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 like those of the YTH family that recruit additional factors to alter splicing, RNA stability or translation. Germline-specific YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here we use a tagged mouse line to identify U-rich motifs as binding sites of YTHDC2 on 3′ UTRs of testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mouse lacking this ability displays no obvious phenotypes. Its 3′→5′ RNA helicase activity is essential for fertility, with the catalytic-dead mutation being dominant-negative. Single-cell transcriptomics reveal that Ythdc2 mutant mitotic testicular germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity. We also reveal a conserved role for YTHDC2 in animal fertility. iCLIP experiment was used to identify the RNA targets of YTHDC2, as well as its binding sites and motifs. RNA sequencing was applied to study the gene expression differences between Ythdc2YTHmut/+ and Ythdc2YTHmut/YTHmut of testicular 2n and 4n cells, Ythdc2+/+, Ythdc2+/cat-dead, and Ythdc2cat-dead/cat-dead.