Regulation of Mammalian mRNA Decapping

描述（由申请人提供）：mRNA稳定性的控制最近成为真核生物基因表达调控中一个关键但知之甚少的决定因素。mRNA稳定性的变化可以产生深远的影响，可能表现为临床表型。原癌基因异常表达引起的恶性肿瘤和珠蛋白基因异常表达引起的地中海贫血是两个例子，表明适当调节mRNA稳定性的重要性。然而，关于控制哺乳动物mRNA转换的成分，我们所知甚少。我们设计了一个体外mRNA衰变实验，概括了在细胞中观察到的受调节的mRNA转换，并表明哺乳动物的mRNA可以从5‘或3’端降解。我们已经确定了两种哺乳动物的脱帽酶，其中一种是酵母Dcp2蛋白的同源物，并在被封盖的mRNA上起作用。第二种是清除物脱帽活性，dcp，它作用于mRNA 3‘到5’衰变产生的残余帽结构。我们已经证明，人Dcp2 （hDcp2）是一种rna结合蛋白，其活性受poly(A)尾巴和一种与智力迟钝有关的有效抑制蛋白的调节。我们还观察到，人类dcp除了在mRNA衰变后水解帽结构外，还具有促进mRNA衰变的新调节作用。这项提议的长期目标是了解决定因素和核酸酶调节mRNA衰变在哺乳动物。本提案的重点是：（AIM 1）研究控制hDcp2脱帽的调控机制；（AIM 2）确定DcpS脱帽酶调控mRNA衰变的机制；和（AIM 3）确定涉及两种脱帽酶的mRNA衰变的不同途径在体内的贡献。这项工作将为基因表达转录后控制的基本机制提供重要的见解，即mRNA的转换，并将为调节治疗干预的基因表达的新方法提供框架。

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.