GSE137925
Global Profiling of RNA-Binding Protein Target Sites by LACE-seq
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 an RBP in those rare cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of cDNA 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 oocytes. Unexpectedly, transcriptomic and proteomic analysis of Ago2-/- oocytes revealed that Ago2 interacted with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes could fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP binding sites in a few cells opens the door for studying the roles of RBPs in embryonic development and reproductive diseases. Overall design: We first developed a linear amplification of cDNA ends and sequencing (LACE-seq) methodology to identify RBP targets at or near the single-cell level. Next, we determined the binding sites for several RBPs, including Argonaute 2 (Ago2), MILI, Ddx4, and PTBP1 (also known as PTB) in single or dozens of fully-grown mouse oocytes. Two biological repeats, labeled as Rep 1 and Rep 2, were generated for each sample. For measuring Ago2 regulated genes in fully-grown oocytes, we also performed single-cell RNA-seq analysis on five control and Ago2-null oocytes. To identify endo-siRNAs loaded into Ago2, we performed single-cell CAS-seq using Ago2 immunoprecipitated small RNAs from MII oocyte and labeled as Ago2_IP_Oocyte. To evaluate the specificity of LACE-seq, we performed Ago2 LACE-seq in oocytes from Ago2 conditional knockout (cKO) mice and PTBP1 LACE-seq in K562 cells treated with PTBP1 shRNA. RNA-seq analysis of Meiosis II oocytes from Ago2 cKO (Ago2 loxP/loxP,Zp3-cre) and Control(Ago2 loxP/+) female mice.