Ribosomal Activity Research Articles

Ribosome Structural Changes Dynamically Affect Ribosome Function.

Ribosomes were known to be multicomponent complexes as early as the 1960s. Nonetheless, the prevailing view for decades considered active ribosomes to be a monolithic population, in which all ribosomes are identical in composition and function. This implied that ribosomes themselves did not actively contribute to the regulation of protein synthesis. In this perspective, I review evidence for a different model, based on results showing that ribosomes can harbor different types of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) and, furthermore, need not contain a complete set of r-proteins. I also summarize recent results favoring the notion that such distinct types of ribosomes have different affinities for specific messenger RNAs and may execute the translation process differently. Thus, ribosomes should be considered active contributors to the regulation of protein synthesis.

Gene expression profiles in placenta and their association with anesthesia, delivery mode and maternal diabetes

IntroductionFetal development is dependent on placenta and affected by multiple factors including maternal diabetes. Here we aimed to identify maternal diabetes-associated changes in placentas and analyzed placental gene expression to understand its modulation by maternal diabetes and birth mode. MethodsPlacental RNAseq transcriptome analyses were performed on maternally-derived decidua and fetal-derived villous tissue from pregnancies of mothers with type 1 diabetes (n=14), gestational diabetes (n=6) and without diabetes (n=14). Information on delivery mode and anesthesia were included as covariables. Analyses were performed separately for decidua and fetal tissues and adjusted for sex. ResultsSubstantial placenta gene expression variation was associated with factors other than maternal diabetes, including site, sex, anesthesia type and delivery mode. Two dominant gene expression clusters aligned to anesthesia and delivery mode were observed for decidua and villous tissue. Upregulation of genes within pathways related to organ morphogenesis and downregulation of immune response to steroid- and hypoxia pathway genes was characteristic of placentas from primary cesarean section deliveries with spinal anesthesia. Opposite profiles were observed for placentas from secondary cesarean and epidural anesthesia deliveries. Placentas from vaginal delivery had intermediate gene expression profiles. More subtle changes were associated with maternal diabetes: upregulation of ribosome activity, down-regulation of maternally-derived decidua chemokine signaling pathways and for gestational diabetes, alteration in hypoxia response genes. DiscussionThe findings reveal suppression of immune pathways and upregulation of ribosome activity in the placenta by maternal diabetes highlighting the importance of confounding factors when examining cell and tissue expression profiles. Further studies should determine whether the observed gene expression differences are related to underlying causes for cesarean section deliveries.

Using Mycobacterium smegmatis as a Bioindicator for Zinc-limited Growth Conditions in Mycobacteria.

Many bacteria build alternative ribosomes in Zn2+-limiting growth conditions by replacing Zn2+-binding ribosomal proteins with Zn2+-independent paralogs. Defining a system to study these alternative ribosomes has proven difficult because Zn2+ contamination in the laboratory is common. To address this issue, chelating agents are sometimes added to growth media, but this approach convolutes the biological response to gradual Zn2+limitation and is associated with ribosome hibernation. Here, detailed instructions are outlined for preparing media and seeding cultures for Zn2+-limited growth without adding chelators. Following this method, the model bacterium, Mycobacterium smegmatis, undergoes morphogenesis, which depends on alternative ribosomes. Because morphogenesis is tractable and only occurs in Zn2+-limiting conditions, M. smegmatis can be used as a bioindicator to verify biologically relevant growth conditions. Three bioindicator phenotypes (cell density, cell length, and coenzyme F420 fluorescence) that indicate Zn2+ limitation in the wild-type are described, and changes in these bioindicators for a deletion mutant that cannot build alternative ribosomes are outlined. Since trace Zn2+ contamination is difficult to control for each batch of media, and precise quantification of Zn2+ in each media preparation is overly burdensome, following this bioindicator phenotype is an accessible way to validate the preparation of Zn2+-limited growth media. To help identify proper conditions for Zn2+-limiting growth and alternative ribosome production, changes in the bioindicator phenotypes were profiled for Zn2+-contaminated or severely Zn2+-depleted preparations of Zn2+-limited media as well. Further details to achieve Zn2+-limiting growth and alternative ribosome production in M. tuberculosis are presented, along with the associated bioindicator phenotype. Overall, the detailed instructions and bioindicator phenotypes described here will help standardize the production of translationally active alternative ribosomes in mycobacteria.

