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Global Profiling of RNA-Binding Protein Target Sites by LACE-seq

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 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.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE137925 | GEO | 2021/04/25

REPOSITORIES: GEO

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