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

• Published in: eLife 2021-09, Vol.10
• Main Authors: Arribas-Hernández, Laura, Rennie, Sarah, Köster, Tino, Porcelli, Carlotta, Lewinski, Martin, Staiger, Dorothee, Andersson, Robin, Brodersen, Peter
• Format: Article
• Language: English
• Subjects: Adenosine; Arabidopsis; Binding sites; Competition; ECT2; Gene regulation; Genetics and Genomics; Homology; hyperTRIBE; iCLIP; Immunoprecipitation; m6A; motif; mRNA; N6-methyladenosine; Phase transitions; Plant Biology; Proteins; RNA-binding protein; RRACH
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.72375

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 ( i ndividual nucleotide resolution c ross l inking and i mmuno p recipitation) and HyperTRIBE ( t argets of R NA-binding proteins i dentified b y e 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(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. Genes are strings of genetic code that contain instructions for producing a cell’s proteins. Active genes are copied from DNA into molecules called mRNAs, and mRNA molecules are subsequently translated to create new proteins. However, the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA. Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production. One of these methods involves adding a chemical tag, a methyl group, onto mRNA while it is being created. These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation. Most eukaryotic species – which include animals, plants and fungi – use the same system to add methyl tags to mRNA molecules. One methyl tag in particular, known as m 6 A, is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants. However, in the fl