Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

Mol Cell. 2019 Feb 7;73(3):505-518.e5. doi: 10.1016/j.molcel.2018.11.005. Epub 2018 Dec 13.

Abstract

Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing ∼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.

Keywords: DGCR8; DROSHA; RNase III; miRNA; microRNA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • HCT116 Cells
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • MicroRNAs / chemistry
  • MicroRNAs / genetics
  • MicroRNAs / metabolism*
  • Models, Molecular
  • Nucleic Acid Conformation
  • Nucleotide Motifs*
  • Protein Interaction Domains and Motifs
  • RNA Processing, Post-Transcriptional*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Structure-Activity Relationship
  • Substrate Specificity

Substances

  • DGCR8 protein, human
  • MicroRNAs
  • RNA-Binding Proteins
  • DROSHA protein, human
  • Ribonuclease III