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

Baron, Francis; Zhang, Mi; Archer, Nathan; Bellows, Eleanor; Knight, Helen M.; Welham, Simon; Rutland, Catrin S.; Mongan, Nigel P.; Hayes, Chris J.; Fray, Rupert G.; Bodi, Zsuzsa

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.

Citation

Baron, F., Zhang, M., Archer, N., Bellows, E., Knight, H. M., Welham, S., …Bodi, Z. (2023). The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken β-actin zipcode. RNA, 29(6), 777-789. https://doi.org/10.1261/rna.079615.123

• Journal Article Type: Article
• Acceptance Date: Feb 6, 2023
• Online Publication Date: Feb 21, 2023
• Publication Date: 2023-06
• Deposit Date: Feb 23, 2023
• Publicly Available Date: Feb 23, 2023
• Journal: RNA
• Print ISSN: 1355-8382
• Electronic ISSN: 1469-9001
• Publisher: Cold Spring Harbor Laboratory Press
• Peer Reviewed: Peer Reviewed
• Volume: 29
• Issue: 6
• Pages: 777-789
• DOI: https://doi.org/10.1261/rna.079615.123
• Keywords: RedBaron method, β-actin localization, m6A site verification, MeRIPSeq, m6A site specific quantification
• Public URL: https://nottingham-repository.worktribe.com/output/17661551
• Publisher URL: https://rnajournal.cshlp.org/content/29/6/777