Cytosolic Translation Research Articles

Cytosolic N6AMT1-dependent translation supports mitochondrial RNA processing

Mitochondrial biogenesis relies on both the nuclear and mitochondrial genomes, and imbalance in their expression can lead to inborn errors of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic methyltransferase that exhibits genetic codependency with mitochondria. We determine transcriptional and translational profiles of N6AMT1 and report that it is required for the cytosolic translation of TRMT10C (MRPP1) and PRORP (MRPP3), two subunits of the mitochondrial RNAse P enzyme. In the absence of N6AMT1, or when its catalytic activity is abolished, RNA processing within mitochondria is impaired, leading to the accumulation of unprocessed and double-stranded RNA, thus preventing mitochondrial protein synthesis and oxidative phosphorylation, and leading to an immune response. Our work sheds light on the function of N6AMT1 in protein synthesis and highlights a cytosolic program required for proper mitochondrial biogenesis.

The Toxoplasma gondii mitochondrial transporter ABCB7L is essential for the biogenesis of cytosolic and nuclear iron-sulfur cluster proteins and cytosolic translation.

Iron-sulfur (Fe-S) clusters are inorganic cofactors of proteins that play key roles in numerous essential biological processes, for example, respiration and DNA replication. Cells possess dedicated biosynthetic pathways to assemble Fe-S clusters, including a pathway in the mitochondrion and cytosol. A single transporter, called ABCB7, connects these two pathways, allowing an essential intermediate generated by the mitochondrial pathway to be used in the cytosolic pathway. Cytosolic and nuclear Fe-S proteins are dependent on the mitochondrial pathway, mediated by ABCB7, in numerous organisms studied to date. Here, we study the role of a homolog of ABCB7, which we name ABCB7-like (ABCB7L), in the ubiquitous unicellular apicomplexan parasite Toxoplasma gondii. We generated a depletion mutant of Toxoplasma ABCB7L and showed its importance for parasite fitness. Using comparative quantitative proteomic analysis and experimental validation of the mutants, we show that ABCB7L is required for cytosolic and nuclear, but not mitochondrial, Fe-S protein biogenesis. Our study supports the conservation of a protein homologous to ABCB7 and which has a similar function in apicomplexan parasites and provides insight into an understudied aspect of parasite metabolism.

Impact of mtDNA-encoded proteins arising from cytosolic translation (mPACT)

Mitochondria are multifunctional organelles that regulate energy homeostasis, apoptosis, cell signaling and many biosynthetic pathways. Mitochondria have their own genomes that reportedly contain 37 well-known genes, including 13 protein-coding genes, 2 genes encoding ribosomal RNAs (rRNAs) and 22 genes encoding transfer RNAs (tRNAs). RNA is essential for most biological functions which can act as a translation template for protein production or perform the function itself to directly regulate cellular processes. In addition to those functioning inside mitochondria, mitochondria can regulate many important biological processes through exporting RNAs including long non-coding mitochondrial RNAs (ncmtRNAs), circRNAs, rRNAs, tRNAs and mRNAs.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

A novel protein CYTB-187AA encoded by the mitochondrial gene CYTB modulates mammalian early development

The mitochondrial genome transcribes 13 mRNAs coding for well-known proteins essential for oxidative phosphorylation. We demonstrate here that cytochrome b (CYTB), the only mitochondrial-DNA-encoded transcript among complex III, also encodes an unrecognized 187-amino-acid-long protein, CYTB-187AA, using the standard genetic code of cytosolic ribosomes rather than the mitochondrial genetic code. After validating the existence of this mtDNA-encoded protein arising from cytosolic translation (mPACT) using mass spectrometry and antibodies, we show that CYTB-187AA is mainly localized in the mitochondrial matrix and promotes the pluripotent state in primed-to-naive transition by interacting with solute carrier family 25 member 3 (SLC25A3) to modulate ATP production. We further generated a transgenic knockin mouse model of CYTB-187AA silencing and found that reduction of CYTB-187AA impairs females' fertility by decreasing the number of ovarian follicles. For the first time, we uncovered the novel mPACT pattern of a mitochondrial mRNA and demonstrated the physiological function of this 14th protein encoded by mtDNA.

Cardiomyocyte-Specific Loss of Glutamyl-prolyl-tRNA Synthetase Leads to Disturbed Protein Homeostasis and Dilated Cardiomyopathy.

