RNA-coupled Coenzymes

烟酰胺腺嘌呤二核苷酸（NAD）是一种被广泛研究的、重要的 辅酶，参与许多关键的生物氧化还原反应。氧化型NAD (NAD) 接受两个电子，而其还原形式 (NADH) 则将两个电子捐赠给 各种基材。最近，NAD 被确定为非规范起始物质。 原核和真核RNA中的核苷酸（NCIN）。由于焦磷酸盐链接 NAD 中的腺嘌呤和烟酰胺核苷酸，这样的 NCIN 类似于 7- 大多数真核生物信使 RNA 的 5' 末端都有甲基鸟苷酸帽。的 RNA 以 NAD 开头的原因尚不清楚，但该领域目前的范式 假设 NCIN 主要是为了调节 RNA 功能而存在（即它们是 “表观转录组修饰”）。该提案检验了另一个假设： 认为事实恰恰相反，即需要 RNA 成分来调节 辅酶活性。如果 RNA 影响辅酶活性，则由以下物质催化的反应 NAD利用酶应以不同的速率或程度进行，并且 RNA 的序列应该会影响反应。第一个预测将通过 比较各种酶利用NAD-、NADP-、NADH-、NADPH-的能力 将RNA加帽以限制其使用游离二核苷酸的能力。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 capped-RNA 的反应，以估计 RNA 亲和力和 RNA特异性。将使用酶动力学和直接结合来监测亲和力 化验。将使用先前报告的细胞库来研究特异性 NAD 加帽 RNA，并通过对与每个 RNA 共免疫沉淀的 RNA 进行测序 酶。初步结果表明LDH使用NAD加帽的RNA作为辅酶， 表明这可能是第一个表明 NCIN 可以参与细胞参与的研究 化学反应，并可能形成一类新的关键核糖核蛋白或 核酶。

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.