Regulation of Mammalian mRNA Decay

项目摘要 MRNA稳定性的控制是真核生物转录后调控的关键决定因素 基因表达。即使是mRNA稳定性的微小变化也会产生深远的后果，并可能 表现为临床表型，表现为原癌基因异常表达的能力 这可能会导致恶性肿瘤。真核生物的mRNAs通常被认为含有N7甲基 鸟苷(M7G)在其5美分末端，以促进其稳定性和翻译。然而，我们最近 证明哺乳动物的mRNAs也可以携带5‘端烟酰胺腺嘌呤二核苷酸 (NAD)帽与m7G帽不同，它促进了mRNA的衰退，为mRNA提供了一个新的范式 5末端加工和核苷酸代谢产物在mRNA周转中的贡献。我们现在 证明NAD的氧化还原状态也可以通过自由NAD功能来调节3‘RNA的衰变 作为促进RNA衰变的辅因子，并潜在地提供了与细胞能量学的联系。此外， 黄素腺嘌呤二磷酸(FAD)也可以作为带有Nudt16和Nudt16的哺乳动物RNA的5‘帽。 在体外，DXO水解酶作为蛋白质发挥作用，可以去除FAD帽(DeFADding)。我们会 在三个具体目标内，在整个提案中建立在这些新发现的基础上。第一个将解决 游离NAD在体外控制3‘端RNA衰变中的作用及其分子描述 参与其刺激腐烂的机制。第二个将推断出mRNA衰变的变化作为 细胞内NAD水平改变的后果，并评估应激条件所赋予的调节作用 通过控制NAD水平来调节RNA的衰变。在最后一个目标中，我们将时尚帽设置为 一种替代的RNA帽，识别FAD帽的RNA并破译FAD帽和 细胞中的脱氢酶。总而言之，拟议的研究将提供对迄今 未知的基本转录后调控机制，将为 控制正常和疾病状态下基因表达的潜在新途径。

Project Summary The control of mRNA stability is a critical determinant in the post-transcriptional regulation of eukaryotic gene expression. Even minor alterations in mRNA stability can have profound consequences and may manifest as clinical phenotypes as illustrated by the ability of aberrantly expressed proto-oncogenes that can give rise to malignancies. Eukaryotic mRNAs are generally thought to possess an N7 methyl guanosine (m7G) cap at their 5¢ end to promote their stability and translation. However, our recent demonstration that mammalian mRNAs can also carry a 5´-end nicotinamide adenine dinucleotide (NAD) cap that in contrast to the m7G cap promotes mRNA decay, provides a new paradigm for mRNA 5´ end processing and the contribution of nucleotide metabolites in mRNA turnover. We now demonstrate that the redox state of NAD can also modulate 3´ RNA decay with free NAD functioning as a cofactor to enhance RNA decay and potentially providing a link to cellular energetics. Moreover, flavin adenine diphosphate (FAD) can also serve as a 5´ cap on mammalian RNAs with Nudt16 and DXO hydrolases functioning as proteins that can remove the FAD cap (deFADding) in vitro. We will build on these novel findings throughout this proposal within three specific aims. The first will address the functional role of free NAD on the control of 3´ end RNA decay in vitro and delineate the molecular mechanism involved in its stimulation of decay. The second will deduce changes in mRNA decay as a consequence of altered NAD levels in cells and assess the regulatory role imparted by stress conditions in modulating RNA decay through the control of NAD levels. In the last aim, we establish FAD cap as an alternative RNA cap, identify FAD-capped RNAs and decipher the role of FAD caps and the deFADding enzymes in cells. Collectively, the proposed studies will provide insight into a heretofore unknown fundamental post-transcriptional regulatory mechanism and will provide the framework for potential novel avenues to control gene expression in normal and disease states.