Translational control of gene expression in fungi

总结 mRNA的翻译是一个中心的细胞过程，但控制它的机制尚未完全了解。 高达50%的真核生物mRNA含有预测的上游开放阅读框（uORF），但 这些uORF中的绝大多数仍然未确定。在某些情况下，我们知道这种额外的翻译 能力在进化上是保守的，并且在控制基因表达中起关键作用。专班 这些保守的uORFs编码的肽，响应于小分子的存在， 代谢物。这些新生的调节肽在核糖体通道内起作用以阻止翻译;通过这样做， 它们控制代谢中重要酶的产生。然而，在以下方面仍存在重大差距： 了解uORF的功能。首先，uORF编码的肽识别小分子的机制， 分子和真核生物核糖体控制基因表达的机制尚不清楚。第二，在多大程度上 uORF在不同条件下在细胞中翻译，uORF在进化上保守的程度， 以及特定uORF的翻译如何以及为什么控制基因表达，或者它们的翻译是否服务于其他目的， 功能，不为人知。为了帮助弥合这些差距，我们将确定一个新发现的 保守的真菌uORF肽命名为肌醇调节肽（IRP）。我们的数据表明， 调节合成重要分子肌醇所必需的第一种酶的表达。我们 我知道IRP虽然起源于真菌，但可以调节哺乳动物细胞和真菌中的报告基因 粗糙脉孢菌（Neurospora crassa），我们首先在其中发现了它。利用真菌和哺乳动物的无细胞翻译系统， 我们获得了IRP控制翻译的直接证据。我们将使用体内和体外方法 以确定IRP功能的作用机制和生理后果。在目标1中，我们将 功能分析以确定IRP的作用机制。在目标2中，我们将执行结构分析 以确定IRP的作用机制。成功完成拟议工作将提供 机械信息，应该大大增加我们对翻译控制机制的理解 一般来说都很重要。它将为调节和代谢途径提供新的见解， 重要的是发展战略，操纵新陈代谢，以改善人类健康和福利。

Summary Translation of mRNA is a central cellular process, but the mechanisms that control it are not fully understood. Up to 50% of eukaryotic mRNAs contain predicted upstream open reading frames (uORFs), but the roles of the vast majority of these uORFs remain undetermined. In some cases, we know that this additional translational capacity is evolutionarily conserved and serves critical functions in controlling gene expression. A special class of these conserved uORFs encodes peptides that stall protein synthesis in response to the presence of small metabolites. These nascent regulatory peptides act within the ribosome tunnel to arrest translation; by doing so, they control the production of enzymes important in metabolism. However, there remain important gaps in knowledge of the functions of uORFs. First, the mechanisms by which uORF-encoded peptides recognize small molecules and stall eukaryotic ribosomes to control gene expression remain unclear. Second, the extent to which uORFs are translated in cells under different conditions, the extent to which uORFs are evolutionarily conserved, and how and why the translation of particular uORFs controls gene expression, or if their translation serves other functions, is not known. To help bridge these gaps, we will determine the functions of a newly discovered conserved fungal uORF peptide named the inositol regulatory peptide (IRP). Our data indicate that the IRP regulates the expression of the first enzyme necessary for the synthesis of the important molecule inositol. We know that the IRP, while fungal in origin, can regulate reporter genes in mammalian cells as well as the fungus Neurospora crassa, in which we first discovered it. Using fungal and mammalian cell-free translation systems, we obtained direct evidence for translational control by the IRP. We will use both in vivo and in vitro approaches to determine the mechanism of action and physiological consequences of IRP function. In Aim 1, we will perform functional analyses to determine the mechanism of action of the IRP. In Aim 2, we will perform structural analyses to determine the mechanism of action of the IRP. Successful completion of the proposed work will provide mechanistic information that should significantly increase our understanding of translational control mechanisms that are generally important. It will provide new insights into regulatory and metabolic pathways that could be important for developing strategies to manipulate metabolism to improve human health and welfare.