An in vitro system to silence mitochondrial gene expression

Cell. 2021 Nov 11;184(23):5824-5837.e15. doi: 10.1016/j.cell.2021.09.033. Epub 2021 Oct 20.


The human mitochondrial genome encodes thirteen core subunits of the oxidative phosphorylation system, and defects in mitochondrial gene expression lead to severe neuromuscular disorders. However, the mechanisms of mitochondrial gene expression remain poorly understood due to a lack of experimental approaches to analyze these processes. Here, we present an in vitro system to silence translation in purified mitochondria. In vitro import of chemically synthesized precursor-morpholino hybrids allows us to target translation of individual mitochondrial mRNAs. By applying this approach, we conclude that the bicistronic, overlapping ATP8/ATP6 transcript is translated through a single ribosome/mRNA engagement. We show that recruitment of COX1 assembly factors to translating ribosomes depends on nascent chain formation. By defining mRNA-specific interactomes for COX1 and COX2, we reveal an unexpected function of the cytosolic oncofetal IGF2BP1, an RNA-binding protein, in mitochondrial translation. Our data provide insight into mitochondrial translation and innovative strategies to investigate mitochondrial gene expression.

Keywords: IGF2BP1; antisense; mitochondria; mitochondrial ribosome; morpholino; oxidative phosphorylation; translation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electron Transport
  • Electron Transport Complex IV / genetics
  • Gene Expression Regulation*
  • Gene Silencing*
  • Genes, Mitochondrial*
  • HEK293 Cells
  • Humans
  • Mitochondrial Proteins / metabolism
  • Oligonucleotides / chemistry
  • Oxidative Phosphorylation
  • Protein Biosynthesis
  • Protein Subunits / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Mitochondrial / metabolism
  • RNA-Binding Proteins / metabolism
  • Ribosomes / metabolism
  • Saccharomyces cerevisiae / metabolism


  • IGF2BP1 protein, human
  • Mitochondrial Proteins
  • Oligonucleotides
  • Protein Subunits
  • RNA, Messenger
  • RNA, Mitochondrial
  • RNA-Binding Proteins
  • Electron Transport Complex IV