Principles of mRNA targeting via the mA-binding protein ECT2.

Specific recognition of -methyladenosine (mA) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The YTH domain protein ECT2 is thought to bind to mRNA at URU(mA)Y sites, yet RR(mA)CH is the canonical mA consensus site in all eukaryotes and ECT2 functions require mA-binding activity. Here, we apply iCLIP (ndividual nucleotide resolution rossinking and mmunorecipitation) and HyperTRIBE (argets of NA-binding proteins dentified y diting) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(mA)CH. Pyrimidine-rich motifs are enriched around, but not at mA sites, reflecting a preference for -adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of mA sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of mA-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.