Principles of mRNA targeting via the Arabidopsis m⁶A-binding protein ECT2

Specific recognition of N6-methyladenosine (m⁶A) 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(m⁶A)Y sites, yet RR(m⁶A)CH is the canonical m⁶A consensus site in all eukaryotes and ECT2 functions require m⁶A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking 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(m⁶A)CH. Pyrimidine-rich motifs are enriched around, but not at m⁶A 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 m⁶A 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 m⁶A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.