m(6)A reader Ythdf proteins control retrotransposon B2 repeat expression and safeguard early embryo development.

N(6)-methyladenosine (m(6)A) and its binding proteins are critical regulators of gene expression and development in mammals. Despite its extent and importance, the regulatory mechanisms of m(6)A-binding reader proteins Ythdf1, Ythdf2, and Ythdf3 during the early stages of mammalian development remain incompletely understood. Here, we show that Ythdf2 and Ythdf3, but not Ythdf1, are required for early embryo development in mice. Mechanically, we demonstrate that all three Ythdf proteins mediate the decay of their target transcripts by binding to similar m(6)A sites, including maternal mRNAs, mid-preimplantation-activated transcripts, and retrotransposon RNAs. Among these, retrotransposon B2 RNAs emerge as one of the primary targets of Ythdf proteins throughout early embryo development, and deficiency in Ythdf1-3 leads to the accumulation of SINE/B2 RNAs, which, in turn, attenuates RNA polymerase II (Pol II) transcription through trans-regulatory mechanisms. In parallel, Ythdf1-3 deficiency represses Pol III-driven B2 transcription, thereby modulating RNA polymerase II activity at genomic regions adjacent to B2 loci via cis-regulatory effects. Together, the coordinated regulatory axis of Ythdf-SINE/B2-gene expression governs a broad transcriptional network that is crucial for embryogenesis.