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

项目3，摘要 转录处理和降解是细胞调节其功能和能力的关键过程 然而，关于这些通路在结核分枝杆菌(Mtb)中是如何发挥作用的，人们知之甚少。 RNA加工和降解机制组件的突变与耐药性有关 在临床菌株以及小鼠和体外耐药的情况下，强调了这些途径与 临床上重要的表型。对分枝杆菌核糖核酸基础生物学知识的缺乏 新陈代谢，包括这些途径的结构和功能后果，代表着一种障碍 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 阐明关键的RNA加工和降解之间的靶标和功能关系 蛋白质，重点是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加工和组织的物理和遗传相互作用 退化途径。 2.确定与耐药相关的RNA降解蛋白的靶点。 3.确定干扰对RNA加工途径的表型后果。 该项目寻求从零敲碎打的努力转向能够阐明 对适应宿主和药物压力很重要的复杂过程的后果。为了实现这一目标， 该项目利用高吞吐量方法以及与其他项目和核心的广泛合作。 生化方法将被用来确定降解途径之间的物理联系 组分及其靶标特异性；TnSeq用于绘制途径结构及其表型结果； 转录组学方法确定RNA代谢途径如何塑造转录组；小鼠 询问RNA加工突变体与体内药物疗效关系的模型；以及代谢- 用脂质组学方法确定RNA加工突变体对代谢状态和通透性的影响。 以有目的和系统的方式结合各种方法和专业知识将使该项目 确定分枝杆菌中RNA加工和降解的机制和后果 在此之前并不可行。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、药物和养生方法。

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.