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Updated: Aug 13, 2026
Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA
Published on: November 14, 2017
5 S rRNA and tRNA import into human mitochondria. Comparison of in vitro requirements
N S Entelis1, O A Kolesnikova, S Dogan
1Formation de Recherche en Evolution 2375, CNRS Modèles d'Etude de Pathologies Humaines, 21 rue René Descartes, 67084 Strasbourg, France.
Abstract:
In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from the cytoplasm. We demonstrated previously that isolated human mitochondria are able to internalize a yeast tRNA(Lys) in the presence of yeast soluble factors. Here, we describe an assay for specific uptake of 5 S rRNA by isolated human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was comparable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble proteins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA uptake by the mitochondria. We show that human mitochondria can also selectively internalize several in vitro synthesized versions of yeast tRNA(Lys) as well as a transcript of the human mitochondrial tRNA(Lys). Either yeast or human soluble proteins can direct this import, suggesting that human cells possess all factors needed for such an artificial translocation. On the other hand, the efficiency of import directed by yeast or human protein factors varies significantly, depending on the tRNA version. Similarly to the yeast system, tRNA(Lys) import into human mitochondria depended on aminoacylation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.
Insights
Human mitochondria import 5S ribosomal RNA (rRNA) but not transfer RNAs (tRNAs) in vivo. This study details 5S rRNA and tRNA import into isolated human mitochondria, revealing distinct requirements and shared import pathways.
Area of Science:
- Mitochondrial biology
- Molecular biology
- RNA transport
Background:
- Human mitochondria import 5S ribosomal RNA (rRNA) but exclude transfer RNAs (tRNAs) from the cytoplasm in vivo.
- Previous studies showed isolated human mitochondria can import yeast tRNA(Lys) with yeast soluble factors.
Purpose of the Study:
- To develop an assay for specific 5S rRNA uptake by isolated human mitochondria.
- To compare the import requirements of 5S rRNA with artificial tRNA import.
- To investigate the role of the pre-protein import channel in RNA uptake.
Main Methods:
- Development of an in vitro assay for isolated human mitochondrial 5S rRNA uptake.
- Comparison of 5S rRNA and tRNA import requirements, including ATP, membrane potential, and soluble factors.
- Investigation of the involvement of the pre-protein import channel using channel blockers.
- Analysis of tRNA import dependency on aminoacylation and lysyl-tRNA synthetase precursor.
Main Results:
- 5S rRNA uptake efficiency by isolated mitochondria mirrored in vivo observations.
- 5S rRNA import required ATP, transmembrane electrochemical potential, and soluble protein factors.
- Inhibition of the pre-protein import channel blocked both 5S rRNA and tRNA internalization, suggesting its involvement in RNA import.
- Human mitochondria could import in vitro synthesized yeast tRNA(Lys) variants and human mitochondrial tRNA(Lys) transcripts, directed by either yeast or human soluble factors.
- tRNA import efficiency varied with tRNA version and depended on aminoacylation and lysyl-tRNA synthetase precursor.
- Soluble factors for 5S rRNA import were distinct from those mediating tRNA internalization.
Conclusions:
- Human mitochondria possess a specific uptake mechanism for 5S rRNA that is energy-dependent and protein-guided.
- The pre-protein import channel appears to play a role in the mitochondrial import of both 5S rRNA and tRNAs.
- Human cells contain the necessary factors for artificial tRNA translocation into mitochondria, although efficiency is tRNA-dependent.
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