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

Abstract

N6-methyladenosine (m⁶A) 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 m⁶A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m⁶A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m⁶A 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 m⁶A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m⁶A site in the chicken β-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 β-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m⁶A may play a role in regulating localized translation of β-actin mRNA, and the ability of m⁶A to enhance or inhibit a reader protein's RNA binding highlights the importance of m⁶A detection at nucleotide resolution.