A Compendium of RNA-Binding Proteins That Regulate MicroRNA Biogenesis

Mol Cell. 2017 Apr 20;66(2):270-284.e13. doi: 10.1016/j.molcel.2017.03.014.

Abstract

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 ∼180 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.

Keywords: LIN28; RNA binding proteins; RNA processing; RNA pull-down; TRIM71; ZC3H10; ZC3H7; microRNA-guided gene silencing; regulation of microRNA biogenesis; siRNA pool.

MeSH terms

  • A549 Cells
  • Binding Sites
  • CRISPR-Cas Systems
  • DEAD-box RNA Helicases / metabolism
  • Databases, Genetic
  • Gene Expression Regulation
  • Genomics / methods
  • HEK293 Cells
  • HeLa Cells
  • Hep G2 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Jurkat Cells
  • MCF-7 Cells
  • MicroRNAs / biosynthesis*
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • Nucleic Acid Conformation
  • Protein Binding
  • Proteomics / methods
  • RNA Interference
  • RNA Precursors / biosynthesis*
  • RNA Precursors / chemistry
  • RNA Precursors / genetics
  • RNA Processing, Post-Transcriptional*
  • RNA, Messenger / biosynthesis
  • RNA, Messenger / genetics
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Ribonuclease III / metabolism
  • Sequence Analysis, RNA
  • Structure-Activity Relationship
  • Transcription, Genetic*
  • Transfection
  • Tripartite Motif Proteins / genetics
  • Tripartite Motif Proteins / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism

Substances

  • MIRN29 microRNA, human
  • MicroRNAs
  • RNA Precursors
  • RNA, Messenger
  • RNA-Binding Proteins
  • Tripartite Motif Proteins
  • TRIM71 protein, human
  • Ubiquitin-Protein Ligases
  • DICER1 protein, human
  • DROSHA protein, human
  • Ribonuclease III
  • DEAD-box RNA Helicases