Regulation of Mammalian mRNA decapping

描述(申请人提供)：在真核基因表达的转录后调控中，信使核糖核酸稳定性的控制是一个关键的决定因素。MRNA稳定性的改变可能会产生深远的后果，并可能表现为临床表型。原癌基因的异常表达可能会导致恶性肿瘤，1和2珠蛋白基因的不平衡表达会导致地中海贫血，这可能是最好的例证。尽管信使核糖核酸的稳定性在控制基因表达方面很重要，但直到最近才在鉴定和表征控制信使核糖核酸周转的成分方面取得了进展。我们一直致力于研究哺乳动物的核酸酶和参与mRNA衰退的途径，特别是两种解帽酶Dcp2和DCPS。Dcp2是一种信使核糖核酸解离酶，作用于被封顶的信使核糖核酸，而当信使核糖核酸在34到54个衰变后，DCP2起到清算所的作用，以水解帽结构。这项建议的长期目标是了解调控哺乳动物mRNA解链和mRNA衰退的机制，并利用这些信息来控制正常和疾病状态下的基因表达。我们已经证明Dcp2是一种转录特异性的解帽蛋白，它不是统一表达的，甚至在某些组织中不能检测到，这表明哺乳动物中存在未知的解帽酶。我们还发现了一种与X连锁精神发育迟滞有关的蛋白质，它是去顶的调节因子。此外，我们还表明，DCPS除了在mRNA周转中的作用外，还是核帽结合蛋白介导的功能的调节器，也是治疗脊髓性肌萎缩的候选药物的靶底物。在这项建议中，i)我们将测试Dcp2及其缺失对Aim I中哺乳动物细胞中非Dcp2信使核糖核酸解离酶的影响；ii)表征解离调节因子影响Aim 2中信使核糖核酸稳定性的机制；以及iii)评估DCPS在目标3中对细胞质功能(包括信使核糖核酸稳定性和翻译)的调节作用。这项工作将为深入了解转录后控制基因表达的基本机制提供依据，并将为人类疾病的治疗干预提供新的方法框架。公共卫生相关性：我们的总体目标是了解涉及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。去核糖核酸是mRNA稳定和最终消亡的关键步骤。我们将继续研究Dcp2和DCPS解帽酶在哺乳动物中的基因表达及其与智力低下和脊髓性肌萎缩症的相关性。我们将确定Dcp2在mRNA衰退中的存在和令人惊讶的自然缺失的影响，它的调节，以及DCPS对细胞质帽子结合蛋白过程的调制功能。这项工作将深入了解转录后基因表达控制的基本机制，并可能为治疗干预的新方法提供一个框架。

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.