Regulation of Mammalian mRNA Decapping

描述（由申请人提供）：mRNA稳定性的控制最近已成为真核基因表达调控中关键但知之甚少的决定因素。mRNA稳定性的变化可能会产生深远的影响，这些影响可能会表现为临床表型。由原癌基因的异常表达引起的恶性肿瘤和由不适当的珠蛋白基因表达引起的地中海贫血是表明适当调节mRNA稳定性的重要性的两个例子。然而，我们对控制哺乳动物mRNA周转的成分知之甚少。我们设计了一种体外mRNA衰变测定法，该测定法概括了在细胞中观察到的受调节的mRNA周转，并表明哺乳动物mRNA可以从5'或3'末端降解。我们已经确定了两种哺乳动物的脱帽酶，其中之一是一个同源的酵母Dcp 2蛋白和功能的加帽mRNA。第二种是清除剂脱帽活性，DcpS，其作用于由mRNA的3'至5'衰变产生的残余帽结构。我们已经证明人Dcp 2（hDcp 2）是一种RNA结合蛋白，其活性受poly（A）尾和一种与智力低下有关的有效抑制蛋白的调节。我们还观察到，人DcpS，除了其在水解帽结构的作用，mRNA衰变后，功能在一个新的调节作用，以促进mRNA衰变。这项计划的长期目标是了解哺乳动物中调节mRNA衰变的决定簇和核酸酶。该提案的重点是：（目的1）研究控制hDcp 2脱帽的调节机制;（目的2）确定DcpS脱帽酶调节mRNA衰变的机制;以及（目的3）确定体内涉及两种脱帽酶的mRNA衰变的不同途径的贡献。这项工作将提供重要的见解，参与基因表达的转录后控制的基本机制，即mRNA周转，并将提供一个新的方法来调节基因表达的治疗干预的框架。

DESCRIPTION (provided by applicant): The control of mRNA stability has recently emerged as a critical yet poorly understood determinant in the regulation of eukaryotic gene expression. Changes of mRNA stability can have profound consequences that may become manifest as clinical phenotypes. Malignancies that arise from aberrant expression of proto-oncogenes, and thalassemias, resulting from improper globin gene expression, are two examples indicating the importance of proper regulation of mRNA stability. Yet very little is known concerning the components that control mammalian mRNA turnover. We have devised an in vitro mRNA decay assay that recapitulates regulated mRNA turnover observed in cells and have shown that mammalian mRNA can be degraded from either the 5' or the 3' end. We have identified the two mammalian decapping enzymes, one of which is a homolog of the yeast Dcp2 protein and functions on capped mRNA. The second is a scavenger decapping activity, DcpS, which functions on the residual cap structure resulting from 3' to 5' decay of an mRNA. We have demonstrated human Dcp2 (hDcp2) is an RNA-binding protein whose activity is regulated by both the poly(A) tail and a potent inhibitor protein implicated in mental retardation. We have also observed that the human DcpS, in addition to its role in hydrolyzing the cap structure following mRNA decay, functions in a novel regulatory role to facilitate mRNA decay. The long term objective of this proposal is to understand the determinants and nucleases that regulate mRNA decay in mammals. The focus of this proposal is to: (AIM 1) investigate the regulatory mechanisms that control hDcp2 decapping; (AIM 2) determine the mechanism by which DcpS decapping enzyme functions to regulate mRNA decay; and (AIM 3) determine the contribution of the different pathways of mRNA decay involving the two decapping enzymes in vivo. This work will provide significant insights into a fundamental mechanism involved in the post transcriptional control of gene expression, that of mRNA turnover, and will provide a framework for novel approaches to regulate gene expression for therapeutic intervention.