Abstract
N6-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.
Highlights
Life-long male fertility relies on spermatogenesis that is responsible for the generation of millions of sperm (Fok et al, 2014)
Depletion of fat mass and obesity-associated protein (FTO) in Spermatogonia Subjected to CRISPR-Cas9 Gene Editing
We detected total m6A level of mRNA extracted from wild type cells and FTO-KO cells
Summary
Life-long male fertility relies on spermatogenesis that is responsible for the generation of millions of sperm (Fok et al, 2014). Spermatogenesis is a complex developmental process that consists of three stages: mitosis of spermatogonia, meiosis of spermatocyte and transformation of sperm from haploid spermatids (Kanatsu-Shinohara and Shinohara, 2013). Spermatogonia are the cornerstone of sperm production (Hamra et al, 2004). M6A is mainly enriched near the stop codon, within the consensus motif DRACH (D = A, G, U; R = A, G; H = A, C, U) (Dominissini et al, 2012). M6A is installed by the “writer” complex that is composed of METTL3, METTL14, WTAP, and several unknown components (Liu et al, 2014). Growing evidences have indicated that m6A is involved in post-transcriptional processes including translation, mRNA degradation, alternative splicing, and microRNA maturation, affecting gene expression
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