The Dominant Role of Polyadenylation in Regulating Maternal mRNA Dynamics in Dormant Oocytes and the Function of ZAR1 in this Process_PAIso-seq2
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: In brief, total RNA was extracted with TRIzol Reagent and Direct-zol RNA MicroPrep (Zymo Research, Cat. no. R2060), and the barcode containing adapter were ligated to the 3'-end of mRNA. After being purified by the RNA Clean & Concentrator-5 kit (Zymo Research, Cat. no. R1016), the RNA were RT into cDNA by the UMI containing template-switching oligo (TSO) primer and the first PCR amplification were proceeded. Then the cDNA from rRNA will be removed by the CRISPR-Cas9 system and the second PCR amplification will be performed to get enough cDNA. Then the cDNA from each sample will be concatenated into long molecules by following the MAS-ISO-seq procedure. Finally, all the samples were mixed up according to the molar ratios of cDNA, and at least 500ng of cDNA was sent for SMRTbell library preparation and sequenced on PacBio Revio platform.