A comparison of poly (A)-associated RNA from synaptosomes and cytoplasmic subcellular fractions of rat brain

Oligo(dT)-cellulose binding poly(A)-RNA from rat brain microsomes, mitochondria and synaptosomes was isolated and analysed. Synaptosomes and mitochondria appeared to contain a higher proportion of such RNA than microsomes. Poly(A)-RNA from the three fractions was then electrophoretically fractionated in two different gel systems. Mitochondrial and microsomal poly(A)-RNA presented different fractionation patterns. Synaptosomal poly(A)-RNA also presented a characteristic patterns of its own which could not be entirely explained as a mixture of the previous two. Its main feature was the presence of a prominent band with an SE value of around 7. Such species, absent in microsomes, was present as well in mitochondria but in smaller concentration. On the basis of the present evidence and of previous results, it appears that such 7 SE species, although a mitochondrial messenger RNA in origin, becomes accumulated in vivo into synaptic membranes.     

Salt hardiness and dye reduction by potato tissue and mitochondrial fractions as influenced by previous storage of the tubers

Oxygen uptake and tetrazolium reduction occurred at higher rates in discs from potato tubers (Solanum tuberosum L.) stored at 0° than in discs from tubers stored at 12.8°. Tetrazolium reduction was at a higher rate in mitochondrial fractions from tubers stored at 0° than in mitochondrial fractions from tubers stored at 12.8°. These physiological activities were more resistant to hypertonic KCl treatments in tissue and mitochondrial fractions from tubers stored at 0° than in tissue and mitochondrial fractions from tubers stored at 12.8°. Inhibition of O₂ uptake and tetrazolium reduction progressively increased with increasing concentrations of KCl for tissue and mitochondrial fractions from tubers stored at 0 and 12.8°, but inhibition was more severe and occurred at lower concentrations of KCl for the material from tubers stored at 12.8°. Tissue from tubers stored at 0° was at the same time more sensitive to hypotonic solutions and more resistant to hypertonic solutions than corresponding tissue from tubers stored at 12.8°. Adaptive changes brought on in the tubers by the stress of cold storage were demonstrated in the discs and mitochondrial fractions prepared from cold-stored tubers.     

The significance of promitochondrial structures in rat liver for mitochondrial biogenesis

1. The heavy, light and fluffy mitochondrial fractions obtained by differential centrifugation were further characterized with respect to their protein synthesizing ability in vitro, their nucleic acid content, buoyant density of their DNA and ultrastructure. 2. The light mitochondrial fraction synthesized proteins in vitro at a rate 4-5 times as high as heavy and fluffy mitochondria. The incorporation ability of this fraction was also maximally affected by the thyroid status of the animal. The radioactivity in leucyl-tRNA of the light mitochondrial fraction was about 3-4 times as high as that of the other two fractions. 3. The heavy, light and fluffy mitochondrial fractions contained small but consistent amounts of RNA and DNA. Although the DNA content was the same in all mitochondria fractions, the light mitochondria contained relatively more RNA. The buoyant density of DNA from all the fractions was 1.701g/cm(3). 4. Electron microscopy revealed that the heavy mitochondria have a typical mitochondrial architecture, with densely packed cristae and a well developed double membrane. Light mitochondria were also surrounded by double membranes, but were smaller in size and contained less cristae. The fluffy fraction consisted of a mixture of well formed mitochondria and those in the process of degradation. 5. The significance of these findings in relation to mammalian mitochondrial genesis is discussed.     

An unmethylated “3 SE” RNA in hamster mitochondria: A 5 S RNA-equivalent?

A novel low molecular weight (“3 S_E”) RNA associated with hamster cell mitochondria has been partially characterized. It was present at approx. 1:1 molar ratio with structural mitochondrial ribosomal RNA; it was unmethylated; and it resembled other mitochondrial RNA fractions in having a low content of G + C. These findings support the idea that 3 S_E RNA is a mitochondrial equivalent of 5 S ribosomal RNA.     

Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions

Semen contains relatively ill-defined regulatory components that likely aid fertilization, but which could also interfere with defense against infection. Each ejaculate contains trillions of exosomes, membrane-enclosed subcellular microvesicles, which have immunosuppressive effects on cells important in the genital mucosa. Exosomes in general are believed to mediate inter-cellular communication, possibly by transferring small RNA molecules. We found that seminal exosome (SE) preparations contain a substantial amount of RNA from 20 to 100 nucleotides (nts) in length. We sequenced 20–40 and 40–100 nt fractions of SE RNA separately from six semen donors. We found various classes of small non-coding RNA, including microRNA (21.7% of the RNA in the 20–40 nt fraction) as well as abundant Y RNAs and tRNAs present in both fractions. Specific RNAs were consistently present in all donors. For example, 10 (of ∼2600 known) microRNAs constituted over 40% of mature microRNA in SE. Additionally, tRNA fragments were strongly enriched for 5’-ends of 18–19 or 30–34 nts in length; such tRNA fragments repress translation. Thus, SE could potentially deliver regulatory signals to the recipient mucosa via transfer of small RNA molecules.     

Seizure-induced changes in mitochondrial redox status

The aim of this study was to determine seizure-induced oxidative stress by measuring hippocampal glutathione (GSH) and glutathione disulfide (GSSG) levels in tissue and mitochondria. Kainate-induced status epilepticus (SE) in rats resulted in a time-dependent decrease of GSH/GSSG ratios in both hippocampal tissue and mitochondria. However, changes in GSH/GSSG ratios were more dramatic in the mitochondrial fractions compared to hippocampal tissue. This was accompanied by a mild increase in glutathione peroxidase activity and a decrease in glutathione reductase activity in hippocampal tissue and mitochondria, respectively. Since coenzyme A (CoASH) and its disulfide with GSH (CoASSG) are primarily compartmentalized within mitochondria, their measurement in tissue was undertaken to overcome problems associated with GSH/GSSG measurement following subcellular fractionation. Hippocampal tissue CoASH/CoASSG ratios were decreased following kainate-induced SE, the time course and magnitude of change paralleling mitochondrial GSH/GSSG levels. Cysteine, a rate-limiting precursor of glutathione was decreased following kainate administration in both hippocampal tissue and mitochondrial fractions. Together these changes in altered redox status provide further evidence for seizure-induced mitochondrial oxidative stress.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.     

Study of the ability of mitochondrial proteins to interact with actin

Using affinity chromatography of F-actin-sepharose 4B, the ability of proteins from rat liver submitochondrial fractions to interact with rabbit skeletal muscle actin was studied. The bulk of the actin-bound components was detected in the soluble compartments of the mitochondria, i.e., mitochondrial matrix and intermembrane space. The interaction was predominantly weak, since the desorption of the proteins from the column occurred at increased ionic strength of the solution. In membrane fractions, four polypeptides with Mr 65 000, 62 000, 59 000 and 10 500 eluting from the column only under effects of denaturating agents were predominant, thus suggesting the specificity of their binding to the immobilized actin. In a model system involving mitochondrial enzyme preparations (cytochrome c, glutamate dehydrogenase, isocitrate dehydrogenase, catalase), the possibility of their adsorption of F-actin-sepharose was investigated. It was shown that the highest adsorption capacity was observed in the case of immobilized actin with respect to catalase, the lowest one-to glutamate dehydrogenase. The data obtained suggest that the interaction of the actin-like mitochondrial protein with the system of solubilized enzymes may serve as a basis for their normal functioning.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Effect of undernutrition on DNA and RNA synthesis in subcellular fractions from different regions of the developing rat brain

The effect of undernutrition on the incorporation of [methyl-³H]thymidine into DNA and of 5-[³H]uridine into RNA of cerebral hemispheres, cerebellum, and brain stem was studied in vivo and in vitro in rats. The labeling of DNA from nuclei and mitochondria and of RNA from nuclei, mitochondria, microsomes, and soluble fractions, was also measured in vitro. The results demonstrate that nucleic acid synthesis is impaired and delayed during undernutrition. Specific effects were observed for the different brain regions and subcellular fractions: at 10 days nuclear and mitochondrial DNA and RNA synthesis was impaired, whereas at 30 days only the mitochondrial nucleic acid synthesis was affected.The delay of DNA and RNA labeling, caused by undernutrition, was most evident in the cerebellum, probably due to its intense cell proliferation during postnatal development. The specific sensitivity of mitochondria as compared to other subcellular fractions, may be due to the intense biogenesis and/or turnover of nucleic acids in brain mitochondria not only during postnatal development, but also in the adult animal.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment.

