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Visualizing Mitochondrial Ribosomal RNA and Mitochondrial Protein Synthesis in Human Cell Lines.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2192, pp. 159-181. - Publication Year :
- 2021
-
Abstract
- Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.
- Subjects :
- Amino Acids chemistry
Cell Line, Tumor
DNA, Mitochondrial genetics
Humans
Microscopy, Fluorescence methods
Oxidative Phosphorylation
Protein Biosynthesis
Ribosomal Proteins metabolism
Click Chemistry methods
Immunohistochemistry methods
In Situ Hybridization, Fluorescence methods
Mitochondrial Proteins metabolism
Mitochondrial Ribosomes metabolism
RNA, Mitochondrial metabolism
RNA, Ribosomal metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 2192
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 33230773
- Full Text :
- https://doi.org/10.1007/978-1-0716-0834-0_13