Global profiling of RNA-binding protein target sites by LACE-seq
Global profiling of RNA-binding protein target sites by LACE-seq
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通过 LACE-seq 对 RNA 结合蛋白靶位点进行全局分析
DOI：
10.1038/s41556-021-00696-9
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发表时间：
2021-06-01
影响因子：
21.3
通讯作者：
Xue, Yuanchao
中科院分区：
文献类型：
--
作者：
Su, Ruibao;Fan, Li-Hua;Xue, Yuanchao
RNA结合蛋白（RBP）在生殖系和早期胚胎发育过程中具有重要的功能。然而，目前的方法无法在这些低丰度细胞中鉴定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，用于在单个卵母细胞水平或接近单个卵母细胞水平识别RBP调节的RNA网络。我们确定了几种RBP，包括Argonaute 2（Ago 2），Mili，Ddx 4和Ptbp 1，在成熟小鼠卵母细胞中的结合位点和调控机制。出乎意料的是，Ago 2 −/−卵母细胞的转录组学和蛋白质组学分析显示，Ago 2与内源性小干扰RNA（endo-siRNA）相互作用，全面抑制mRNA翻译。此外，Ago 2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。
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 ofAgo2−/−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.