A nuclear role for the Argonaute protein AGO2 in mammalian gametogenesis

The reduced sperm count observed in Ago2 cKO mice implies that AGO2 has non-redundant functions in the male germ line. Because AGO2 is a key protein in the RNA interference (RNAi) pathway, we first postulated that AGO2 loss disrupts normal transcriptional and translational dynamics of target mRNAs relevant to sperm maturation. To address this hypothesis, we took advantage of the apparently normal spermatogenesis in Ago2 cKO mice, which allowed us to purify matched meiotic and post-meiotic germ cells from Ago2 cKO and wild type controls. We examined changes in the transcriptome and proteome of these two spermatogenic stages in Ago2 cKO relative to control mice using RNA-seq and quantitative mass spectrometry (MS). To further examine if the changes in mRNA and protein levels detected in Ago2 cKO germ cells was due to a loss of regulation by the canonical AGO2-miRNA pathway, we characterized AGO2 protein interactors by AGO2 immunoprecipitation-mass spectrometry (IP-MS) in cytoplasmic and nuclear fractions of post-meiotic cells

在Ago2条件性敲除（cKO）小鼠中观察到的精子计数减少现象，提示AGO2在雄性生殖系中具有非冗余的功能。由于AGO2是RNA干扰（RNA interference, RNAi）通路中的关键蛋白，我们首先提出假说：AGO2的缺失会破坏与精子成熟相关的靶mRNA的正常转录及翻译动态调控过程。为验证该假说，我们利用了Ago2 cKO小鼠中精子发生过程看似正常的特点，得以从Ago2 cKO小鼠与野生型对照小鼠中分离得到匹配的减数分裂期及减数分裂后生殖细胞。我们通过RNA测序（RNA-seq）与定量质谱（mass spectrometry, MS）技术，分析了Ago2 cKO小鼠相较于对照小鼠，这两个生精阶段的转录组与蛋白质组变化情况。为进一步探究Ago2 cKO生殖细胞中检测到的mRNA与蛋白质水平变化是否源于经典AGO2-微小RNA（microRNA, miRNA）通路调控的缺失，我们通过AGO2免疫沉淀-质谱（immunoprecipitation-mass spectrometry, IP-MS）技术，对减数分裂后细胞的细胞质与细胞核组分中的AGO2蛋白互作因子进行了表征。

搜集汇总

背景与挑战

背景概述

该数据集研究AGO2蛋白在哺乳动物配子发生中的核内作用，重点关注其对精子成熟的影响。通过比较Ago2条件性敲除小鼠与野生型对照的生殖细胞，结合转录组和蛋白质组学分析，揭示AGO2在调控基因表达和蛋白质动态中的非冗余功能。实验进一步利用AGO2免疫沉淀质谱探究其与细胞质和核内蛋白的互作，以阐明AGO2在RNA干扰途径中的具体机制。