Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide

Zylbee, Ester; Vesco, Cesare; Penman, Sheldon

doi:10.1016/0022-2836(69)90414-8

Cited by 299 publications

(80 citation statements)

References 16 publications

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“…This is consistent with the improved protection of the atfs-1(et15) gof mutant against ethidium bromide (EtBr) (Fig. S4 A-C), which impairs replication and transcription of the mitochondrial genome in cultured cells thus reducing the synthesis of mitochondrially encoded polypeptides (16)(17)(18)(19), and the improved respiration of this mutant when challenged with statin (see below).…”

Section: Resultssupporting

confidence: 80%

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

Rauthan

Ranji

Pradenas

et al. 2013

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

117

137

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Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction.

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Section: Resultssupporting

confidence: 80%

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

Rauthan

Ranji

Pradenas

et al. 2013

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

117

137

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“…1). Ethidium bromide is specific for mitochondrial RNA under these conditions (22). The molecular weight nuclear RNA but allows synthesis of mitochondrial RNA (23)(24)(25).…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial Protein Synthesis: RNA with the Properties of Eukaryotic Messenger RNA

Perlman

Abelson

Penman

1973

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

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A heterogeneous RNA fraction with properties resembling those of messenger RNA was identified in mammalian mitochondria. Synthesis of contaminating RNA of nuclear origin was suppressed by treatment with camptothecin. Labeling of the messenger-like RNA is completely inhibited by ethidium bromide, a specific inhibitor of mitochondrial functions.Although mitochondrial protein synthesis resembles that of prokaryotes in several regards, the messenger-like RNA is covalently linked to poly(adenylic acid) [poly(A)J. Poly(A) has thus far been found only in eukaryotic cells. The poly(A) segment has a gel electrophoretic mobility of about 4 S, corresponding to a length of 50-80 nucleotides, and thus resembles in size the poly(A) found in some mammalian viral RNAs. The messenger RNA can be released from the mitochondrial protein-synthesizing structure by treatment with puromycin.Mitochondria of eukaryotic cells have a distinct system of protein synthesis that is quite dissimilar from that found in the cellular cytoplasm. The ribosome-like structure of mitochondria is relatively small (60 S) (1-4). Mitochondrial protein synthesis is sensitive to chloramphenicol, an inhibitor of prokaryotic ribosomal function, and insensitive to cycloheximide, an inhibitor of eukaryotic protein synthesis. NFormyl-methionine has been found in mitochondria and is presumed to initiate polypeptide synthesis as in bacteria (5, 6).These properties of mitochondrial protein synthesis suggested a similarity to prokaryotic rather than eukaryotic mechanisms, and there has been speculation about a possible origin of mitochondria in a primordial prokaryotic symbiont.In this report, we describe an RNA species synthesized in mitochondria with properties that suggest it serves as messenger RNA for mitochondrial protein synthesis. Surprisingly, this RNA contains polyadenylate segments, which have, so far, been found only in the messenger RNA of eukaryotic cells (7)(8)(9)(10)(11) Analysis of Total Mitochondrial RNA. Cellular extracts were prepared and analyzed as described (13). Briefly, cells were washed and then suspended in 1 ml of RSB buffer [10 mM NaCl-1.5 mM MgCl2-10 mM Tris* HCO (pH 7.4)] before they were broken in a Dounce homogenizer. Nuclei were removed by centrifugation at 800 X g for 2 min. The supernatant was again centrifuged at 7700 X g for 8 min. This pellet (mitochondrial fraction) was resuspended in sodium dodecyl sulfate (SDS) buffer and extracted by the hot phenol-SDS method (14). The RNA was analyzed by electrophoresis on 2.7% acrylamide gels for 3.5 hr at 7.5 V/cm. Gels were fractionated as described (15).Analysis of Poly(A)-Containing RNA. Mitochondrial RNA was labeled in the presence of camptothecin, and a crude mitochondrial fraction was prepared. Mitochondria were suspended in MSB buffer [100 mM NaCl-10 mM MgCl2-10 mM Tris*HCl (pH 7.4)] and lysed with 0.5% sodium deoxycholate-0.5% Brij 58. (Brij 58 was obtained from the Hercules Powder, Co.) The lysed mitochondria were layered on a 15-30% MBS-sucrose gradients and centrifuged ...

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“…1 16 in the cell at the time the drug was administered (3) . In animal cells, EBr has been shown to inhibit mitochondrial protein synthesis (4,5) and mitochondrial RNA synthesis (6,7) .…”

Section: Introductionmentioning

confidence: 99%

THE EFFECT OF ETHIDIUM BROMIDE ON MITOCHONDRIAL DNA SYNTHESIS AND MITOCHONDRIAL DNA STRUCTURE IN HELA CELLS

Leibowitz

1971

The Journal of Cell Biology

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The synthesis of mitochondrial DNA (mDNA) in HeLa cells is selectively inhibited by relatively low concentrations of ethidium bromide . After exposure of cells to strongly inhibitory concentrations of the drug, the apparent superhelix density of mDNA is rapidly increased, as judged by its buoyant density in CsC1 in the presence of ethidium bromide . Mitochondrial DNA synthesized in the presence of partially inhibitory concentrations of ethidium bromide is also altered in its buoyant density in the presence of the dye, but is more heterogeneous in this respect . However, the change in buoyant density of newly synthesized mDNA may be explained by changes in structure other than a change in superhelix density, as indicated by its increased resistance to digestion by pancreatic DNase .

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Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide

Cited by 299 publications

References 16 publications

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway

Mitochondrial Protein Synthesis: RNA with the Properties of Eukaryotic Messenger RNA

THE EFFECT OF ETHIDIUM BROMIDE ON MITOCHONDRIAL DNA SYNTHESIS AND MITOCHONDRIAL DNA STRUCTURE IN HELA CELLS

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