物理学家 并有开发尖端方法的记录，以满足他在基因调控方面的独特观点。 格罗斯曼博士是一位著名的细菌遗传学家，有三十年的教授指导经验。 许多博士后学员获得了独立的研究职位。组合环境 由这两个人在麻省理工学院培养的目的是为了 这项研究建议并帮助我作为一名独立研究员的发展。

PROJECT SUMMARY/ABSTRACT Transcription and RNA turnover shape the pool of transcripts in the cell. Regulation of RNA degradation, however, remains elusive when compared to the enumerable mechanisms of transcriptional control. In bacteria, mRNA decay is thought to be initiated by a few endoribonucleases that have promiscuous activities, yet mRNA half-lives vary greatly. Here I propose to globally define the set of adaptors for endoribonucleases that instruct mRNA decay, akin to transcription factors modulating the activity of RNA polymerase. Only a few adaptors of degradation have been discovered in bacteria, and they were all found serendipitously. To provide a far more comprehensive view, I will develop several general methodologies that will be translatable to other organisms. My first approach will identify classes of RNAs potentially targeted by the same adaptor, using their dependence on nuclease levels as a classifier. Demonstration of this top-down method utilizes the central endonuclease RNase Y from the gram-positive bacterium Bacillus subtilis. Completion of the first part of this aim will be the first high-resolution study of RNA degradation in B. subtilis and demonstrates a general strategy to define the direct targets of promiscuous central endonucleases. Secondly, to complement the systems-wide approach, I have designed both genetic and biochemical approaches to identify the molecular constituents of adaptors of degradation. Discoveries of novel adaptors for mRNA decay will expand our understanding of gene regulation and provide transformative tools for molecular biology. Together, this set of top-down and bottom-up approaches will transform our understanding of endonuclease specificity in bacteria and provide a general strategy for studying the post-transcriptional control of gene expression. The fellowship training plan leverages complementary expertise and experiences of two mentors, Dr. Gene- Wei Li and Dr. Alan Grossman. Technical training within the research strategy consists of (1) quantitative modeling and next-generation sequencing techniques from my sponsor Dr. Li and (2) methodologies in forward genetics assays and nucleic acid biochemistry from my co-sponsor Dr. Grossman. Dr. Li is a trained physicist and has a record of developing cutting-edge approaches to address his unique perspective on gene regulation. Dr. Grossman is a renowned bacterial geneticist and has thirty years of mentorship experience as a professor with many postdoctoral trainees obtaining independent research positions. The combined environment fostered by these two individuals at the Massachusetts Institute of Technology serves to address the aims of the research proposed and to aid in my development as an independent researcher.