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5--6-fold higher in the parenchymal cells.     

Kinetics of synthesis of cytoplasmic messenger-like RNA not associated with ribosomes in HeLa cells

The turn-over of cytoplasmic messenger-like RNA not associated with polyribosomes as well as that of polyribosomal mRNA was investigated by labelling with [3H]uridine in conditions of arrested ribosomal RNA and mitochondrial RNA synthesis. The synthesis of ribosomal RNA was inhibited with toyokamycin and that of mitochondrial RNA with ethidium bromide. In both accumulation kinetics and actinomycin-D-chase experiments, cytoplasmic messenger-like ribonucleoprotein particles and polyribosomes were fractionated by buoyant density centrifugation in CsCl gradients. The half-life of free m1RNA was found to be of 1--2 h whereas the bulk of polyribosomal mRNA was stable over the time period considered (up to 8 h) but with a minor short-lived component. Purification of RNA from polyribosomes labelled under the same conditions and fractionation of it into polyadenylated and non-polyadenylated fractions showed that this short-lived minor component of half-life less than 1 h is non-polyadenylated.     

The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P

The mitochondrion-associated RNase P activity (mtRNase P) was extensively purified from HeLa cells and shown to reside in particles with a sedimentation constant ( approximately 17S) very similar to that of the nuclear enzyme (nuRNase P). Furthermore, mtRNase P, like nuRNase P, was found to process a mitochondrial tRNA(Ser(UCN)) precursor [ptRNA(Ser(UCN))] at the correct site. Treatment with micrococcal nuclease of highly purified mtRNase P confirmed earlier observations indicating the presence of an essential RNA component. Furthermore, electrophoretic analysis of 3'-end-labeled nucleic acids extracted from the peak of glycerol gradient-fractionated mtRNase P revealed the presence of a 340-nucleotide RNA component, and the full-length cDNA of this RNA was found to be identical in sequence to the H1 RNA of nuRNase P. The proportions of the cellular H1 RNA recovered in the mitochondrial fractions from HeLa cells purified by different treatments were quantified by Northern blots, corrected on the basis of the yield in the same fractions of four mitochondrial nucleic acid markers, and shown to be 2 orders of magnitude higher than the proportions of contaminating nuclear U2 and U3 RNAs. In particular, these experiments revealed that a small fraction of the cell H1 RNA (of the order of 0.1 to 0.5%), calculated to correspond to approximately 33 to approximately 175 intact molecules per cell, is intrinsically associated with mitochondria and can be removed only by treatments which destroy the integrity of the organelles. In the same experiments, the use of a probe specific for the RNA component of RNase MRP showed the presence in mitochondria of 6 to 15 molecules of this RNA per cell. The available evidence indicates that the levels of mtRNase P detected in HeLa cells should be fully adequate to satisfy the mitochondrial tRNA synthesis requirements of these cells.     

Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA

In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5%. In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA. The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked. The primary sequence of ms RNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA. In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA. However, ms RNA was detected mainly in the mitochondrial 25?000?×?g supernatant fraction which was devoid of ribosomes. It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.     

Subcellular localization of aldosterone-induced proteins in toad urinary bladders

Paired toad urinary hemibladders were incubated with [35S]methionine in the presence (experimental) or absence (control) of aldosterone. Short-circuit current was used to monitor aldosterone-induced Na+ transport. Protein synthesis in epithelial cell subcellular fractions (cytosolic, microsomal, mitochondrial) was evaluated by gradient polyacrylamide gel electrophoresis and autoradiography. Aldosterone-induced proteins were identified in the cytosolic and microsomal fractions (70 000 and 15 000 daltons, respectively). These results represent the first demonstration of aldosterone-induced proteins in subcellular fractions of epithelial cells derived from single toad urinary hemibladders.     

A Fox2-Dependent Fatty Acid ?-Oxidation Pathway Coexists Both in Peroxisomes and Mitochondria of the Ascomycete Yeast Candida lusitaniae

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying ¹⁴C_α-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.