Global analysis of mRNA decay in Bacillus subtilis

枯草芽孢杆菌 mRNA 衰减的整体分析

• 批准号: 8515472
• 负责人: DAVID H BECHHOFER
• 金额: $ 32.25万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8515472
• 关键词: 5' -exoribonuclease; Address; Affect; Bacillus subtilis; Bacteria; Collection; Complement; DNA; Data; Environment; Enzymes; Escherichia coli; Exoribonucleases; Family; France; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Germany; Gram-Positive Bacteria; Half-Life; Laboratories; Lead; Length; Libraries; Light; Maps; Messenger RNA; Microarray Analysis; Modeling; Modification; Molecular Biology; Natural regeneration; Nature; Organism; Pathway interactions; Phosphodiesterase I; Play; Polyribonucleotide Nucleotidyltransferase; Process; Proteins; Protocols documentation; RNA; RNA Decay; RNA Processing; RNA Sequences; RNA analysis; RNA chemical synthesis; Reading; Recycling; Relative (related person); Reproducibility; Resistance; Ribonuclease III; Ribonucleases; Ribonucleotides; Role; Site; Specific qualifier value; Specificity; Structure; Technology; Testing; Transcription Process; Transcriptional Regulation; Translations; Work; antibiotic design; antimicrobial drug; base; cell growth; density; design; endonuclease; environmental change; flexibility; human tissue; interest; microorganism; mutant; new technology; poly A specific exoribonuclease; prevent; programs; public health relevance; research study; ribonuclease B; ribonuclease R; spleen exonuclease; transcriptome sequencing; tripolyphosphate

DESCRIPTION (provided by applicant): Our laboratory seeks to understand the process of messenger RNA decay in the model Gram-positive bacterium, Bacillus subtilis. While much is known about the mechanisms and regulation of transcription of DNA into mRNA and translation of mRNA into protein, relatively little is known about the intermediate step in gene expression - degradation of mRNA. Earlier work identified two major 3' exoribonucleases in B. subtilis, PNPase and RNase R, which can efficiently degrade mRNA decay intermediates subsequent to a decay-initiating endonuclease cleavage. More recently, two new classes of bacterial ribonucleases, the RNase J and RNase Y families, were discovered in B. subtilis. One of the B. subtilis RNase J enzymes, RNase J1, is a particular focus of interest as it specifies a 5'-to-3' exonuclease activity, an activity that is not present in more well-studied bacteria, such as E. col. The presence of a 5' exoribonuclease expands the scope of possible mechanisms by which mRNA can be degraded. New studies of RNase Y, an endonuclease, suggest that it plays a key role in determining the half-life of many mRNAs. This proposal applies the power of RNA-Seq (deep RNA sequencing) technology to explore mechanisms of mRNA decay at the transcriptome level. In particular, application of standard RNA-Seq to exoribonuclease mutant strains will be used to examine the role of 3' exonucleases, primarily PNPase and RNase R, in the turnover of mRNA decay intermediates. These studies will shed light on the question of ribonuclease redundancy, and whether specific ribonucleases act in the turnover of particular mRNAs. In addition, standard RNA-Seq will be used to study the role of RNase J1 5' exonuclease activity in the turnover of 3'-terminal fragments that contain the RNase-resistant transcription terminator structure. A modified RNA-Seq protocol called PARE (parallel analysis of RNA ends), which has been used by a number of laboratories to study eukaryotic mRNA processing, will be used to map 5'-monophosphate ends that arise in the course of mRNA decay. This protocol will be used to study the nature and distribution of cleavages by RNase Y, the major B. subtilis endonuclease, as well as by RNase III, a narrow-specificity endonuclease that is essential in B. subtilis. PARE will also be used to detect conversion of the native transcription product 5'-triphosphate end to a 5'- monophosphate end. This conversion may be a precursor step in the degradation of full-length mRNAs. RELEVANCE: Degradation of messenger RNA is an important step in regulating gene expression and is an essential function of bacteria. A thorough understanding of the mechanism of mRNA decay will enable design of antimicrobial agents that disrupt this process and thereby interfere with bacterial cell growth.

描述（由申请人提供）：我们的实验室旨在了解模型革兰氏阳性细菌枯草芽孢杆菌的信使RNA衰变过程。虽然人们对DNA转录成mRNA和mRNA翻译成蛋白质的机制和调控了解很多，但对基因表达的中间步骤- mRNA的降解知之甚少。早期的研究发现枯草芽孢杆菌中有两种主要的3'外核糖核酸酶，PNPase和RNase R，它们可以在启动衰变的内切酶切割后有效地降解mRNA衰变中间体。最近，在枯草芽孢杆菌中发现了两种新的细菌核糖核酸酶，RNase J和RNase Y家族。其中一种枯草芽孢杆菌RNase J酶RNase J1是人们特别感兴趣的焦点，因为它指定了一种5‘至3’外切酶活性，这种活性在更深入研究的细菌（如大肠杆菌）中不存在。5'外核糖核酸酶的存在扩大了mRNA降解可能机制的范围。核糖核酸内切酶RNase Y的新研究表明，它在决定许多mrna的半衰期中起着关键作用。本研究利用RNA- seq（深度RNA测序）技术在转录组水平上探索mRNA衰变的机制。特别是，将标准RNA-Seq应用于外核糖核酸酶突变株，将用于检测3'外切酶，主要是PNPase和RNase R，在mRNA衰变中间体翻转中的作用。这些研究将阐明核糖核酸酶冗余的问题，以及特定的核糖核酸酶是否在特定mrna的周转中起作用。此外，标准RNA-Seq将用于研究RNase j15 ‘外切酶活性在含有RNase抗性转录终止体结构的3’末端片段的周转中的作用。一种被称为PARE （RNA末端平行分析）的改进RNA- seq协议，已被许多实验室用于研究真核mRNA加工，将用于绘制mRNA衰变过程中出现的5'-单磷酸端。该方案将用于研究主要的枯草芽孢杆菌内切酶RNase Y以及枯草芽孢杆菌必需的窄特异性内切酶RNase III切割的性质和分布。PARE还将用于检测天然转录产物5'-三磷酸端到5'-单磷酸端的转化。这种转化可能是全长mrna降解的前驱步骤。相关性：信使RNA的降解是调节基因表达的重要步骤，是细菌的基本功能。对mRNA衰变机制的透彻理解将有助于设计出破坏这一过程的抗菌药物，从而干扰细菌细胞的生长。