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Global profiling of RNA-binding protein target sites by LACE-seq, Nat Cell Biol, 9 Jun 2021

发布时间:2021年06月09日

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Nature Cell Biology, 9 June, 2021, DOI:黄金城游戏免费注册

Global profiling of RNA-binding protein target sites by LACE-seq

Ruibao Su, Li-Hua Fan, Changchang Cao, Lei Wang, Zongchang Du, Zhaokui Cai, Ying-Chun Ouyang, Yue Wang, Qian Zhou, Ligang Wu, Nan Zhang, Xiaoxiao Zhu, Wen-Long Lei, Hailian Zhao, Yong Tian, Shunmin He, Catherine C. L. Wong, Qing-Yuan Sun & Yuanchao Xue 

Abstract

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.

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