N6-methyladenosine (m6A) has been demonstrated to be ubiquitous in several types of eukaryotic RNAs, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), and small nuclear RNA (snRNA)1. The recent discoveries of RNA m6A methyltransferase complex METTL3/METTL14/WTAP and demethylases FTO and ALKBH5 prove the reversibility of m6A modification2,3,4,5,6. This modification plays important roles in various biological processes, including circadian rhythms7, RNA splicing8, yeast meiosis9, and embryonic stem cell self-renewal10. Two recent studies show that YTH domain family 2 (YTHDF2) and other YTHDF proteins preferentially bind to m6A-containing mRNA in vivo and in vitro and regulate localization and stability of the bound mRNA8,11. YTHDF2 is also known to be involved in development of acute myeloid leukemia12. YTHDC1 (splicing factor YT521-B), another YTH domain-containing protein, is known to play an important role in Emery-Dreifuss muscular dystrophy. While the function of YTHDF2 in the regulation of mRNA stability has been explored, the molecular mechanism for specific recognition of m6A by the YTH domain remains elusive.

To verify the specific recognition of m6A-RNA by YTHYTHDF2, we performed fluorescence polarization (FP) assays and electrophoretic mobility shift assays (EMSA) using purified YTHYTHDF2, unmodified RNA (A-RNA) and m6A-RNA. Interestingly, it appears that YTHYTHDF2 binds to A-RNA in a one-step binding mode (with the goodness-of-fit R2 value of 0.9943), but to m6A-RNA in a two-step binding mode (with R2 of 0.9987, while the R2 value is 0.9843 in one-step binding mode.) (Figure 1B, Supplementary information, Figure S1A and Table S1A). Both FP and EMSA experiments show that YTHYTHDF2 bound to m6A-RNA with much higher binding affinity than to A-RNA (Figure 1B and 1C). Note that 30 pmol of YTHYTHDF2 led to an almost complete shift of m6A-RNA (lane 8), while 100 pmol of YTHYTHDF2 only shifted less than half of the A-RNA (lane 5) (Figure 1C). A similar two-step binding mode was observed in other RNA-binding proteins13. It is likely that binding to RNA facilitates the further recognition of m6A.

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