Institute of Biochemistry, Department of Chemistry, University of Münster, 48149 Münster, Germany.
RNA. 2019 Oct;25(10):1311-1323. doi: 10.1261/rna.070706.119. Epub 2019 Jul 1.
-methyladenosine (mA) is the most common internal modification in eukaryotic mRNA and associated with numerous cellular processes in health and disease. Up- and down-regulation of its "writer" or "eraser" proteins alter the global mA level; however, modifying distinct mA sites has remained elusive. We genetically fused the dioxygenase FTO responsible for mA demethylation to RCas9 as an RNA-targeting module. The resulting RCas9-FTO retained demethylation activity and bound to RNA in a sequence-specific manner depending on the sgRNA and PAMmer. Using SCARLET analysis, we quantified the mA level at a specific site and analyzed the effect of the PAM-to-mA distance on activity. Sequence-specific demethylation by RCas9-FTO was tested on different RNA combinations and showed up to 15-fold sequence preference for target RNA compared to off-target RNA. Taken together, RCas9-FTO represents a new tool for sequence-specific demethylation of mA in RNA that can be readily adapted to any given RNA sequence and opens the door to studying the function of distinct mA sites.
N6-甲基腺苷（m6A）是真核 mRNA 中最常见的内部修饰，与健康和疾病中的许多细胞过程有关。其“书写者”或“擦除者”蛋白的上调和下调会改变全局 m6A 水平；然而，修饰不同的 m6A 位点仍然难以捉摸。我们将负责 m6A 去甲基化的双加氧酶 FTO 基因融合到作为 RNA 靶向模块的 RCas9 中。所得的 RCas9-FTO 保留了去甲基化活性，并根据 sgRNA 和 PAMmer 以序列特异性的方式结合到 RNA 上。我们使用 SCARLET 分析在特定位置定量 m6A 水平，并分析 PAM 到 m6A 距离对活性的影响。RCas9-FTO 对不同的 RNA 组合进行了序列特异性去甲基化测试，与非靶 RNA 相比，对靶 RNA 的序列偏好性高达 15 倍。总之，RCas9-FTO 代表了一种用于 RNA 中 m6A 序列特异性去甲基化的新工具，可轻松适用于任何给定的 RNA 序列，并为研究不同 m6A 位点的功能开辟了道路。
