Principles of mRNA targeting via the Arabidopsis m ⁶ A-binding protein ECT2

Specific recognition of N6 -methyladenosine (m ⁶ A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH-domain protein ECT2 is thought to bind to mRNA at URU(m ⁶ A)Y sites, yet RR(m ⁶ A)CH is the canonical m ⁶ A consensus site in all eukaryotes and ECT2 functions require m ⁶ A binding activity. Here, we apply iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) 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(m ⁶ A)CH. Pyrimidine-rich motifs are enriched around, but not at m ⁶ A-sites, reflecting a preference for N6 -adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m ⁶ A 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 m ⁶ A-YTH function in plants, and reveals new insight into the mode of RNA recognition by YTH-domain-containing proteins.