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, whilst methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localised 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.

Direct m6A recognition by IMP1 underlays an alternative model of target selection for non-canonical methyl-readers

“…This reinforces a model where the selectivity of m6A regulation of IMP1 likely depends on multiple variables, and, rather than providing an on/off molecular switch, enhances target selectivity in specific cellular settings. Consistent with this hypothesis, while finalizing this work, a manuscript was published proposing that, as for other m6A regulators, the in cell structure of the RNA may also play a role in the interaction ( 62 ).…”