Global Rates Of Protein Synthesis Research Articles

Translational control of gene expression noise and its relationship to ageing in yeast

Gene expression noise influences organism evolution and fitness but is poorly understood. There is increasing evidence that the functional roles of components of the translation machinery influence noise intensity. In addition, modulation of the activities of at least some of these same components affects the replicative lifespan of a broad spectrum of organisms. In a novel comparative approach, we modulate the activities of the translation initiation factors eIFG1 and eIF4G2, both of which are involved in the process of recruiting ribosomal 43S preinitiation complexes to the 5' end of eukaryotic mRNAs. We show that tagging of the cell wall using a fluorescent dye allows us to follow gene expression noise as different yeast strains progress through successive cycles of replicative ageing. This procedure reveals a relationship between global protein synthesis rate and gene expression noise (cell-to-cell heterogeneity), which is accompanied by a parallel correlation between gene expression noise and the replicative age of mother cells. An alternative approach, based on microfluidics, confirms the interdependence between protein synthesis rate, gene expression noise and ageing. We additionally show that it is important to characterize the influence of the design of the microfluidic device on the nutritional state of the cells during such experiments. Analysis of the noise data derived from flow cytometry and fluorescence microscopy measurements indicates that both the intrinsic and the extrinsic noise components increase as a function of ageing.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C.elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

1016 – REGULATION OF RIBOSOME BIOGENESIS IN HEMATOPOIETIC STEM CELLS

Dysregulation of ribosome biogenesis is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. As prominently illustrated by one type of ribosomopathies, Shwachman-Diamond Syndrome (SDS), germline biallelic mutations in three assembly factors essential for the maturation of the 60S ribosome subunit result in progressive hematopoietic stem and progenitor cell (HSPC) failure, suggesting that HSPCs are sensitive to perturbations in ribosome assembly. However, how ribosome assembly is regulated in HSPCs remains poorly understood, as are approaches to restore in hematopoietic stem cell (HSC) function in the context of defective ribosome biogenesis. Here we uncover a novel role for the E3 ubiquitin ligase, HectD1, in regulating HSC function via ribosome biogenesis. HSCs from hematopoietic-specific <i>Hectd1</i> knockout mice exhibit a striking defect in transplantation ability and self-renewal. <i>Hectd1</i>-deficient HSCs fail to be maintained ex vivo, concomitant with a reduction in global protein synthesis and growth rate. The mechanism underlying HSC dysfunction upon <i>Hectd1</i> deficiency is directly linked to aberrant ribosome assembly. We demonstrate that HectD1 ubiquitinates and degrades an assembly factor for the polypeptide exit tunnel of the 60S ribosome in the cytoplasm. Depletion of <i>HectD1</i> led to an accumulation of the anti-association factor eIF6 on the 60S and decreased 80S to 60S ratio, consistent with a subunit joining defect associated with SDS-like diseases. Importantly, we found that restoration of ribosome assembly in <i>HectD1</i>-deficient cells promoted protein synthesis and enhanced HSC reconstitution capacity. These findings highlight the importance of ribosome assembly in HSC regeneration and represent a rare <i>in vivo</i> example of genetic suppression of HSC defects associated with dysfunctional ribosome biogenesis.

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice

The pathogenesis of Alzheimer’s disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER- and mitochondria-targeted aequorin-based Ca2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca2+ while Ca2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8–10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.

Effects of Plantar Mechanical Stimulation on Anabolic and Catabolic Signaling in Rat Postural Muscle Under Short-Term Simulated Gravitational Unloading.

It is known that plantar mechanical stimulation (PMS) is able to attenuate unloading-induced skeletal muscle atrophy and impaired muscle function. However, molecular mechanisms underlying the effect of PMS on skeletal muscle during unloading remain undefined. The aim of the study was to evaluate the effects of PMS on anabolic and catabolic signaling pathways in rat soleus at the early stages of mechanical unloading. Wistar rats were randomly assigned to ambulatory control, hindlimb suspension (HS) for 1 or 3 days, and HS for 1 or 3 days with PMS. The key anabolic and catabolic markers were assessed by western blotting and RT-PCR. Protein synthesis (PS) rate was estimated using SUnSET technique. PMS attenuated a 1-day HS-induced decrease in 4E-BP1, GSK-3β, and AMPK phosphorylation. PMS also partially prevented a decrease in PS, phosphorylation of GSK-3β, nNOS, and an increase in eEF2 phosphorylation after 3-day HS. PMS during 1- and 3-day HS prevented MuRF-1, but not MAFbx, upregulation but did not affect markers of ribosome biogenesis (18S + 28S rRNA, c-myc) as well as AKT phosphorylation. Thus, PMS during 3-day HS partially prevented a decrease in the global rate of PS in rat soleus muscle, which was accompanied by attenuation of MuRF-1 mRNA expression as well as changes in GSK-3β, nNOS, and eEF2 phosphorylation.

