Eif1ad3 as a key regulator during zygotic genome activation

Eif1ad3 with its homologs, including Eif1ad4, Eif1ad6, Eif1ad7, etc., which are barely studied before, are found to be highly translated during ZGA and regulating the translational activity of critical genes, especially for ribosome assembly. Knocking down Eif1ad3 leads to defective ZGA and further preimplantation development. These data identified Eif1ad3 as an indispensable factor governing mouse embryo development at ZGA. For Eif1ad3 and its variance knockdown experiments, 1-Cell embryo were collected from CF1 (envigo) microinjected with control (only RfxCas13d-IVT mRNA) or Eif1adKD (gRNA+RfxCas13d-IVT mRNA). Embryos from control and knockdown groups were further cultured until blastocyst to observe the development. For RNA-Seq, 2-cell stage (control and knockdown groups) were collected. To generate Eif1ad3-6xHis mRNA, total RNA from 2-Cell embryos was extracted from a pool of mouse embryos, followed by first strand cDNA synthesis. To overexpress Eif1ad3, in vitro transcribed mRNAs (IVT-mRNAs) were microinjected into zygotes at final concentration of 200 ng/uL. RIP experiments were conducted using the EZ-Magna RIP Kit (Millipore Corporation, Billerica, MA). Briefly, 2-Cell stage embryos (over expressed 200 cells and control 200 cell per replicate) were harvested, lysed and then incubated with RIP buffer containing magnetic beads conjugated with anti-His tag antibody (Millipore) or corresponding negative control IgG (Millipore). After the antibody was recovered using protein A/G beads, the purified RNA were used to perform RNA-seq.

Eif1ad3及其同源基因（包括此前鲜有研究的Eif1ad4、Eif1ad6、Eif1ad7等）被证实于合子基因组激活（zygotic genome activation, ZGA）阶段呈现高翻译水平，并调控关键基因的翻译活性，尤其在核糖体装配过程中发挥关键作用。敲低Eif1ad3会导致合子基因组激活缺陷，并进一步阻碍小鼠胚胎的植入前发育。本研究数据证实Eif1ad3是调控小鼠胚胎合子基因组激活阶段发育的不可或缺的核心因子。针对Eif1ad3及其变异体的敲低实验，研究人员从CF1（Envigo）品系小鼠中收集1-细胞期胚胎，分别显微注射对照试剂（仅含RfxCas13d体外转录mRNA，即RfxCas13d-IVT mRNA）或Eif1adKD试剂（gRNA+RfxCas13d-IVT mRNA）。将对照组与敲低组的胚胎继续体外培养至囊胚阶段，以观察胚胎发育情况。针对RNA测序实验，研究人员收集了对照组与敲低组的2-细胞期胚胎。为制备Eif1ad3-6xHis mRNA，研究人员从批量合并的小鼠2-细胞期胚胎中提取总RNA，随后进行第一链cDNA合成。为过表达Eif1ad3，研究人员将体外转录的mRNA（IVT-mRNA）以200 ng/μL的终浓度显微注射至受精卵中。RNA免疫沉淀（RNA immunoprecipitation, RIP）实验采用EZ-Magna RIP试剂盒（Millipore Corporation, 马萨诸塞州比勒里卡市）完成。简要操作流程如下：收集2-细胞期胚胎（每个重复样本包含过表达组200个胚胎与对照组200个胚胎），裂解后与结合有抗His标签抗体（Millipore）或相应阴性对照IgG（Millipore）的磁珠悬浮于RIP缓冲液中孵育。使用蛋白A/G磁珠回收抗体后，提取纯化得到的RNA用于RNA测序实验。