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Mitochondria contain their own gene expression machinery, which synthesizes core subunits of the oxidative phosphorylation system. Monitoring mitochondrial translation within spatial compartments of cells is difficult. Here we describe a method to visualize mitochondrial translation within defined parts of cells, using a click chemistry approach. This method can be applied to different cell types such as neurons and allows detection of newly synthesized mitochondrial proteins in spatial resolution using microscopy techniques. Furthermore, using click chemistry, mitochondrial translation can also be monitored by standard SDS-PAGE. The described method avenues the analysis of newly synthesized mitochondrial encoded proteins in the cellular context, by avoiding the usage of radioactive components., (Copyright © 2024. Published by Elsevier Inc.)

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. [Display omitted] • Precursor-morpholino chimera can be imported into mitochondria in vitro • Translation of individual mRNAs can be efficiently blocked • Translation of bicistronic mRNAs requires translation of upstream open reading frame • Cytosolic IGF2BP1 also localizes to mitochondria and is required for translation The mitochondrial genome encodes core subunits of the OXPHOS system. Imported precursor-morpholino chimera efficiently blocks the translation of mitochondrial mRNAs. Translation of bicistronic mRNAs was shown to depend on translation of the upstream open reading frame. IGF2BP1 represents a mitochondrial RNA- and ribosome-interacting protein required for mitochondrial translation. [ABSTRACT FROM AUTHOR]