Divergent transcriptomic profiles in depressed individuals with hyper- and hypophagia implicating inflammatory status

BackgroundMajor Depressive Disorder (MDD) is a heterogenous and etiologically complex disease often presenting with divergent appetitive phenotypes including Hyperphagic MDD (characterized by an increased appetite) and Hypophagic MDD (characterized by a decrease in appetite) which are closely related to comorbidities, including cardiometabolic disorders. Hyperphagia is associated with atypical depression, decreased stress-hormone signaling, a pro-inflammatory status, hypersomnia, and poorer clinical outcomes. Yet, our understanding of associated biological correlates of Hyperphagic and Hypophagic MDD remain fragmented. MethodsWe performed an exploratory study on peripheral blood RNA profiling using bulk RNAseq in unmedicated individuals with Hyperphagic and Hypophagic MDD (n = 7 and n = 13, respectively). ResultsAt baseline, we discovered an increased expression of TADA2B in hyperphagic MDD with the significant enrichment of 72 gene ontology pathways mainly related to inflammation. In addition, we used the Maastricht Acute Stress Task to uncover stress-related transcriptomic profiles in Hyper- and Hypophagic MDD and discovered the upregulation of CCDC196 and the downregulation of SPATA33 in hyperphagic MDD. Gene ontology enrichment analysis after stress exposure showed pathways related to ribosomal activity. Limitations: The present findings are tempered primarily by the limited sample size, which requires independent replication of this exploratory study. However, stringent methods controlling for false positive findings mitigate the risk associated with sample size limitations. DiscussionLimitations notwithstanding, findings suggest that hyper- and hypophagic MDD is associated with divergent RNA expression profiles in peripheral blood that are amplified by exposure to a controlled stress test. Our findings in a well-controlled study provide evidence for peripheral markers of a relevant endophenotype of MDD.

Time-resolved multi-omics reveals diverse metabolic strategies of Salmonella during diet-induced inflammation.

With a rise in antibiotic resistance and chronic infection, the metabolic response of Salmonella enterica serovar Typhimurium to various dietary conditions over time remains an understudied avenue for novel, targeted therapeutics. Elucidating how enteric pathogens respond to dietary variation not only helps us decipher the metabolic strategies leveraged for expansion but also assists in proposing targets for therapeutic interventions. In this study, we use a multi-omics approach to identify the metabolic response of Salmonella enterica serovar Typhimurium in mice on both a fibrous diet and high-fat diet over time. When comparing Salmonella gene expression between diets, we found a preferential use of respiratory electron acceptors consistent with increased inflammation in high-fat diet mice. Looking at the high-fat diet over the course of infection, we noticed heterogeneity in samples based on Salmonella ribosomal activity, which is separated into three infection phases: early, peak, and late. We identified key respiratory, carbon, and pathogenesis gene expressions descriptive of each phase. Surprisingly, we identified genes associated with host cell entry expressed throughout infection, suggesting subpopulations of Salmonella or stress-induced dysregulation. Collectively, these results highlight not only the sensitivity of Salmonella to its environment but also identify phase-specific genes that may be used as therapeutic targets to reduce infection.IMPORTANCEIdentifying novel therapeutic strategies for Salmonella infection that occur in relevant diets and over time is needed with the rise of antibiotic resistance and global shifts toward Western diets that are high in fat and low in fiber. Mice on a high-fat diet are more inflamed compared to those on a fibrous diet, creating an environment that results in more favorable energy generation for Salmonella. We observed differential gene expression across infection phases in mice over time on a high-fat diet. Together, these findings reveal the metabolic tuning of Salmonella to dietary and temporal perturbations. Research like this, which explores the dimensions of pathogen metabolic plasticity, can pave the way for rationally designed strategies to control disease.

Drosophila Clueless ribonucleoprotein particles display novel dynamics that rely on the availability of functional protein and polysome equilibrium.