P11.36 Ribosome hydroxylase Mina53 is required for Glioblastoma and is involved in regulation of translation rateand fidelity by regulating ribosomal biogenesis

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive types of tumors with a poor response to standard treatment and a median 5-year survival of less than 5%. Therefore, there is an urgent need for new treatments. Recently, a large number of genome-wide studies have shown that the epigenetic modifiers are frequently deregulated in cancer. Using a mouse GBM model, we performed in vitro and in vivo shRNA screens to identify epigenetic regulators required for the tumorigenic process in GBM. Among these regulators is a ribosome hydroxylase Mina53 which hydroxylates His-39 of ribosomal protein, RPL27a. We have found that the knock-down (KD) of Mina53 reduces the in vitro proliferation and colony forming ability of mouse glioma initiating cells (mGIC) and this is dependent on the catalytic activity of Mina. Knock-down of Mina resulted into a small but significant reduction in the global protein synthesis rate. A tandem affinity purification experiment to identify proteins associated with Mina revealed that it is associated mainly with ribosomal proteins, including its substrate RPL27a. Global proteomic analyses revealed that final amounts and de novo protein synthesis of many ribosomal proteins were reduced upon Mina depletion. Isolation and identification of different polysome fraction bound mRNAs using high-throughput sequencing found that mRNAs encoding many ribosomal proteins have lower number of ribosomes loaded on them in the Mina depleted samples compared to the control. Taken together, this study has found that Mina53 is required for glioblastoma and it regulates translation through regulation of ribosomal biogenesis

MTOR-mediated inactivation of 4E-BP1, an inhibitor of translation, precedes cartilage degeneration in rat osteoarthritic knees.

Proper control of protein synthesis is vital for tissue homeostasis and its deregulation is characteristic of many disorders including osteoarthritis (OA). The objectives of this work were to analyze and correlate changes in activity of the translation apparatus associated with cartilage degeneration in an animal model of OA. Osteoarthritis was induced surgically in rats by anterior cruciate ligament transection (ACLT). Using a modified Mankin scoring system and analysis of protein expression we demonstrated, that mechanistic target of rapamycin complex 1 (mTORC1)-mediated 4E-BP1 phosphorylation was detected significantly earlier than other mTORC1-mediated modifications, such as p70S6K and ULK1 phosphorylation. 4E-BP1 is an inhibitor of cap-dependent translation those functions are inhibited by mTORC1 mediated phosphorylation. This signaling event not only preceded prominent signs of cartilage degeneration but also the increase in global protein synthesis rate. These results suggest that abnormal mTORC1 activity is one of the primary dysregulations observed in OA cartilage. Importantly, it is distributed disproportionately between targets, with 4E-BP1 being phosphorylated earlier than other downstream targets. Thus, our work provides new insights into the sequence of molecular events leading to cartilage destruction in OA and identifies translational control as an important regulatory hub involved in initiating OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2728-2735, 2018.

TAp73 contributes to the oxidative stress response by regulating protein synthesis

TAp73 is a transcription factor that plays key roles in brain development, aging, and cancer. At the cellular level, TAp73 is a critical homeostasis-maintaining factor, particularly following oxidative stress. Although major studies focused on TAp73 transcriptional activities have indicated a contribution of TAp73 to cellular metabolism, the mechanisms underlying its role in redox homeostasis have not been completely elucidated. Here we show that TAp73 contributes to the oxidative stress response by participating in the control of protein synthesis. Regulation of mRNA translation occupies a central position in cellular homeostasis during the stress response, often by reducing global rates of protein synthesis and promoting translation of specific mRNAs. TAp73 depletion results in aberrant ribosomal RNA (rRNA) processing and impaired protein synthesis. In particular, polysomal profiles show that TAp73 promotes the integration of mRNAs that encode rRNA-processing factors in polysomes, supporting their translation. Concurrently, TAp73 depletion causes increased sensitivity to oxidative stress that correlates with reduced ATP levels, hyperactivation of AMPK, and translational defects. TAp73 is important for maintaining active translation of mitochondrial transcripts in response to oxidative stress, thus promoting mitochondrial activity. Our results indicate that TAp73 contributes to redox homeostasis by affecting the translational machinery, facilitating the translation of specific mitochondrial transcripts. This study identifies a mechanism by which TAp73 contributes to the oxidative stress response and describes a completely unexpected role for TAp73 in regulating protein synthesis.

