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FTO Knockout Causes Chromosome Instability and G2/M Arrest in Mouse GC-1 Cells
Tao Huang1, Qiang Gao1, Tongying Feng1
1Laboratory of Reproductive Biology and Cell Engineering, College of Animal Science and Technology, Northwest A&F University, Xianyang, China.
Abstract:
N 6-methyladenosine (m6A) is the most abundant modification on eukaryotic mRNA. m6A plays important roles in the regulation of post-transcriptional RNA splicing, translation, and degradation. Increasing studies have uncovered the significance of m6A in various biological processes such as stem cell fate determination, carcinogenesis, adipogenesis, stress response, etc, which put forwards a novel conception called epitranscriptome. However, functions of the fat mass and obesity-associated protein (FTO), the first characterized m6A demethylase, in spermatogenesis remains obscure. Here we reported that depletion of FTO by CRISPR/Cas9 induces chromosome instability and G2/M arrest in mouse spermatogonia, which was partially rescued by expression of wild type FTO but not demethylase inactivated FTO. FTO depletion significantly decreased the expression of mitotic checkpoint complex and G2/M regulators. We further demonstrated that the m6A modification on Mad1, Mad2, Bub1b, Cdk1, and Ccnb2 were directly targeted by FTO. Therefore, FTO regulates cell cycle and mitosis checkpoint in spermatogonia because of its m6A demethylase activity. The findings give novel insights into the role of RNA methylation in spermatogenesis.
Insights
The fat mass and obesity-associated protein (FTO) is crucial for mouse spermatogonia cell cycle regulation. FTO
Area of Science:
- Molecular Biology
- Epigenetics
- Reproductive Biology
Background:
- N6-methyladenosine (m6A) is a key mRNA modification regulating gene expression.
- The fat mass and obesity-associated protein (FTO) is the primary m6A demethylase.
- The role of FTO in spermatogenesis is currently unknown.
Purpose of the Study:
- To investigate the function of FTO in mouse spermatogonia.
- To determine if FTO's demethylase activity is essential for its role in spermatogenesis.
Main Methods:
- CRISPR/Cas9 was used to deplete FTO in mouse spermatogonia.
- Wild-type and demethylase-inactivated FTO were expressed to assess rescue effects.
- Expression levels of cell cycle and mitotic checkpoint regulators were analyzed.
- m6A modification on specific target genes was assessed.
Main Results:
- FTO depletion caused chromosome instability and G2/M cell cycle arrest.
- These effects were partially rescued by wild-type FTO but not by inactive FTO.
- FTO depletion reduced the expression of mitotic checkpoint and G2/M regulators.
- FTO directly targets m6A modification on Mad1, Mad2, Bub1b, Cdk1, and Ccnb2.
Conclusions:
- FTO's m6A demethylase activity is essential for regulating cell cycle and mitosis checkpoint in spermatogonia.
- FTO plays a critical role in maintaining genomic stability during spermatogenesis.
- This study highlights the importance of RNA methylation in male reproductive processes.
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