The cytoplasm is populated with many ribonucleoprotein (RNP) particles that post-transcriptionally regulate mRNAs. These membraneless organelles assemble and disassemble in response to stress, performing functions such as sequestering stalled translation pre-initiation complexes or mRNA storage, repression and decay. Drosophila Clueless (Clu) is a conserved multi-domain ribonucleoprotein essential for mitochondrial function that forms dynamic particles within the cytoplasm. Unlike well-known RNP particles, stress granules and Processing bodies, Clu particles completely disassemble under nutritional or oxidative stress. However, it is poorly understood how disrupting protein synthesis affects Clu particle dynamics, especially since Clu binds mRNA and ribosomes. Here, we capitalize on ex vivo and in vivo imaging of Drosophila female germ cells to determine what domains of Clu are necessary for Clu particle assembly, how manipulating translation using translation inhibitors affects particle dynamics, and how Clu particle movement relates to mitochondrial association. Using Clu deletion analysis and live and fixed imaging, we identified three protein domains in Clu, which are essential for particle assembly. In addition, we demonstrated that overexpressing functional Clu disassembled particles, while overexpression of deletion constructs did not. To examine how decreasing translation affects particle dynamics, we inhibited translation in Drosophila germ cells using cycloheximide and puromycin. In contrast to stress granules and Processing bodies, cycloheximide treatment did not disassemble Clu particles yet puromycin treatment did. Surprisingly, cycloheximide stabilized particles in the presence of oxidative and nutritional stress. These findings demonstrate that Clu particles have novel dynamics in response to altered ribosome activity compared to stress granules and Processing bodies and support a model where they function as hubs of translation whose assembly heavily depends on the dynamic availability of polysomes.

Endothelial Cell Transcription Modulation in Cerebral Aneurysms After Endovascular Flow Diversion.

Flow diverting stents (FDS) are used to treat cerebral aneurysms, by promoting thrombosis and occlusion of the aneurysm sac. However, retreatment is required in some cases, and the biologic basis behind treatment outcome is not known. The goal of this study was to understand how changes in hemodynamic flow after FDS placement affect aneurysmal endothelial cell (EC) activity. Three-dimensional models of patient-specific aneurysms were created to quantify the EC response to FDS placement. Computational fluid dynamic simulations were used to determine the hemodynamic impact of FDS. Two identical models were created for each patient; into one a FDS was inserted. Each model was then populated with human carotid ECs and subjected to patient-specific pulsatile flow for 24 h. ECs were isolated from aneurysm dome from each model and bulk RNA sequencing was performed. Paired untreated and treated models were created for four patients. Aneurysm dome EC analysis revealed 366 (2.6%) significant gene changes between the untreated and FDS conditions, out of 13909 total expressed genes. Gene set enrichment analysis of the untreated models demonstrated enriched gene ontology terms related to cell adhesion, growth/tensile activity, cytoskeletal organization, and calcium ion binding. In the FDS models, enriched terms were related to cellular proliferation, ribosomal activity, RNA splicing, and protein folding. Treatment of cerebral aneurysms with FDS induces significant EC gene transcription changes related to aneurysm hemodynamics in patient-specific in vitro 3D-printed models subjected to pulsatile flow. Further investigation is needed into the relationship between transcriptional change and treatment outcome.

Abstract We008: Hallmark Maturation of the Human Pluripotent Stem Cells-derived Cardiovascular Progenitors in Myocardial Infarcted Large Animal Model

