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Updated: Jan 21, 2026
A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Meclofenamic acid represses spermatogonial proliferation through modulating m6A RNA modification
Tao Huang1, Jiayin Guo1, Yinghua Lv1
1Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100 Shaanxi China.
Background:
N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m6A associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) and YTH domain containing 2 (YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m6A demethylase, fat mass and obesity associate protein (FTO), in germ cells remains elusive. Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction.
Methods:
Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular m6A and m6Am level were analyzed through high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through EdU and western blot. The mRNA level of core cyclin dependent kinases (CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the m6A modification at 3'UTR.
Results:
MA2 significantly increased the cellular m6A level and down-regulated the expression of CDK1, CDK2, CDK6 and CdC25a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 mRNA stability. Additionally, mutation of the predicted m6A sites in the Cdk2-3'UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment.
Conclusion:
MA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.
Insights
Fat mass and obesity associated protein (FTO) inhibition using MA2 repressed spermatogonial proliferation by increasing m6A levels and degrading CDK mRNA. This study elucidates FTO’s role in germ cell regulation and mammalian reproduction.
Area of Science:
- Epigenetics and Gene Regulation
- Reproductive Biology
- Molecular Mechanisms of Cell Division
Background:
- N6-Methyladenosine (m6A) is a prevalent mRNA modification crucial for biological processes.
- While several m6A-binding proteins regulate germ cell development, the role of the m6A demethylase FTO in germ cells is largely unknown.
- Understanding FTO's function in germ cells is vital for insights into mammalian reproduction.
Purpose of the Study:
- To investigate the role of the m6A demethylase FTO in spermatogonial proliferation.
- To elucidate the molecular mechanisms underlying FTO's regulation of germ cell fate.
Main Methods:
- Inhibition of FTO demethylase activity in mouse GC-1 spg cells using meclofenamic acid (MA2).
- Quantification of cellular m6A and m6Am levels via HPLC/MS-MS.
- Assessment of cell apoptosis, proliferation, mRNA stability, and cyclin-dependent kinases (CDKs) expression.
Main Results:
- MA2 treatment significantly increased cellular m6A levels and downregulated CDK1, CDK2, CDK6, and CdC25a expression.
- FTO inhibition led to G1/S phase arrest, decreased cell proliferation, and reduced CDK2 mRNA stability.
- Mutation of predicted m6A sites in the CDK2 3'UTR partially rescued mRNA degradation following MA2 treatment.
Conclusions:
- MA2 affects CDK expression via an m6A-dependent mRNA degradation pathway.
- FTO inhibition represses spermatogonial proliferation by modulating CDK expression and mRNA stability.
- This study highlights FTO's critical role in regulating spermatogonial proliferation through epigenetic mechanisms.
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