Glutamyl-prolyl-tRNA synthetase (EPRS1), an aminoacyl-tRNA synthetase (ARS) ligating glutamic acid and proline to their corresponding tRNAs, plays an essential role in decoding proline codons during translation elongation. The physiological function of EPRS1 in cardiomyocytes (CMs) and the potential effects of the CM-specific loss of Eprs1 remain unknown. Here, we found that heterozygous Eprs1 knockout in CMs does not cause any significant changes in CM hypertrophy induced by pressure overload, while homozygous knockout leads to dilated cardiomyopathy, heart failure, and lethality at around 1 month after Eprs1 deletion. The transcriptomic profiling of early-stage Eprs1 knockout hearts suggests a significantly decreased expression of multiple ion channel genes and an increased gene expression in proapoptotic pathways and integrated stress response. Proteomic analysis shows decreased protein expression in multi-aminoacyl-tRNA synthetase complex components, fatty acids, and branched-chain amino acid metabolic enzymes, as well as a compensatory increase in cytosolic translation machine-related proteins. Immunoblot analysis indicates that multiple proline-rich proteins were reduced at the early stage, which might contribute to the cardiac dysfunction of Eprs1 knockout mice. Taken together, this study demonstrates the physiological and molecular outcomes of loss-of-function of Eprs1 in vivo and provides valuable insights into the potential side effects on CMs, resulting from the EPRS1-targeting therapeutic approach.

NTRC and thioredoxins m1/m2 underpin the light acclimation of plants on proteome and metabolome levels.

During photosynthesis, plants must manage strong fluctuations in light availability on different time scales, leading to long-term acclimation and short-term responses. However, little is known about the regulation and coordination of these processes and the modulators involved. In this study, we used proteomics, metabolomics, and reverse genetics to investigate how different light environmental factors, such as intensity or variability, affect long-term and short-term acclimation responses of Arabidopsis (Arabidopsis thaliana) and the importance of the chloroplast redox network in their regulation. In the wild type, high light, but not fluctuating light, led to large quantitative changes in the proteome and metabolome, accompanied by increased photosynthetic dynamics and plant growth. This finding supports light intensity as a stronger driver for acclimation than variability. Deficiencies in NADPH-thioredoxin reductase C (NTRC) or thioredoxins m1/m2, but not thioredoxin f1, almost completely suppressed the re-engineering of the proteome and metabolome, with both the induction of proteins involved in stress and redox responses and the repression of those involved in cytosolic and plastid protein synthesis and translation being strongly attenuated. Moreover, the correlations of protein or metabolite levels with light intensity were severely disturbed, suggesting a general defect in the light-dependent acclimation response, resulting in impaired photosynthetic dynamics. These results indicate a previously unknown role of NTRC and thioredoxins m1/m2 in modulating light acclimation at proteome and metabolome levels to control dynamic light responses. NTRC, but not thioredoxins m1/m2 or f1, also improves short-term photosynthetic responses by balancing the Calvin-Benson cycle in fluctuating light.

Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins.

A complex interplay between mRNA translation and cellular respiration has been recently unveiled, but its regulation in humans is poorly characterized in either health or disease. Cancer cells radically reshape both biosynthetic and bioenergetic pathways to sustain their aberrant growth rates. In this regard, we have shown that the molecular chaperone TRAP1 not only regulates the activity of respiratory complexes, behaving alternatively as an oncogene or a tumor suppressor, but also plays a concomitant moonlighting function in mRNA translation regulation. Herein, we identify the molecular mechanisms involved, showing that TRAP1 (1) binds both mitochondrial and cytosolic ribosomes, as well as translation elongation factors; (2) slows down translation elongation rate; and (3) favors localized translation in the proximity of mitochondria. We also provide evidence that TRAP1 is coexpressed in human tissues with the mitochondrial translational machinery, which is responsible for the synthesis of respiratory complex proteins. Altogether, our results show an unprecedented level of complexity in the regulation of cancer cell metabolism, strongly suggesting the existence of a tight feedback loop between protein synthesis and energy metabolism, based on the demonstration that a single molecular chaperone plays a role in both mitochondrial and cytosolic translation, as well as in mitochondrial respiration.

Dysfunctional Autophagy, Proteostasis, and Mitochondria as a Prelude to Age-Related Macular Degeneration.

