Project 3. Defining the RNA processing and degradation pathways of Mtb.

项目3，摘要 转录物加工和降解是细胞调节其功能能力的关键过程， 生理状态，但很少有人知道这些途径如何在结核分枝杆菌（Mtb）的功能。 RNA加工和降解机制的组成部分的突变与耐药性有关 在临床菌株和小鼠和体外药物耐受性，突出了这些途径的相关性， 临床上重要的表型。对分枝杆菌RNA的基础生物学知识的缺乏 代谢，包括途径结构和这些途径的功能后果，代表了一个障碍， 了解临床上重要的耐药途径。拟议的项目通过以下方式弥补这一差距： 阐明关键RNA加工和降解之间的目标和功能关系 蛋白质，专注于这些过程如何改变药物疗效。具体目标是： 1.确定定义和组织RNA加工的物理和遗传相互作用， 降解途径 2.确定与耐药性相关的RNA降解蛋白的靶点。 3.确定干扰RNA加工途径的表型后果。 该项目力求从零敲碎打的努力转向能够阐明 复杂过程的后果对适应宿主和药物压力很重要。为了实现这一点， 该项目利用高通量方法并与其他项目和核心进行广泛合作。 生物化学方法将用于确定降解途径之间的物理联系 TnSeq用于绘制途径结构及其表型结果; 确定RNA代谢途径如何塑造转录组的转录组学方法;小鼠 研究RNA加工突变体与体内药物功效之间关系的模型;以及代谢- 脂质组学方法来确定RNA加工突变体对代谢状态和渗透性的影响。 以一种有意的和系统的方式结合各种方法和专业知识， 明确分枝杆菌中RNA加工和降解的机制和后果， 以前是不可行的。从长远来看，所获得的知识将有助于设计更多的 有效的诊断、药物和治疗方案。

Project 3, Abstract Transcript processing and degradation are key processes by which the cell regulates its functional capacity and physiological state, yet little is known about how these pathways function in Mycobacterium tuberculosis (Mtb). Mutations in components of the RNA processing and degradation machinery are associated with drug resistance in clinical strains and with drug tolerance in mice and in vitro, highlighting the relevance of these pathways for clinically important phenotypes. The lack of knowledge about the fundamental biology of mycobacterial RNA metabolism, including pathway structure and functional consequences of these pathways, represent a barrier to understanding clinically important routes to drug resistance. The proposed project addresses this gap by elucidating the targets and functional relationships between the critical RNA processing and degradation proteins, focusing on how these processes modify drug efficacy. The specific aims are to: 1. Determine the physical and genetic interactions that define and organize RNA processing and degradation pathways. 2. Define the targets of RNA degradation proteins that are associated with drug resistance. 3. Determine the phenotypic consequences of perturbations to RNA processing pathways. The project seeks to move from piecemeal efforts to a pathways-directed approach capable of elucidating the consequences of complex processes important to adaptation to the host and to drug pressure. To achieve this, the project leverages high-throughput methodologies and extensive collaboration with other projects and cores. Biochemical approaches will be used to define the physical associations between degradation pathway components and their target specificities; TnSeq to map pathway structure and its phenotypic consequences; transcriptomics approaches to determine how RNA metabolism pathways shape the transcriptome; murine models to interrogate the relationship between RNA processing mutants and drug efficacy in vivo; and metabo- lipidomics approaches to determine the impact of RNA processing mutants on metabolic status and permeability. Combining a variety of approaches and expertise in an intentional and systematic fashion will allow the project to define the mechanisms and consequences of RNA processing and degradation in mycobacteria in a way that has not previously been feasible. In the long term, the knowledge obtained will inform efforts to design more effective diagnostics, drugs and regimens.