Global analysis of mRNA decay in Bacillus subtilis

描述(申请人提供)：我们实验室试图了解模式革兰氏阳性细菌枯草芽孢杆菌中信使RNA的衰退过程。虽然人们对DNA转录为信使核糖核酸和将信使核糖核酸翻译为蛋白质的机制和调控已知很多，但对基因表达的中间步骤--信使核糖核酸的降解知之甚少。早期的工作发现枯草杆菌中有两种主要的3‘外切核糖核酸酶，即PNPase和RNaseR，它们可以有效地降解衰退启动的内切酶切割后的mRNA衰退中间产物。最近，在枯草杆菌中发现了两类新的细菌核糖核酸酶，即RNaseJ和RNaseY家族。枯草杆菌核糖核酸酶J酶之一，核糖核酸酶J1，是一个特别令人感兴趣的焦点，因为它规定了5‘至3’核酸外切酶活性，这种活性在更多研究较好的细菌中不存在，如E.col.5‘外切核糖核酸酶的存在扩大了信使核糖核酸降解的可能机制的范围。对核酸内切酶RNaseY的最新研究表明，它在决定许多mRNAs的半衰期方面发挥着关键作用。该建议应用RNA-Seq(深度RNA测序)技术在转录组水平上探索mRNA衰退的机制。特别是，将标准RNA-Seq应用于外切核酸酶突变株，将被用来研究3‘外切酶，主要是PNPase和RNaseR，在mRNA衰变中间产物周转中的作用。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，标准的RNA-Seq将被用来研究RNase J15‘外切酶活性在含有RNase抗性转录终止子结构的3’末端片段转换中的作用。一种被称为PARE(并行分析RNA末端)的改进的RNA-Seq协议，已经被许多实验室用于研究真核mRNA的加工，将被用来定位在mRNA衰退过程中出现的5‘-单磷酸末端。这一方法将被用于研究枯草杆菌的主要内切酶RNaseY和RNaseIII的切割性质和分布，RNaseIII是枯草杆菌必需的一种窄特异性内切酶。PARE还将用于检测天然转录产物5‘-三磷酸末端向5’-单磷酸末端的转化。这种转换可能是全长mRNAs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，也是细菌的一项基本功能。彻底了解信使核糖核酸衰变的机制将有助于设计抗菌剂来扰乱这一过程，从而干扰细菌细胞的生长。 与公共健康相关：信使RNA(信使RNA)是合成蛋白质的模板分子，需要快速翻转以适应不断变化的环境，并回收合成新RNA所需的核糖核酸池。在模式微生物枯草杆菌中，将使用有助于全球观察RNA过程的深层RNA测序技术，详细研究mRNA降解的机制。阐明mRNA衰退的机制可能导致设计出抑制这一必要过程的抗生素，从而防止细菌在人体组织中的定植。

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. PUBLIC HEALTH RELEVANCE: Messenger RNA (mRNA), the template molecule upon which proteins are synthesized, needs to be turned over rapidly to adapt to changing environments and to recycle the pool of ribonucleotides needed for new RNA synthesis. The mechanisms by which mRNA is degraded will be studied in detail in the model microorganism, Bacillus subtilis, using deep RNA sequencing technology that facilitates global views of RNA processes. Elucidating the mechanism of mRNA decay could lead to the design of antibiotics that inhibit this essential process and thereby prevent bacterial colonization of human tissues.