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控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，但许多机制尚不清楚。我们将研究选择性的信使核糖核酸降解，即特定的信使核糖核酸从细胞中被主动清除的过程。受调控的mRNAs周转深刻影响基因表达，我们的广泛目标是了解这种调控的机制和后果。我们专注于包含提前翻译终止密码子的mRNAs的加速降解。含有提前终止密码子的信使RNA比它们的野生型信使RNA降解得更快，这种现象被称为“无意义介导的信使RNA衰退”(NMD)。NMD存在于所有被测试的真核生物中，它的作用对人类遗传病有很大影响。在所有导致人类遗传病的测序点突变中，大约有三分之一是无稽之谈或移码突变，其中大多数已知或预测会引起突变mRNA的NMD。我们将研究NMD所需的7个线虫基因(SMG-1至SMG-7)，并研究它们的遗传和分子特性。我们的工作分为三大类：(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mRNPs的结构。 这种mRNPs不同于那些不含过早终止密码子的mRNPs，它选择性地结合SMG-2蛋白。 2.研究SMG-2的磷酸化和去磷酸化在NMD中的作用。 3.我们将调查最近发现的NMD和RNAi之间的关系。我们将测试一下 NMD通过内源性RNAi参与基因沉默。 4.我们将确定NMD的大部分或全部内源性mRNA靶点。 我们的长期目标是了解信使核糖核酸周转的分子机制和 国家导弹防御系统的生物学作用。我们的实验通过识别NMD所需的蛋白质，通过描述它们在体内的生化活动，以及通过研究NMD在正常生长和发育中的作用，为这些目标做出了贡献。

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.