Specific recognition between YTHDF3 and m⁶A‐modified RNA: An all‐atom molecular dynamics simulation study

Abstract

The YTH domain of YTHDF3 belongs to a class of protein "readers" recognizing the N6-methyladenosine (m 6 A) modification in mRNA. Although static crystal structure reveals m 6 A recognition by a conserved aromatic cage, the dynamic process in recognition and importance of aromatic cage residues are not completely clear. Here, molecular dynamics (MD) simulations are performed to explore the issues and negative selectivity of YTHDF3 toward unmethylated substrate. Our results reveal that there exist conformation selectivity and induced-fit in YTHDF3 binding with m 6 Amodified RNA, where recognition loop and loop6 play important roles in the specific recognition. m 6 A modification enhances the stability of YTHDF3 in complex with RNA. The methyl group of m 6 A, like a warhead, enters into the aromatic cage of YTHDF3, where Trp492 anchors the methyl group and constraints m 6 A, making m 6 A further stabilized by π-π stacking interactions from Trp438 and Trp497. In addition, the methylation enhances the hydrophobicity of adenosine, facilitating water molecules excluded out of the aromatic cage, which is another reason for the specific recognition and stronger intermolecular interaction. Finally, the comparative analyses of hydrogen bonds and binding free energy between the methylated and unmethylated complexes reveal the physical basis for the preferred recognition of m 6 A-modified RNA by YTHDF3. This study sheds light on the mechanism by which YTHDF3 specifically recognizes m 6 A-modified RNA and can provide important information for structure-based drug design.

Characterization of epitranscriptome reader proteins experimentally and in silico: Current knowledge and future perspectives beyond the YTH domain

“…Although these binding experiments with the wild-type protein sequences demonstrate the selectivity of the human YTH proteins and–more specifically–their domains for m 6 A, the key regions responsible for this selectivity needed to be determined. To elucidate the protein features responsible for the selective recognition of m 6 A containing transcripts, crystal structures were resolved in tandem with binding characterization efforts for all human YTH domains [39] , [40] , [41] , [42] , [43] , [45] , [55] , [56] , [57] , [58] , [59] , [60] .…”

“…In addition to the work that has been performed to identify interacting proteins with m 6 A modified RNAs, similar mass spectrometry techniques have been used to identify proteins interacting with m 1 A [28] , m 5 C [29] , and 8-oxoG [30] ( Table 2 , [5] , [7] , [11] , [17] , [21] , [28] , [29] , [30] , [37] , [39] , [40] , [45] , [56] , [58] , [59] , [62] , [76] , [77] , [78] , [79] , [80] , [81] , [82] , [83] ). Stable isotope labeling by amino acid in cell culture (SILAC) [84] has been used to identify proteins interacting with m 1 A with a 34-mer RNA probe designed to carry a portion of the SOX18 gene known to be modified in vivo [28] , [36] , [85] .…”