RESUMO
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.
Assuntos
MicroRNAs/biossíntese , Precursores de RNA/biossíntese , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Células A549 , Sítios de Ligação , Sistemas CRISPR-Cas , RNA Helicases DEAD-box/metabolismo , Bases de Dados Genéticas , Regulação da Expressão Gênica , Genômica/métodos , Células HEK293 , Células HeLa , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Proteômica/métodos , Interferência de RNA , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Relação Estrutura-Atividade , Transfecção , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.