Ribosome Density Research Articles

Ultrastructural insights into cellular organization, energy storage and ribosomal dynamics of an ammonia-oxidizing archaeon from oligotrophic oceans.

Nitrososphaeria, formerly known as Thaumarchaeota, constitute a diverse and widespread group of ammonia-oxidizing archaea (AOA) inhabiting ubiquitously in marine and terrestrial environments, playing a pivotal role in global nitrogen cycling. Despite their importance in Earth's ecosystems, the cellular organization of AOA remains largely unexplored, leading to a significant unanswered question of how the machinery of these organisms underpins metabolic functions. In this study, we combined spherical-chromatic-aberration-corrected cryo-electron tomography (cryo-ET), scanning transmission electron microscopy (STEM), and energy dispersive X-ray spectroscopy (EDS) to unveil the cellular organization and elemental composition of Nitrosopumilus maritimus SCM1, a representative member of marine Nitrososphaeria. Our tomograms show the native ultrastructural morphology of SCM1 and one to several dense storage granules in the cytoplasm. STEM-EDS analysis identifies two types of storage granules: one type is possibly composed of polyphosphate and the other polyhydroxyalkanoate. With precise measurements using cryo-ET, we observed low quantity and density of ribosomes in SCM1 cells, which are in alignment with the documented slow growth of AOA in laboratory cultures. Collectively, these findings provide visual evidence supporting the resilience of AOA in the vast oligotrophic marine environment.

Translation-dependent and -independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T cell activation.

mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality-control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions, and the recruitment of specific proteins to ribosomes is an important determinant of mRNA turnover. However, the extent to which translation-dependent mRNA decay (TDD) and translation-independent mRNA decay (TID) pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes, and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3- and AU3-rich codons. Altogether, our data show a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.

Ribosome profiling reveals downregulation of UMP biosynthesis as the major early response to phage infection.

The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Riboformer: a deep learning framework for predicting context-dependent translation dynamics

Translation elongation is essential for maintaining cellular proteostasis, and alterations in the translational landscape are associated with a range of diseases. Ribosome profiling allows detailed measurements of translation at the genome scale. However, it remains unclear how to disentangle biological variations from technical artifacts in these data and identify sequence determinants of translation dysregulation. Here we present Riboformer, a deep learning-based framework for modeling context-dependent changes in translation dynamics. Riboformer leverages the transformer architecture to accurately predict ribosome densities at codon resolution. When trained on an unbiased dataset, Riboformer corrects experimental artifacts in previously unseen datasets, which reveals subtle differences in synonymous codon translation and uncovers a bottleneck in translation elongation. Further, we show that Riboformer can be combined with in silico mutagenesis to identify sequence motifs that contribute to ribosome stalling across various biological contexts, including aging and viral infection. Our tool offers a context-aware and interpretable approach for standardizing ribosome profiling datasets and elucidating the regulatory basis of translation kinetics.

Physical modeling of ribosomes along messenger RNA: Estimating kinetic parameters from ribosome profiling experiments using a ballistic model.

Gene expression is the synthesis of proteins from the information encoded on DNA. One of the two main steps of gene expression is the translation of messenger RNA (mRNA) into polypeptide sequences of amino acids. Here, by taking into account mRNA degradation, we model the motion of ribosomes along mRNA with a ballistic model where particles advance along a filament without excluded volume interactions. Unidirectional models of transport have previously been used to fit the average density of ribosomes obtained by the experimental ribo-sequencing (Ribo-seq) technique in order to obtain the kinetic rates. The degradation rate is not, however, accounted for and experimental data from different experiments are needed to have enough parameters for the fit. Here, we propose an entirely novel experimental setup and theoretical framework consisting in splitting the mRNAs into categories depending on the number of ribosomes from one to four. We solve analytically the ballistic model for a fixed number of ribosomes per mRNA, study the different regimes of degradation, and propose a criterion for the quality of the inverse fit. The proposed method provides a high sensitivity to the mRNA degradation rate. The additional equations coming from using the monosome (single ribosome) and polysome (arbitrary number) ribo-seq profiles enable us to determine all the kinetic rates in terms of the experimentally accessible mRNA degradation rate.

Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

Exclusion model of mRNA translation with collision-induced ribosome drop-off

The translation of messenger RNA transcripts to proteins is commonly modeled as a one-dimensional totally asymmetric exclusion process with extended particles. Here we focus on the effects of premature termination of translation through the irreversible detachment of ribosomes. We consider a model where the detachment is induced by the unsuccessful attempt to move to an occupied site. The model is exactly solvable in a simplified geometry consisting of the translation initiation region followed by a single slow site representing a translation bottleneck. In agreement with recent experimental and computational studies we find a non-monotonic dependence of the ribosome current on the initiation rate, but only if the leading particle in a colliding pair detaches. Simulations show that the effect persists for larger lattices and extended bottlenecks. In the homogeneous system the ribosome density decays asymptotically as the inverse square root of the distance to the initiation site.

