The Function of Processing Bodies in Human mRNA Turnover

描述(申请人提供)：信使核糖核酸周转是基因表达调控的关键步骤。它的不当监管与一系列人类疾病有关。我的实验室的长期目标是了解人类信使核糖核酸周转的机制和调节。通过去帽过程去除mRNA5‘端是mRNA5’端转运的重要步骤。最近的证据表明，负责解离的蛋白质复合体与其他参与从5‘端降解信使核糖核酸的因子共定位于被称为加工小体的亚细胞质病灶中。此外，令人信服的数据表明，加工体在信使核糖核酸的衰变中非常活跃。这表明，信使核糖核酸的衰退可能是一个局部的过程。在这里，我们将测试人类处理机构在三个特定的mRNA衰退过程中所扮演的角色，这三个过程在人类基因表达和疾病中发挥关键作用：全富元素(Are)介导的mRNA衰退、无义介导的衰退(NMD)和由微小(Mi)RNA引发的mRNA衰退。我们将使用原位杂交、间接免疫荧光、共免疫沉淀和信使核糖核酸衰变试验来测试这些信使核糖核酸衰变途径是如何与加工体结合的。此外，我们将测试PBS的催化成分当定位在PBS之外时是否以蛋白质降解为目标，在那里它们可能刺激混杂的RNA衰退。这些实验旨在解决以下问题：i)不稳定的mRNAs是如何针对加工体的，以及ii)为什么mRNA衰变酶集中在PBS而不是整个细胞质？这一建议与公共健康相关，因为对编码蛋白质的mRNAs降解的错误调控与一系列人类疾病有关。了解信使核糖核酸转换背后的机制将有助于制定抗击此类疾病的策略。

DESCRIPTION (provided by applicant): mRNA turnover is a key step in regulation of gene expression. Its mis-regulation has been linked to a range of human disorders. The long-term goal of my laboratory is to understand the mechanism and regulation of mRNA turnover in humans. Removal of the mRNA 5' cap by the process of decapping is an important step in mRNA turnover. Recent evidence has shown that the protein complex responsible for decapping co-localizes with other factors involved in degradation of mRNA from the 5' end in sub-cytoplasmic foci termed processing bodies. Moreover, compelling data shows that processing bodies are active in mRNA decay. This suggests that mRNA decay can be a localized process. Here we will test the role of human processing bodies in three specific mRNA decay processes that play a key role in human gene expression and disease: All-rich element (ARE)-mediated mRNA decay, nonsense-mediated decay (NMD) and mRNA decay triggered by micro (mi)RNAs. We will employ in situ hybridization, indirect immunofluorescence, co- immunoprecipitation and mRNA decay assays to test how these mRNA decay pathways interface with processing bodies. In addition, we will test if catalytic components of PBs are targeted for proteolysis when localized outside of PBs where they may stimulate promiscuous RNA decay. These experiments are aimed at addressing the following questions: i) How are unstable mRNAs targeted to processing bodies and ii) why are mRNA decay enzymes concentrated in PBs rather than throughout the cytoplasm? This proposal is relevant to public health because mis-regulation of the degradation of protein-encoding mRNAs has been linked to a range of human disorders. Understanding the mechanism behind mRNA turnover will help develop strategies to fight such diseases.