Department of Epigenetics and Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Department of Medical Microbiology and Immunology, and Program in Bioinformatics, The University of Toledo College of Medicine and Life Sciences, Toledo, Ohio, USA.
J Biol Chem. 2021 Jan-Jun;296:100270. doi: 10.1016/j.jbc.2021.100270. Epub 2021 Jan 9.
RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3' UTR of the S-adenosyl-l-methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h μM on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h μM acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity (K) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both (K >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4-9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.
各种 RNA 种类（mRNA、tRNA、rRNA、非编码 RNA）的 RNA 甲基化生成一系列修饰核苷酸，包括 N6-甲基腺苷。在这里，我们研究了三种人类 RNA 甲基转移酶的酶学特性，这些酶在不同的环境中甲基化腺苷的氨基，具体情况包括：mRNA 帽结构后的第一个转录核苷酸（PCIF1）、18S rRNA 的第 1832 位（MettL5-Trm112 复合物）以及 S-腺苷甲硫氨酸合成酶（MettL16）3'UTR 中的发夹结构。在这三种酶中，催化效率从 PCIF1 开始，其对 mRNA 帽类似物的最高周转率超过 230 h μM，而 MettL16 的效率最低，约为 3 h μM，作用于 RNA 发夹。PCIF1 和 MettL5 对 SAM 和 RNA 这两种底物的结合亲和力（K）均约为 1 μM 或更低，而 MettL16 对这两种底物的结合亲和力均显著降低（K >0.4 mM 用于 SAM，K >10 μM 用于 RNA）。这三种酶在较宽的 pH 范围（约 5.4-9.4）内均具有活性，并且对离子强度具有不同的偏好。200 mM 氯化钠明显降低了 MettL5-Trm112 复合物的甲基化活性，而 MettL16 在 200 至 500 mM NaCl 范围内具有更高的活性。锌离子抑制了所有三种酶的活性。综上所述，这些结果说明了 RNA 腺苷甲基转移酶在其酶学机制和底物特异性方面的多样性，并强调了在研究中优化测定方法的必要性。