Retinal pigment epithelial (RPE) cell dysfunction is a key driving force of AMD. RPE cells form a metabolic interface between photoreceptors and choriocapillaris, performing essential functions for retinal homeostasis. Through their multiple functions, RPE cells are constantly exposed to oxidative stress, which leads to the accumulation of damaged proteins, lipids, nucleic acids, and cellular organelles, including mitochondria. As miniature chemical engines of the cell, self-replicating mitochondria are heavily implicated in the aging process through a variety of mechanisms. In the eye, mitochondrial dysfunction is strongly associated with several diseases, including age-related macular degeneration (AMD), which is a leading cause of irreversible vision loss in millions of people globally. Aged mitochondria exhibit decreased rates of oxidative phosphorylation, increased reactive oxygen species (ROS) generation, and increased numbers of mitochondrial DNA mutations. Mitochondrial bioenergetics and autophagy decline during aging because of insufficient free radical scavenger systems, the impairment of DNA repair mechanisms, and reductions in mitochondrial turnover. Recent research has uncovered a much more complex role of mitochondrial function and cytosolic protein translation and proteostasis in AMD pathogenesis. The coupling of autophagy and mitochondrial apoptosis modulates the proteostasis and aging processes. This review aims to summarise and provide a perspective on (i) the current evidence of autophagy, proteostasis, and mitochondrial dysfunction in dry AMD; (ii) current in vitro and in vivo disease models relevant to assessing mitochondrial dysfunction in AMD, and their utility in drug screening; and (iii) ongoing clinical trials targeting mitochondrial dysfunction for AMD therapeutics.

Structure of the actively translating plant 80S ribosome at 2.2 Å resolution

In plant cells, translation occurs in three compartments: the cytosol, the plastids and the mitochondria. While the structures of the (prokaryotic-type) ribosomes in plastids and mitochondria are well characterized, high-resolution structures of the eukaryotic 80S ribosomes in the cytosol have been lacking. Here the structure of translating tobacco (Nicotiana tabacum) 80S ribosomes was solved by cryo-electron microscopy with a global resolution of 2.2 Å. The ribosome structure includes two tRNAs, decoded mRNA and the nascent peptide chain, thus providing insights into the molecular underpinnings of the cytosolic translation process in plants. The map displays conserved and plant-specific rRNA modifications and the positions of numerous ionic cofactors, and it uncovers the role of monovalent ions in the decoding centre. The model of the plant 80S ribosome enables broad phylogenetic comparisons that reveal commonalities and differences in the ribosomes of plants and those of other eukaryotes, thus putting our knowledge about eukaryotic translation on a firmer footing.

Rfoot-seq: Transcriptomic RNase Footprinting for Mapping Stable RNA-Protein Complexes and Rapid Ribosome Profiling.

Ribosome profiling isolates ribosome-protected fragments for sequencing and is a valuable method for studying different aspects of RNA translation. However, conventional protocols require millions of input cells and time-consuming steps to isolate translating ribosome complexes using ultracentrifugation or immunoprecipitation. These limitations have prevented their application to rare physiological samples. To address these technical barriers, we developed an RNase footprinting approach named Rfoot-seq to map stable transcriptomic RNA-protein complexes that allows rapid ribosome profiling using low-input samples (Li, Yang, Stroup, Wang, & Ji, 2022). In this assay, we treat a cell lysate with concentrated RNase without complex crosslinking and retained only RNA footprints associated with stable complexes for sequencing. The footprints in coding regions represent ribosome-protected fragments and can be used to study cytosolic and mitochondrial translation simultaneously. Rfoot-seq achieves comparable results to conventional ribosome profiling to quantify ribosome occupancy and works robustly for various cultured cells and primary tissue samples. Moreover, Rfoot-seq maps RNA fragments associated with stable non-ribosomal RNA-protein complexes in noncoding domains of small noncoding RNAs and some long noncoding RNAs. Taken together, Rfoot-seq opens an avenue to quantify transcriptomic translation and characterize functional noncoding RNA domains using low-input samples. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Harvesting and lysing adherent cells Alternate Protocol 1: Harvesting and lysing suspension cells Alternate Protocol 2: Harvesting and lysing primary tissue samples Basic Protocol 2: RNase treatment and footprint purification for low-input samples Alternate Protocol 3: RNase treatment and footprint purification for ultra-low-input samples Basic Protocol 3: Library preparation for high-throughput sequencing Support Protocol: Preparation of dsDNA markers for library size selection Basic Protocol 4: Data analysis and quality control after sequencing.

