Search Results (10)

Disturbance or insufficiency of the tear film challenges the regulatory systems of the ocular surfaces. The reaction of the surfaces includes temporary mechanisms engaged in the preservation of homeostasis. However, strong or persisting challenges can lead to the potential exhaustion of the coping capacity. This again activates the vicious circle with chronic inflammation and autocatalytic deterioration. Hence, the factors challenging the homeostasis should be addressed in time. Amongst them are a varying osmolarity, constant presence of small lesions at the epithelium, acidification, attrition with mechanical irritation, and onset of pain and discomfort. Each of them and, especially when occurring simultaneously, impose stress on the coping mechanisms and lead to a stress response. Many stressors can culminate, leading to an exhaustion of the coping capacity, outrunning normal resilience. Reaching the limits of stress tolerance leads to the manifestation of a lubrication deficiency as the disease we refer to as dry eye disease (DED). To postpone its manifestation, the avoidance or amelioration of stress factors is one key option. In DED, this is the target of lubrication therapy, substituting the missing tear film or its components. The latter options include the management of secondary sequelae such as the inflammation and activation of reparative cascades. Preventive measures include the enhancement in resilience, recovery velocity, and recovery potential. The capacity to handle the external load factors is the key issue. The aim is to guard homeostasis and to prevent intercellular stress responses from being launched, triggering and invigorating the vicious circle. Considering the dilemma of the surface to have to cope with increased time of exposure to stress, with simultaneously decreasing time for cellular recovery, it illustrates the importance of the vicious circle as a hub for ocular surface stress. The resulting imbalance triggers a continuous deterioration of the ocular surface condition. After an initial phase of the reaction and adaption of the ocular surface to the surrounding challenges, the normal coping capacity will be exhausted. This is the time when the integrated stress response (ISR), a protector for cellular survival, will inevitably be activated, and cellular changes such as altered translation and ribosome pausing are initiated. Once activated, this will slow down any recovery, in a phase where apoptosis is imminent. Premature senescence of cells may also occur. The process of prematurization due to permanent stress exposures contributes to the risk for constant deterioration. The illustrated flow of events in the development of DED outlines that the ability to cope, and to recover, has limited resources in the cells at the ocular surface. The reduction in and amelioration of stress hence should be one of the key targets of therapy and begin early. Here, lubrication optimization as well as causal treatment such as the correction of anatomical anomalies (leading to anatomical dry eye) should be a prime intent of any therapy. The features of cellular stress as a key hub for the vicious circle will be outlined and discussed. Full article

The unique amino acid hypusine [N^ε-(4-amino-2-hydroxybutyl)lysine] is exclusively formed on the translational regulator eukaryotic initiation factor 5A (eIF5A) via a process coined hypusination. Hypusination is mediated by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH), and hypusinated eIF5A (eIF5A^Hyp) promotes translation elongation by alleviating ribosome pauses at amino acid motifs that cause structural constraints, and it also facilitates translation initiation and termination. Accordingly, eIF5A^Hyp has diverse biological functions that rely on translational control of its targets. Homozygous deletion of Eif5a, Dhps, or Dohh in mice leads to embryonic lethality, and heterozygous germline variants in EIF5A and biallelic variants in DHPS and DOHH are associated with rare inherited neurodevelopmental disorders, underscoring the importance of the hypusine circuit for embryonic and neuronal development. Given the pleiotropic effects of eIF5A^Hyp, a detailed understanding of the cell context-specific intrinsic roles of eIF5A^Hyp and of the chronic versus acute effects of eIF5A^Hyp inhibition is necessary to develop future strategies for eIF5A^Hyp-targeted therapy to treat various human health problems. Here, we review the most recent studies documenting the intrinsic roles of eIF5A^Hyp in different tissues/cell types under normal or pathophysiological conditions and discuss these unique aspects of eIF5A^Hyp-dependent translational control. Full article

Regulation of translation is a crucial step in gene expression. Developmental signals and environmental stimuli dynamically regulate translation via upstream small open reading frames (uORFs) and ribosome pausing. Recent studies have revealed many plant genes that are specifically regulated by uORF translation following changes in growth conditions, but ribosome-pausing events are less well understood. In this study, we performed ribosome profiling (Ribo-seq) of etiolated maize (Zea mays) seedlings exposed to light for different durations, revealing hundreds of genes specifically regulated at the translation level during the early period of light exposure. We identified over 400 ribosome-pausing events in the dark that were rapidly released after illumination. These results suggested that ribosome pausing negatively regulates translation from specific genes, a conclusion that was supported by a non-targeted proteomics analysis. Importantly, we identified a conserved nucleotide motif downstream of the pausing sites. Our results elucidate the role of ribosome pausing in the control of gene expression in plants; the identification of the cis-element at the pausing sites provides insight into the mechanisms behind translation regulation and potential targets for artificial control of plant translation. Full article

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA. Full article

Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA–ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings. Full article

