4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila.

Dietary restriction (DR) extends lifespan in multiple species. To examine the mechanisms of lifespan extension upon DR, we assayed genome-wide translational changes in Drosophila. A number of nuclear encoded mitochondrial genes, including those in Complex I and IV of the electron transport chain, showed increased ribosomal loading and enhanced overall activity upon DR. We found that various mitochondrial genes possessed shorter and less structured 5'UTRs, which were important for their enhanced mRNA translation. The translational repressor 4E-BP, the eukaryotic translation initiation factor 4E binding protein, was upregulated upon DR and mediated DR dependent changes in mitochondrial activity and lifespan extension. Inhibition of individual mitochondrial subunits from Complex I and IV diminished the lifespan extension obtained upon DR, reflecting the importance of enhanced mitochondrial function during DR. Our results imply that translational regulation of nuclear-encoded mitochondrial gene expression by 4E-BP plays an important role in lifespan extension upon DR. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

A Novel Caenorhabditis Elegans Proteinopathy Model Shows Changes in mRNA Translational Frameshifting During Aging.

BACKGROUND/AIMS: Regulation of mRNA translation is central to protein homeostasis and is optimized for speed and accuracy. Spontaneous recoding events occur virtually at any codon but at very low frequency and are commonly assumed to increase as the cell ages. METHODS: Here, we leveraged the polyglutamine(polyQ)-frameshifting model of huntingtin exon 1 with CAG repeat length in the pathological range (Htt51Q), which undergoes enhanced non-programmed translational -1 frameshifting. RESULTS: In body muscle cells of Caenorhabditis elegans, -1 frameshifting occured at the onset of expression of the zero-frame product, correlated with mRNA level of the non-frameshifted expression and formed aggregates correlated with reduced motility in C. elegans. Spontaneous frameshifting was modulated by IFG-1, the homologue of the nutrient-responsive eukaryotic initiation factor 4G (eIF4G), under normal growth conditions and NSUN-5, a conserved ribosomal RNA methyltransferase, under osmotic stress. CONCLUSION: Our results suggest that frameshifting and aggregation occur at even early stages of development and, because of their intrinsic stability, may persist and accelerate the onset of age-related proteinopathies.

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms.

Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR-/+, HER2-/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.

Physiological Consequences of Nonsense-Mediated Decay and Its Role in Adaptive Responses.

The evolutionarily conserved nonsense-mediated mRNA decay (NMD) pathway is a quality control mechanism that degrades aberrant mRNA containing one or more premature termination codons (PTCs). Recent discoveries indicate that NMD also differentially regulates mRNA from wild-type protein-coding genes despite lacking PTCs. Together with studies showing that NMD is involved in development and adaptive responses that influence health and longevity, these findings point to an expanded role of NMD that adds a new layer of complexity in the post-transcriptional regulation of gene expression. However, the extent of its control, whether different types of NMD play different roles, and the resulting physiological outcomes remain unclear and need further elucidation. Here, we review different branches of NMD and what is known of the physiological outcomes associated with this type of regulation. We identify significant gaps in the understanding of this process and the utility of genetic tools in accelerating progress in this area.

Stress-induced Eukaryotic Translational Regulatory Mechanisms.

The eukaryotic protein synthesis process entails intricate stages governed by diverse mechanisms to tightly regulate translation. Translational regulation during stress is pivotal for maintaining cellular homeostasis, ensuring the accurate expression of essential proteins crucial for survival. This selective translational control mechanism is integral to cellular adaptation and resilience under adverse conditions. This review manuscript explores various mechanisms involved in selective translational regulation, focusing on mRNA-specific and global regulatory processes. Key aspects of translational control include translation initiation, which is often a rate-limiting step, and involves the formation of the eIF4F complex and recruitment of mRNA to ribosomes. Regulation of translation initiation factors, such as eIF4E, eIF4E2, and eIF2, through phosphorylation and interactions with binding proteins, modulates translation efficiency under stress conditions. This review also highlights the control of translation initiation through factors like the eIF4F complex and the ternary complex and also underscores the importance of eIF2α phosphorylation in stress granule formation and cellular stress responses. Additionally, the impact of amino acid deprivation, mTOR signaling, and ribosome biogenesis on translation regulation and cellular adaptation to stress is also discussed. Understanding the intricate mechanisms of translational regulation during stress provides insights into cellular adaptation mechanisms and potential therapeutic targets for various diseases, offering valuable avenues for addressing conditions associated with dysregulated protein synthesis.

