Regulation of Mammalian mRNA decapping

描述（由申请人提供）：mRNA稳定性的控制是真核基因表达转录后调控的关键决定因素。mRNA稳定性的变化可能产生深远的影响，并可能表现为临床表型。这可能是最好的说明异常表达的原癌基因的能力，可以引起恶性肿瘤和1和2珠蛋白基因表达不平衡，导致地中海贫血。尽管mRNA稳定性在控制基因表达中非常重要，但直到最近才在控制mRNA周转的成分的鉴定和表征方面取得了进展。我们一直致力于研究哺乳动物中核酸酶和mRNA降解途径，特别是两种去帽酶Dcp 2和DcpS。Dcp 2是对加帽mRNA起作用的mRNA去帽酶，而DcpS起交换所的作用，以在mRNA的34至54次衰变后水解所得帽结构。该提案的长期目标是了解调节哺乳动物mRNA脱帽和mRNA衰变的机制，并利用这些信息来控制正常和疾病状态下的基因表达。我们已经表明，Dcp 2是一种转录特异性的脱帽蛋白，它不是均匀表达的，甚至在某些组织中检测不到，这表明哺乳动物中存在未知的脱帽酶。我们还确定了一种与X连锁精神发育迟滞有关的蛋白质作为脱帽的调节剂。此外，我们已经表明，DcpS是一种调节剂的核帽结合蛋白介导的功能，除了其在mRNA的营业额以及治疗脊髓性肌萎缩症的候选药物的靶底物的作用。在该提案中，i）我们将测试Dcp 2及其缺失对mRNA衰变的影响，并在目的I中鉴定哺乳动物细胞中的非Dcp 2 mRNA脱帽酶; ii）表征脱帽调节剂影响目的2中mRNA稳定性的机制; iii）评估DcpS对细胞质功能的调节作用，包括目的3中mRNA稳定性和翻译。这项工作将提供一个基本的机制，参与基因表达的转录后控制的见解，并将提供一个框架，为人类疾病的治疗干预的新方法。公共卫生相关性：我们的总体目标是了解mRNA周转的精确控制，并利用这些信息来调节正常和疾病状态下的基因表达。去帽是mRNA稳定和最终死亡的关键步骤。我们将建立在我们正在进行的哺乳动物基因表达中的Dcp 2和DcpS脱帽酶的功能研究及其与精神发育迟滞和脊髓性肌萎缩症的相关性。我们将确定Dcp 2在mRNA衰变中的存在和令人惊讶的自然缺失的影响，其调节和DcpS对细胞质帽结合蛋白过程的调节功能。这项工作将提供一个基本的机制，参与基因表达的转录后控制，并可能提供一个框架，在新的方法进行治疗干预。

DESCRIPTION (provided by applicant): The control of mRNA stability is a critical determinant in the post-transcriptional regulation of eukaryotic gene expression. Changes in mRNA stability can have profound consequences and may manifest as clinical phenotypes. This is perhaps best illustrated by the ability of aberrantly expressed proto-oncogenes that can give rise to malignancies and by the imbalanced 1 and 2 globin gene expression resulting in thalassemias. Despite the importance of mRNA stability in the control of gene expression, progress has only recently been made in the identification and characterization of the components that control mRNA turnover. We have focused our efforts on the study of nucleases and the pathways involved in mRNA decay in mammals, in particular, the two decapping enzymes, Dcp2 and DcpS. Dcp2 is the mRNA decapping enzyme that functions on capped mRNA while DcpS functions as the clearinghouse to hydrolyze the resulting cap structure following 34 to 54 decay of an mRNA. The long-term objective of this proposal is to understand the mechanisms that regulate mRNA decapping and mRNA decay in mammals and to utilize this information to control gene expression under normal and disease states. We have shown that Dcp2 is a transcript-specific decapping protein that is not uniformly expressed and is even undetectable in certain tissues indicating that there are as yet unknown decapping enzymes in mammals. We have also identified a protein implicated in X-linked mental retardation as a regulator of decapping. Furthermore, we have shown that DcpS is a modulator of the nuclear cap binding protein-mediated functions in addition to its role in mRNA turnover as well as the target substrate of a drug candidate for the treatment of spinal muscular atrophy. In this proposal, i) we will test the impact of Dcp2 and its absence on mRNA decay and identify the non-Dcp2 mRNA decapping enzyme in mammalian cells in Aim I; ii) characterize the mechanism by which the decapping regulator influences mRNA stability in Aim 2; and iii) assess the modulatory role of DcpS on cytoplasmic functions including mRNA stability and translation in Aim 3. This work will provide insights into a fundamental mechanism involved in the post-transcriptional control of gene expression and will provide a framework for novel approaches for therapeutic intervention in human disorders. PUBLIC HEALTH RELEVANCE: Our overall objective is to understand the precise controls involved in mRNA turnover and to utilize this information to regulate gene expression under normal and disease states. Decapping is a key step in the stability and ultimate demise of an mRNA. We will build on our ongoing functional studies of the Dcp2 and DcpS decapping enzymes in mammalian gene expression and their correlation to both mental retardation and spinal muscular atrophy. We will determine the impact of the presence, and surprising natural absence, of Dcp2 in mRNA decay, its regulation and the modulatory function of DcpS on cytoplasmic cap-binding protein processes. This work will provide insight into a fundamental mechanism involved in the post-transcriptional control of gene expression and could provide a framework in novel approaches for therapeutic intervention.