DEAD-box RNA helicases are ATP-dependent RNA binding proteins and RNA-dependent ATPases that possess weak, nonprocessive unwinding activity in vitro, but they can form long-lived complexes on RNAs when the ATPase activity is inhibited. Ded1 is a yeast DEAD-box protein, the functional ortholog of mammalian DDX3, that is considered important for the scanning efficiency of the 48S pre-initiation complex ribosomes to the AUG start codon. We used a modified PAR-CLIP technique, which we call quicktime PAR-CLIP (qtPAR-CLIP), to crosslink Ded1 to 4-thiouridine-incorporated RNAs in vivo using UV light centered at 365 nm. The irradiation conditions are largely benign to the yeast cells and to Ded1, and we are able to obtain a high efficiency of crosslinking under physiological conditions. We find that Ded1 forms crosslinks on the open reading frames of many different mRNAs, but it forms the most extensive interactions on relatively few mRNAs, and particularly on mRNAs encoding certain ribosomal proteins and translation factors. Under glucose-depletion conditions, the crosslinking pattern shifts to mRNAs encoding metabolic and stress-related proteins, which reflects the altered translation. These data are consistent with Ded1 functioning in the regulation of translation elongation, perhaps by pausing or stabilizing the ribosomes through its ATP-dependent binding. Full article

The SARS-CoV-2 Spike glycoprotein (S protein) acquired a unique new 4 amino acid -PRRA- insertion sequence at amino acid residues (aa) 681–684 that forms a new furin cleavage site in S protein as well as several new glycosylation sites. We studied various statistical properties of the -PRRA- insertion at the RNA level (CCUCGGCGGGCA). The nucleotide composition and codon usage of this sequence are different from the rest of the SARS-CoV-2 genome. One of such features is two tandem CGG codons, although the CGG codon is the rarest codon in the SARS-CoV-2 genome. This suggests that the insertion sequence could cause ribosome pausing as the result of these rare codons. Due to population variants, the Nextstrain divergence measure of the CCU codon is extremely large. We cannot exclude that this divergence might affect host immune responses/effectiveness of SARS-CoV-2 vaccines, possibilities awaiting further investigation. Our experimental studies show that the expression level of original RNA sequence “wildtype” spike protein is much lower than for codon-optimized spike protein in all studied cell lines. Interestingly, the original spike sequence produces a higher titer of pseudoviral particles and a higher level of infection. Further mutagenesis experiments suggest that this dual-effect insert, comprised of a combination of overlapping translation pausing and furin sites, has allowed SARS-CoV-2 to infect its new host (human) more readily. This underlines the importance of ribosome pausing to allow efficient regulation of protein expression and also of cotranslational subdomain folding. Full article

Present-day chloroplast and mitochondrial genomes contain only a few dozen genes involved in ATP synthesis, photosynthesis, and gene expression. The proteins encoded by these genes are only a small fraction of the many hundreds of proteins that act in chloroplasts and mitochondria. Hence, the vast majority, including components of organellar gene expression (OGE) machineries, are encoded by nuclear genes, translated into the cytosol and imported to these organelles. Consequently, the expression of nuclear and organellar genomes has to be very precisely coordinated. Furthermore, OGE regulation is crucial to chloroplast and mitochondria biogenesis, and hence, to plant growth and development. Notwithstanding, the molecular mechanisms governing OGE are still poorly understood. Recent results have revealed the increasing importance of nuclear-encoded modular proteins capable of binding nucleic acids and regulating OGE. Mitochondrial transcription termination factor (mTERF) proteins are a good example of this category of OGE regulators. Plant mTERFs are located in chloroplasts and/or mitochondria, and have been characterized mainly from the isolation and analyses of Arabidopsis and maize mutants. These studies have revealed their fundamental roles in different plant development aspects and responses to abiotic stress. Fourteen mTERFs have been hitherto characterized in land plants, albeit to a different extent. These numbers are limited if we consider that 31 and 35 mTERFs have been, respectively, identified in maize and Arabidopsis. Notwithstanding, remarkable progress has been made in recent years to elucidate the molecular mechanisms by which mTERFs regulate OGE. Consequently, it has been experimentally demonstrated that plant mTERFs are required for the transcription termination of chloroplast genes (mTERF6 and mTERF8), transcriptional pausing and the stabilization of chloroplast transcripts (MDA1/mTERF5), intron splicing in chloroplasts (BSM/RUG2/mTERF4 and Zm-mTERF4) and mitochondria (mTERF15 and ZmSMK3) and very recently, also in the assembly of chloroplast ribosomes and translation (mTERF9). This review aims to provide a detailed update of current knowledge about the molecular functions of plant mTERF proteins. It principally focuses on new research that has made an outstanding contribution to unravel the molecular mechanisms by which plant mTERFs regulate the expression of chloroplast and mitochondrial genomes. Full article

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome. Full article

Proteins in the cell are synthesized by a ribosome translating the genetic information encoded on the single-stranded messenger RNA (mRNA). It has been shown that the ribosome can also translate through the duplex region of the mRNA by unwinding the duplex. Here, based on our proposed model of the ribosome translation through the mRNA duplex we study theoretically the distribution of dwell times of the ribosome translation through the mRNA duplex under the effect of a pulling force externally applied to the ends of the mRNA to unzip the duplex. We provide quantitative explanations of the available single molecule experimental data on the distribution of dwell times with both short and long durations, on rescuing of the long paused ribosomes by raising the pulling force to unzip the duplex, on translational arrests induced by the mRNA duplex and Shine-Dalgarno(SD)-like sequence in the mRNA. The functional consequences of the pauses or arrests caused by the mRNA duplex and the SD sequence are discussed and compared with those obtained from other types of pausing, such as those induced by “hungry” codons or interactions of specific sequences in the nascent chain with the ribosomal exit tunnel. Full article