RESUMEN
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify â¼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
Asunto(s)
MicroARNs/biosíntesis , Precursores del ARN/biosíntesis , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Transcripción Genética , Células A549 , Sitios de Unión , Sistemas CRISPR-Cas , ARN Helicasas DEAD-box/metabolismo , Bases de Datos Genéticas , Regulación de la Expresión Génica , Genómica/métodos , Células HEK293 , Células HeLa , Células Hep G2 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Jurkat , Células MCF-7 , MicroARNs/química , MicroARNs/genética , Conformación de Ácido Nucleico , Unión Proteica , Proteómica/métodos , Interferencia de ARN , Precursores del ARN/química , Precursores del ARN/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Relación Estructura-Actividad , Transfección , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Argonaute proteins interact with small RNAs that guide them to complementary target RNAs, thus leading to inhibition of gene expression. Some but not all Argonaute proteins are endonucleases and can cleave the complementary target RNA. Here, we have mutated inactive human Ago1 and Ago3 and generated catalytic Argonaute proteins. We find that two short sequence elements at the N terminus are important for activity. In addition, PIWI-domain mutations in Ago1 may misarrange the catalytic center. Our work helps in understanding of the structural requirements that make an Argonaute protein an active endonucleolytic enzyme.