For the goal of regenerative cardiology, cellular therapy may be used to restore injured heart muscle. The molecular mechanisms behind the engraftment and maturation of these transplanted cells, however, remain poorly understood. Thus, to obtain a comprehensive understanding of the complex transcriptional landscapes within their transplanted spatial context, we utilized spatial transcriptomics in this study. We have previously demonstrated the safety and effectiveness of transplanting our cardiovascular progenitors (CVPs) produced from laminin-221 into pig hearts that had suffered myocardial infarction (MI). Here, we undertake 10x spatial transcriptomics in chronic MI pig models at 1-, 4-, and 12 weeks post-transplantation to further our understanding of these cells in vivo. With confidence, we can distinguish between the transcripts from the pigs and the humans in the infarcted region. By using human-specific anti-Ku80 antibodies for immunohistochemical labeling of the pig tissue samples, this in silico prediction was verified. The CVPs' engraftment and longevity in the infracted region were shown by the long-term identification and staining of human cells at various time points. It is interesting to note that after 12 weeks, ribosomal and mitochondrial activity were higher than at 1 and 2 weeks. These results are consistent with the hallmarks of cardiac maturation, in which cells transit from immature fetal-like cells to mature cardiomyocytes (CMs) by switching their metabolic energy source from glucose to fatty acids. Also, by comparing the heart-specific genes, including MYH6 and MYH7, we were able to quantify the myosin isoform switches in the xenograft over time. Furthermore, to delineate the fate of the transplanted cells, we performed a ligand-receptor interaction analysis. Intriguing, we identified the midkine protein as a possible secreted human paracrine factor that enhances angiogenesis in the graft. In conclusion, we have shown the successful engraftment and maturation of human CVPs toward CMs using spatial transcriptomic technology. These data will be used to understand other aspects of regenerative cardiology such as the mechanism of VTs and in vivo cellular repair mechanisms. A shiny app atlas has been generated to share our transcriptomics data for all to explore gene expression of human transplanted cells in myocardial infarcted pig hearts.

A Scaled Proteomic Discovery Study for Prostate Cancer Diagnostic Markers Using ProteographTM and Trapped Ion Mobility Mass Spectrometry.

There is a significant unmet need for clinical reflex tests that increase the specificity of prostate-specific antigen blood testing, the longstanding but imperfect tool for prostate cancer diagnosis. Towards this endpoint, we present the results from a discovery study that identifies new prostate-specific antigen reflex markers in a large-scale patient serum cohort using differentiating technologies for deep proteomic interrogation. We detect known prostate cancer blood markers as well as novel candidates. Through bioinformatic pathway enrichment and network analysis, we reveal associations of differentially abundant proteins with cytoskeletal, metabolic, and ribosomal activities, all of which have been previously associated with prostate cancer progression. Additionally, optimized machine learning classifier analysis reveals proteomic signatures capable of detecting the disease prior to biopsy, performing on par with an accepted clinical risk calculator benchmark.

The Beak of Eukaryotic Ribosomes: Life, Work and Miracles.

Ribosomes are not totally globular machines. Instead, they comprise prominent structural protrusions and a myriad of tentacle-like projections, which are frequently made up of ribosomal RNA expansion segments and N- or C-terminal extensions of ribosomal proteins. This is more evident in higher eukaryotic ribosomes. One of the most characteristic protrusions, present in small ribosomal subunits in all three domains of life, is the so-called beak, which is relevant for the function and regulation of the ribosome's activities. During evolution, the beak has transitioned from an all ribosomal RNA structure (helix h33 in 16S rRNA) in bacteria, to an arrangement formed by three ribosomal proteins, eS10, eS12 and eS31, and a smaller h33 ribosomal RNA in eukaryotes. In this review, we describe the different structural and functional properties of the eukaryotic beak. We discuss the state-of-the-art concerning its composition and functional significance, including other processes apparently not related to translation, and the dynamics of its assembly in yeast and human cells. Moreover, we outline the current view about the relevance of the beak's components in human diseases, especially in ribosomopathies and cancer.

The global landscapes of lysine crotonylation in pseudorabies virus infection

Lysine crotonylation is a common occurrence in eukaryotic cells, regulating various physiological functions, including chromatin remodeling, cellular growth, and development. However, its involvement in viral infections has rarely been documented. In this study, we reveal that pseudorabies virus (PRV) infection significantly alters the global lysine crotonylation levels in porcine kidney PK-15 cells. Specifically, we identified a few viral proteins, including UL54, gM, gD, UL19, UL37, and UL46, which undergo crotonylation modification. Our observations indicate that at 20 h post-infection (hpi), 551 crotonylation sites were reduced across 345 proteins, while 47 new sites emerged in 37 proteins compared to the control group. By 40 hpi, 263 sites had decreased in 190 proteins, while 389 new sites appeared in 240 proteins. Deeper analysis revealed that the proteins with altered crotonylation levels were primarily involved in binding, catalytic activity, biosynthetic processes, ribosome activity, and metabolic processes. Additionally, our findings underscored the significance of ribosomes and the endoplasmic reticulum (ER), which were enriched with proteins exhibiting altered crotonylation. Overall, our study for the first time offers new insights into the relationship between crotonylation and herpes virus infection, paving the way for future investigations into the role of crotonylation in viral infections.

