RNA-coupled Coenzymes

烟酰胺腺嘌呤二核苷酸(NAD)是一种研究广泛、重要的 辅酶，参与许多关键的生物氧化还原反应。氧化型NAD (NAD+)接受两个电子，而它的还原形式(NADH)给一个 基材种类繁多。最近，NAD已被确定为一种非规范的启动 原核和真核RNA中的核苷酸(NCIN)。因为焦磷酸盐连接 NAD中的腺嘌呤和烟酰胺核苷酸，这样的NCIN类似于7- 大多数真核信使RNA的5‘端都有甲基鸟苷帽。这个 RNA始于NAD的原因尚不清楚，但该领域目前的范式 假设NCIN主要存在于调节RNA功能(即，它们是 “表位转录修改”)。这项提议检验了另一个假设，即 主张相反的情况是正确的，即需要RNA成分来调节 辅酶活性。如果RNA影响辅酶活性，那么由 利用NAD的酶应该以不同的速度或程度进行，并且 RNA的序列会影响反应的进行。第一个预测将通过以下方式进行验证 比较不同酶利用NAD+-、NADP+-、NADH-、NADPH-的能力 限制了RNA使用游离二核苷酸的能力。选择用于分析的酶 3-磷酸甘油醛脱氢酶、乳酸脱氢酶、 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、 多聚(ADP-核糖)聚合酶和依赖NAD的脱乙酰酶。每一种酶 将更详细地研究与Caped-RNA的反应，以估计RNA亲和力和 RNA特异性。亲和力将使用酶动力学和直接结合进行监测 化验。特异性将使用先前报道的细胞库进行研究 NAD-帽RNA，并通过对与每个RNA共同免疫沉淀的RNA进行测序 酵素。初步结果显示，乳酸脱氢酶使用NAD封端的RNA作为辅酶， 这表明这可能是第一个表明NCIN可以参与细胞内 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.