Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression

Question Dependent Recurrent Entity Network for Question Answering

Question Answering is a task which requires building models capable of providing answers to questions expressed in human language. Full question answering involves some form of reasoning ability. We introduce a neural network architecture for this task, which is a form of $Memory\ Network$, that recognizes entities and their relations to answers through a focus attention mechanism. Our model is named $Question\ Dependent\ Recurrent\ Entity\ Network$ and extends $Recurrent\ Entity\ Network$ by exploiting aspects of the question during the memorization process. We validate the model on both synthetic and real datasets: the $bAbI$ question answering dataset and the $CNN\ \&\ Daily\ News$$reading\ comprehension$ dataset. In our experiments, the models achieved a State-of-The-Art in the former and competitive results in the latter.Comment: 14 page

Expression of the Mitochondrial Genome in HeLa Cells. XV. Effect of Inhibition of Mitochondrial Protein Synthesis on Mitochondrial Formation

The effect of selective inhibition of mitochondrial protein synthesis by chloramphenicol at 40 or 200 µg/ml on the formation of mitochondria in HeLa cells was investigated. HeLa cells, under the conditions used in the present work, grow at a decreasing rate for at least four cell generations in the presence of 40 µg/ml chloramphenicol, and for two generations in the presence of 200 µg/ml chloramphenicol. The progressive cell growth inhibition which begins after 2 days of exposure of the cells to 40 µg/ml chloramphenicol is immediately or gradually reversible, upon removal of the drug, for periods up to at least 8 days of treatment, though there is a progressive loss of cloning efficiency. In cells which have been treated for 6–7 days with 40 or 200 µg/ml of chloramphenicol, mitochondrial protein synthesis occurs at a normal or near-normal rate 1 h after removal of the drug. Mitochondria increase normally in number and show a normal size and amount of cristae in the presence of either concentration of drug. However, in 4–5% of the mitochondrial profiles the cristae appear to be arranged in unusual, circular, looped or whorled configuration

Metabolic properties of the products of mitochondrial protein synthesis in HeLa cells

The metabolic behavior of the mitochondrial protein synthesis products has been investigated in HeLa cells. Particular attention was given to the four major electrophoretic components (designated as Nos. 2, 3, 5, and 8) of the 10 previously identified as organelle-specific products. Inhibition of cytoplasmic protein synthesis with emetine or cycloheximide causes a rapid decline in the rate of mitochondrial protein synthesis, with an estimated half-life of 1 to 2 h, affecting in a parallel way all the discrete components. About 30% of the original synthetic activity appears to be resistant to emetine treatment for at least 24 h; however, all the polypeptides synthesized after the first 4 h of cell exposure to emetine are metabolically unstable, possibly because of lack of integration into the inner mitochondrial membrane. An analysis of the stability of newly synthesized products of mitochondrial protein synthesis pulse-labeled in the presence of cycloheximide and then chased in the absence of the drug (i.e. under conditions of resumed cytoplasmic protein synthesis) has revealed marked differences among the various discrete components. In particular, about three-fourths of the radioactivity associated with components 3 and 5 decays within 4 h of chase, the remainder being substantailly stable afterwards; by contrast, the radioactivity in components 2 and 8 shows only a slow decline during a 3-day chase. If the chase is carried out under conditions of a persistent block of cytoplasmic protein synthesis, as is the situation after a pulse labeling in the presence of emetine, all newly synthesized components appear to be destablized in various degrees, with the exception of component 5, which is to a great extent stabilized. Inhibition of mitochondrial protein synthesis with chloramphenicol has a progressive stabilizing effect on most of the discrete components newly synthesized after removal of the drug; this effect is especially striking in the case of component 5 which, in experiments of continuous labeling in the presence of emetine after prolonged chloramphenicol treatment, becomes, after 24 h of labeling or more, the only recognizable peak in the electrophoretic pattern of the sodium dodecyl sulfate-lysed mitochondrial fraction. The results of the kinetic experiments described here are interpreted in terms of two roles of cytoplasmically synthesized proteins, one required for the synthesis of polypeptides within the organelles, the other necessary for the stabilization of the mitochondrial products

Differential regulation of expression of the multiple ADP/ATP translocase genes in human cells

