Base editing mechanisms are being investigated as potential therapeutic tools to alleviate genetic diseases. Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine base editing tools are capable of altering RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyzes C-to-U edits and likely uses the canonical deamination mechanism. Proteins developed from consensus sequences from the DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into mechanistic insights into U-to-C editing. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids . Despite addition of 14 different putative amine/amide donors , U-to-C editing could not be observed. C-to-U editing was found not to be concomitant with U-to-C editing, thus discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were covalently crosslinked to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed an additional mass of 319 Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319 Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA–protein conjugate. Sequenced RT-PCR products from the fern indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

中文翻译：

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合成 DYW-KP 碱基编辑器交联碱基修饰的 RNA 表明酶促赖氨酸作为 U 至 C RNA 编辑的氮供体


碱基编辑机制正在被研究作为减轻遗传疾病的潜在治疗工具。序列特异性胞苷至尿苷（C 至 U）和腺苷至肌苷碱基编辑工具能够改变 RNA 和 DNA 序列，并利用需要活性位点锌离子和谷氨酸残基的水解脱氨机制。在植物细胞器中，含有 DYW-PG 结构域的酶催化 C 到 U 编辑，并可能使用规范的脱氨基机制。从 DYW-KP 结构域家族的共有序列开发的蛋白质催化了最初看似尿苷到胞苷（U-to-C）的编辑，从而导致了对 U-to-C 编辑机制的研究。发现合成的 DYW-KP 酶 KP6 足以进行通过添加羧酸刺激的 C-to-U 编辑活性。尽管添加了 14 种不同的推定胺/酰胺供体，但无法观察到 U-to-C 编辑。发现C-to-U编辑与U-to-C编辑并不同时进行，因此忽略了嘧啶转氨酶机制。含有碱基修饰的 RNA 与 KP6、KP2 和 KP3 蛋白共价交联。纯化的 KP2 和 KP6 蛋白的质谱分析显示额外质量为 319 Da。 U-to-C 交联机制预计可以解释交联、RNA 碱基变化和~319 Da 质量之间的联系。在此模型中，酶促赖氨酸攻击尿苷的 C4，形成席夫碱 RNA-蛋白质缀合物。来自蕨类植物的 RT-PCR 测序产物表明，U-to-C 碱基编辑不会保留植物中的蛋白质交联。假设质子化席夫碱缀合物水解释放胞苷来解释植物中的完整途径。