Unwinding of RNA G-quadruplexes induces mouse and human totipotency

Abstract Totipotent stem cells (TotiSCs) have significant application prospects in regenerative medicine and assisted reproductive technologies. The mRNAs of totipotency-related genes are rich in G-quadruplex (G4) structures, while the G4 density of pluripotency-related genes mRNAs is relatively low, suggesting a potential functional link between RNA G4s and the totipotent state. Nevertheless, how G4s are involved in the establishment or maintenance of mouse and human totipotency remains poorly understood. Here, we demonstrate that FC3, a small-molecule RNA G4 unwinder, enables efficient induction and maintenance of mouse and human TotiSCs (mG4TotiSCs and hG4TotiSCs). These FC3-induced TotiSCs closely recapitulate the molecular features of 2-cell-stage blastomeres via transcriptomic and epigenomic profiling. Functionally, both mG4TotiSCs and hG4TotiSCs exhibit totipotent capacity: they differentiate into embryonic and extraembryonic lineages in vitro and in chimeric embryos; moreover, they autonomously self-organize into blastocyst-like structures without exogenous signalling. Mechanistically, we identify that Gata2 mRNA, a G4-containing transcript, is essential for FC3-mediated reprogramming. Genetic disruption of Gata2 would abolish totipotency induction. Collectively, RNA G4 unwinding is a new regulatory axis governing totipotency acquisition and maintenance, providing insight into the functional interplay between nucleic acid secondary structures and cell fate plasticity.