Meclofenamic acid represses spermatogonial proliferation through modulating m 6 A RNA modification

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Abstract

Background

N6-Methyladenosine (m ⁶A), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several m ⁶A 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 ⁶A and m ⁶A _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 ⁶A modification at 3’UTR.

Results

MA2 significantly increased the cellular m ⁶A 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 ⁶A sites in the Cdk2–3’UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment.

Conclusion

MA2 affected CDKs expression through the m ⁶A-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.

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Most cited references 25

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The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.

Thomas Gerken, Christophe A J Girard, Yi-Chun Tung … (2007)

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

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Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis

Yueshuai Guo, Chuan Sheng He, Bin Shen … (2017)

N 6 -methyladenosine (m 6 A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m 6 A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m 6 A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m 6 A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m 6 A-binding protein that plays critical roles during spermatogenesis.

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Inactivation of the Fto gene protects from obesity.

Julia Fischer, Linda Koch, Christian Emmerling … (2009)

Several independent, genome-wide association studies have identified a strong correlation between body mass index and polymorphisms in the human FTO gene. Common variants in the first intron define a risk allele predisposing to obesity, with homozygotes for the risk allele weighing approximately 3 kilograms more than homozygotes for the low risk allele. Nevertheless, the functional role of FTO in energy homeostasis remains elusive. Here we show that the loss of Fto in mice leads to postnatal growth retardation and a significant reduction in adipose tissue and lean body mass. The leanness of Fto-deficient mice develops as a consequence of increased energy expenditure and systemic sympathetic activation, despite decreased spontaneous locomotor activity and relative hyperphagia. Taken together, these experiments provide, to our knowledge, the first direct demonstration that Fto is functionally involved in energy homeostasis by the control of energy expenditure.

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Author and article information

Contributors

Tao Huang: huangtao_199112@163.com

Jiayin Guo: guojiayin89kza@163.com

Yinghua Lv: hongriyinghua@163.com

Yi Zheng: y.zheng@nwafu.edu.cn

Tongying Feng: fengtongying1994@163.com

Qiang Gao: gaoqiang1151866427@126.com

Wenxian Zeng:

ORCID: http://orcid.org/0000-0002-2912-8943

+86 029 87092120 , 15229247918 , zengwenxian2015@126.com

Journal

Journal ID (nlm-ta): J Anim Sci Biotechnol

Journal ID (iso-abbrev): J Anim Sci Biotechnol

Title: Journal of Animal Science and Biotechnology

Publisher: BioMed Central (London )

ISSN (Print): 1674-9782

ISSN (Electronic): 2049-1891

Publication date (Electronic): 11 July 2019

Publication date PMC-release: 11 July 2019

Publication date Collection: 2019

Volume: 10

Electronic Location Identifier: 63

Affiliations

ISNI 0000 0004 1760 4150, GRID grid.144022.1, Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, , Northwest A&F University, ; Yangling, 712100 Shaanxi China

Author information

Wenxian Zeng http://orcid.org/0000-0002-2912-8943

Article

Publisher ID: 361

DOI: 10.1186/s40104-019-0361-6

PMC ID: 6621992

PubMed ID: 31333841

SO-VID: c93bb426-266d-4279-8623-7859fb8f5980

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 22 August 2018

Date accepted : 10 May 2019

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;

Award ID: 31572401

Award Recipient : Wenxian Zeng

Custom metadata

ScienceOpen disciplines: Animal science & Zoology

Keywords: cell cycle,fto,meclofenamic acid,n6-methyladenosine,spermatogonial proliferation

Data availability:

ScienceOpen disciplines: Animal science & Zoology

Keywords: cell cycle, fto, meclofenamic acid, n6-methyladenosine, spermatogonial proliferation