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single-Cell Level.

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single-cell level are still lacking. To address this need, we apply O-propargyl-puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single-cell level. In conclusion, OPP-labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.

Hyperammonemia and proteostasis in cirrhosis.

Skeletal muscle loss or sarcopenia is a frequent complication of cirrhosis that adversely affects clinical outcomes. As skeletal muscle is the largest store of proteins in the body, proteostasis or protein homeostasis is required for maintenance of muscle mass. This review will focus on disordered skeletal muscle proteostasis in liver disease. Increased skeletal muscle uptake of ammonia initiates responses that result in disordered proteostasis including impaired protein synthesis and increased autophagy. The cellular response to the stress of hyperammonemia (hyperammonemic stress response, HASR) involves the coordinated action of diverse signaling pathways targeting the molecular mechanisms of regulation of protein synthesis. Transcriptional upregulation of myostatin, a TGFβ superfamily member, results in impaired mTORC1 signaling. Phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) also relates to decreased global protein synthesis rates and mTORC1 signaling. Ammonia also causes mitochondrial and bioenergetic dysfunction because of cataplerosis of α-ketoglutarate. Lowering ammonia, targeting components of HASR and regulating cellular amino acid levels can potentially restore proteostasis. Signaling via myostatin and eIF2α phosphorylation causes decreases in protein synthesis and mTORC1 activity with a parallel mitochondrial dysfunction and increased autophagy contributing to proteostasis perturbations during skeletal muscle hyperammonemia of liver disease.

Attenuated Protein Synthesis Drives the Hematopoietic Defects in Shwachman-Diamond Syndrome

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure and a striking propensity to develop poor prognosis myelodysplastic syndrome and acute myeloid leukemia. In 90 % of cases the disease is caused by biallelic mutations in the gene encoding SBDS. We have shown previously that SBDS is a cytoplasmic ribosome assembly factor that catalyzes the release of the eukaryotic initiation factor 6 (eIF6) from the subunit joining interface of 60S ribosomal subunit (Menne et al, 2007; Finch et al, 2011). Deficiency of SBDS therefore results in aberrant retention of eIF6 on the 60S subunits that in turn perturbs ribosomal subunit joining and the formation of translation-competent 80S ribosomes. However, the mechanism linking defective ribosome assembly to marrow failure and leukemia in SDS remain poorly understood.Lack of viable mouse models presents a barrier to progress in understanding SDS disease pathophysiology and to evaluate novel therapies. We hypothesized that induced overexpression of eIF6 would mimic the consequences of SBDS deficiency by reducing the cytoplasmic pool of free 60S subunits and impairing translation. To test this hypothesis we have generated a novel transgenic eIF6 mouse model for SDS using KH2 embryonic stem cells that constitutively express the M2-reverse tetracycline transactivator at the Rosa26 locus with the EIF6 gene targeted downstream of the Col1a1 locus. This strategy permits systemic doxycycline-inducible and graded overexpression of eIF6 through control of the transgene copy number.We have validated that eIF6 overexpression promotes an increase in eIF6-bound cytoplasmic 60S subunits with a concomitant reduction in 80S ribosomes and polysomes in c-kit+ hematopoietic progenitor cells isolated from the transgenic eIF6 mice, thereby recapitulating the ribosomal subunit joining defect observed in patients with SDS. In vitro, the hematopoietic progenitor cells exhibit a strict eIF6 dose-dependent expansion defect. In vivo, mice with graded eIF6 overexpression are viable but develop macrocytic anemia with reticulocytopenia, thrombocytosis and mild leukopenia. Bone marrow transfer experiments demonstrate that the phenotype is autonomous to the hematopoietic system. Longitudinal phenotypic analyses in primary and transplanted animals are ongoing. Flow cytometric analysis of the bone marrow from transgenic eIF6 mice reveals a significant increase in the frequencies of preCFU-E and CFU-E erythroid progenitor cells and erythroblasts, but a significant reduction in the frequency of reticulocytes. Furthermore, we observe a striking accumulation of abnormal orthochromatic erythroblast-like cells that appear to have failed to enucleate, comprising approximately 1.5 % of the total bone marrow cells. Amnis ImageStream analysis, which combines flow cytometry with fluorescent microscopy, reveals a significant decrease in the frequency of erythroblasts that are able to complete the enucleation process. To address the underlying mechanism, we hypothesized that by impairing the formation of translation-competent 80S ribosomes, eIF6 overexpression would reduce the global rate of protein synthesis. Indeed, O-propargyl-puromycin incorporation assays established that the erythroblasts from the transgenic eIF6 mice have an approximately 3-fold reduction in global protein synthesis rate. Furthermore, our preliminary data suggest that the erythroid phenotype is p53-independent. Finally, erythroblasts from the transgenic eIF6 mice show a significant increase in levels of reactive oxygen species, but the functional significance of this finding remains unclear.We conclude that reduced rates of global translation drive defective hematopoiesis in the transgenic eIF6 mice. Importantly, eIF6 overexpression in vivo phenocopies SBDS depletion in human CD34+ cells (Sen et al, 2011). Together with the recent discovery of DNAJC21 (the human homologue of the 60S ribosomal assembly factor JJJ1 in yeast) as an SDS disease gene, our data support the hypothesis that deregulated cytoplasmic 60S subunit maturation and reduced translation are the primary drivers of the hematopoietic defect in SDS. Our viable transgenic eIF6 mouse model provides a unique tool to further dissect the mechanisms that underlie bone marrow failure and malignant transformation in SDS and for the development of novel therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Structural asymmetry in the eukaryotic Elongator complex.

