The Dominant Role of Polyadenylation in Regulating Maternal mRNA Dynamics in Dormant Oocytes and the Function of ZAR1 in this Process_LACE-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: The LACE-seq method can be found in the work by Su Ruibao et.al. Briefly, 2 µg per sample ZAR1 antibody was coupled to the activated protein A/G magnetic beads. Then 30 GV stage oocytes were harvested from WT and Zar1/2-/- female mice as described and were irradiated twice with UV-C light on ice at 400 mJ for RBP-RNA crosslinking. Afterwards, the cells were lysed on ice, and the antibody-coupled beads were added to the lysate, and the ZAR1 target RNA was then immunoprecipitated by beads and fragmented by MNase. Then the RNA dephosphorylation and the first-strand synthesis of RT were both performed on beads, and the 3' linker and biotin-carried T7-RT primer w be added to the RNA at this step. Then the first-strand cDNA was released and captured by streptavidin beads, and the followed polyA tailing and pre-amplification were both performed on beads, and the products were purified for further in vitro transcription (IVT). After IVT amplificating, the DNA template would be removed, and the remaining RNA was purified for further RT and PCR barcoding. The barcoded libraries were sequenced on the Illumina HiSeq 2500 at Novogene.