RESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , TranscriptomaRESUMO
Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.