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. 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.

在减数分裂过程中，卵母细胞会长期处于休眠状态，直至合子基因组激活（zygotic genome activation, ZGA）时转录才会被启动，因此母源转录组的动态变化与稳态平衡亟待深入探究。既往研究表明，利用Smart-seq2技术可检测到母源转录本显著下调。但本研究发现，由于寡聚d(T)引物的存在，Smart-seq2的检测结果会受到mRNA polyA尾长度的影响而产生偏差。与之相反，利用全转录组RNA测序（total RNA-seq）检测到的母源转录本下调幅度相对较小，而polyA尾长度的动态变化则更为显著。

ZAR1是一种已被报道可稳定母源mRNA的RNA结合蛋白（RNA-binding protein, RBP）。但在Zar1/2基因敲除的卵母细胞中，利用total RNA-seq和Smart-seq2检测到的母源转录本差异表达结果并不一致，这提示多聚腺苷酸化过程受到了干扰。本研究结合全转录组RNA测序、LACE-seq、PAIso-seq2以及免疫沉淀-质谱联用（IP-MS, immunoprecipitation-mass spectrometry）技术，发现ZAR1可靶向结合母源转录本的编码区（CDS, coding sequence），在GV期卵母细胞中调控其稳定性，并通过与其他蛋白质相互作用，调节mRNA的多聚腺苷酸化过程。

本研究通过多组学联合分析，探讨了Smart-seq2技术在卵母细胞研究中的局限性，重新探究了母源转录组的动态变化，并揭示了ZAR1在调控母源转录组中的新功能。

简言之，本研究使用TRIzol试剂与Direct-zol RNA MicroPrep试剂盒（Zymo Research，货号：R2060）提取总RNA，并将带有条形码的接头连接至mRNA的3'端。经RNA Clean & Concentrator-5试剂盒（Zymo Research，货号：R1016）纯化后，利用带有唯一分子标识符（Unique Molecular Identifier, UMI）的模板转换寡核苷酸（template-switching oligo, TSO）引物将RNA反转录为cDNA，并完成第一轮PCR扩增。随后利用CRISPR-Cas9系统去除核糖体RNA（ribosomal RNA, rRNA）对应的cDNA，再进行第二轮PCR扩增以获得足量的cDNA。随后按照MAS-ISO-seq流程，将每个样本的cDNA拼接为长链分子。最后，按照cDNA的摩尔比将所有样本混合，取至少500ng的cDNA用于构建SMRTbell文库，并在PacBio Revio平台上进行测序。