RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2^−/− oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.

RNA 结合蛋白 (RBP) 在种系和早期胚胎发育过程中具有重要功能。然而，目前的方法无法在这些低丰度细胞中识别 RBP 的体内靶标。在这里，通过将 RBP 介导的逆转录终止与互补 DNA 末端的线性扩增和测序相结合，我们提出了 LACE-seq 方法，用于在单卵母细胞水平或附近识别 RBP 调节的 RNA 网络。我们确定了成熟小鼠卵母细胞中几种 RBP 的结合位点和调节机制，包括 Argonaute 2 (Ago2)、Mili、Ddx4 和 Ptbp1。出乎意料的是， Ago2的转录组学和蛋白质组学分析^-/-卵母细胞显示 Ago2 与内源性小干扰 RNA (endo-siRNA) 相互作用，以全面抑制 mRNA 翻译。此外，Ago2 和 endo-siRNA 复合物通过切割长末端重复逆转录转座子衍生的嵌合转录物来微调转录组。低输入细胞中 RBP 结合位点的精确定位为研究 RBP 在胚胎发育和生殖疾病中的作用打开了大门。