Cytidine acetylation (ac4C) of RNA is a post-transcriptional modification catalyzed by Nat10. Recently, an approach termed RedaC:T was employed to map ac4C in human mRNA, relying on detection of C>T mutations in WT but not in Nat10-KO cells. RedaC:T suggested widespread ac4C presence. Here, we reanalyze RedaC:T data. We find that mismatch signatures are not reproducible, as C>T mismatches are nearly exclusively present in only one of two biological replicates. Furthermore, all mismatch types—not only C>T—are highly enriched in WT samples, inconsistent with an acetylation signature. We demonstrate that the originally observed enrichment in mutations in one of the WT samples is due to its low complexity, resulting in the technical amplification of all classes of mismatch counts. Removal of duplicate reads abolishes the skewed mismatch patterns. These analyses account for the irreproducible mismatch patterns across samples while failing to find evidence for acetylation of RedaC:T sites.

RNA 的胞苷乙酰化 (ac4C) 是 Nat10 催化的转录后修饰。最近，采用一种称为 RedaC:T 的方法来绘制人类 mRNA 中的 ac4C，依赖于检测 WT 中的 C>T 突变，而不是 Nat10-KO 细胞中的 C>T 突变。 RedaC:T 表明 ac4C 广泛存在。在这里，我们重新分析 RedaC:T 数据。我们发现错配特征是不可重复的，因为 C>T 错配几乎只存在于两个生物重复之一中。此外，所有错配类型（不仅是 C>T）在 WT 样品中高度富集，与乙酰化特征不一致。我们证明，最初观察到的 WT 样本之一中突变的富集是由于其复杂性低，导致所有类别的错配计数在技术上放大。删除重复读取消除了倾斜的不匹配模式。这些分析解释了样本之间不可重现的错配模式，但未能找到 RedaC:T 位点乙酰化的证据。