Ribosome profiling analysis of human skeletal muscle identifies reduced translation of mitochondrial proteins with age.
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ABSTRACT:
SUBMITTER: Tharakan R
PROVIDER: S-EPMC8583053 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mitochondria are essential cellular organelles for generation of energy and their dysfunction may cause diabetes, Parkinson's disease and multi-systemic failure marked by failure to thrive, gastrointestinal problems, lactic acidosis and early lethality. Disease-associated mitochondrial mutations often affect components of the mitochondrial translation machinery. Here we perform ribosome profiling to measure mitochondrial translation at nucleotide resolution. Using a protocol optimized for the retrieval of mitochondrial ribosome protected fragments (RPFs) we show that the size distribution of wild-type mitochondrial RPFs follows a bimodal distribution peaking at 27 and 33 nucleotides, which is distinct from the 30-nucleotide peak of nuclear RPFs. Their cross-correlation suggests generation of mitochondrial RPFs during ribosome progression. In contrast, RPFs from patient-derived mitochondria mutated in tRNA-Tryptophan are centered on tryptophan codons and reduced downstream, indicating ribosome stalling. Intriguingly, long RPFs are enriched in mutated mitochondria, suggesting they characterize stalled ribosomes. Our findings provide the first model for translation in wild-type and disease-triggering mitochondria.
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Project description:Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.
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Project description:The rising prevalence of gestational diabetes mellitus (GDM) affects up to 18% of pregnant women with immediate and long-term metabolic consequences for both mother and infant. Abnormal glucose uptake and lipid oxidation are hallmark features of GDM prompting us to use an exploratory proteomics approach to investigate the cellular mechanisms underlying differences in skeletal muscle metabolism between obese pregnant women with GDM (OGDM) and obese pregnant women with normal glucose tolerance (ONGT). Functional validation was performed in a second cohort of obese OGDM and ONGT pregnant women. Quantitative proteomic analysis in rectus abdominus skeletal muscle tissue collected at delivery revealed reduced protein content of mitochondrial complex I (C-I) subunits (NDUFS3, NDUFV2) and altered content of proteins involved in calcium homeostasis/signaling (calcineurin A, ?1-syntrophin, annexin A4) in OGDM (n?=?6) vs. ONGT (n?=?6). Follow-up analyses showed reduced enzymatic activity of mitochondrial complexes C-I, C-III, and C-IV (-60-75%) in the OGDM (n?=?8) compared with ONGT (n?=?10) subjects, though no differences were observed for mitochondrial complex protein content. Upstream regulators of mitochondrial biogenesis and oxidative phosphorylation were not different between groups. However, AMPK phosphorylation was dramatically reduced by 75% in the OGDM women. These data suggest that GDM is associated with reduced skeletal muscle oxidative phosphorylation and disordered calcium homeostasis. These relationships deserve further attention as they may represent novel risk factors for development of GDM and may have implications on the effectiveness of physical activity interventions on both treatment strategies for GDM and for prevention of type 2 diabetes postpartum.
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Project description:BackgroundSkeletal muscle growth and development from embryo to adult consists of a series of carefully regulated changes in gene expression. Understanding these developmental changes in agriculturally important species is essential to the production of high quality meat products. For example, consumer demand for lean, inexpensive meat products has driven the turkey industry to unprecedented production through intensive genetic selection. However, achievements of increased body weight and muscle mass have been countered by an increased incidence of myopathies and meat quality defects. In a previous study, we developed and validated a turkey skeletal muscle-specific microarray as a tool for functional genomics studies. The goals of the current study were to utilize this microarray to elucidate functional pathways of genes responsible for key events in turkey skeletal muscle development and to compare differences in gene expression between two genetic lines of turkeys. To achieve these goals, skeletal muscle samples were collected at three critical stages in muscle development: 18d embryo (hyperplasia), 1d post-hatch (shift from myoblast-mediated growth to satellite cell-modulated growth by hypertrophy), and 16 wk (market age) from two genetic lines: a randombred control line (RBC2) maintained without selection pressure, and a line (F) selected from the RBC2 line for increased 16 wk body weight. Array hybridizations were performed in two experiments: Experiment 1 directly compared the developmental stages within genetic line, while Experiment 2 directly compared the two lines within each developmental stage.ResultsA total of 3474 genes were differentially expressed (false discovery rate; FDR < 0.001) by overall effect of development, while 16 genes were differentially expressed (FDR < 0.10) by overall effect of genetic line. Ingenuity Pathways Analysis was used to group annotated genes into networks, functions, and canonical pathways. The expression of 28 genes involved in extracellular matrix regulation, cell death/apoptosis, and calcium signaling/muscle function, as well as genes with miscellaneous function was confirmed by qPCR.ConclusionsThe current study identified gene pathways and uncovered novel genes important in turkey muscle growth and development. Future experiments will focus further on several of these candidate genes and the expression and mechanism of action of their protein products.
