Global analysis of mRNA decay in Bacillus subtilis

枯草芽孢杆菌 mRNA 衰减的整体分析

基本信息

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
项目类别：
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&apos -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

项目摘要

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）：我们的实验室致力于了解模型革兰氏阳性菌枯草芽孢杆菌中信使 RNA 的衰变过程。虽然人们对 DNA 转录为 mRNA 以及 mRNA 翻译为蛋白质的机制和调控了解很多，但对于基因表达的中间步骤——mRNA 的降解却知之甚少。早期的工作在枯草芽孢杆菌中鉴定出两种主要的 3' 核糖核酸外切酶：PNPase 和 RNase R，它们可以在引发衰变的核酸内切酶裂解后有效降解 mRNA 衰变中间体。最近，在枯草芽孢杆菌中发现了两类新的细菌核糖核酸酶：RNase J 和 RNase Y 家族。枯草芽孢杆菌 RNase J 酶之一 RNase J1 是人们特别关注的焦点，因为它具有 5' 至 3' 核酸外切酶活性，而这种活性在经过深入研究的细菌（如大肠杆菌）中并不存在。上校5' 核糖核酸外切酶的存在扩大了 mRNA 降解的可能机制的范围。对 RNase Y（一种核酸内切酶）的新研究表明，它在确定许多 mRNA 的半衰期中发挥着关键作用。该提案应用 RNA-Seq（深度 RNA 测序）技术的力量来探索转录组水平上 mRNA 衰减的机制。特别是，标准RNA-Seq对核糖核酸外切酶突变株的应用将用于检查3'核酸外切酶（主要是PNPase和RNase R）在mRNA衰变中间体周转中的作用。这些研究将揭示核糖核酸酶冗余的问题，以及特定核糖核酸酶是否在特定 mRNA 的周转中发挥作用。此外，标准 RNA-Seq 将用于研究 RNase J1 5' 核酸外切酶活性在包含 RNase 抗性转录终止子结构的 3' 末端片段周转中的作用。一种名为 PARE（RNA 末端并行分析）的改良 RNA 测序方案已被许多实验室用于研究真核 mRNA 加工，该方案将用于绘制 mRNA 衰变过程中出现的 5'-单磷酸末端图谱。该方案将用于研究 RNase Y（主要的枯草芽孢杆菌内切核酸酶）以及 RNase III（枯草芽孢杆菌中必需的窄特异性内切核酸酶）切割的性质和分布。 PARE 还将用于检测天然转录产物 5'-三磷酸末端到 5'-单磷酸末端的转化。这种转变可能是全长 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，也是细菌的一项重要功能。对 mRNA 降解机制的透彻了解将有助于设计出破坏这一过程的抗菌剂，从而干扰细菌细胞的生长。