Profiling Analysis of N6-Methyladenosine mRNA Methylation Reveals Differential m6A Patterns during the Embryonic Skeletal Muscle Development of Ducks

SIMPLE SUMMARY: Recent studies show that N6-methyladenosine (m6A) modification, the most common RNA chemical modification, influences the modification, processing, transport, and translation of RNA. N6-methyladenosine is an epigenetic modification that influences skeletal myogenesis and skeletal muscle development. However, the N6-methyladenosine modification profile and its function during poultry muscle development is unclear, and there is only one report about m6A modification in ducks, which focuses on duck hepatitis A virus infection. Here, we compared the m6A modification profiles between E13 (embryonic day 13) and E19 (embryonic day 19) in duck breast muscle differentiation using MeRIP-seq, and evaluated the expression profile of the methyl transferase METTL14 and its cofactors during breast muscle development. This is the first study of N6-methyladenosine modification patterns in duck muscle tissue. The current study not only elucidates the regulation mechanisms of duck skeletal muscle development, but also lays the groundwork for studying the role of RNA modification in poultry muscle development. ABSTRACT: N6-Methyladenosine is a reversible epigenetic modification that influences muscle development. However, the m6A modification profile during poultry skeletal muscle development is poorly understood. Here, we utilized m6A-specific methylated RNA immunoprecipitation sequencing to identify m6A sites during two stages of breast muscle development in ducks: embryonic days 13 (E13) and E19. MeRIP-seq detected 19,024 and 18,081 m6A peaks in the E13 and E19 groups, respectively. Similarly to m6A distribution in mammalian transcripts, our results revealed GGACU as the main m6A motif in duck breast muscle; they also revealed that m6A peaks are mainly enriched near the stop codons. In addition, motif sequence analysis and gene expression analysis demonstrated that m6A modification in duck embryo skeletal muscles may be mediated by the methyltransferase-like 14. GO and KEGG analysis showed that m6A peaks containing genes at E19 were mainly enriched in muscle-differentiation- and muscle-growth-related pathways, whereas m6A peaks containing genes in E13 were mainly enriched in embryonic development and cell proliferation pathways. Combined analysis of MeRIP-seq and RNA-seq showed that the mRNA expression may be affected by m6A modification. Moreover, qRT-PCR analysis of the expression of METTL14 and its cofactors (WTAP, ZC3H13, RBM15 and VIRMA) during duck embryonic skeletal muscle development in breast and leg muscle samples revealed a significant downward trend as the developmental age progressed. Our results demonstrated that m6A mRNA methylation modifications control muscle development in ducks. This is the first study of m6A modification patterns in duck muscle tissue development, and it lays the foundation for the study of the effects of RNA modification on poultry skeletal muscle development.