Influence of Extreme Factors of High Mountains on the Ultrastructure of Rat Blood Leukocytes

This paper presents the results of a study on the ultrastructure of white blood cells under the influence of high-altitude factors in combination with cold. Materials and methods: 70 outbred male laboratory rats weighing 200-300 g; method of electron microscopic examination, statistical methods. Research results. It has been established that in rats, during adaptation to high altitude conditions, in combination with exposure to cold, there are sharp changes in the ultrastructure of leukocytes. In experimental animals on the 3rd day of observation, the level of volumetric density of mitochondria increased almost 2 times in neutrophils, 1.5 times in eosinophils, and 1.4 times in monocytes and medium lymphocytes. With an increase in the length of stay in the highlands in combination with cold exposure, the volumetric density of mitochondria continued to increase in all types of white blood cells, and on the 30th day of adaptation it remained statistically significantly higher both in comparison with the control and in comparison, with the indices of the animal group on the 3rd and day of adaptation. The volumetric density of ribosomes also increased statistically significantly on the 3rd day of adaptation to high mountains in combination with cold in neutrophils, eosinophils, and lymphocytes, and continued to increase by the 30th day of observation. A trend towards an increase in bulk density was also noted in cell vacuoles: on the 3rd day of adaptation, an increase in the volumetric density of vacuoles was recorded compared to the control in neutrophils. By the 30th day of adaptation, the bulk density of vacuoles, compared with animals on the 3rd day of adaptation, statistically significantly decreased, although it did not reach the indicators of animals in the control group. The bulk density of specific granites on the 3rd day of stay in the mountains in neutrophils and eosinophils decreased by almost 100%.

Structural and Functional State of Leukocytes During Long-term Adaptation of Experimental Animals to the Climatic and Geographical Conditions of High Mountains

This study presents the results of studies of the structural and functional state of leukocytes during long-term adaptation of experimental animals to high altitude conditions, obtained using the electron microscopic method of morphometry. It has been shown that under the influence of extreme factors of high altitude in the process of adaptation in experimental animals there is a statistically significant increase in the volume density of mitochondria and ribosomes in neutrophils, lymphocytes, eosinophils, monocytes, which can be regarded as an indicator of activation of the synthesis of nucleic acids and proteins, activation of the biosynthesis of mitochondrial structures and increase energy production in white blood cells. The volumetric value of specific granules also increased. The volume density of vacuoles in all white blood cells by the 30th day of stay at altitude was statistically significantly reduced, however, in neutrophils, eosinophils and lymphocytes, this indicator did not reach the values of animals in the control group. Thus, adaptation to high mountains has a certain training effect, the state of white blood cells under these conditions is a kind of marker of adaptive processes occurring in the body during the stay of humans and animals in high mountains.

Distal nucleotides affect the rate of stop codon read‐through

BackgroundA key step in gene expression is the recognition of the stop codon to terminate translation at the correct position. However, it has been observed that ribosomes can misinterpret the stop codon and continue the translation in the 3′UTR region. This phenomenon is called stop codon read‐through (SCR). It has been suggested that these events would occur on a programmed basis, but the underlying mechanisms are still not well understood.MethodsHere, we present a strategy for the comprehensive identification of SCR events in the Drosophila melanogaster transcriptome by evaluating the ribosomal density profiles. The associated ribosomal leak rate was estimated for every event identified. A statistical characterization of the frequency of nucleotide use in the proximal region to the stop codon in the sequences associated to SCR events was performed.ResultsThe results show that the nucleotide usage pattern in transcripts with the UGA codon is different from the pattern for those transcripts ending in the UAA codon, suggesting the existence of at least two mechanisms that could alter the translational termination process. Furthermore, a linear regression models for each of the three stop codons was developed, and we show that the models using the nucleotides at informative positions outperforms those models that consider the entire sequence context to the stop codon.ConclusionsWe report that distal nucleotides can affect the SCR rate in a stop‐codon dependent manner.

Optimization of ribosome utilization in Saccharomyces cerevisiae.

