PABPN1 couples the polyadenylation and translation of maternal transcripts to mouse oocyte meiotic maturation

In the process of mammalian maternal-to-zygotic transition (MZT), the degradation of zygotic decay (Z-decay) mRNA is later than that of maternal factor-mediated mRNA decay (M-decay) mRNA. The regulatory mechanism and physiological significance of Z-decay mRNA to maintain stability during meiosis are urgent scientific questions. In this study, we identified that PAPα worked together with poly(A)-bound RNA binding protein PABPN1 to promote the polyadenylation and translation of maternal transcripts, leading to rupture of germinal vesicles in mammalian oocytes. The oocytes specifically knocked out Pabpn1 at the primary follicle stage could develop into the fully grown (FGO) stage, but hardly could enter to the meiotic process. Furthermore, PABPN1 maintains the stability of Z-decay maternal transcripts during oocyte meiotic maturation. A number of Z-decay transcripts and the translational activity in Pabpn1-null oocytes was significantly lower than that of wild-type oocytes during meiosis. Therefore, during meiotic maturation of oocytes, PABPN1 maintains the stability of Z-decay transcripts and promotes the degradation of some M-decay transcripts, ensuring normal GVBD and polar body-1 emission. The construction of PAIso-seq libraries (WT FGO, WT MII oocytes, zKO FGO and zKO MII oocytes, 100K oocyes per sample) followed the protocol published before[1] Briefly, total RNA was extracted by TRIzol reagent (Invitrogen, #15596026) and chloroform (Sinopharm, #10006818), and precipitated with isopropanol (Sinopharm, #80109218) and dissolved with nuclease-free water. Purified total RNA were used for templated end extension by dU-containing end-extension oligo[1] and Klenow fragment 3’-5’ exo- (NEB, #M0212L). Then the templated RNA were cleaned by USER enzyme (NEB, #M5505S) and RNA Clean & Concentrator-5 kit (Zymo Research, #R1016). Purified templated RNA were retro-transcripted into cDNA by using SuperScript Ⅱ reverse transcriptase (Invitrogen, #18064-014) and TSO primer. The full-length cDNA were further pre-amplified by using 2×KAPA HiFi HotStart ReadyMix (KAPA Biosystems, #KK2601) and cleaned by SPRIselect beads (Beckman Coulter, #B23318). For each sample, 20 ng purified cDNA were used for final amplification and cleaning as pre-amplification. Finally, cDNAs from different samples were mixed together according to the concentration and fragments length, and 500ng mixed cDNA were used for SMRTbell template library and sequencing by using Pacbio sequell Ⅱ.