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

肌苷单磷酸脱氢酶在 mRNA 调节中的作用：鉴定 mRNA 结合和功能后果

• 批准号: 10796269
• 负责人: Sarah Fitzgerald Mitchell
• 金额: $ 42.49万
• 依托单位: LOYOLA MARYMOUNT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-11 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796269
• 关键词: Address; Anabolism; Area; Autoimmune Diseases; Binding; Binding Proteins; Biological; Biological Assay; Biology; Blindness; Cell Proliferation; Cell physiology; Cells; Complex; DNA; Data; Development; Disease; Enzymes; Gene Expression; Gene Expression Regulation; Goals; Guanine; Guanine Nucleotides; Health; Homeostasis; Human; Human Activities; IMPDH1 gene; IMPDH2 gene; Immune System Diseases; Inherited; Inosine Monophosphate; Knowledge; Link; Malignant Neoplasms; Measures; Messenger RNA; Metabolic; Mutation; Northern Blotting; Nucleic Acid Binding; Nucleotide Biosynthesis; Nucleotides; Outcome; Oxidoreductase; Pathway interactions; Play; Post-Transcriptional Regulation; Procedures; Process; Production; Protein Biosynthesis; Proteins; Public Health; Publishing; RNA immunoprecipitation sequencing; Regulation; Reporter; Research; Research Personnel; Retina; Retinitis Pigmentosa; Rhodopsin; Role; Scientist; Surveys; System; Testing; Transcript; Translating; Translational Repression; Translations; Western Blotting; Work; Yeasts; anticancer research; autosome; cancer therapy; career; design; experience; experimental study; human disease; innovation; insight; mRNA Decay; mRNA Stability; mRNA Translation; mutant; nervous system disorder; next generation; nucleotide metabolism; protein expression; protein function; targeted treatment; undergraduate student; yeast genome

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.

项目概要/摘要 肌苷单磷酸脱氢酶 (IMPDH) 催化鸟嘌呤核苷酸的限速步骤 事实上，人类 IMPDH（IMPDH1 和 IMPDH1）的突变对于细胞的正常功能至关重要。 IMPDH2）已知会引起疾病，例如常染色体显性遗传性视网膜色素变性（adRP）。 它还具有一个令人兴奋的、未开发的功能：它与 mRNA 结合。该项目的长期目标是阐明。 未表征的mRNA结合蛋白的功能和机制，揭示基因的新原理 该应用程序的总体目标是表征人类疾病的表达和信息研究。 IMPDH 在 mRNA 结合和调节中的作用，并确定 adRP 突变引起的影响 核心假设是 IMPDH 与一组选定的 mRNA 结合并调节它们的翻译。 提出这一假设的原因是：1) 酵母 IMPDH 酶被可靠地鉴定为 mRNA 结合 与多个 mRNA 结合的蛋白质；2) 已发表的数据证明在翻译过程中存在人 IMPDH 复合物；3）我们的初步数据显示了对蛋白质水平的影响，从而支持了其在蛋白质合成中的作用。 该提案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途径的理解，并支持新的策略来解决 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） 识别酵母 IMPDH 酶结合的 mRNA；2) 确定 IMPDH 如何调节 mRNA 生物学；3) 第一个目标是 RIP-seq，描述由 adRP 引起的突变对 mRNA 调控的破坏。 将对所有三种酵母 IMPDH 酶（Imd2、Imd3 和 Imd4）进行检测，以鉴定结合的 mRNA。 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 blotting 和 Western blotting 分析报告基因 mRNA 和内源 mRNA 的调控 IMPDH 的结合伙伴将通过重复这些测定来研究 mRNA 调节的机制。 在缺乏翻译和/或 mRNA 衰变的关键调节因子的酵母菌株中，第三个目标是已知的突变。 导致 adRP 将被插入到酵母基因组中，并对 mRNA 结合和调节产生影响 可以使用 RIP-seq、蛋白质印迹法和北方印迹法进行观察。 申请人的意见，因为它代表着对现状的实质性背离，将重点放在 IMPDH 上 mRNA 功能的调节剂并研究结合的 mRNA 的身份及其后果 所提出的研究具有重要意义，因为它将揭示一种新的机制。 mRNA 调节并定义关键酶 IMPDH 的未探索功能这项工作将由 PI 和一支完全由本科生研究人员组成的团队，为以下领域提供变革性的研究经验： 下一代科学家。