Meclofenamic acid represses spermatogonial proliferation through modulating m6A RNA modification

Tao Huang,Wenxian Zeng,Yi Zheng,Jiayin Guo,Tongying Feng,Yinghua Lv,Qiang Gao

doi:10.1186/s40104-019-0361-6

Tao Huang, Wenxian Zeng + Show 5 more

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https://doi.org/10.1186/s40104-019-0361-6

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Abstract

BackgroundN6-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.MethodsMouse 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.ResultsMA2 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.ConclusionMA2 affected CDKs expression through the m6A-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation.

Highlights

N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes
Over 140 chemical modifications have been discovered in RNA, of which N6-methyladenosine (m6A) is the most abundant one that distributes extensively in about 60% of mammalian mRNA. m6A mainly occurs at a common RR (m6A) CH motif, in the 3’UTR and near the stop codon [1, 2]. m6A modification is catalyzed by the methyltransferase complex composed by methyltransferase like 3 (METTL3)
We found that mRNA of CDK1, CDK2 and CDK4 could be detected in the IP RNA, while CDK5, CDK6 and CDK7 could not (Fig. 6b and c), indicating that fat mass and obesity associate protein (FTO) directly interacted with CDK1, CDK2 and CDK4

Summary

Introduction

N6-Methyladenosine (m6A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Spermatogenesis is a complex process that contains proliferation and differentiation of spermatogonia, meiosis of spermatocytes and post-meiotic spermiogenesis of round spermatids, resulting in generating millions of spermatozoa daily in mammalian testes [22]. This process requires accurately, spatially and temporally regulated patterns of gene expression. Investigating the underlying mechanisms of spermatogenesis will provide theoretical basis for development of novel approaches for male infertility therapy and male contraception Epigenetic factors, such as histone modifications and DNA modifications, have been reported to play important roles in spermatogenesis [23,24,25,26]. Simultaneously deletion of METTL14 and METTL3 in advanced mouse germ cells resulted in decrease of sperm motility and increase of abnormal sperm ratio, indicating the significance of m6A in spermiogenesis [20]

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