Regulation of Messenger RNA by DEAD-box Proteins

 描述(申请人提供)：DEAD-box蛋白(DBP)是细胞内RNA结构的关键调节者，通常被认为是依赖于ATP的RNA解旋酶。尽管DBP参与了广泛的细胞过程，但这些因子在细胞内发挥作用的机制尚不清楚。这项拟议工作的目的是阐明和表征来自大肠杆菌的两个DBP，Dead和SrmB的体内功能。已发表的数据和我们实验室的初步结果表明，这两个因子都参与了细胞内RNA代谢的多个部分重叠的方面。为了详细定义它们的细胞功能，我们提出了三个具体目标。首先，SrmB和Dead都与核糖体组装有关。通过对基因抑制基因的鉴定，我们发现该蛋白在核糖体中的作用是刺激一种关键的核糖体蛋白L13的产生。在这个目的中提出的实验将检验这样的假设，即SrmB调节编码L13的mRNA的结构，从而影响L13的产生。我们还将分析缺乏DEAD的细胞的遗传抑制因子，以阐明这种DBP调节核糖体组装的机制。在第二个目标中，我们将在全球范围内分析大肠杆菌中mRNAs的调控。我们的初步数据表明，任何一种DBP的缺失都会影响数百个mRNAs的丰度。将进行研究，以检验DBP介导的结构变化在转录终止、稳定性或翻译水平上影响RNA表达的假设。我们还将调查DBP的不依赖于ATPase的活动，如我们最近发现的那些，是否对细胞中的转录调控重要。在第三个目标中，我们将应用一种有效的交联方法来鉴定DBP与mRNAs的结合SIES，并确定RNA结合基序。阐明DBP介导的RNA识别的基础对于理解RNA调控的基本基础至关重要。总之，这些研究将为这一重要的RNA调节家族的功能、它们与RNA相互作用的基础以及调节RNA底物的机制提供多种见解。

 DESCRIPTION (provided by applicant): DEAD-box proteins (DBPs) are key regulators of RNA structure inside the cell and are generally considered to function as ATP-dependent RNA helicases. Although DBPs are implicated in a wide range of cellular processes, the mechanisms by which these factors function inside the cell are not well understood. The objective of the proposed work is elucidate and characterize the in vivo functions of two DBPs from E. coli, DeaD and SrmB. Published data and preliminary results from our laboratory indicate that both factors participate in multiple, partially overlapping aspects of RNA metabolism in the cell. To define their cellular functions in detail, we propose three specific aims. First, both SrmB and DeaD have been implicated in ribosome assembly. Through the identification of genetic suppressors that alleviate the cellular defects caused by an absence of SrmB, we have identified the ribosomal role of this protein is to stimulate the production of a key ribosomal protein, L13. The experiments proposed in this aim will test the hypothesis that SrmB regulates the structure of the mRNA that encodes L13, and thereby, affects L13 production. We will also analyze genetic suppressors of cells that lack DeaD to elucidate the mechanism by which this DBP regulates ribosome assembly. In the second aim, we will globally analyze the regulation of mRNAs in E. coli. Our preliminary data indicate that an absence of either DBP affects the abundance of hundreds of mRNAs. Studies will be performed to test the hypothesis that DBP-mediated structural changes affect RNA expression at the level of transcription termination, stability or translation. We will also investigate whether ATPase-independent activities of the DBPs, such as the ones we have recently identified, are important for transcript regulation in the cell. In the third aim, we will apply a validated cross-linking approach to identify the binding sies of the DBPs on mRNAs and determine RNA binding motifs. Elucidating the basis for DBP- mediated recognition of RNA is critical to understand the underlying basis for RNA regulation. Collectively, these studies will provide multiple insights into the functions of this important famly of RNA regulators, the basis for their interaction with RNA and the mechanisms through which RNA substrates are regulated.