Modulation of ribosomal subunit associations by eIF6 is critical for mitotic exit and cancer progression.

Moderating the pool of active ribosomal subunits is critical for maintaining global translation rates. A factor crucial for modulating the 60S ribosomal subunits is eukaryotic translation initiation factor 6. Release of eIF6 from 60S is essential to permit 60S interactions with 40S. Here, using the N106S mutant of eIF6, we show that disrupting eIF6 interaction with 60S leads to an increase in vacant 80S. It further highlights a dichotomy in the anti-association activity of eIF6 that is distinct from its role in 60S biogenesis and shows that the nucleolar localization of eIF6 is not dependent on uL14-BCCIP interactions. Limiting active ribosomal pools markedly deregulates translation especially in mitosis and leads to chromosome segregation defects, mitotic exit delays and mitotic catastrophe. Ribo-Seq analysis of the eIF6-N106S mutant shows a significant downregulation in the translation efficiencies of mitotic factors and specifically transcripts with long 3'UTRs. eIF6-N106S mutation also limits cancer invasion, and this role is correlated with the overexpression of eIF6 only in high-grade invasive cancers suggesting that deregulation of eIF6 is probably not an early event in cancers. Thus, this study highlights the segregation of eIF6 functions and its role in moderating 80S availability for mitotic translation and cancer progression.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

Diversity of ribosomes at the level of rRNA variation associated with human health and disease.

Ribosomal DNA and RNA (rDNA and rRNA) sequences are usually discarded from sequencing analyses. But with hundreds of copies of rDNA genes it is unknown whether they possess sequence variations that form different types of ribosomes that affect human physiology and disease. Here, we developed an algorithm for variant-calling between paralog genes (termed RGA) and compared rDNA variations found in short- and long-read sequencing data from the 1,000 Genomes Project (1KGP) and Genome In A Bottle (GIAB). We additionally developed a novel protocol for long-read sequencing full-length rRNA (RIBO-RT) from actively translating ribosomes. Our analyses identified hundreds of rDNA variants, most of which, surprisingly, are short insertion-deletions (indels) and dozens of highly abundant rRNA variants that are incorporated into translationally active ribosomes. To visualize variant ribosomes at the single cell level, we developed an in-situ rRNA sequencing method (SWITCH-seq) which revealed that variants are co-expressed within individual cells. Strikingly, by analyzing rDNA, we found that variants assemble into distinct ribosome subtypes. We discovered that these subtypes acquire different rRNA structures by successfully employing dimethyl sulfate (DMS) probing of full length rRNA. With this atlas we investigated rRNA variation changes across human tissues and cancer types. This revealed tissue-specific rRNA subtype expression in endoderm/ectoderm-derived tissues. In cancer, low abundant rRNA variants can become highly expressed, which suggests the presence of cancer-specific ribosomes. Together, this study identifies and comprehensively characterizes the diversity of ribosomes at the level of rRNA variants which is dominated by indel variants, their chromosomal location and unique structure as well as the association of ribosome variation with tissue-specific biology and cancer.

Sulfur Dioxide Alleviates Organ Damage and Inflammatory Response in Cecal Ligation and Puncture-Induced Sepsis Rat.

