The Role of Inosine Monophosphate Dehydrogenase in mRNA Regulation: Identification of mRNAs Bound and Functional Consequences

项目摘要/摘要 肌苷单磷酸脱氢酶(IMPDH)催化鸟嘌呤核苷酸的限速步骤 合成，因此对正常的细胞功能至关重要。事实上，人类IMPDH(IMPDH1和IMPDH)突变 已知会导致疾病，如常染色体显性遗传性视网膜色素变性(ADRP)。这种酶 它还有一个令人兴奋的、未知的功能：它结合信使核糖核酸。拟议项目的长期目标是澄清 未鉴定的mRNA结合蛋白的功能和机制揭示新的基因原理 对人类疾病的表达和信息研究。这个应用程序的总体目标是描述 IMPDH在mRNA结合和调控中的作用，并确定ADRP引起的突变对 那个活动。中心假设是，IMPDH与选定的一组mRNAs结合，并调节它们的翻译。 这一假说的提出是因为：1)酵母IMPDH酶被确凿地鉴定为mrna结合。 与多个mRNAs结合的蛋白质；2)已发表的数据表明，人类IMPDH在翻译过程中存在 3)我们的初步数据显示了对蛋白质水平的影响，从而支持在蛋白质合成中发挥作用。 这一建议背后的理论基础是理解核苷酸生物合成和基因之间的联系 表达将加深我们对中央生物途径的理解，并支持新的应对策略 包括遗传性失明在内的疾病。核心假设将通过追求三个具体目标来检验：1) 确定酵母IMPDH酶结合的mRNAs；2)确定IMPDH如何调节mRNA生物学；以及3) 描述由adrp引起的突变对信使核糖核酸调节的破坏。在第一个目标下，RIP-seq 将对所有三种酵母IMPDH酶(Imd2、Imd3和Imd4)进行检测，以鉴定结合的mRNAs。 对于第二个目标，将使用以下方法确定IMPDH结合对mRNA稳定性和翻译的影响 Northern印迹和Western印迹分析报告基因和内源基因的调控 IMPDH的结合伙伴。我们将通过重复这些检测来研究mrna的调控机制。 在缺乏翻译和/或信使核糖核酸衰变关键调节因子的酵母菌株中。对于第三个目标，已知的突变 导致adrp将被插入酵母基因组并对mRNA的结合和调节产生影响 用RIP-seq、Western blotting和Northern blotting观察。拟议的研究是创新的，在 申请人的意见，因为这代表着实质上背离现状，把提高妇女地位方案作为 信使核糖核酸的功能调控和结合信使核糖核酸的同一性及其后果的研究 基因表达的相互作用。这项拟议的研究具有重要意义，因为它将揭示一种新的 并定义了一种未知的关键酶IMPDH的功能。这项工作将由 PI和一个专门的本科生研究团队，为以下项目提供变革性的研究经验 下一代科学家。

Project Summary/Abstract Inosine Monophosphate Dehydrogenase (IMPDH) catalyzes the rate limiting step in guanine nucleotide synthesis and is thus critical for proper cellular function. Indeed, mutations in human IMPDH (IMPDH1 and IMPDH2) are known to cause diseases, such as autosomal dominant Retinitis Pigmentosa (adRP). This enzyme also has an exciting, unexplored function: it binds mRNA. The long-term goal of the proposed project is to elucidate the functions and mechanisms of uncharacterized mRNA binding proteins to reveal new principles of gene expression and inform research on human disease. The overall objective for this application is to characterize the role of IMPDH in binding and regulation of mRNA, and to identify the impact that adRP causing mutations have on that activity. The central hypothesis is that IMPDH binds to a select group of mRNAs and regulates their translation. This hypothesis has been formulated because: 1) yeast IMPDH enzymes were robustly identified as mRNA binding proteins that bind to multiple mRNAs; 2) published data demonstrated the presence of human IMPDH in translating complexes; and 3) our preliminary data shows effects on protein levels, thus supporting a role in protein synthesis. The rationale behind this proposal is that understanding the connection between nucleotide biosynthesis and gene expression will deepen our understanding of central biological pathways and support new strategies to address diseases including inherited blindness. The central hypothesis will be tested by pursuing three specific aims: 1) Identify mRNAs bound by yeast IMPDH enzymes; 2) Determine how IMPDH regulates mRNA biology; and 3) Characterize disruptions to mRNA regulation caused by adRP causing mutations. Under the first aim, RIP-seq will be performed on all three of the yeast IMPDH enzymes (Imd2, Imd3, and Imd4) to identify bound mRNAs. For the second aim, consequences of IMPDH binding on mRNA stability and translation will be identified using northern blotting and western blotting to analyze regulation of both a reporter mRNA and endogenous mRNA binding partners of IMPDH. The mechanism of mRNA regulation will be investigated by repeating these assays in yeast strains deficient in key regulators of translation and/or mRNA decay. For the third aim, mutations known to cause adRP will be inserted into the yeast genome and consequences on mRNA binding and regulation will be observed using RIP-seq, western blotting, and northern blotting. The proposed research is innovative, in the applicant’s opinion, because it represents a substantive departure from the status quo by focusing on IMPDH as a regulator of mRNA function and investigating the identity of bound mRNAs as well as the consequences of this interaction on gene expression. The proposed research is significant because it will reveal a new mechanism of mRNA regulation and define an unexplored function of the key enzyme IMPDH. This work will be performed by the PI and a team of exclusively undergraduate researchers, providing transformative research experiences for the next generation of scientists.