Nucleoside modifications in tRNA anticodons regulate ribosome dynamics during translation elongation and, thereby, fine-tune global protein synthesis rates. The highly conserved eukaryotic Elongator complex conducts specific C5-substitutions in tRNA wobble base uridines. It harbors two copies of each of its six individual subunits, which are all equally important for its activity. Here, we summarize recent developments focusing on the architecture of the Elongator complex, showing an asymmetric subunit arrangement, and its functional implications. In addition, we discuss the role of its proposed active site, its individual subunits and temporarily associated regulatory factors. Finally, we aim to provide mechanistic explanations for the link between mutations in Elongator subunits and the onset of several severe human pathologies.

Abstract IA18: The post-transcriptional response to bulky-adduct DNA damage

Abstract Under conditions of physiological cell stress, including those that are found within a tumor cell and following treatment with chemotoxic drugs that cause DNA damage, post-transcriptional control pathways are initiated to aid cell survival. Global rates of protein synthesis are reduced by altering the phosphorylation status of eukaryotic initiation factors (eIFs), leading to decreases in the levels of ternary complex (required to bring the initiator Met-tRNAi to the 40S ribosomal subunit) and eIF4F complex (which binds to the 7methyl G cap). In parallel with protein synthesis shut down there is reprogramming of the translatome to allow the translation of “survival” mRNAs, which is mediated by RNA motifs within their 5' and 3' untranslated regions (UTRs) and their interaction with RNA binding proteins. Interestingly, the data show that this re-programming of the translatome is dependent upon the type of DNA damage initiated. For example, we have shown that following treatment with agents that cause bulky-adduct DNA damage, such a platinum-based compounds (cisplatin, oxaliplatin) or mitomycin C, the polysomes are enriched with mRNAs that encode enzymes that function in nucleotide excision repair. We have shown that these mRNAs contain regulatory upstream open reading frames (uORFs). Importantly, we have identified a common polymorphism in an uORF in the 5' UTR of the critical DNA repair enzyme ERCC5 and show that this determines sensitivity to platinum-based chemotherapy by controlling ERCC5 translation. Signaling from DNA-PKcs to eIF2 is required as part of this response, and modulation of this pathway alters the synthesis of critical repair enzymes and can chemosensitize cells to treatment with platinum-based compounds. We have used RNA-interactome capture to examine changes in the interactions of RNA binding proteins with their target mRNAs following bulky adduct-DNA damage and have identified a cytoplasmic RNA-BP complex that responds specifically to this type of DNA damage. Citation Format: Anne E. Willis. The post-transcriptional response to bulky-adduct DNA damage. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA18.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

Embryonic stem cells: a novel paradigm to study proteostasis?

Embryonic stem cells (ESCs) exhibit a striking ability to replicate continuously in the absence of senescence. Despite the knowledge gained into ESC biology and cell reprogramming, the mechanisms that regulate their pluripotency, self-renewal, and differentiation remain largely unknown. Recently, cumulative evidence has highlighted the importance of protein homeostasis, or proteostasis, in the maintenance of ESC function. These findings indicate that ESCs exhibit intrinsic differences in the regulation and activity of key nodes of the proteostasis network such as global protein synthesis, folding, and degradation rates. Here, we review new insights into proteostasis of ESCs and the questions raised by these findings. In addition, we discuss the potential of these discoveries to be applied into aging and cancer research.

