Defining Adaptors for mRNA Degradation in Bacteria

定义细菌中 mRNA 降解的接头

• 批准号: 10132728
• 负责人: Matthew Tien
• 金额: $ 6.86万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10132728
• 关键词: Adaptor Signaling Protein; Address; Affect; Affinity Chromatography; Animal Model; Bacillus subtilis; Bacteria; Bacterial Model; Behavior; Binding; Biochemical; Biological Assay; Categories; Cells; Complex; DNA-Directed RNA Polymerase; Dependence; Development; Dose; Endoribonucleases; Environment; Event; Fellowship; Fostering; Foundations; Future; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Gram-Positive Bacteria; Individual; Infection; Institutes; Invaded; Knowledge; Massachusetts; Measures; Mediating; Mentors; Mentorship; Messenger RNA; Methodology; Methods; Microbial Biofilms; Modeling; Molecular; Molecular Biology; Mutagenesis; Nucleic Acid Biochemistry; Organism; Pathway interactions; Play; Positioning Attribute; Post-Transcriptional Regulation; Protein Isoforms; Proteins; RNA; RNA Degradation; Regulation; Research; Research Personnel; Resolution; Ribonucleases; Role; Shapes; Site; Small RNA; Specific qualifier value; Specificity; Staphylococcus aureus; Streptococcus pyogenes; Techniques; Technology; Testing; Titrations; Toxin; Training; Transcript; Transcriptional Regulation; Virulence; complement system; crosslink; design; endonuclease; experience; experimental study; falls; forward genetics; genetic approach; genome-wide; in vivo; in vivo monitoring; mRNA Decay; mRNA Transcript Degradation; mutant; next generation sequencing; novel; nuclease; overexpression; pathogen; pathogenic bacteria; professor; response; tool; transcription factor; transcriptome; transcriptome sequencing

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.

项目总结/摘要 转录和RNA周转形成细胞中的转录物库。RNA降解的调节， 然而，当与转录控制的可解释机制相比时，仍然是难以捉摸的。在 在细菌中，mRNA衰变被认为是由一些具有混杂活性的核糖核酸内切酶引发的， 然而mRNA的半衰期变化很大。在这里，我建议全局定义内切核糖核酸酶的衔接子集 这类转录因子类似于调节RNA聚合酶活性的转录因子。 在细菌中只发现了几种降解的适配器，而且它们都是偶然发现的。 为了提供一个更全面的观点，我将开发几个通用的方法， 可转化为其他生物。我的第一种方法将识别可能被靶向的RNA类别。 相同的衔接子，使用它们对核酸酶水平的依赖性作为分类器。这种自上而下的演示 该方法利用来自革兰氏阳性细菌枯草芽孢杆菌的中心核酸内切酶RNase Y。 这一目标的第一部分的完成将是对B中RNA降解的第一次高分辨率研究。枯草 并展示了定义混杂中心核酸内切酶的直接靶的一般策略。 其次，为了补充系统范围的方法，我设计了遗传和生物化学 鉴定降解衔接子分子成分的方法。新型适配器的发现 mRNA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工具。 生物学总之，这套自上而下和自下而上的方法将改变我们对 核酸内切酶特异性的细菌，并提供了一个通用的策略，研究转录后控制 的基因表达。 研究金培训计划利用了两位导师的互补专业知识和经验，吉恩博士- 李伟和艾伦·格罗斯曼博士。研究战略中的技术培训包括（1）定量 建模和下一代测序技术，从我的赞助商李博士和（2）方法，在未来 遗传学检测和核酸生物化学的报告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，以解决他对基因调控的独特观点。 博士格罗斯曼是一位著名的细菌遗传学家，有三十年的指导经验， 许多博士后获得了独立的研究职位。组合环境 由这两个人在马萨诸塞州理工学院培育，旨在解决的目标， 研究建议，并在我的发展作为一个独立的研究人员援助。