The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition

The functional consequence of N⁶-methyladenosine (m⁶A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m⁶A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that YTHDC2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that YTHDC2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.