Neuronal CBP-1 is required for enhanced body muscle proteostasis in response to reduced translation downstream of mTOR.

Background: The ability to maintain muscle function decreases with age and loss of proteostatic function. Diet, drugs, and genetic interventions that restrict nutrients or nutrient signaling help preserve long-term muscle function and slow age-related decline. Previously, it was shown that attenuating protein synthesis downstream of the mechanistic target of rapamycin (mTOR) gradually increases expression of heat shock response (HSR) genes in a manner that correlates with increased resilience to protein unfolding stress. Here, we investigate the role of specific tissues in mediating the cytoprotective effects of low translation. Methods: This study uses genetic tools (transgenic C. elegans , RNA interference and gene expression analysis) as well as physiological assays (survival and paralysis assays) in order to better understand how specific tissues contribute to adaptive changes involving cellular cross-talk that enhance proteostasis under low translation conditions. Results: We use the C. elegans system to show that lowering translation in neurons or the germline increases heat shock gene expression and survival under conditions of heat stress. In addition, we find that low translation in these tissues protects motility in a body muscle-specific model of proteotoxicity that results in paralysis. Low translation in neurons or germline also results in increased expression of certain muscle regulatory and structural genes, reversing reduced expression normally observed with aging in C. elegans . Enhanced resilience to protein unfolding stress requires neuronal expression of cbp-1 . Conclusion: Low translation in either neurons or the germline orchestrate protective adaptation in other tissues, including body muscle.

Neuronal CBP-1 is Required for Enhanced Body Muscle Proteostasis in Response to Reduced Translation Downstream of mTOR.

BACKGROUND: The ability to maintain muscle function decreases with age and loss of proteostatic function. Diet, drugs, and genetic interventions that restrict nutrients or nutrient signaling help preserve long-term muscle function and slow age-related decline. Previously, it was shown that attenuating protein synthesis downstream of the mechanistic target of rapamycin (mTOR) gradually increases expression of heat shock response (HSR) genes in a manner that correlates with increased resilience to protein unfolding stress. Here, we investigate the role of specific tissues in mediating the cytoprotective effects of low translation. METHODS: This study uses genetic tools (transgenic Caenorhabditis elegans (C. elegans), RNA interference and gene expression analysis) as well as physiological assays (survival and paralysis assays) in order to better understand how specific tissues contribute to adaptive changes involving cellular cross-talk that enhance proteostasis under low translation conditions. RESULTS: We use the C. elegans system to show that lowering translation in neurons or the germline increases heat shock gene expression and survival under conditions of heat stress. In addition, we find that low translation in these tissues protects motility in a body muscle-specific model of proteotoxicity that results in paralysis. Low translation in neurons or germline also results in increased expression of certain muscle regulatory and structural genes, reversing reduced expression normally observed with aging in C. elegans. Enhanced resilience to protein unfolding stress requires neuronal expression of cbp-1. CONCLUSIONS: Low translation in either neurons or the germline orchestrate protective adaptation in other tissues, including body muscle.

The integrated stress response protects against ER stress but is not required for altered translation and lifespan from dietary restriction in Caenorhabditis elegans.

The highly conserved integrated stress response (ISR) reduces and redirects mRNA translation in response to certain forms of stress and nutrient limitation. It is activated when kinases phosphorylate a key residue in the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). General Control Nonderepressible-2 (GCN2) is activated to phosphorylate eIF2α by the presence of uncharged tRNA associated with nutrient scarcity, while protein kinase R-like ER kinase-1 (PERK) is activated during the ER unfolded protein response (UPRER). Here, we investigated the role of the ISR during nutrient limitation and ER stress with respect to changes in protein synthesis, translationally driven mRNA turnover, and survival in Caenorhabditis elegans. We found that, while GCN2 phosphorylates eIF2α when nutrients are restricted, the ability to phosphorylate eIF2α is not required for changes in translation, nonsense-mediated decay, or lifespan associated with dietary restriction (DR). Interestingly, loss of both GCN2 and PERK abolishes increased lifespan associated with dietary restriction, indicating the possibility of other substrates for these kinases. The ISR was not dispensable under ER stress conditions, as demonstrated by the requirement for PERK and eIF2α phosphorylation for decreased translation and wild type-like survival. Taken together, results indicate that the ISR is critical for ER stress and that other translation regulatory mechanisms are sufficient for increased lifespan under dietary restriction.

Early-adulthood spike in protein translation drives aging via juvenile hormone/germline signaling.