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Project description:Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner-membrane proteins were cotranslationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein-folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.
| S-EPMC4263316 | biostudies-literature
Project description:Skeletal muscle from sedentary older adults exhibits reduced mitochondrial abundance and oxidative capacity.The primary objective was to determine whether 8 weeks of combined training (CT) has a more robust effect than endurance training (ET) or resistance training (RT) on mitochondrial physiology in healthy young (18-30 years) and older (? 65 years) adults.Thirty-four young and 31 older adults were randomly assigned to 8 weeks of ET, RT, and control/CT. Control subjects completed 8 weeks of no exercise (control) followed by 8 weeks of CT. Body composition, skeletal muscle strength, and peak oxygen uptake were measured before and after the intervention. Vastus lateralis muscle biopsy samples were obtained before and 48 hours after the intervention. Mitochondrial physiology was evaluated by high-resolution respirometry and expression of mitochondrial proteins and transcription factors by quantitative PCR and immunoblotting.ET and CT significantly increased oxidative capacity and expression of mitochondrial proteins and transcription factors. All training modalities improved body composition, cardiorespiratory fitness, and skeletal muscle strength. CT induced the most robust improvements in mitochondria-related outcomes and physical characteristics despite lower training volumes for the ET and RT components. Importantly, most of the adaptations to training occurred independent of age.Collectively, these results demonstrate that both ET and CT increase muscle mitochondrial abundance and capacity although CT induced the most robust improvements in the outcomes measured. In conclusion, CT provides a robust exercise regimen to improve muscle mitochondrial outcomes and physical characteristics independent of age.
| S-EPMC4399307 | biostudies-literature
Project description:Translation is one of the fundamental processes of life. It comprises the assembly of polypeptides whose amino acid sequence corresponds to the codon sequence of an mRNA's ORF. Translation is performed by the ribosome; therefore, in order to understand translation and its regulation we must be able to determine the numbers and locations of ribosomes on mRNAs in vivo. Furthermore, we must be able to examine their redistribution in different physiological contexts and in response to experimental manipulations. The ribosome profiling method provides us with an opportunity to learn these locations, by sequencing a cDNA library derived from the short fragments of mRNA covered by the ribosome. Since its original description, the ribosome profiling method has undergone continuing development; in this article we describe the method's current state. Important improvements include: the incorporation of sample barcodes to enable library multiplexing, the incorporation of unique molecular identifiers to enable to removal of duplicated sequences, and the replacement of a gel-purification step with the enzymatic degradation of unligated linker.
| S-EPMC5582988 | biostudies-literature
Project description:Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods.
| S-EPMC4176156 | biostudies-literature
Project description:Ribosome profiling via high-throughput sequencing (ribo-seq) is a promising new technique for characterizing the occupancy of ribosomes on messenger RNA (mRNA) at base-pair resolution. The ribosome is responsible for translating mRNA into proteins, so information about its occupancy offers a detailed view of ribosome density and position which could be used to discover new translated open reading frames (ORFs), among other things. In this work, we propose Rp-Bp, an unsupervised Bayesian approach to predict translated ORFs from ribosome profiles. We use state-of-the-art Markov chain Monte Carlo techniques to estimate posterior distributions of the likelihood of translation of each ORF. Hence, an important feature of Rp-Bp is its ability to incorporate and propagate uncertainty in the prediction process. A second novel contribution is automatic Bayesian selection of read lengths and ribosome P-site offsets (BPPS). We empirically demonstrate that our read length selection technique modestly improves sensitivity by identifying more canonical and non-canonical ORFs. Proteomics- and quantitative translation initiation sequencing-based validation verifies the high quality of all of the predictions. Experimental comparison shows that Rp-Bp results in more peptide identifications and proteomics-validated ORF predictions compared to another recent tool for translation prediction.
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Project description:BackgroundMitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells.AimsTranslate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages.MethodsWe developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency.ResultsAnalysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age-on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach.Discussion/conclusionsThe exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.
| S-EPMC7985047 | biostudies-literature