Environmental Toxicology Graduate Program, University of California Riverside, Riverside, California 92521-0403, United States.
Department of Chemistry, University of California Riverside, Riverside, California 92521-0403, United States.
Anal Chem. 2022 Sep 20;94(37):12559-12564. doi: 10.1021/acs.analchem.2c03549. Epub 2022 Sep 9.
-Methyladenosine (mA) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. Aside from mA, RNA is known to carry many other types of chemical modifications; no systematic investigations, however, have been conducted about the crosstalk between mA and other modified nucleosides in RNA. Here, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding mA eraser proteins (i.e., , ) and the catalytic subunit of the major mA writer complex (i.e., ) being individually ablated. Notably, eight proteins, including writer proteins for 5-methylcytidine and pseudouridine, were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of and . Analysis of previously published mA mapping results revealed the presence of mA in the corresponding mRNAs for four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between mA and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.
甲基腺苷（mA）及其读写擦蛋白（RWE）在调节 mRNA 的剪接、稳定性和翻译中发挥着关键作用。除了 mA，RNA 已知还携带许多其他类型的化学修饰；然而，目前还没有关于 mA 与 RNA 中其他修饰核苷之间相互作用的系统研究。在这里，我们通过将稳定同位素标记（SIL）肽作为内标或替代标准纳入我们最近建立的液相色谱-平行反应监测（LC-PRM）方法，对其进行了修改，以分析 RNA 的转录后修饰 RWE 蛋白。我们能够在分别敲除 mA 擦除蛋白（即， 和 ）和主要 mA 写入复合物的催化亚基（即， ）的基因的 HEK293T 细胞中重复地检测到总共 114 种 RWE 蛋白。值得注意的是，在耗尽 和 后，八种蛋白质，包括 5-甲基胞嘧啶和假尿嘧啶的写入蛋白，其表达量发生了超过 1.5 倍的变化。在 HEK293T 细胞中，分析先前发表的 mA 映射结果表明，这四种蛋白质的相应 mRNA 中存在 mA。总的来说，我们将 SIL 肽整合到我们的 LC-PRM 方法中，用于定量转录后修饰 RWE 蛋白，我们的工作揭示了 mA 与其他转录后修饰之间的潜在相互作用。我们使用 SIL 肽修改后的 LC-PRM 方法应该适用于其他细胞类型和组织中转录后修饰 RWE 蛋白的高通量分析。
