Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

Abstract

Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA. As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA. For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant. We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts. We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified. INTRODUCTIONMitochondria are organelles present in virtually all eukaryotic cells, responsible for most of the energy production required for normal cellular homeostasis. Human mitochondria possess their own DNA (mtDNA), which encodes the two RNA species present in mitochondrial ribosomes (12S and 16S rRNAs), a full set of transfer RNAs (tRNAs) (22 genes) required for protein synthesis (O'Brien et al., 1990), and 13 polypeptides, all constituents of respiratory chain complexes (Anderson et al., 1981).Because mitochondria possess a fully functional genetic apparatus capable of replication, transcription, and translation, they are often considered to be intracellular organelles endowed with a partial autonomy. This autonomy, however, is more apparent than real; in addition to the components encoded by mtDNA, all of the remaining enzymes required for proper functioning of the mitochondrion's genetic machinery (such as DNA and RNA polymerases, ribosomal proteins, aminoacyl tRNA synthetases, etc.) are encoded by nuclear DNA (nDNA), synthesized in the cytosol, and imported into the organelle (Schatz and Dobberstein, 1996;Neupert, 1997). The same is true for all enzymes involved in the myriad metabolic pathways that take place in the mitochondrial environment.Interestingly, at least two mitochondrial enzymes, RNase MRP (a site-specific endoribonuclease involved in primer RNA metabolism in mammalian mitochondria [Chang and Clayton, 1987;Topper and Clayton, 1990;Li et al., 1994]) and RNase P (an endoribonuclease involved in tRNA processing [Doerson et al., 1985]), are ribonucleoproteins that contain an RNA moiety that is encoded by nDNA and is imported into the organelle. However, unlike the mechanisms for protein import into mitochondria, the mechanisms of RNA import into mitochondria are poorly understood.The importation of RNA into mitochondria was first postulated over 30 years ago, as a corollary to mitochondrial protein synthesis and the lack of a full set of tRNA genes in the mitochondrial genome of Tetrahymena (Suyama and Eyer, 1967). This postulate was proven recently (Rusconi and Cech, 1996), and the import of tRNAs into mitochondria has now been (Schneider, 1994;Kaz...

CHCM1/CHCHD6, Novel Mitochondrial Protein Linked to Regulation of Mitofilin and Mitochondrial Cristae Morphology

“…Mitochondria from these cells were isolated and gradient-purified as described previously (18). Briefly, cells were harvested by low speed centrifugation and resuspended in homogenization buffer (10 mM NaCl, 1.5 mM MgCl 2 , and 10 mM Tris-HCl, pH 7.5).…”

Does Human mtDNA Recombine?

“…Experiments in yeast have shown that DNA can leave the mitochondria at a rate of ∼2 × 10 −5 but no flow of DNA was detected in the opposite direction (Thorsness and Fox 1993). However, it is clear that the mitochondria of a wide range of eukaryotes, including humans, import various RNAs (Doersen et al 1985;Li et al 1994;Magalhaes et al 1998) and even viral RNA (Somasundaran et al 1994). …”

Peptide nucleic acid delivery to human mitochondria

“…25,26 Schon and colleagues have recently reported that a 5S ribosomal RNA may be imported, but its function has yet to be determined. 27 It is therefore possible that mammalian mitochondria do not possess an efficient import mechanism for nucleic acids. Vestweber and Schatz, 28 however, were able to demonstrate that by subjecting import-competent, isolated mitochondria to DNA-conjugated mitochondrial precursor proteins, the DNA became nuclease or phosphodiesterase resistant, consistent with the mitochondria importing nucleic acids via the general protein import pathway.…”