Life in the cold: links between mammalian hibernation and longevity.

The biological process of aging is the primary determinant of lifespan, but the factors that influence the rate of aging are not yet clearly understood and remain a challenging question. Mammals are characterized by >100-fold differences in maximal lifespan, influenced by relative variances in body mass and metabolic rate. Recent discoveries have identified long-lived mammalian species that deviate from the expected longevity quotient. A commonality among many long-lived species is the capacity to undergo metabolic rate depression, effectively re-programming normal metabolism in response to extreme environmental stress and enter states of torpor or hibernation. This stress tolerant phenotype often involves a reduction in overall metabolic rate to just 1-5% of the normal basal rate as well as activation of cytoprotective responses. At the cellular level, major energy savings are achieved via coordinated suppression of many ATP-expensive cell functions; e.g. global rates of protein synthesis are strongly reduced via inhibition of the insulin signaling axis. At the same time, various studies have shown activation of stress survival signaling during hibernation including up-regulation of protein chaperones, increased antioxidant defenses, and transcriptional activation of pro-survival signaling such as the FOXO and p53 pathways. Many similarities and parallels exist between hibernation phenotypes and different long-lived models, e.g. signal transduction pathways found to be commonly regulated during hibernation are also known to induce lifespan extension in animals such as Drosophila melanogaster and Caenorhabditis elegans. In this review, we highlight some of the molecular mechanisms that promote longevity in classic aging models C. elegans, Drosophila, and mice, while providing a comparative analysis to how they are regulated during mammalian hibernation.

Key Markers of mTORC1-Dependent and mTORC1-Independent Signaling Pathways Regulating Protein Synthesis in Rat Soleus Muscle During Early Stages of Hindlimb Unloading

Background/Aims: The purpose of the study was to assess the amount of rRNA and phosphorylation status of the key markers of mTORC1-dependent (70s6k, 4E-BP1) and mTORC1-independent (GSK-3β, AMPK) signaling pathways controlling protein synthesis in rat soleus during early stages of mechanical unloading (hindlimb suspension (HS) for 1-, 3- and 7 days). Methods: The content of the key signaling molecules of various anabolic signaling pathways was determined by Western-blotting. The amount of 28S rRNA was evaluated by RT-PCR. The rate of protein synthesis was assessed using in-vivo SUnSET technique. Results: HS for 3 and 7 days induced a significant (p<0.05) decrease in the rate of global protein synthesis in soleus muscle in comparison with control. HS within 24 hours resulted in a significant (p<0.05) decrease in p-4E-BP1 content, p-AMPK content and increase in p-p70s6k content in rat soleus muscle. Following three days of HS the content of p-AKT was decreased (p<0.05). After 7 days of HS the phosphorylation level of AKT and GSK-3beta was significantly reduced (p<0.05) compared to control. We also observed a significant decrease in the amount of 28S rRNA in rat soleus following 1, 3 and 7 days of HS. Conclusion: Taken together, the results of our study suggest that a decline in the global rate of protein synthesis in rat soleus during early stages of simulated microgravity is associated with impaired ribosome biogenesis as well as reduced activity of mTORC1-independent signaling pathways.

Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins.

A central question in cell and developmental biology is how the information encoded in the genome is differentially interpreted to generate a diverse array of cell types. A growing body of research on posttranscriptional gene regulation is revealing that both global protein synthesis rates and the translation of specific mRNAs are highly specialized in different cell types. How this exquisite translational regulation is achieved is the focus of this review. Two levels of regulation are discussed: the translation machinery and cis-acting elements within mRNAs. Recent evidence shows that the ribosome itself directs how the genome is translated in time and space and reveals surprising functional specificity in individual components of the core translation machinery. We are also just beginning to appreciate the rich regulatory information embedded in the untranslated regions of mRNAs, which direct the selective translation of transcripts. These hidden RNA regulons may interface with a myriad of RNA-binding proteins and specialized translation machinery to provide an additional layer of regulation to how transcripts are spatiotemporally expressed. Understanding this largely unexplored world of translational codes hardwired in the core translation machinery is an exciting new research frontier fundamental to our understanding of gene regulation, organismal development, and evolution.

The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice.

ABSTRACTThe maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading.