Meiotic Maturation-associated RNA G-quadruplexes removal is Crucial for Translational Activation in Mouse Oocytes [LACE-seq]

Following meiosis resumption, the transcription was silencing; and large number of maternal mRNA was actively translated and degraded during oocyte maturation. However, due to the limitation of sample number and technology, the dynamic distribution of RNA structure and its correlation with RNA dynamic change have never been revealed. RNA G-quadruplex (rG4) was a four-stranded RNA secondary structure related to translation regulation and RNA stability. In this study, we developed a low-input technique of rG4 detection (G4-lace-seq), and found that rG4s were highly abundant in fully grown oocytes and reduced during oocyte maturation. We ingeniously applied rG4 stable ligand BYBX in aim to eliminate the DNA G-quadruplex obstruction, and phenotypic analysis showed that rG4 accumulation severely impaired oocytes maturation. Above all, RBPs proteomic spectra revealed RNA structure change caused by G4 accumulation directly weakened the binding of RBPs to RNA, especially the enrichment of G4s affect the RBP binding associated with RNA metabolism, and G4s on the 5′-UTR hindered 40s ribosome movement and binding of 60s ribosome eukaryotic translation initiation factors to RNA. Combined analysis of G4-Lace-seq with Ribo-lite and RNA-seq, further proved that rG4s accumulation on UTRs disrupted translational efficiency and maternal mRNA degradation during oocyte maturation. Overexpression of DHX36, a G4s helicase, rescued the defects caused by BYBX to oocytes and restored the development rates. Jointly analyzing the function and distribution of rG4s and the metabolic signature of maternal mRNA, we hypothesized that a large amount of rG4s present before meiosis resumption is necessary to protect long-term mRNA stability from degradation in a state of poor efficiency translation. Following meiosis resumption, rG4s were cleaned up, translation efficiency increased, and the mRNA became unstable and was rapidly degraded. Collectively, our results announced for the first time that G4, as a secondary structure, had a hand in in the special dynamic rule of RNA content and translation. rG4s clearance determined the fate of cytoplasmic maturation during oocyte maturation. The 4-week-old female mice were injected with 5 IU of pregnant mare’s serum gonadotropin (PMSG, Ningbo Sansheng Pharmaceutical) and humanely euthanized 44h later. Oocytes were obtained in 37°C pre-warmed M2 medium (M7167; Sigma-Aldrich) at the GV stage and cultured in M16 medium (M7292; Sigma-Aldrich) covered with mineral oil (M5310; Sigma-Aldrich) at 37°C in a 5% CO2 atmosphere. BYBX (5 mM) was diluted to a gradient concentration by DMSO and added to M16 medium. Milrinone (2 μM) was applied with the culture medium to inhibit spontaneous GVBD in some experiments. G4-LACE-seq experiments were improved according to published procedure. Briefly, 50 oocytes were gathered in 1.5 ml Eppendorf LoBind microcentrifuge tubes and swiftly settled at the base using a mini centrifuge.