m(6)A methylation controls pluripotency of porcine induced pluripotent stem cells by targeting SOCS3/JAK2/STAT3 pathway in a YTHDF1/YTHDF2-orchestrated manner

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease treatment, and organ transplantation. As the ethical issue of human ESCs and similarity of pig in human genome and physiological characteristics, the porcine iPSCs (piPSCs) have become an ideal alternative study model. N(6)-methyladenosine (m(6)A) methylation is the most prevalent modification in eukaryotic mRNAs, regulating the self-renewal and differentiation of pluripotency stem cells. However, the explicit m(6)A-regulating machinery remains controversial. Here, we demonstrate that m(6)A modification and its modulators play a crucial role in mediating piPSCs pluripotency. In brief, loss of METTL3 significantly impairs self-renewal and triggers differentiation of piPSCs by interfering JAK2 and SOCS3 expression, further inactivating JAK2–STAT3 pathway, which then blocks the transcription of KLF4 and SOX2. We identify that both of JAK2 and SOSC3 have m(6)A modification at 3′UTR by m(6)A-seq analysis. Dual-luciferase assay shows that METTL3 regulates JAK2 and SOCS3 expression in an m(6)A-dependent way. RIP-qPCR validates JAK2 and SOCS3 are the targets of YTHDF1 and YTHDF2, respectively. SiMETTL3 induced lower m(6)A levels of JAK2 and SOCS3 lead to the inhibition of YTHDF1-mediated JAK2 translation and the block of YTHDF2-dependent SOCS3 mRNA decay. Subsequently, the altered protein expressions of JAK2 and SOCS3 inhibit JAK2–STAT3 pathway and then the pluripotency of piPSCs. Collectively, our work uncovers the critical role of m(6)A modification and its modulators in regulating piPSCs pluripotency and provides insight into an orchestrated network linking the m(6)A methylation and SOCS3/JAK2/STAT3 pathway in pluripotency regulation.