Protein translation (PT) declines with age in invertebrates, rodents, and humans. It has been assumed that elevated PT at young ages is beneficial to health and PT ends up dropping as a passive byproduct of aging. In Drosophila, we show that a transient elevation in PT during early-adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later-life. Blocking the early-life PT elevation robustly improves life-/health-span and prevents age-related protein aggregation, whereas transiently inducing an early-life PT surge in long-lived fly strains abolishes their longevity/proteostasis benefits. The early-life PT elevation triggers proteostatic dysfunction, silences stress responses, and drives age-related functional decline via juvenile hormone-lipid transfer protein axis and germline signaling. Our findings suggest that PT is adaptively suppressed after early-adulthood, alleviating later-life proteostatic burden, slowing down age-related functional decline, and improving lifespan. Our work provides a theoretical framework for understanding how lifetime PT dynamics shape future aging trajectories.

Exportin 1 modulates life span by regulating nucleolar dynamics via the autophagy protein LGG-1/GABARAP.

Altered nucleolar and ribosomal dynamics are key hallmarks of aging, but their regulation remains unclear. Building on the knowledge that the conserved nuclear export receptor Exportin 1 (XPO-1/XPO1) modulates proteostasis and life span, we systematically analyzed the impact of nuclear export on protein metabolism. Using transcriptomic and subcellular proteomic analyses in nematodes, we demonstrate that XPO-1 modulates the nucleocytoplasmic distribution of key proteins involved in nucleolar dynamics and ribosome function, including fibrillarin (FIB-1/FBL) and RPL-11 (RPL11). Silencing xpo-1 led to marked reduction in global translation, which was accompanied by decreased nucleolar size and lower fibrillarin levels. A targeted screen of known proteostatic mediators revealed that the autophagy protein LGG-1/GABARAP modulates nucleolar size by regulating RPL-11 levels, linking specific protein degradation to ribosome metabolism. Together, our study reveals that nucleolar size and life span are regulated by LGG-1/GABARAP via ribosome protein surveillance.

Anabolic Function Downstream of TOR Controls Trade-offs Between Longevity and Reproduction at the Level of Specific Tissues in C. elegans.

As the most energetically expensive cellular process, translation must be finely tuned to environmental conditions. Dietary restriction attenuates signaling through the nutrient sensing mTOR pathway, which reduces translation and redirects resources to preserve the soma. These responses are associated with increased lifespan but also anabolic impairment, phenotypes also observed when translation is genetically suppressed. Here, we restricted translation downstream of mTOR separately in major tissues in C. elegans to better understand their roles in systemic adaptation and whether consequences to anabolic impairment were separable from positive effects on lifespan. Lowering translation in neurons, hypodermis, or germline tissue led to increased lifespan under well-fed conditions and improved survival upon withdrawal of food, indicating that these are key tissues coordinating enhanced survival when protein synthesis is reduced. Surprisingly, lowering translation in body muscle during development shortened lifespan while accelerating and increasing reproduction, a reversal of phenotypic trade-offs associated with systemic translation suppression. Suppressing mTORC1 selectively in body muscle also increased reproduction while slowing motility during development. In nature, this may be indicative of reduced energy expenditure related to foraging, acting as a "GO!" signal for reproduction. Together, results indicate that low translation in different tissues helps direct distinct systemic adaptations and suggest that unknown endocrine signals mediate these responses. Furthermore, mTOR or translation inhibitory therapeutics that target specific tissues may achieve desired interventions to aging without loss of whole-body anabolism.

The ribosomal RNA m5C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans.

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans. Here, we show that NSUN-1 is writing the second known 26S rRNA m5C at position C2982. Depletion of nsun-1 or nsun-5 improved thermotolerance and slightly increased locomotion at midlife, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodeled leading to reduced production of collagens, loss of structural integrity of the cuticle, and impaired barrier function. We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal development and physiology.

Dietary restriction induces posttranscriptional regulation of longevity genes.

Dietary restriction (DR) increases life span through adaptive changes in gene expression. To understand more about these changes, we analyzed the transcriptome and translatome of Caenorhabditis elegans subjected to DR. Transcription of muscle regulatory and structural genes increased, whereas increased expression of amino acid metabolism and neuropeptide signaling genes was controlled at the level of translation. Evaluation of posttranscriptional regulation identified putative roles for RNA-binding proteins, RNA editing, miRNA, alternative splicing, and nonsense-mediated decay in response to nutrient limitation. Using RNA interference, we discovered several differentially expressed genes that regulate life span. We also found a compensatory role for translational regulation, which offsets dampened expression of a large subset of transcriptionally down-regulated genes. Furthermore, 3' UTR editing and intron retention increase under DR and correlate with diminished translation, whereas trans-spliced genes are refractory to reduced translation efficiency compared with messages with the native 5' UTR. Finally, we find that smg-6 and smg-7, which are genes governing selection and turnover of nonsense-mediated decay targets, are required for increased life span under DR.

