Meclofenamic acid represses spermatogonial proliferation through modulating m6A RNA modification

Abstract

Background N6-Methyladenosine (m 6 A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m 6 A 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 m 6 A and m 6 A m 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 m 6 A modification at 3’UTR. Results MA2 significantly increased the cellular m 6 A 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 m 6 A sites in the Cdk2–3’UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment. Conclusion MA2 affected CDKs expression through the m 6 A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation. Electronic supplementary material The online version of this article (10.1186/s40104-019-0361-6) contains supplementary material, which is available to authorized users.

The role of m6A modification in physiology and disease

“… ALKBH5 Pre-mRNA splicing Spermatocytes and round spermatids Controls splicing and stability of long 3′-UTR mRNAs in male germ cells 142 . FTO mRNA decay Hela cells Inhibited by MA2 is accompanied with elevated m6A levels in cyclin-dependent kinases, accelerating their destabilization and impairing the cell cycle progression 143 . YTHDC2 mRNA decay Spermatogonia Facilitates a clean switch from mitosis to meiosis in mouse germ cells by downregulating genes expression that are important for mitosis 144 – 147 .…”

The Effect of Huyang Yangkun Formula on N6-methyladenosine Modification in 4-vinylcyclohexene diepoxide Induced Premature Ovarian Insufficiency Rats

“…Unlike other regulatory factors, the expression of FTO increased by VCD and then decreased after HYYKF treatment, which may not be consistent with other studies. One study has reported that after inhibiting the activity of FTO, the proliferation of mouse spermatogonia cells was decreased (Huang et al, 2019). In addition, a case-controlled study showed that the FTO protein expression level was significantly lower in POI patients than in normal people (Ding et al, 2018).…”

Function and evolution of RNA N6-methyladenosine modification

“…Inhibiting ALKBH5 results in aberrant splicing and shorter transcripts of mRNAs, ultimately developing meiosis abnormity of spermatocytes and male infertility in mice [77,78]. Meclofenamic acid suppresses cell viability and blocks G1/S progression in spermatogonia of mice through accelerating the degradation of CDK2 mRNA via specifically decreasing FTO and elevating the m 6 A content of 3'UTR [79].…”