scholarly journals Differential stability of mitochondrial mRNA in HeLa cells.

2005 ◽  
Vol 53 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Janusz Piechota ◽  
Rafał Tomecki ◽  
Kamil Gewartowski ◽  
Roman Szczesny ◽  
Aleksandra Dmochowska ◽  
...  
Keyword(s):  
Stop Codon ◽  
Careful Analysis ◽  
Mrna Turnover ◽  
Cyt B ◽  
Mitochondrial Translation ◽  
Transcription Process ◽  
Steady State Levels ◽  
Mitochondrial Transcripts ◽  
Differential Stability ◽  
Rna Precursor

The physiological significance and metabolism of oligoadenylated and polyadenylated human mitochondrial mRNAs are not known to date. After study of eight mitochondrial transcripts (ND1, ND2, ND3, ND5, CO1, CO2, ATP6/8 and Cyt. b) we found a direct correlation between the half-lives of mitochondrial mRNAs and their steady-state levels. Investigation of the mt-mRNA decay after thiamphenicol treatment indicated that three transcripts (ND2, ND3 and Cyt. b) are significantly stabilized after inhibition of mitochondrial translation. Careful analysis one of them, ND3, showed that inaccurate processing of the H-strand RNA precursor may occasionally occur between the ND3 and tRNA(Arg) locus leading to synthesis of ND3 mRNAs lacking the STOP codon. However, analysis of the oligo(A) fraction observed in case of the ND3 indicates that partially polyadenylated mRNAs are linked rather to the transcription process than to the translation-dependent deadenylation. Analysis of ND3 mRNA turnover in cells with siRNA-mediated knock-down of the mitochondrial poly(A) polymerase shows that strongly decreased polyadenylation does not markedly affect the decay of this transcript. We present a model where oligoadenylated mitochondrial transcripts are precursors of molecules containing full length poly(A) tails.

Defects in mitochondrial protein synthesis and respiratory chain activity segregate with the tRNA(Leu(UUR)) mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes

1992 ◽  
Vol 12 (2) ◽  
pp. 480-490
Author(s):  
M P King ◽  
Y Koga ◽  
M Davidson ◽  
E A Schon
Keyword(s):  
Steady State ◽  
Lactic Acidosis ◽  
Respiratory Chain ◽  
Mitochondrial Myopathy ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Morphological Characteristics ◽  
Nucleotide Position ◽  
Mitochondrial Translation ◽  
Steady State Levels

Cytoplasts from two unrelated patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) harboring an A----G transition at nucleotide position 3243 in the tRNA(Leu(UUR)) gene of the mitochondrial genome were fused with human cells lacking endogenous mitochondrial DNA (mtDNA) (rho 0 cells). Selected cybrid lines, containing less than 15 or greater than or equal to 95% mutated genomes, were examined for differences in genetic, biochemical, and morphological characteristics. Cybrids containing greater than or equal to 95% mutant mtDNA, but not those containing normal mtDNA, exhibited decreases in the rates of synthesis and in the steady-state levels of the mitochondrial translation products. In addition, NADH dehydrogenase subunit 1 (ND 1) exhibited a slightly altered mobility on polyacrylamide gel electrophoresis. The mutation also correlated with a severe respiratory chain deficiency. A small but consistent increase in the steady-state levels of an RNA transcript corresponding to 16S rRNA + tRNA(Leu(UUR)) + ND 1 genes was detected. However, there was no evidence of major errors in processing of the heavy-strand-encoded transcripts or of altered steady-state levels or ratios of mitochondrial rRNAs or mRNAs. These results provide evidence for a direct relationship between the tRNALeu(UUR) mutation and the pathogenesis of this mitochondrial disease.

scholarly journals Using mitoribosomal profiling to investigate human mitochondrial translation

2018 ◽  
Vol 2 ◽  
pp. 116
Author(s):  
Fei Gao ◽  
Maria Wesolowska ◽  
Reuven Agami ◽  
Koos Rooijers ◽  
Fabricio Loayza-Puch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Codon Position ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Synthesis ◽  
Mitochondrial Translation ◽  
Base Pairs ◽  
Suboptimal Structure ◽  
Human Counterpart ◽  
Steady State Levels

