The importance of m⁶A topology in chicken embryo mRNA: a precise mapping of m⁶A at the conserved chicken -actin zipcode

N6-methyladenosine (m(6)A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m(6)A-specific methylated RNA immuno-precipitation with next-generation sequencing (MeRIPSeq) or m(6)A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m(6)A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m(6)A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m(6)A site in the chicken beta-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the beta-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m(6)A may play a role in regulating localized translation of beta-actin mRNA, and the ability of m(6)A to enhance or inhibit a reader protein's RNA binding highlights the importance of m(6)A detection at nucleotide resolution.