N6-methylations of adenine are recognized explicitly by proteins using adjacent aromatic amino acid side chains

N6-methyladenosine (m6A) is the most abundant nucleotide modification in eukaryotic mRNA. Changes in the levels of m6A are implicated in many human diseases and conditions such as various types of cancer, diabetes, neurological diseases, male infertility, and obesity. However, molecular mechanisms for m6A recognition by various readers are not well. We used phage display to identify key sequence requirements of methyl readers for m6A recognition. The RNA sequence as well as the secondary structure in which m6A is located was shown to influence its recognition by methyl readers. Further, adjacent aromatic residues are involved in m6A recognition, possibly in a manner similar to that of YTH-domain containing m6A readers. RNA pulldown assays identified many candidates that could possibly be novel m6A readers. Among them, hnRNPA1 was identified as a promising m6A reader. Alignment of hnRNAPA1 along with the known m6A readers of hnRNP family of proteins against the most abundant peptide selected from phage display, shows the conservation of adjacent aromatic residues. These aromatic residues are located in between the regions known to be important for RNA binding by hnRNPA1. We believe these aromatic residues and m6A increase the specificity of RNA recognition by hnRNPA1. This information on the evolutionary pressure exerted by m6A on its readers can be used to discover novel m6A readers