Shimanski Brittany, Marin Juan F, Warminski Marcin, McKeon Riley M, Kowalska Joanna, Jemielity Jacek, Hadden-Perilla Jodi A, Mugridge Jeffrey S
Department of Chemistry & Biochemistry, University of Delaware, Newark, DE 19716, United States.
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 02-089 Warsaw, Poland.
bioRxiv. 2025 May 10:2025.05.09.653100. doi: 10.1101/2025.05.09.653100.
The RNA demethylase FTO acts as a methyl 'eraser' to remove either internal -methyladenosine (mA) or 5' end -2'-O-dimethyladenosine (mA) modifications on mRNA. FTO has an intrinsic preference and significantly faster demethylation rates for mA modifications located at the 5' mRNA cap structure, but the structural basis for FTO's ability to discriminate mA versus mA modifications has remained unknown. Here we utilize molecular dynamics simulations of FTO-RNA cap complexes to identify conserved aromatic residues on the surface of FTO involved in 5' cap recognition. Subsequent mutagenesis and enzymology experiments validate the specificity of these residues in engaging the 5' cap structure to promote mA demethylation. We also identify a nonpolar surface on FTO that interacts with the 2'-O-methyl group of mA to impact demethylation kinetics. This work provides the first structural insights into how FTO selectively catalyzes mA versus mA demethylation on mRNA, suggests why FTO is sensitive to different 5' cap modifications, and furthers our understanding of how FTO activity is regulated by diverse mechanisms to help control the epitranscriptome.
RNA去甲基化酶FTO作为一种甲基“擦除器”，可去除mRNA上的内部N6-甲基腺苷（m6A）或5'端N6,2'-O-二甲基腺苷（m6Am）修饰。FTO对位于5'mRNA帽结构处的m6A修饰具有内在偏好且去甲基化速率明显更快，但FTO区分m6A与m6Am修饰的结构基础仍不清楚。在此，我们利用FTO-RNA帽复合物的分子动力学模拟来鉴定FTO表面参与5'帽识别的保守芳香族残基。随后的诱变和酶学实验验证了这些残基与5'帽结构结合以促进m6A去甲基化的特异性。我们还在FTO上鉴定出一个与m6Am的2'-O-甲基相互作用以影响去甲基化动力学的非极性表面。这项工作首次提供了关于FTO如何在mRNA上选择性催化m6A与m6Am去甲基化的结构见解，揭示了FTO为何对不同的5'帽修饰敏感，并进一步加深了我们对FTO活性如何通过多种机制进行调节以帮助控制表观转录组的理解。
