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Updated: Jul 25, 2025
A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin
Jinfu Zhou1,2, Sining Liao1,3, Chenran Zhang1,3
1Department of Preventive Medicine, School of Public Health, Fujian Medical University, Fuzhou, 350122, Fujian Province, China.
Background:
Abnormal methylation of N6-methyladenosine (m6A) is reportedly associated with central nervous system disorders. However, the role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research.
Methods:
Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m6A levels were measured using an m6A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m6A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq).
Results:
Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m6A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m6A levels in PC12 cells. Furthermore, 1533 m6A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m6A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m6A peaks and differentially expressed mRNA levels were identified.
Conclusion:
Our study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.
Insights
Unconjugated bilirubin (UCB) alters N6-methyladenosine (m6A) mRNA methylation in neuronal cells. This m6A modification is a key factor in UCB-induced neurotoxicity.
Area of Science:
- Neuroscience
- Epigenetics
- Molecular Biology
Background:
- Abnormal N6-methyladenosine (m6A) methylation is linked to central nervous system disorders.
- The specific role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity is not well understood.
Purpose of the Study:
- To investigate the impact of UCB on m6A mRNA methylation in neuronal cells.
- To explore the potential involvement of m6A modifications in UCB-induced neurotoxicity.
Main Methods:
- Utilized rat pheochromocytoma PC12 cells as an in vitro model for UCB neurotoxicity.
- Measured total RNA m6A levels and quantified the expression of m6A-modifying enzymes (demethylases and methyltransferases).
- Employed methylated RNA immunoprecipitation sequencing (MeRIP-seq) to profile m6A methylation patterns in UCB-treated cells.
Main Results:
- UCB treatment altered the expression of key m6A regulators, decreasing ALKBH5 and increasing METTL3/METTL14, leading to elevated total m6A levels.
- Identified significant changes in m6A peak distribution, with thousands of peaks either increased or decreased upon UCB exposure.
- Genes with differential m6A modifications were enriched in pathways critical for cellular function, including protein processing, proteolysis, cell cycle, and endocytosis.
Conclusions:
- UCB exposure significantly modulates m6A mRNA methylation patterns in neuronal cells.
- These m6A methylation changes are implicated as a significant mechanism contributing to UCB neurotoxicity.
