RNA m⁶A modification regulates L1 retrotransposons in human spermatogonial stem cell differentiation in vitro and in...

The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m⁶A) plays an essential role in regulating various biological processes. However, the function of m⁶A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m⁶A methyltransferase METTL3 or the m⁶A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m⁶A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m⁶A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m⁶A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m⁶A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m⁶A modification in restricting L1 retrotransposition.