Genetic & Molecular Studies of Nematode mRNA Degradation

描述（由申请人提供）：我们研究的主要目的是了解通过转录后过程调节真核基因表达的分子机制和生理后果。大量重要的生物调节发生在转录后，但许多机制还不清楚。我们将研究选择性mRNA降解，通过该过程，特定的mRNA被积极地从细胞中消除。mRNA的调节周转深刻地影响基因表达，我们的广泛目标是了解这种调节的机制和后果。我们专注于加速降解的mRNA，包含过早的翻译终止密码子。含有提前终止密码子的信使RNA比它们的野生型对应物降解得更快，这种现象称为“无义介导的mRNA衰变”（NMD）。NMD发生在所有测试的真核生物中，并且其作用实质上影响人类遗传疾病。导致人类遗传疾病的所有测序点突变中约有三分之一是无义突变或移码突变，并且已知或预测这些突变中的大多数会引起突变mRNA的NMD。我们将学习7个C。线虫NMD所需的基因（smg-1至smg-7），并将研究其遗传和分子特性。我们的工作福尔斯三大类：（1）研究NMD的分子机制;（2）研究NMD与RNA干扰的关系;（3）研究NMD在野生型动物中的生物学功能。我们的具体目标是： 1.我们将研究含有提前翻译终止密码子的mRNP的结构。 这种mRNP与不含提前终止密码子的mRNP的不同之处在于选择性结合SMG-2蛋白。 2.我们将研究SMG-2磷酸化和去磷酸化在NMD中的作用。 3.我们将研究最近发现的NMD和RNAi之间的关系。我们将测试 NMD通过内源性RNAi促进基因沉默。 4.我们将鉴定NMD的大部分或全部内源性mRNA靶点。 我们的长期目标是了解mRNA周转的分子机制和基因表达的变化。 NMD的生物学作用。我们的实验有助于这些目标，通过确定所需的NMD的蛋白质，通过描述其在体内的生化活性，并通过调查NMD在正常生长和发育过程中的作用。

DESCRIPTION (provided by applicant): The broad aims of our research are to understand the molecular mechanisms and physiological consequences of regulating eukaryotic gene expression by post-transcriptional processes. A large amount of important biological regulation occurs post-transcriptionally, but many of the mechanisms are not well understood. We will study selective mRNA degradation, the processes by which specific mRNAs are actively eliminated from cells. Regulated turnover of mRNAs profoundly influences gene expression, and our broad goals are to understand the mechanisms and consequences of that regulation. We focus on the accelerated degradation of mRNAs that contain premature translation termination codons. Messenger RNAs that contain premature stop codons are degraded more rapidly than their wild-type counterparts, a phenomenon termed "nonsense-mediated mRNA decay" (NMD). NMD occurs in all eukaryotes tested, and its action substantially impacts human genetic disease. Approximately one third of all sequenced point mutations that cause human genetic disease are nonsense or frameshift mutations, and most of these are known or predicted to elicit NMD of the mutant mRNA. We will study seven C. elegans genes required for NMD (smg-1 through smg-7) and will investigate their genetic and molecular properties. Our work falls into three broad categories: (1) investigating the molecular mechanisms of NMD; (2) investigating the relationship of NMD to RNA interference; and (3) investigating the biological function of NMD in wild-type animals. Our Specific Aims are: 1. We will investigate the structure of mRNPs containing premature translation termination codons. Such mRNPs differ from those not containing premature stop codons by selectively binding the SMG-2 protein. 2. We will study the roles of SMG-2 phosphorylation and dephosphorylation in NMD. 3. We will investigate a recently discovered relationship between NMD and RNAi. We will test whether NMD contributes to gene silencing via endogenous RNAi. 4. We will identify most or all of the endogenous mRNA targets of NMD. Our long-range goals are to understand both the molecular mechanisms of mRNA turnover and the biological roles of NMD. Our experiments contribute to these goals by identifying proteins that are required for NMD, by describing their biochemical activities in vivo, and by investigating the role of NMD during normal growth and development.