Ssu72 phosphatase is essential for thermogenic adaptation by regulating cytosolic translation

Brown adipose tissue (BAT) plays a pivotal role in maintaining body temperature and energy homeostasis. BAT dysfunction is associated with impaired metabolic health. Here, we show that Ssu72 phosphatase is essential for mRNA translation of genes required for thermogenesis in BAT. Ssu72 is found to be highly expressed in BAT among adipose tissue depots, and the expression level of Ssu72 is increased upon acute cold exposure. Mice lacking adipocyte Ssu72 exhibit cold intolerance during acute cold exposure. Mechanistically, Ssu72 deficiency alters cytosolic mRNA translation program through hyperphosphorylation of eIF2α and reduces translation of mitochondrial oxidative phosphorylation (OXPHOS) subunits, resulting in mitochondrial dysfunction and defective thermogenesis in BAT. In addition, metabolic dysfunction in Ssu72-deficient BAT returns to almost normal after restoring Ssu72 expression. In summary, our findings demonstrate that cold-responsive Ssu72 phosphatase is involved in cytosolic translation of key thermogenic effectors via dephosphorylation of eIF2α in brown adipocytes, providing insights into metabolic benefits of Ssu72.

Comparative oxidation proteomics analyses suggest redox regulation of cytosolic translation in rice leaves upon Magnaporthe oryzae infection

Pathogen attack can increase plant levels of reactive oxygen species (ROS), which act as signaling molecules to activate plant defense mechanisms. Elucidating these processes is crucial for understanding redox signaling pathways in plant defense responses. Using an iodo-tandem mass tag (TMT)-based quantitative proteomics approach, we mapped 3362 oxidized cysteine sites in 2275 proteins in rice leaves. Oxidized proteins were involved in gene expression, peptide biosynthetic processes, stress responses, ROS metabolic processes, and translation pathways. Magnaporthe oryzae infection led to increased oxidative modification levels of 512 cysteine sites in 438 proteins, including many transcriptional regulators and ribosomal proteins. Ribosome profiling (Ribo-seq) analysis revealed that the oxidative modification of ribosomal proteins promoted the translational efficiency of many mRNAs involved in defense response pathways, thereby affecting rice immunity. Our results suggest that increased oxidative modification of ribosomal proteins in rice leaves promotes cytosolic translation, thus revealing a novel function of post-translational modifications. Furthermore, the oxidation-sensitive proteins identified here provide a valuable resource for research on protein redox regulation and can guide future mechanistic studies.

Anti-retroviral treatment with zidovudine alters pyrimidine metabolism, reduces translation, and extends healthy longevity via ATF-4

The human population is aging, and the need for interventions to slow progression of age-related diseases (geroprotective interventions) is growing. Repurposing compounds already used clinically, usually at modified doses, allows rapid implementation of geroprotective pharmaceuticals. Here we find the anti-retroviral nucleoside reverse transcriptase inhibitor (NRTI) zidovudine robustly extends lifespan and health span in C.elegans, independent of electron transport chain impairment or ROS accumulation. Rather, zidovudine treatment modifies pyrimidine metabolism and transcripts related to proteostasis. Testing regulators of mitochondrial stress and proteostasis shows that lifespan extension is dependent on activating transcription factor 4 (ATF-4). ATF-4 regulates longevity induced by mitochondrial stress, specifically communication between mitochondrial and cytosolic translation. Translation is reduced in zidovudine-treated worms, also dependent on ATF-4. Finally, we show ATF-4-dependent lifespan extension induced by didanosine, another NRTI. Altogether, our work elucidates the geroprotective effects of NRTIs such as zidovudine invivo, via reduction of translation and ATF-4.

Pentatricopeptide Protein PTCD2 Regulates COIII Translation in Mitochondria of the HeLa Cell Line.

Protein biosynthesis in mitochondria is tightly coupled with assembly of inner membrane complexes and therefore must be coordinated with cytosolic translation of the mRNAs corresponding to the subunits which are encoded in the nucleus. Molecular mechanisms underlying the regulation of mitochondrial translation remain unclear despite recent advances in structural biology. Until now, only one translational regulator of protein biosynthesis in mammalian mitochondria is known-protein TACO1, which regulates translation of COI mRNA. Here we describe the function of pentatricopeptide-containing protein PTCD2 as a translational regulator of another mitochondrially encoded subunit of cytochrome c oxidase-COIII in the HeLa cell line. Deletion of the PTCD2 gene leads to significant decrease in COIII translation efficiency and impairment in CIV activity. Additionally, we show that PTCD2 protein is partially co-sedimentates with associated mitochondrial ribosome and associates with mitochondrial ribosome proteins in pull-down assays. These data allow concluding that PTCD2 is a specific translational regulator of COIII which attracts the mRNA to the mitochondrial ribosome.

