Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates
Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates
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FTO 多种 RNA 底物去甲基化催化机制的结构见解
DOI：
10.1073/pnas.1820574116
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发表时间：
2019-02-19
影响因子：
11.1
通讯作者：
Jia, Guifang
中科院分区：
文献类型：
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作者：
Zhang, Xiao;Wei, Lian-Huan;Jia, Guifang
FTO使内部N-6-甲基腺苷（m(6)A）和n -6,2'- o -二甲基腺苷（m(6)A）去甲基化；在体内和体外均有证据表明，它还能使n -6-甲基脱氧腺苷（(6)mA）、3-甲基胸腺嘧啶（3mT）和3-甲基尿嘧啶（m(3)U）去甲基化。然而，目前尚不清楚FTO如何识别和催化这些不同的底物。在这里，我们在体外和体内证明，FTO在内部m(6)A和cap m(6)A(m)上都具有广泛的去甲基化酶活性。考虑到(6)mA、m(6)A和m(6)A(m)都具有相同的核碱基，我们展示了人类FTO与(6)mA修饰的ssDNA结合的晶体结构，揭示了FTO对多种RNA底物催化去甲基化的分子基础。我们发现(i) n -6-甲基腺嘌呤是FTO最有利的核碱基底物，（ii） FTO对相同RNA序列中的m(6)A和m(6)A(m)具有相同的去甲基化活性，这表明FTO的底物特异性主要来自于催化口袋中残基与核碱基（而不是核糖环）的相互作用，以及（iii） RNA的序列和三级结构可以影响FTO的催化活性。我们的发现为理解FTO脱甲基化其多种底物的催化机制提供了结构基础，并为功能研究和潜在治疗应用的选择性化学品的结构指导设计铺平了道路。
FTO demethylates internal N-6-methyladenosine (m(6)A) and N-6,2'-O-dimethyladenosine (m(6)A(m); at the cap + 1 position) in mRNA, m(6)A and m(6)A(m) in snRNA, and N-1-methyladenosine (m(1)A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N-6-methyldeoxyadenosine ((6)mA), 3-methylthymine (3mT), and 3-methyluracil (m(3)U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m(6)A and cap m(6)A(m). Considering that (6)mA, m(6)A, and m(6)A(m) all share the same nucleobase, we present a crystal structure of human FTO bound to (6)mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N-6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m(6)A andm(6)A(m) in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.