Background: Gene expression in human mitochondria has various idiosyncratic features. One of these was recently revealed as the unprecedented recruitment of a mitochondrially-encoded tRNA as a structural component of the large mitoribosomal subunit. In porcine particles this is mt-tRNAPhe whilst in humans it is mt-tRNAVal. We have previously shown that when a mutation in mt-tRNAVal causes very low steady state levels, there is preferential recruitment of mt-tRNAPhe. We have investigated whether this altered mitoribosome affects intra-organellar protein synthesis. Methods: By using mitoribosomal profiling we have revealed aspects of mitoribosome behaviour with its template mt-mRNA under both normal conditions as well as those where the mitoribosome has incorporated mt-tRNAPhe. Results: Analysis of the mitoribosome residency on transcripts under control conditions reveals that although mitochondria employ only 22 mt-tRNAs for protein synthesis, the use of non-canonical wobble base pairs at codon position 3 does not cause any measurable difference in mitoribosome occupancy irrespective of the codon. Comparison of the profile of aberrant mt-tRNAPhe containing mitoribosomes with those of controls that integrate mt-tRNAVal revealed that the impaired translation seen in the latter was not due to stalling on triplets encoding either of these amino acids. The alterations in mitoribosome interactions with start codons was not directly attributable to the either the use of non-cognate initiation codons or the presence or absence of 5’ leader sequences, except in the two bicistronic RNA units, RNA7 and RNA14 where the initiation sites are internal. Conclusions: These data report the power of mitoribosomal profiling in helping to understand the subtleties of mammalian mitochondrial protein synthesis. Analysis of profiles from the mutant mt-tRNAVal cell line suggest that despite mt-tRNAPhe being preferred in the porcine mitoribosome, its integration into the human counterpart results in a suboptimal structure that modifies its interaction with mt-mRNAs.

scholarly journals A mutation in the tRNA nucleotidyltransferase gene promotes stabilization of mRNAs in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5778-5784
Author(s):  
S W Peltz ◽  
J L Donahue ◽  
A Jacobson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Steady State ◽  
Relative Number ◽  
Mrna Turnover ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Yeast Saccharomyces Cerevisiae ◽  
Temperature Sensitive Mutants ◽  
Steady State Levels

To identify trans-acting factors involved in mRNA decay in the yeast Saccharomyces cerevisiae, we have begun to characterize conditional lethal mutants that affect mRNA steady-state levels. A screen of a collection of temperature-sensitive mutants identified ts352, a mutant that accumulated moderately stable and unstable mRNAs after a shift from 23 to 37 degrees C (M. Aebi, G. Kirchner, J.-Y. Chen, U. Vijayraghavan, A. Jacobson, N.C. Martin, and J. Abelson, J. Biol. Chem. 265:16216-16220, 1990). ts352 has a defect in the CCA1 gene, which codes for tRNA nucleotidyltransferase, the enzyme that adds 3' CCA termini to tRNAs (Aebi et al., J. Biol. Chem., 1990). In a shift to the nonpermissive temperature, ts352 (cca1-1) cells rapidly cease protein synthesis, reduce the rates of degradation of the CDC4, TCM1, and PAB1 mRNAs three- to fivefold, and increase the relative number of ribosomes associated with mRNAs and the overall size of polysomes. These results were analogous to those observed for cycloheximide-treated cells and are generally consistent with models that invoke a role for translational elongation in the process of mRNA turnover.

scholarly journals Schizosaccharomyces pombe Ppr10 is required for mitochondrial translation

2020 ◽  
Vol 367 (19) ◽  
Author(s):  
Zecheng Liu ◽  
Yan Li ◽  
Wanqiu Xie ◽  
Ying Huang
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Critical Role ◽  
Growth Defect ◽  
Pentatricopeptide Repeat ◽  
Mitochondrial Translation ◽  
Mitochondrial Functions ◽  
Oxphos System ◽  
Mitochondrial Transcripts ◽  
The Stability ◽  
Mitochondrial Introns