Cytosolic and mitochondrial tRNA synthetase inhibitors increase lifespan in a GCN4/atf-4-dependent manner

Deletion of genes encoding ribosomal proteins extends lifespan in yeast. This increases translation of the functionally conserved transcription factor Gcn4, and lifespan extension in these mutants is GCN4-dependent. Gcn4 is also translationally upregulated by uncharged tRNAs, as are its C aenorhabditis elegans and mammalian functional orthologs. Here, we show that cytosolic tRNA synthetase inhibitors upregulate Gcn4 translation and extend yeast lifespan in a Gcn4-dependent manner. This cytosolic tRNA synthetase inhibitor is also able to extend the lifespan of C. elegans in an atf-4-dependent manner. We show that mitochondrial tRNA synthetase inhibitors greatly extend the lifespan of C. elegans, and this depends on atf-4. This suggests that perturbations of both cytosolic and mitochondrial translation may act in part via the same downstream pathway. These findings establish GCN4 orthologs as conserved longevity factors and, as long-lived mice exhibit elevated ATF4, leave open the possibility that tRNA synthetase inhibitors could also extend lifespan in mammals.

Host adaptive mutations in the 2009 H1N1 pandemic influenza A virus PA gene regulate translation efficiency of viral mRNAs via GRSF1

Avian species are the major natural reservoir from which pandemic influenza A viruses can be introduced to humans. Avian influenza A virus genes, including the three viral polymerase genes, PA, PB1 and PB2, require host-adaptive mutations to allow for viral replication and transmission in humans. Previously, PA from the 2009 pH1N1 viral polymerase was found to harbor host-adaptive mutations leading to enhanced viral polymerase activity. By quantifying translation and mRNA transcription, we found that the 2009 pH1N1 PA, and the associated host-adaptive mutations, led to greater translation efficiency. This was due to enhanced cytosolic accumulation of viral mRNA, which was dependent on the host RNA binding protein GRSF1. Mutations to the GRSF1 binding site in viral mRNA, as well as GRSF1 knockdown, reduced cytosolic accumulation and translation efficiency of viral mRNAs. This study identifies a previously unrecognized mechanism by which host-adaptive mutations in PA regulate viral replication and host adaptation. Importantly, these results provide greater insight into the host adaptation process of IAVs and reveal the importance of GRSF1 in the lifecycle of IAV.

Correlated retrograde and developmental regulons implicate multiple retrograde signals as coordinators of chloroplast development in maize.

Signals emanating from chloroplasts influence nuclear gene expression, but roles of retrograde signals during chloroplast development are unclear. To address this gap, we analyzed transcriptomes of non-photosynthetic maize mutants and compared them to transcriptomes of stages of normal leaf development. The transcriptomes of two albino mutants lacking plastid ribosomes resembled transcriptomes at very early stages of normal leaf development, whereas the transcriptomes of two chlorotic mutants with thylakoid targeting or plastid transcription defects resembled those at a slightly later stage. We identified ∼2,700 differentially expressed genes, which fall into six major categories based on the polarity and mutant-specificity of the change. Downregulated genes were generally expressed late in normal development and were enriched in photosynthesis genes, whereas upregulated genes act early and were enriched for functions in chloroplast biogenesis and cytosolic translation. We showed further that target-of-rapamycin (TOR) signaling was elevated in mutants lacking plastid ribosomes and declined in concert with plastid ribosome buildup during normal leaf development. Our results implicate three plastid signals as coordinators of photosynthetic differentiation. One signal requires plastid ribosomes and activates photosynthesis genes. A second signal reflects attainment of chloroplast maturity and represses chloroplast biogenesis genes. A third signal, the consumption of nutrients by developing chloroplasts, represses TOR, promoting termination of cell proliferation during leaf development.

Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants.

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or “super-resistance”, to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.

Corrigendum: Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.

Corrigendum: Low-input RNase footprinting for simultaneous quantification of cytosolic and mitochondrial translation.