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been investigated in cultured cell systems under different experimental conditions, using isoform- specific probes. In several human cell lines tested, i.e. HeLa, Hep3B, 143B, HL60, the T3 gene is expressed as a single 1300-nucleotide mRNA, whereas the T2 gene produces two species of mRNA, 1450 and 1600 nucleotides in size. These two species, which are present in HeLa cells in approximately equivalent amounts, were shown to derive from the use of two different polyadenylation signals. The gene for the muscle-specific isoform of ADP/ATP translocase, T1, was not found to be expressed in any of the cell lines investigated. The levels of T2 and T3 mRNAs in HeLa cells are differentially affected by the growth conditions. In fact, the T2 mRNA level remains relatively constant throughout the exponential and stationary phases, whereas the T3 mRNA level decreases progressively in the second half of the exponential phase and in the stationary phase down to less than 50%. This difference in quantitative behavior of the two mRNAs must reflect changes in their rates of synthesis, since their half-lives are very similar (t^1/2 = 5-6 h), with no significant growth-related differences. Treatment of HL60 cells with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid, two agents which induce cessation of cell proliferation and cell differentiation, resulted in a marked decrease in both T2 and T3 mRNA levels. Exposure of HeLa cells to chloramphenicol produced a pronounced decrease in the levels of both T2 and T3 mRNAs after 48 to 72 h of treatment. Half-life time measurements strongly suggested that this decrease reflected a reduction in the rate of synthesis of the two transcripts. Treatment of HeLa cells with dinitrophenol also produced a dramatic decrease in the steady state levels of both T2 and T3 mRNA, which, however, in contrast to the just mentioned situation, could be accounted for by a decrease in their metabolic stability. Control experiments indicated that the chloramphenicol- and dinitrophenol-induced changes were not a nonspecific consequence of mitochondrial dysfunction. The observations reported here clearly demonstrate that the expression of the multiple ADP/ATP translocase genes in human cells is sensitive to the cell physiological conditions, responding to the varying cellular demands by changes in the rate of synthesis or stability of their mRNAs

Distinctive pattern and translational control of mitochondrial protein synthesis in rat brain synaptic endings

Mitochondrial gene expression has been investigated in synaptic endings from rat cerebral cortex isolated at various stages during the postnatal development and maturation of the animal. The pattern of the mitochondrial translation products labeled in vitro in rat brain synaptosomes revealed some distinctive features when compared with the pattern observed in a rat fibroblast cell line, the most remarkable being the apparent absence of labeling of the ND5 product. This absence contrasted with the presence in synaptosomes of an amount of ND5 mRNA comparable with that found in the rat fibroblast cell line. The rate of mitochondrial protein synthesis per unit amount of mtDNA inbrain synaptosomes showed a characteristic reproducible burst at 10-13 days after birth, thereafter declining sharply in the 3rd week to reach a level that remained constant over a 2-year period. The postnatal burst of mitochondrial protein synthesis coincided with a sharp increase in cytochrome c oxidase activity, pointing to a phase of rapid assembly of respiratory complexes. A comparison of the levels of mitochondrial mRNAs with the corresponding rates of protein synthesis during the animal development and maturation showed a lack of correlation. These observations, together with the apparent lack of translation of the ND5 mRNA, indicate that translational control plays a major role in the regulation of gene expression in rat brain synaptic mitochondria

Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells

In human mitochondrial DNA (mtDNA), the tRNA genes are located in three different transcription units that are transcribed at three different rates. To analyze the regulation of tRNA formation by the three transcription units, we have examined the steady-state levels and metabolic properties of the tRNAs of HeLa cell mitochondria. DNA excess hybridization experiments utilizing separated strands of mtDNA and purified tRNA samples from exponential cells long term labeled with [32P]orthophosphate have revealed a steady-state level of 6 x 10(5) tRNA molecules/cell, with three-fourths being encoded in the H-strand and one-fourth in the L-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNA(Phe) and tRNA(Val) are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs isolated from cells labeled with very short pulses of [5-3H]uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady-state amounts. Therefore, the approximately 25-fold higher rate of transcription of the tRNA(Phe) and tRNA(Val) genes relative to the other H-strand tRNA genes and the 10-16-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes are not reflected in the steady-state levels or the rates of formation of the corresponding tRNAs. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters

Markedly different ATP requirements for rRNA synthesis and mtDNA light strand transcription versus mRNA synthesis in isolated human mitochondria

In isolated mitochondria from HeLa cells, the ATP requirements for mitochondral DNA (mtDNA) transcription and RNA processing can be satisfied by either endogenous synthesis, mainly through oxidative- phosphorylation, or by exogenous supply. The pattern of RNA synthesis changes dramatically depending upon the level of ATP available. At the low intramitochondrial ATP levels produced from endogenous ADP in the presence of an oxidizable substrate and phosphate, the mRNA species are labeled to a substantial extent, whereas there is only a marginal labeling of the rRNA species and light (L) strand transcripts. By contrast, high ATP levels, either provided exogenously or produced endogenously in the presence of an oxidizable substrate, phosphate, and exogenous ADP, strongly stimulate rRNA synthesis (about 10-fold) and light (L) strand transcription (greater than 10-fold), with only a slight increase in mRNA synthesis

Rate-limiting Step Preceding Cytochrome c Release in Cells Primed for Fas-mediated Apoptosis Revealed by Analysis of Cellular Mosaicism of Respiratory Changes

In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an ~30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost ~40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane