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A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Profiling Analysis of N6-Methyladenosine mRNA Methylation Reveals Differential m6A Patterns during the Embryonic
Biao Chen1,2, Shuibing Liu1,2, Wentao Zhang1,2
1College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China.
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.
Insights
N6-Methyladenosine (m6A) RNA methylation regulates duck skeletal muscle development. This study mapped m6A sites, revealing its role in muscle growth and differentiation, providing foundational insights for poultry research.
Area of Science:
- Epigenetics
- Molecular Biology
- Developmental Biology
Background:
- N6-Methyladenosine (m6A) is a crucial reversible epigenetic modification impacting gene expression and cellular processes.
- The role and patterns of m6A modification in poultry skeletal muscle development remain largely unexplored.
- Understanding m6A dynamics is vital for elucidating mechanisms of muscle growth and differentiation.
Purpose of the Study:
- To investigate the m6A modification profile during duck skeletal muscle development.
- To identify key m6A sites, motifs, and associated genes during embryonic muscle development.
- To explore the functional implications of m6A modification in poultry muscle growth and differentiation.
Main Methods:
- Utilized m6A-specific methylated RNA immunoprecipitation sequencing (MeRIP-seq) to map m6A sites in duck breast muscle at embryonic days 13 (E13) and E19.
- Performed motif sequence analysis, gene expression analysis (RNA-seq), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
- Conducted quantitative real-time PCR (qRT-PCR) to validate the expression of m6A-related genes.
Main Results:
- Identified 19,024 and 18,081 m6A peaks in E13 and E19 duck breast muscle, respectively.
- The primary m6A motif was GGACU, with peaks predominantly located near stop codons, similar to mammalian patterns.
- m6A-modified genes were enriched in muscle differentiation and growth pathways at E19, and in embryonic development and cell proliferation at E13.
- Expression of key m6A regulators (METTL14, WTAP, ZC3H13, RBM15, VIRMA) showed a decreasing trend with developmental progression.
Conclusions:
- m6A mRNA methylation modifications play a significant role in controlling duck skeletal muscle development.
- This study provides the first comprehensive analysis of m6A patterns in duck muscle development, establishing a basis for future research in poultry.
- m6A modification influences distinct developmental stages, impacting pathways related to cell proliferation, embryonic development, muscle growth, and differentiation.
