Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines

Proc. Natl. Acad. Sci. U.S.A.

et al. 2021

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Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multisubunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

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Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

High-resolution imaging reveals compartmentalization of mitochondrial protein synthesis in cultured human cells

Zorkau

Proc. Natl. Acad. Sci. U.S.A.

et al. 2021

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“…RNA FISH/IF was carried out as in [20] with minor modifications: Permeabilisation was carried out for 10 min, followed by incubation with respective antibodies (as above) in PBS. Stellaris Custom RNA FISH probe sets for rRNA and mRNA were designed with high stringency (masking level 5 and 4) respectively using the Stellaris Designer tool (https://www.biosearchtech.com/stellaris-designer).…”

Section: Rna Fish / Immunofluorescent Cytochemistrymentioning

confidence: 99%

“…Initial data from mitochondrial FUNCAT visualising mitochondrial protein synthesis in fibroblasts, U2OS and HeLa cells had suggested an enrichment of translation in the perinuclear region of the cell compared to the periphery when normalized to a marker of mitochondrial mass [19]. To extend this analysis we subjected human cultured U2OS cells to mitochondrial FUNCAT as previously described [19,20]. All cell images were manually masked such that signals were assigned either to the perinuclear or peripheral regions of each cell.…”

Section: Mitochondrial Translation Occurs Preferentially In the Perinuclear Networkmentioning

confidence: 99%

“…A B A similar protocol that had been used to visualize mitoribosomal rRNA by RNA FISH [20] was employed to detect the mt-mRNA that encodes COXII of complex IV. As shown in Figure 3A, once again there was a noticeable decrease in signal towards the periphery of the cell (35%) even after normalization to the mitochondrial network marker…”

Section: Differential Distribution Of Mt-protein Synthesis Is Not Reflective Of the Mt-mrna Distributionmentioning

confidence: 99%

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Mitochondrial translation occurs preferentially in the peri-nuclear mitochondrial network of cultured human cells

Caroline

et al. 2021

Preprint

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Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

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Four-Color STED Super-Resolution RNA Fluorescent In Situ Hybridization and Immunocytochemistry to Visualize Mitochondrial mRNAs in Context with Mitochondrial Ribosomes

Methods in Molecular Biology

Chrzanowska-Lightowlers

et al. 2023