Translational Regulation of Non-autonomous Mitochondrial Stress Response Promotes Longevity.

Reduced mRNA translation delays aging, but the underlying mechanisms remain underexplored. Mutations in both DAF-2 (IGF-1 receptor) and RSKS-1 (ribosomal S6 kinase/S6K) cause synergistic lifespan extension in C. elegans. To understand the roles of translational regulation in this process, we performed polysomal profiling and identified translationally regulated ribosomal and cytochrome c (CYC-2.1) genes as key mediators of longevity. cyc-2.1 knockdown significantly extends lifespan by activating the intestinal mitochondrial unfolded protein response (UPRmt), mitochondrial fission, and AMP-activated kinase (AMPK). The germline serves as the key tissue for cyc-2.1 to regulate lifespan, and germline-specific cyc-2.1 knockdown non-autonomously activates intestinal UPRmt and AMPK. Furthermore, the RNA-binding protein GLD-1-mediated translational repression of cyc-2.1 in the germline is important for the non-autonomous activation of UPRmt and synergistic longevity of the daf-2 rsks-1 mutant. Altogether, these results illustrate a translationally regulated non-autonomous mitochondrial stress response mechanism in the modulation of lifespan by insulin-like signaling and S6K.

Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans.

Protein synthesis is a regulated cellular process that links nutrients in the environment to organismal growth and development. Here we examine the role of genes that regulate mRNA translation in determining growth, reproduction, stress resistance and lifespan. Translational control of protein synthesis by regulators such as the cap-binding complex and S6 kinase play an important role during growth. We observe that inhibition of various genes in the translation initiation complex including ifg-1, the worm homologue of eIF4G, which is a scaffold protein in the cap-binding complex; and rsks-1, the worm homologue of S6 kinase, results in lifespan extension in Caenorhabditis elegans. Inhibition of ifg-1 or rsks-1 also slows development, reduces fecundity and increases resistance to starvation. A reduction in ifg-1 expression in dauers was also observed, suggesting an inhibition of protein translation during the dauer state. Thus, mRNA translation exerts pleiotropic effects on growth, reproduction, stress resistance and lifespan in C. elegans.

Assessing Health Span in Caenorhabditis elegans: Lessons From Short-Lived Mutants.

Genetic changes resulting in increased life span are often positively associated with enhanced stress resistance and somatic maintenance. A recent study found that certain long-lived Caenorhabditis elegans mutants spent a decreased proportion of total life in a healthy state compared with controls, raising concerns about how the relationship between health and longevity is assessed. We evaluated seven markers of health and two health-span models for their suitability in assessing age-associated health in invertebrates using C elegans strains not expected to outperform wild-type animals. Additionally, we used an empirical method to determine the transition point into failing health based on the greatest rate of change with age for each marker. As expected, animals with mutations causing sickness or accelerated aging had reduced health span when compared chronologically to wild-type animals. Physiological health span, the proportion of total life spent healthy, was reduced for locomotion markers in chronically ill mutants, but, surprisingly, was extended for thermotolerance. In contrast, all short-lived mutants had reduced "quality-of-life" in another model recently employed for assessing invertebrate health. Results suggest that the interpretation of physiological health span is not straightforward, possibly because it factors out time and thus does not account for the added cost of extrinsic forces on longer-lived strains.

Reducing translation through eIF4G/IFG-1 improves survival under ER stress that depends on heat shock factor HSF-1 in Caenorhabditis elegans.

