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.
Information on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling ...coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae . Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially the duration of the elongation step. We identified proteins whose synthesis was regulated rapidly by hydrogen peroxide posttranscriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. These data define the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggest that potentiation (coregulation of the transcript level and translation) is a feature of oxidative stress.
Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A ...critical issue in ribosome profiling is nuclease treatment of ribosome-mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems.
Abstract
There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live ...animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.
Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this ...question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5′-terminal oligopyrimidine (5′-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age.
Reduced methionine (Met) intake can extend lifespan of rodents; however, whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met ...restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino-acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino-acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signalling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino-acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension.
Several pharmacological, dietary, and genetic interventions that increase mammalian lifespan are known, but general principles of lifespan extension remain unclear. Here, we performed RNA sequencing ...(RNA-seq) analyses of mice subjected to 8 longevity interventions. We discovered a feminizing effect associated with growth hormone regulation and diminution of sex-related differences. Expanding this analysis to 17 interventions with public data, we observed that many interventions induced similar gene expression changes. We identified hepatic gene signatures associated with lifespan extension across interventions, including upregulation of oxidative phosphorylation and drug metabolism, and showed that perturbed pathways may be shared across tissues. We further applied the discovered longevity signatures to identify new lifespan-extending candidates, such as chronic hypoxia, KU-0063794, and ascorbyl-palmitate. Finally, we developed GENtervention, an app that visualizes associations between gene expression changes and longevity. Overall, this study describes general and specific transcriptomic programs of lifespan extension in mice and provides tools to discover new interventions.
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•Sex-specific differences are decreased in response to longevity interventions•Many interventions, but not rapamycin, exhibit similar transcriptomic responses•Certain gene expression changes are associated with longevity across interventions•Longevity signatures may be used to discover new lifespan-extending interventions
Tyshkovskiy et al. performed a comprehensive analysis of 17 known lifespan-extending interventions in mice at the level of gene expression to better understand general principles of lifespan control and generate gene expression signatures associated with longevity. They applied these signatures to predict new candidate compounds for lifespan extension.
Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading ...misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced ...aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.
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•Genome of the Damaraland mole rat and improved genome assembly of the naked mole rat•Transcriptomes of subterranean rodents and comparative genome analyses•Common adaptations for subterranean life: arginase, globins, and Na(V)1.7•Unique NMR adaptations: UCP1, 28S rRNA processing, melatonin, actin, and pain systems
Subterranean rodents thrive in harsh underground environments. Many are long-lived and hold promise as animal models of successful aging and sustained good health. Here, Fang et al. sequence the genome of the Damaraland mole rat (Fukomys damarensis), improve the genome assembly of the naked mole rat (Heterocephalus glaber), and compare the transcriptomes of subterranean rodents. Comparative analyses reveal candidate molecular adaptations for subterranean life and longevity, as well as traits unique to the naked mole rat, including unusual thermogenesis and novel processing of 28S rRNA.
The naked mole-rat (NMR) is an exceptionally long-lived rodent that shows no increase of mortality with age, defining it as a demographically non-aging mammal. Here, we perform bisulfite sequencing ...of the blood of > 100 NMRs, assessing > 3 million common CpG sites. Unsupervised clustering based on sites whose methylation correlates with age reveals an age-related methylome remodeling, and we also observe a methylome information loss, suggesting that NMRs age. We develop an epigenetic aging clock that accurately predicts the NMR age. We show that these animals age much slower than mice and much faster than humans, consistent with their known maximum lifespans. Interestingly, patterns of age-related changes of clock sites in Tert and Prpf19 differ between NMRs and mice, but there are also sites conserved between the two species. Together, the data indicate that NMRs, like other mammals, epigenetically age even in the absence of demographic aging of this species.
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