RNA-coupled Coenzymes

RNA偶联辅酶

• 批准号: 10359222
• 负责人: David N Frick
• 金额: $ 44.29万
• 依托单位: UNIVERSITY OF WISCONSIN MILWAUKEE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359222
• 关键词: Academic Research Enhancement Awards; Adenine; Affinity; Aging; Animals; Bacteria; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Blindness; Calorimetry; Catalytic RNA; Cells; Coenzyme A; Coenzymes; Coupled; DNA Ligases; DNA biosynthesis; Data; Deacetylase; Diabetes Mellitus; Dihydrofolate Reductase; Dinucleoside Phosphates; Diphosphates; Education; Electrons; Enzyme Kinetics; Enzymes; Epigenetic Process; Equilibrium; Fatty Liver; Future; Gene Expression; Genome; Genomics; Glucosephosphate Dehydrogenase; Glutamate Dehydrogenase; Glyceraldehyde-3-Phosphate Dehydrogenases; Goals; Heart failure; Histone Deacetylase; Hydrogenase; Hypertension; In Vitro; Institution; Isocitrate Dehydrogenase; Isocitrates; Kidney Diseases; Kinetics; Lactate Dehydrogenase; Libraries; Life; Ligase; Link; Literature; Liver diseases; Longevity; Maintenance; Malate Dehydrogenase; Measures; Messenger RNA; Metabolic; Metabolism; Modernization; Modification; Monitor; Mono(ADP-Ribose) Transferases; NADH; NADP; Nerve Degeneration; Niacinamide; Nicotinamide adenine dinucleotide; Nucleotides; Oxidation-Reduction; Oxides; Poly(ADP-ribose) Polymerases; Positioning Attribute; Process; Proteins; Pyruvate; RNA; RNA Binding; RNA Caps; RNA Sequences; Reaction; Reporting; Ribonucleoproteins; Role; Sirtuins; Specificity; Students; Techniques; Testing; Thermodynamics; Titrations; Untranslated RNA; Work; Yeasts; adenylate; alpha ketoglutarate; base; chemical reaction; cofactor; enzyme substrate; epitranscriptomics; exercise capacity; hearing impairment; ketoglutarate dehydrogenase; lactate dehydrogenase A; oxidation; protein function; pyruvate dehydrogenase; transcription factor; transcriptome sequencing; undergraduate student

Nicotinamide adenine dinucleotide (NAD) is a widely studied, important coenzyme, which is involved in many critical biological redox reactions. Oxidized NAD (NAD+) accepts two electrons while its reduced form (NADH) donates two electrons to a variety of substrates. Recently, NAD has been identified as a non-canonical initiating nucleotide (NCIN) in both prokaryotic and eukaryotic RNA. Since a pyrophosphate links the adenine and nicotinamide nucleotides in NAD, such an NCIN resembles the 7- methylguanylate cap found on the 5’ end of most eukaryotic messenger RNAs. The reason that RNA starts with NAD is not clear, but the current paradigm in the field assumes the NCINs primarily exist to modulate RNA function (i.e. they are “epitranscriptomic modifications”). This proposal examines another hypothesis that contends the opposite is true, i.e., that the RNA component is needed to modulate coenzyme activity. If the RNA influences coenzyme activity then reactions catalyzed by the NAD-utilizing enzymes should proceed at different rates or extents, and the sequence of the RNA should influence the reaction. The first prediction will be tested by comparing the ability of various enzymes to use NAD+-, NADP+-, NADH-, NADPH- capped RNA to their ability to use free dinucleotides. The enzymes chosen for analysis are glyceraldehyde 3-phosphate dehydrogenase, lactate dehydrogenase (LDH), pyruvate dehydrogenase, isocitrate dehydrogenase, a-ketoglutarate dehydrogenase; malate dehydrogenase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, dihydrofolate reductase, DNA ligase, mono-ADP ribosyltransferase, poly (ADP-ribose) polymerase, and NAD-dependent deacetylase. Each enzyme that reacts with capped-RNA will be examined in more detail to estimate the RNA affinity and RNA specificity. Affinity will be monitored using enzyme kinetics and direct binding assays. Specificity will be investigated using a library of previously reported cellular NAD-capped RNAs, and by sequencing RNAs that co-immuno-precipitate with each enzyme. Preliminary results show LDH uses NAD-capped RNA as a coenzyme, suggesting that this could be the first study to show NCINs can participate in cellular chemical reactions, and possibly form a new class of key ribonucleoproteins or ribozymes.

烟酰胺腺嘌呤二核苷酸（NAD）是一种研究广泛、重要的 辅酶，参与许多重要的生物氧化还原反应。氧化NAD (NAD+）接受两个电子，而其还原形式（NADH）则将两个电子提供给 各种基板。最近，NAD已被确定为非典型启动 核苷酸（NCIN）在原核和真核RNA。因为焦磷酸盐 NAD中的腺嘌呤和烟酰胺核苷酸，这样的NCIN类似于7- 在大多数真核生物信使RNA的5'末端发现的甲基鸟苷酸帽。的 RNA从NAD开始的原因尚不清楚，但该领域目前的范式 假设NCIN的存在主要是为了调节RNA功能（即它们是 “epitranscriptomic修饰”）。这一建议审查了另一个假设， 认为相反的是正确的，即，需要RNA成分来调节 辅酶活性如果RNA影响辅酶活性， 利用NAD的酶应该以不同的速率或程度进行， RNA的序列应该影响反应。第一个预测将由 比较各种酶利用NAD+-、NADP+-、NADH-、NADPH- 加帽的RNA与它们使用游离二核苷酸的能力有关。选择用于分析的酶 是甘油醛3-磷酸脱氢酶，乳酸脱氢酶（LDH）， 丙酮酸脱氢酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶; 苹果酸脱氢酶，谷氨酸脱氢酶，葡萄糖-6-磷酸 脱氢酶，二氢叶酸还原酶，DNA连接酶，单ADP核糖基转移酶， 聚（ADP-核糖）聚合酶和NAD依赖性脱乙酰酶。每种酶， 将更详细地检查与加帽RNA的反应，以估计RNA亲和力， RNA特异性将使用酶动力学和直接结合监测亲和力 分析。将使用先前报告的细胞库研究特异性 NAD加帽的RNA，并通过测序与每个RNA共免疫沉淀的RNA， 酵素初步结果显示LDH使用NAD加帽的RNA作为辅酶， 这表明这可能是第一项研究表明NCIN可以参与细胞凋亡， 化学反应，并可能形成一类新的关键核糖核蛋白， 核酶