RTCB is Essential for Early Mouse Embryogenesis

Abstract RTCB (RNA 2′,3′-cyclic phosphate and 5′-OH ligase) is a unique 3′–5′ RNA ligase with diverse physiological functions in metazoans. To further explore the role of RTCB in reproduction, we generated a Rtcb conditional knockout mouse model using the Ddx4-Cre system. The complete absence of viable Rtcb-/- offspring indicated embryonic lethality. Integrated analyses of histology, immunohistochemistry, quantitative PCR, and published single-cell RNA sequencing (scRNA-seq) data revealed that Rtcb is expressed throughout embryogenesis, with pronounced upregulation during gastrulation, and that Rtcb knockout resulted in gastrulation failure around embryonic day 6.5 (E6.5), which was accompanied by decreased proliferation and increased apoptosis of embryonic cells. Notably, NUSAP1 (nucleolar and spindle-associated protein 1) is vital for gastrulation as it participates in cell division. In NIH 3T3 cells, knockdown of Rtcb led to destabilization of Nusap1 mRNA, suggesting that NUSAP1 might function downstream of RTCB. Moreover, Rtcb is a target gene of YY1 (Yin-Yang-1), a transcription factor crucial for gastrulation. Expression profiling through scRNA-seq revealed spatiotemporal coordination among Yy1, Rtcb, and Nusap1 between E3.5 and E7.5. Collectively, our findings demonstrate that RTCB is essential for early mouse embryogenesis and propose the presence of a YY1–RTCB–NUSAP1 axis that maintains proper cell proliferation for successful gastrulation.