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

项目概要/摘要 肌苷一磷酸脱氢酶（IMPDH）催化鸟嘌呤核苷酸的限速步骤 合成，因此对适当的细胞功能至关重要。事实上，人IMPDH（IMPDH 1和IMPDH 2）中的突变是不稳定的。 已知IMPDH 2）引起疾病，例如常染色体显性色素性视网膜炎（adRP）。这种酶 它还有一个令人兴奋的未被探索的功能：它与mRNA结合。该项目的长期目标是阐明 未鉴定的mRNA结合蛋白的功能和机制，以揭示基因表达的新原理。 对人类疾病的表达和信息研究。本申请的总体目标是表征 IMPDH在mRNA结合和调节中的作用，并确定引起突变的adRP对 这种活动。核心假设是IMPDH与一组选定的mRNA结合并调节其翻译。 这一假设已经被制定，因为：1）酵母IMPDH酶被稳健地鉴定为mRNA结合 2）已发表的数据表明，在翻译过程中存在人IMPDH， 复合物;和3）我们的初步数据显示对蛋白质水平的影响，从而支持蛋白质合成中的作用。 这一建议背后的基本原理是，了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心生物学途径的理解，并支持新的战略， 包括遗传性失明在内的疾病中心假设将通过追求三个具体目标来检验：1） 鉴定由酵母IMPDH酶结合的mRNA; 2）确定IMPDH如何调节mRNA生物学;和3） 表征由adRP引起的突变引起的mRNA调控的破坏。在第一个目标下，RIP-seq 将对所有三种酵母IMPDH酶（Imd 2、Imd 3和Imd 4）进行分析，以鉴定结合的mRNA。 对于第二个目的，将使用以下方法鉴定IMPDH结合对mRNA稳定性和翻译的影响： 北方印迹和西方印迹分析报告mRNA和内源mRNA的调节 IMPDH的结合伙伴。将通过重复这些测定来研究mRNA调节的机制 在翻译和/或mRNA衰变的关键调节因子缺陷的酵母菌株中。对于第三个目标，已知的突变 导致adRP将被插入酵母基因组，对mRNA结合和调节的后果将 使用RIP-seq、蛋白质印迹和北方印迹进行观察。该研究具有创新性， 申请人的意见，因为它代表了一个实质性的偏离现状，侧重于IMPDH， 一种mRNA功能的调节剂，并研究结合mRNA的身份以及这种作用的后果。 基因表达的相互作用。这项研究意义重大，因为它将揭示一种新的机制， mRNA的调节和定义一个未开发的功能的关键酶IMPDH。这项工作将由 PI和一个完全由本科生研究人员组成的团队，为以下人员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.