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.

项目摘要/摘要 肌苷一磷酸脱氢酶（IMPDH）催化鸟嘌呤核苷酸的速率限制步骤 合成，因此对于正确的细胞功能至关重要。确实，人类Impdh中的突变（impdh1和 已知IMPDH2）会引起疾病，例如色素体炎（ADRP）。这种酶 还具有令人兴奋的意外功能：它结合mRNA。拟议项目的长期目标是阐明 未表征mRNA结合蛋白的功能和机制揭示了基因的新原理 表达并告知人类疾病的研究。该应用程序的总体目的是表征 IMPDH在mRNA的结合和调节中的作用，并确定引起突变对ADRP的影响 那个活动。中心假设是IMPDH与精选的mRNA结合并调节其翻译。 该假设已被提出，因为：1）酵母菌IMPDH酶被牢固地识别为mRNA结合 与多个mRNA结合的蛋白质； 2）已发布的数据证明了人类在翻译时的存在 复合物； 3）我们的初步数据显示对蛋白质水平的影响，从而支持在蛋白质合成中的作用。 该提议背后的理由是了解核苷酸生物合成与基因之间的联系 表达将加深我们对中央生物学途径的理解，并支持新的策略来解决 包括遗传失明在内的疾病。中央假设将通过追求三个具体目标来检验：1） 鉴定由酵母菌IMPDH酶结合的mRNA； 2）确定IMPDH如何调节mRNA生物学； 3） 表征由ADRP引起突变引起的mRNA调节的中断。在第一个目标下，Rip-seq 将对所有三种酵母IMPDH酶（IMD2，IMD3和IMD4）进行识别mRNA。 对于第二个目标，将使用IMPDH结合对mRNA稳定性和翻译的后果。 北印迹和蛋白质印迹以分析记者mRNA和内源mRNA的调节 Impdh的约束伙伴。通过重复这些测定法，将研究mRNA调节的机制 在酵母菌菌株中缺乏翻译和/或mRNA衰减的关键调节剂。对于第三目的，突变已知 引起ADRP将插入酵母基因组中，对mRNA结合和调节的后果将 可以使用RIP-Seq，Western印迹和Northern印迹观察。拟议的研究是创新的，在 申请人的意见，因为它通过专注于Impdh作为现状的实质性偏离了现状 mRNA功能的调节剂，并研究了绑定mRNA的身份以及此的后果 基因表达的相互作用。拟议的研究很重要，因为它将揭示出一种新的机制 mRNA调节并定义了关键酶ImPDH的意外功能。这项工作将由 PI和由本科研究人员组成的团队，为变革性研究经验 下一代科学家。