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; 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革兰氏阳性细菌枯草芽孢杆菌中的使者RNA衰变的过程。尽管对将DNA转录到mRNA和将mRNA转化为蛋白质的机理和调节知之甚少，但对基因表达的中间步骤 - mRNA的降解相对较少。较早的工作确定了枯草芽孢杆菌，PNPase和RNase R中的两个主要3'驱虫酶，它们可以在衰减发射的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种新的细菌核糖核酸酶，即RNase J和RNase Y家族。枯草芽孢杆菌RNase J酶之一RNase J1是一个特别的关注点，因为它指定了5'至3'核酸外切酶活性，这种活性在更精心研究的细菌中不存在，例如Col。 5'驱核核酸酶的存在扩大了可能降解mRNA的可能机制的范围。 RNase Y（一种核酸内切酶）的新研究表明，它在确定许多mRNA的半衰期中起着关键作用。该提案应用RNA-Seq（深RNA测序）技术的力量来探索转录组水平上mRNA衰变的机制。特别是，将标准RNA-SEQ应用于驱虫核酸酶突变菌株将用于检查3'外丝丝（主要是PNPase和RNase R）在mRNA衰变中间体的营业层中的作用。这些研究将阐明核糖核酸酶冗余的问题，以及特定的核糖珠是否在特定mRNA的营业额中起作用。此外，标准RNA-SEQ将用于研究RNase J1 5'Exoniclea酶活性在包含抗RNase耐药转录终结剂结构的3'-末端片段中的营业额中的作用。一种称为PARE的改良RNA-SEQ方案（RNA末端的平行分析），该协议已被许多实验室用于研究真核mRNA处理，将用于绘制在mRNA衰变过程中出现的5'单磷酸末端。该方案将用于研究RNase Y的裂解的性质和分布，主要枯草芽孢杆菌核酸芽孢杆菌以及RNase III（RNase III），这是枯草芽孢杆菌必不可少的窄特异性内切酶。 PARE还将用于检测5'-三磷酸末端的天然转录产物转化为5'-单磷酸端。这种转换可能是全长mRNA降解的前体步骤。相关性：信使RNA的降解是调节基因表达的重要步骤，并且是细菌的重要功能。对mRNA衰变机制的透彻理解将使设计破坏该过程并从而干扰细菌细胞生长的抗菌剂。