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.
Conclusion
MA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.
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.