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.

Figure 1

Scheme for the stringent purification of mitochondrial fractions. See text for details.

Figure 2

(A) Map of the insert of plasmid pT7.5S.Dra. (B) Northern analysis. Total mitochondrial nucleic acids from highly purified rat liver mitochondria (lane 2) were electrophoresed in parallel with T7 runoff transcripts from DraI-digested pT7.5S.Dra (lane 1) and were hybridized with a probe specific for 5S rRNA.

Figure 3

RT-PCR analyses. (A) Analysis of 5S rRNA, COX I mRNA, and COX VIIc mRNA transcripts in total cellular RNA isolated from human 143B.TK⁻ cells. RNA was subjected to RT-PCR in the presence (+) or absence (-) of reverse transcriptase activity. M1, markers of HaeIII-digested ΦX174 DNA. Expected sizes of RT-PCR products, in bp, are shown on the right. (B) Analysis described in A except that RNA from highly purified RNAse A–treated mitochondria was used as the template after DNase I treatment. M2, 100-bp ladder (Genosys Biotechnologies, The Woodlands, TX). (C) Analysis described in B except that RNA from highly purified RNase A–treated mitoplasts (obtained by the digitonin method) was used as the template. Mitoplasts were also lysed with 0.5% SDS in the presence of RNase A and subjected to RT-PCR to amplify 5S rRNA (5S) and COX I mRNA (CI). (D) Analysis of 5S rRNA, COX I mRNA, and 5.8S rRNA transcripts in total RNA isolated from rat liver. Methods and notation are described in A. M3, marker XIII (Boehringer Mannheim). (E) Analysis described in D except that RNA from highly purified RNAse A–treated rat liver mitochondria was used as the template.

Figure 4

Importation of synthetic 5S rRNA into human mitochondria. (A) Map of the insert of p5SSE. The two sets of primer pairs (short single-headed arrows) used to analyze the endogenous and synthetic variants and their corresponding SmaI-digested PCR products (double-headed arrows with the expected sizes above the arrows, in bp) are indicated. The 5S rRNA gene is in black with the known Pol III control elements (Willis, 1993) in the shaded boxes. The relevant restriction sites are indicated as follows: B, BamHI; E, EcoRI; S, SmaI; and Sc, SacI. (B) RT-PCR and RFLP analysis of total RNA from 293T cells that had been transfected transiently with p5SSE or pSV2neo, by the use of primers 5S-F and 5Sm-R, and electrophoresed through a 12% nondenaturing polyacrylamide gel. Note that the sizes of the SmaI digestions of RT-PCR products correspond exactly to the PCR products from pHU5S1 (wild-type) and p5SSE (A–C transversion) standards. Sizes of bands, in bp, are shown on the right. Sizes of M2 markers, in bp, are shown on the left. The 16-bp fragment is not resolved in this gel system. (C) RT-PCR and RFLP analysis of RNA isolated from highly purified mitochondria (Mt) and mitoplasts (Mp; obtained by the swell-contract method) from 293T cells transfected with p5SSE or pSV2neo, by the use of primers 5S-C and 5Sm-R, and electrophoresed through a 20% nondenaturing polyacrylamide gel. Note that the diagnostic 41-bp fragment is present in the p5SSE samples (lanes 4 and 5; the 16-bp fragments were diffuse and barely visible but were discernible in extremely dark exposures), with a size identical to that obtained in a PCR and RFLP from the p5SSE standard (lane 8), but is absent in the pSV2neo control samples (lanes 6 and 7). All RT-PCR species were abolished when mitochondria were treated with RNase A in the presence of SDS (lanes 2 and 3).