Although certain methods of lowering and/or altering mRNA translation are associated with increased lifespan, the mechanisms underlying this effect remain largely unknown. We previously showed that the increased lifespan conferred by reducing expression of eukaryotic translation initiation factor 4G (eIF4G/IFG-1) enhances survival under starvation conditions while shifting protein expression toward factors involved with maintaining ER-dependent protein and lipid balance. In this study, we investigated changes in ER homeostasis and found that lower eIF4G/IFG-1 increased survival under conditions of ER stress. Enhanced survival required the ER stress sensor gene ire-1 and the ER calcium ATPase gene sca-1 and corresponded with increased translation of chaperones that mediate the ER unfolded protein response (UPRER ). Surprisingly, the heat-shock transcription factor gene hsf-1 was also required for enhanced survival, despite having little or no influence on the ability of wild-type animals to survive ER stress. The requirement for hsf-1 led us to re-evaluate the role of eIF4G/IFG-1 on thermotolerance. Results show that lowering expression of this translation factor enhanced thermotolerance, but only after prolonged attenuation, the timing of which corresponded to increased transcription of heat-shock factor transcriptional targets. Results indicate that restricting overall translation through eIF4G/IFG-1 enhances ER and cytoplasmic proteostasis through a mechanism that relies heavily on hsf-1.

Function of ruminant gammadelta T cells is defined by WC1.1 or WC1.2 isoform expression.

WC1 is a transmembrane glycoprotein and member of the scavenger receptor cysteine rich (SRCR) family that is uniquely expressed on gammadelta T cells. The WC1 isoforms referred to as WC1.1, WC1.2, and WC1.3 are expressed on discrete subpopulations of gammadelta T cells with WC1.1 and WC1.2 expressed on mostly nonoverlapping gammadelta T cell populations. Studies have demonstrated a potential role for WC1 in modulating the response of gammadelta T cells but have not converged into a single accepted paradigm. Recent investigations that examined changing representation among mononuclear cells with age and patterns of proliferation and cytokine production by subsets bearing one or more of the previously identified variants of the WC1 molecule are summarized here. While the decrease in percentages within blood in the first year of life was found to be precipitous for WC1.1+ gammadelta T cells it was not for WC1.2+ cells. While both populations proliferated to mitogen stimulation there was a bias towards responses by WC1.2+ cells. In leptospira antigen-stimulated cultures and autologous mixed lymphocyte reaction (AMLR) cultures WC1.1+ cells proliferated and produced interferon-gamma (IFN-gamma) while WC1.2+ cells did to a much lower extent. This suggested functional differences related to the isoform of WC1 expressed. Under Th1-polarizing conditions, the WC1.1+ cells also made IFN-gamma whereas the vast majority of cells expressing WC1.2 did not. Despite the difference in IFN-gamma production, cells bearing either WC1 isoform had similar transcription levels of the high affinity IL-12 receptor subunit (IL-12Rbeta2) as well as of the transcription factors T-bet and GATA-3 when cultured with IL-12. Both populations transcribed low levels of IL-10 mRNA under Th1-polarizing conditions and TGF-beta transcripts were ubiquitously expressed by each of these cell types. Cloning and sequencing of the cytoplasmic tails of the WC1 isoforms revealed a consensus ITAM in all three isoforms but a DENY sequence adjacent to one of the SH-2 binding sites of WC1.1 only. The results suggest that WC1+ gammadelta T cells differentiated on the basis of WC1 isoform expression play distinct roles in immune responses that may be dictated by WC1 intracellular signaling.

Partial cDNA sequences of bovine CD72 and CD166/ALCAM, ligands for SRCR-family accessory molecules CD5 and CD6.

Accessory/co-stimulatory molecules on the surface of T cells are capable of regulating activation signals. Two of these, CD5 and CD6, are molecules from the scavenger receptor cysteine rich (SRCR) superfamily. Partial sequences for the ligands of these molecules, known as CD72 and CD166 (or ALCAM), respectively, are provided for Bos taurus in this communication. Using highly conserved regions between the corresponding human and mouse genes, primers were designed and reverse transcription polymerase chain reaction was used to generate cDNA from bovine PBMC RNA. cDNA clones of several hundred base pairs in length were created and sequenced. The results showed 81% homology between bovine and human CD72 nucleotide sequences and 93% homology for the CD166 sequences. Similar levels of homology are seen between the corresponding human and mouse cDNA sequences.

Role of translation initiation factor 4G in lifespan regulation and age-related health.

Inhibiting expression of eukaryotic translation initiation factor 4G (eIF4G) arrests normal development but extends lifespan when suppressed during adulthood. In addition to reducing overall translation, inhibition alters the stoichiometry of mRNA translation in favor of genes important for responding to stress and against those associated with growth and reproduction in C. elegans. In humans, aberrant expression of eIF4G is associated with certain forms of cancer and neurodegeneration. Here we review what is known about the roles of eIF4G in molecular, cellular, and organismal contexts. Also discussed are the gaps in understanding of this factor, particularly with regard to the roles of specific forms of expression in individual tissues and the importance of understanding eIF4G for development of potential therapeutic applications.