Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs.
Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs.
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DOI：
10.1016/j.jbc.2022.101751
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Cheng X
中科院分区：
文献类型：
--
作者：
Yu D;Dai N;Wolf EJ;Corrêa IR Jr;Zhou J;Wu T;Blumenthal RM;Zhang X;Cheng X
磷酸化RNA聚合酶II CTD相互作用因子1（PCIF 1）是一种甲基转移酶，当Am是帽近端核苷酸时，将甲基添加到2′ O-甲基腺苷（Am）的N6位，生成N6，2′ O-二甲基腺苷（m6 Am）。此外，PCIF 1对内部腺苷（A和Am）具有辅助甲基化活性，尽管相对于其优选的帽底物具有低得多的催化效率。与未修饰的A核苷相比，PCIF 1对2′ O-甲基化Am的偏好主要是由于对Am的结合亲和力增加。重要的是，最近报道PCIF 1可以甲基化病毒RNA。尽管一些病毒RNA可以在没有帽的情况下翻译，但目前还不清楚PCIF 1修饰在病毒RNA的功能中可能发挥什么作用。在这里，我们使用结合和甲基转移的体外测定表明，PCIF 1以与帽类似物大致相同的亲和力（KM = 0.4 vs 0.3 μM）结合未加帽的5′-Am寡核苷酸。此外，PCIF 1甲基化未封端的5′-Am，与其优选的封端底物相比，活性仅降低5 - 6倍。我们最后讨论了PCIF 1催化的RNA甲基化之间的关系，这里显示出比以前认识到的具有更广泛的底物特异性，以及RNA去甲基化酶脂肪质量和肥胖相关蛋白（FTO），这表明PCIF 1对加帽RNA的相反活性。
The phosphorylated RNA polymerase II CTD interacting factor 1 (PCIF1) is a methyltransferase that adds a methyl group to the N6-position of 2′O-methyladenosine (Am), generating N6, 2′O-dimethyladenosine (m6Am) when Am is the cap-proximal nucleotide. In addition, PCIF1 has ancillary methylation activities on internal adenosines (both A and Am), although with much lower catalytic efficiency relative to that of its preferred cap substrate. The PCIF1 preference for 2′O-methylated Am over unmodified A nucleosides is due mainly to increased binding affinity for Am. Importantly, it was recently reported that PCIF1 can methylate viral RNA. Although some viral RNA can be translated in the absence of a cap, it is unclear what roles PCIF1 modifications may play in the functionality of viral RNAs. Here we show, using in vitro assays of binding and methyltransfer, that PCIF1 binds an uncapped 5′-Am oligonucleotide with approximately the same affinity as that of a cap analog (KM = 0.4 versus 0.3 μM). In addition, PCIF1 methylates the uncapped 5′-Am with activity decreased by only fivefold to sixfold compared with its preferred capped substrate. We finally discuss the relationship between PCIF1-catalyzed RNA methylation, shown here to have broader substrate specificity than previously appreciated, and that of the RNA demethylase fat mass and obesity-associated protein (FTO), which demonstrates PCIF1-opposing activities on capped RNAs.
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