The study aimed to elucidate the mechanisms by which sulfur dioxide (SO2) alleviates organ damage during sepsis using RNA-Seq technology. A cecal ligation and puncture (CLP) sepsis model was established in rats, and the effects of SO2 treatment on organ damage were assessed through histopathological examinations. RNA-Seq was performed to analyze differentially expressed genes (DEGs), and subsequent functional annotations and enrichment analyses were conducted. The CLP model successfully induced sepsis symptoms in rats. Histopathological evaluation revealed that SO2 treatment considerably reduced tissue damage across the heart, kidney, liver, and lungs. RNA-Seq identified 950 DEGs between treated and untreated groups, with significant enrichment in genes associated with ribosomal and translational activities, amino acid metabolism, and PI3K-Akt signaling. Furthermore, gene set enrichment analysis (GSEA) showcased enrichments in pathways related to transcriptional regulation, cellular migration, proliferation, and calcium-ion binding. In conclusion, SO2 effectively mitigates multi-organ damage induced by CLP sepsis, potentially through modulating gene expression patterns related to critical biological processes and signaling pathways. These findings highlight the therapeutic promise of SO2 in managing sepsis-induced organ damage.

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling.

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general activity-based arginylation profiling (ABAP) platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. ABAP has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 μg sample input, with 229 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

Adaptation of a fluoroquinolone-sensitive Shigella sonnei to norfloxacin exposure.

Shigella causes shigellosis that requires antibiotic treatment in severe cases. Sublethal antibiotic concentrations can promote resistance, but their effect on antibiotic-sensitive bacteria before resistance development is unclear. This study investigated the effects of sublethal norfloxacin (NOR) challenges on a NOR-sensitive strain, Shigella sonnei UKMCC1015. Firstly, the whole genome of S. sonnei UKMCC1015 was assembled, and 45 antimicrobial resistance (AMR) genes were identified. Interestingly, transcriptomic analysis showed that low NOR levels do not change either the expression of the AMR genes or NOR targets such as gyrA. Instead, multiple ribosomal protein genes were downregulated, which could be attributed to decreased ribosomal protein promoter activity, modulated by elevated guanosine pentaphosphate and tetraphosphate (ppGpp) levels. This alarmone is involved in the bacterial stringent response during environmental stress, and it is mainly produced from the ppGpp synthetase (relA). Additionally, we observed that a relA overexpression (prolonged period of elevated ppGpp levels) may negatively affect the NOR tolerance of the bacteria. In conclusion, this study revealed that a NOR-sensitive strain responds differently to sublethal NOR than commonly reported in resistant strains.

Ribosomal Dysregulation in Metastatic Laryngeal Squamous Cell Carcinoma: Proteomic Insights and CX-5461's Therapeutic Promise.

One of the main barriers to the successful treatment of laryngeal squamous cell carcinoma (LSCC) is postoperative progression, primarily due to tumor cell metastasis. To systematically investigate the molecular characteristics and potential mechanisms underlying the metastasis in laryngeal cancer, we carried out a TMT-based proteomic analysis of both cancerous and adjacent non-cancerous tissues from 10 LSCC patients with lymph node metastasis (LNM) and 10 without. A total of 5545 proteins were quantified across all samples. We identified 57 proteins that were downregulated in LSCC with LNM, which were enriched in cell adhesion pathways, and 69 upregulated proteins predominantly enriched in protein production pathways. Importantly, our data revealed a strong correlation between increased ribosomal activity and the presence of LNM, as 18 ribosomal subunit proteins were found to be upregulated, with RPS10 and RPL24 being the most significantly overexpressed. The potential of ribosomal proteins, including RPS10 and RPL24, as biomarkers for LSCC with LNM was confirmed in external validation samples (six with LNM and six without LNM) using Western blotting and immunohistochemistry. Furthermore, we have confirmed that the RNA polymerase I inhibitor CX-5461, which impedes ribosome biogenesis in LSCC, also decreases the expression of RPS10, RPL24, and RPS26. In vitro experiments have revealed that CX-5461 moderately reduces cell viability, while it significantly inhibits the invasion and migration of LSCC cells. It can enhance the expression of the epithelial marker CDH1 and suppress the expression of the mesenchymal markers CDH2, VIM, and FN at a dose that does not affect cell viability. Our study broadens the scope of the proteomic data on laryngeal cancer and suggests that ribosome targeting could be a supplementary therapeutic strategy for metastatic LSCC.

Loss of Conserved rRNA Modifications in the Peptidyl Transferase Center Leads to Diminished Protein Synthesis and Cell Growth in Budding Yeast.

Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.

Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of Rubisco. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.