ABSTRACT The mitochondrial genome encodes key components of the oxidative phosphorylation (OXPHOS) system, whose expression is essential for mitochondrial functions. We have previously shown that deletion of the Schizosaccharomyces pombe ppr10 encoding a pentatricopeptide repeat protein severely reduces the mature levels of intron-containing mitochondrial transcripts cox1 and cob1, and severely impairs mitochondrial translation. In this study, we examined the possibility that the reduced levels of Cox1 and Cob1 proteins in cells were due to lowered levels of cox1 and cob1 mRNAs. We found that deletion of ppr10 did not affect the levels of mature cox1 and cob1 mRNAs in a mitochondrial intronless background. However, synthesis of Cox1 and Cob1 proteins were still severely affected by deletion of ppr10 in a mitochondrial intronless background. Consistent with this, we found that deletion of mitochondrial introns could not rescue the respiratory growth defect of Δppr10 cells. Our results reveal that Ppr10 is not required for the stability of cox1 and cob1 mRNAs, and provide further support for the idea that Ppr10 plays a critical role in mitochondrial translation.

scholarly journals Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes

2019 ◽  
Vol 47 (20) ◽  
pp. 10543-10552 ◽  
Author(s):  
Alexander Donath ◽  
Frank Jühling ◽  
Marwa Al-Arab ◽  
Stephan H Bernhart ◽  
Franziska Reinhardt ◽  
...  
Keyword(s):  
De Novo ◽  
Stop Codon ◽  
Difficult Problem ◽  
Mitochondrial Genomes ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Genetic Codes ◽  
Annotation Server ◽  
Codon Positions ◽  
Mitochondrial Transcripts

Abstract With the rapid increase of sequenced metazoan mitochondrial genomes, a detailed manual annotation is becoming more and more infeasible. While it is easy to identify the approximate location of protein-coding genes within mitogenomes, the peculiar processing of mitochondrial transcripts, however, makes the determination of precise gene boundaries a surprisingly difficult problem. We have analyzed the properties of annotated start and stop codon positions in detail, and use the inferred patterns to devise a new method for predicting gene boundaries in de novo annotations. Our method benefits from empirically observed prevalances of start/stop codons and gene lengths, and considers the dependence of these features on variations of genetic codes. Albeit not being perfect, our new approach yields a drastic improvement in the accuracy of gene boundaries and upgrades the mitochondrial genome annotation server MITOS to an even more sophisticated tool for fully automatic annotation of metazoan mitochondrial genomes.

scholarly journals In VivoRegulation of Oxidative Phosphorylation in Cells Harboring a Stop-codon Mutation in Mitochondrial DNA-encoded CytochromecOxidase Subunit I

2001 ◽  
Vol 276 (50) ◽  
pp. 46925-46932 ◽  
Author(s):  
Marilena D'Aurelio ◽  
Francesco Pallotti ◽  
Antoni Barrientos ◽  
Carl D. Gajewski ◽  
Jennifer Q. Kwong ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Oxidative Phosphorylation ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Atp Synthesis ◽  
Stop Codon Mutation ◽  
Oxidase Subunit ◽  
Steady State Levels ◽  
Codon Mutation ◽  
In Cells

The mechanisms that regulate oxidative phosphorylation in mammalian cells are largely unknown. To address this issue, cybrids were generated by fusing osteosarcoma cells devoid of mitochondrial DNA (mtDNA) with platelets from a patient with a stop-codon mutation in cytochromecoxidase subunit I (COX I). The molecular and biochemical characteristics of cybrids harboring varying levels of mutated mitochondrial DNA were studied. We found a direct correlation between the levels of mutated COX I DNA and mutated COX I mRNA, whereas the levels of COX I total mRNA were unchanged. COX I polypeptide synthesis and steady-state levels were inversely proportional to mutation levels. Cytochromecoxidase subunit II was reduced proportionally to COX I, indicating impairment in complex assembly. COX enzymatic activity was inversely proportional to the levels of mutated mtDNA. However, both cell respiration and ATP synthesis were preserved in cells with lower proportions of mutated genomes, with a threshold at ∼40%, and decreased linearly with increasing mutated mtDNA. These results indicate that COX levels in mutated cells were not regulated at the transcriptional, translational, and post-translational levels. Because of a small excess of COX capacity, the levels of expression of COX subunits exerted a relatively tight control on oxidative phosphorylation.

