The Dominant Role of Polyadenylation in Regulating Maternal mRNA Dynamics in Dormant Oocytes and the Function of ZAR1 in this Process_total RNA-seq
During the meiosis, the oocytes will keep dormant for a long time that the transcription will not be activated until zygotic genome activation (ZGA), thus the dynamic and homeostasis of maternal transcriptome deserved to be explored. In the previous researches, the maternal transcripts were reported to be drastically down-regulated by using Smart-seq2. However, we found out that the detection of Smart-seq2 can be biased by the polyA tail length of mRNAs due to the oligo-d(T) primer. In contrast, the down-regulation of maternal transcripts detected by total RNA-seq was relatively small, and the dynamic of polyA tail length were much acuter. ZAR1 is an RBP that had been reported to be important to stablize maternal mRNA. However, the differential expression of maternal transcripts in Zar1/2-/- oocytes were also different when detected by total RNA-seq and Smart-seq2, which hinted an interruption of polyadenylation. By combining total RNA-seq, LACE-seq, PAIso-seq2 and IP-MS and found out that ZAR1 may target the CDS of maternal transcripts and regulate its stability in GV stage oocytes and by interacting with other proteins to regulate the polyadenylation of mRNAs. In this research, by jointly analyzing multi-omics data, we discussed the limitation of Smart-seq2 on oocytes, reexplored the dynamic of maternal transcriptome and reported the new roles of ZAR1 on regulating maternal transriptome. Overall design: 30 GV or MII stage oocytes were harvested from WT and Zar1/2-/- female mice as described for one group and three groups per sample were collected for replication. RNA of oocytes were extracted by TRIzol, and ERCC were added as spike-in. Extracted RNA were used for total RNA-seq library construction by rRNA deletion (rRNA Depletion Module (H/M/R), ABclonal, Cat. no. RK20348) and library construction (Fast RNA-seq Lib Prep Kit V2, ABclonal, Cat# RK20306).