Principles of mRNA targeting via the <i>Arabidopsis</i> m⁶A-binding protein ECT2.

Abstract

Specific recognition of <i>N6</i>-methyladenosine (m⁶A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The <i>Arabidopsis</i> 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 (<i>i</i>ndividual nucleotide resolution <i>c</i>ross<i>l</i>inking and <i>i</i>mmuno<i>p</i>recipitation) and HyperTRIBE (<i>t</i>argets of <i>R</i>NA-binding proteins <i>i</i>dentified <i>b</i>y <i>e</i>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(m⁶A)CH. Pyrimidine-rich motifs are enriched around, but not at m⁶A sites, reflecting a preference for <i>N6</i>-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.