scholarly journals Impact of Methods on the Measurement of mRNA Turnover

2017 ◽  
Author(s):  
Takeo Wada ◽  
Attila Becskei
Keyword(s):  
Mrna Stability ◽  
Rna Degradation ◽  
Mrna Turnover ◽  
Gene Control ◽  
Cellular Activity ◽  
Transcription Inhibition ◽  
Rna Molecules ◽  
Metabolic Labelling ◽  
Differential Stability ◽  
Made In

The turnover of the RNA molecules is determined by the rates of transcription and RNA degradation. Several methods have been developed to study mRNA turnover since the beginnings of molecular biology. Here we summarize the main methods to measure RNA half-life: transcription inhibition, gene control and metabolic labelling. These methods were used to detect the cellular activity of the mRNAs degradation machinery, including the exo-ribonuclease Xrn1 and the exosome. Less progress has been made in the study of the differential stability of mature RNAs because the different methods have often yielded inconsistent results so that an mRNA considered to be stable can be classified as unstable by another method. Recent advances in the systematic comparison of different method variants in yeast have permitted the identification of the least invasive methodologies that reflect half-lives the most faithfully, which is expected to open the way for a consistent quantitative analysis of the determinants of mRNA stability.

scholarly journals Cardiomyopathy-associated mutation in the ADP/ATP carrier reveals translation-dependent regulation of cytochromecoxidase activity

2018 ◽  
Vol 29 (12) ◽  
pp. 1449-1464 ◽  
Author(s):  
Oluwaseun B. Ogunbona ◽  
Matthew G. Baile ◽  
Steven M. Claypool
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Mitochondrial Genome ◽  
Cytochrome C Oxidase ◽  
Critical Role ◽  
State Level ◽  
Mitochondrial Translation ◽  
Complex Iv ◽  
Respiratory Supercomplexes ◽  
Steady State Levels

How the absence of the major mitochondrial ADP/ATP carrier in yeast, Aac2p, results in a specific defect in cytochrome c oxidase (COX; complex IV) activity is a long-standing mystery. Aac2p physically associates with respiratory supercomplexes, which include complex IV, raising the possibility that its activity is dependent on its association with Aac2p. Here, we have leveraged a transport-dead pathogenic AAC2 point mutant to determine the basis for the reduced COX activity in the absence of Aac2p. The steady-state levels of complex IV subunits encoded by the mitochondrial genome are significantly reduced in the absence of Aac2p function, whether its association with respiratory supercomplexes is preserved or not. This diminution in COX amounts is not caused by a reduction in the mitochondrial genome copy number or the steady-state level of its transcripts, and does not reflect a defect in complex IV assembly. Instead, the absence of Aac2p activity, genetically or pharmacologically, results in an aberrant pattern of mitochondrial translation. Interestingly, compared with the complete absence of Aac2p, the complex IV–related defects are greater in mitochondria expressing the transport-inactive Aac2p mutant. Our results highlight a critical role for Aac2p transport in mitochondrial translation whose disturbance uniquely impacts cytochrome c oxidase.

A splicing defect in the mouse transferrin gene leads to congenital atransferrinemia

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 482-486 ◽  
Author(s):  
JI Huggenvik ◽  
CM Craven ◽  
RL Idzerda ◽  
S Bernstein ◽  
J Kaplan ◽  
...  
Keyword(s):  
Southern Blot Analysis ◽  
Mouse Liver ◽  
Iron Homeostasis ◽  
Mrna Processing ◽  
Serum Transferrin ◽  
Mouse Line ◽  
Mature Mrna ◽  
Transferrin Gene ◽  
Steady State Levels ◽  
Rna Precursor

Abstract We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5′ and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.

scholarly journals Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

2012 ◽  
Vol 32 (6) ◽  
pp. 549-557 ◽  
Author(s):  
J. Ignacio Moreno ◽  
Babu Patlolla ◽  
Kerry R. Belton ◽  
Brenita C. Jenkins ◽  
Polina V. Radchenkova ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Small Subunit ◽  
The Novel ◽  
Pentatricopeptide Repeat ◽  
Mitochondrial Translation ◽  
Translational Machinery ◽  
Moonlighting Protein ◽  
Steady State Levels ◽  
Novel Strategy

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.

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