Resource optimization in protein synthesis is often looked at from the perspective of translation efficiency-the rate at which proteins are synthesized from a single transcript. The higher the rate of protein synthesis, the more efficiently a transcript is translated. However, the production of a ribosome consumes significantly more cellular resources than an mRNA molecule. Therefore, there should be a stronger selection pressure for optimizing ribosome usage than translation efficiency. This paper reports strong evidence of such optimization which becomes more prominent in highly expressed transcripts that consume a significant amount of cellular resources. The ribosome usage is optimized by the biases in codon usage and translation initiation rates. This optimization significantly reduces the ribosome requirement in Saccharomyces cerevisiae. We also find that a low ribosome density on mRNA transcripts helps optimize ribosome utilization. Therefore, protein synthesis occurs in a low ribosome density regime where translation-initiation is the rate-limiting step. Our results suggest that optimizing ribosome usage is one of the major forces shaping evolutionary selection pressure, and thus provide a new perspective to resource optimization in protein synthesis.

Modeling mRNA Translation With Ribosome Abortions.

We derive a deterministic mathematical model for the flow of ribosomes along a mRNA called the ribosome flow model with extended objects and abortions (RFMEOA). This model incorporates important cellular features such as every ribosome covers several codons and they may detach from various regions along the track due to more realistic biological situations including phenomena of ribosome-ribosome collisions. We prove that the ribosome density profile along the mRNA in the RFMEOA and in particular, the protein production rate converge to a unique steady-state. Simulations of the RFMEOA demonstrate a surprising result that an increase in the initiation rate may sometimes lead to a decrease in the production rate. We believe that this model could be helpful to provide insight into the effects of premature termination on the protein expression and be useful for understanding and re-engineering the translation process.

Translation kinetics and diffusive timescales regulate mitochondrial localization of mRNAs in yeast and mammalian cells.

Eukaryotes employ various regulatory mechanisms to maintain subcellular organization in dynamic environments. Localization of nuclear-encoded mitochondrial mRNAs attunes the nuclear genome to mitochondrial protein levels and metabolic needs. Targeting nuclear-encoded mitochondrial mRNAs to the mitochondrial surface is crucial for cotranslational import and proper folding of mitochondrial membrane proteins. Hundreds of mitochondrial mRNAs contain a Mitochondria Targeting Sequence (“MTS”) and preferentially localize to mitochondria using nascent peptide-mediated interactions with mitochondrial import machinery. Recent studies in brewer's yeast have illuminated MTS-mediated localization as a post-transcriptional mechanism of protein production, identifying two broad classes of mRNA localization patterns--constitutively localized or condition dependent. Our stochastic simulation incorporates gene-specific translation kinetics, MTS exposure and maturation, and diffusive search for mitochondria to explore and predict the localization patterns of mRNAs in brewer's yeast without the need for mRNA-specific binding partners. Our results point to the interplay of global and gene-specific mechanisms for tuning mRNA localization in response to changing metabolic conditions. Interestingly, conditional and constitutive localization patterns are largely conserved for homologous mRNAs from brewer's yeast to mammalian cells despite differences in cell size and gene-specific translation parameters. Notably, the conditional class is enriched for crucial TCA cycle genes, making these metabolic mRNAs sensitive to metabolic conditions in yeast and mammalian cells. Mammalian cell types also exhibit similar patterns in gene-specific kinetics for conditional and constitutive mRNAs. Both classes have similar ribosome densities whereas conditional mRNAs have significantly faster initiation and elongation rates. Given that patterns in mRNA localization and translation kinetics are conserved from brewer's yeast to mammals, we posit that the physical mechanism of mRNA localization is conserved as well.

Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles.

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.

Quinolinic Acid Induces Alterations in Neuronal Subcellular Compartments, Blocks Autophagy Flux and Activates Necroptosis and Apoptosis in Rat Striatum.

Quinolinic acid (QUIN) is an agonist of N-methyl-D-aspartate receptor (NMDAr) used to study the underlying mechanism of excitotoxicity in animal models. There is evidence indicating that impairment in autophagy at early times contributes to cellular damage in excitotoxicity; however, the status of autophagy in QUIN model on day 7 remains unexplored. In this study, the ultrastructural analysis of subcellular compartments and the status of autophagy, necroptosis, and apoptosis in the striatum of rats administered with QUIN (120nmol and 240nmol) was performed on day 7. QUIN induced circling behavior, neurodegeneration, and cellular damage; also, it promoted swollen mitochondrial crests, spherical-like morphology, and mitochondrial fragmentation; decreased ribosomal density in the rough endoplasmic reticulum; and altered the continuity of myelin sheaths in axons with separation of the compact lamellae. Furthermore, QUIN induced an increase and a decrease in ULK1 and p-70-S6K phosphorylation, respectively, suggesting autophagy activation; however, the increased microtubule-associated protein 1A/1B-light chain 3-II (LC3-II) and sequestosome-1/p62 (SQSTM1/p62), the coexistence of p62 and LC3 in the same structures, and the decrease in Beclin 1 and mature cathepsin D also indicates a blockage in autophagy flux. Additionally, QUIN administration increased tumor necrosis factor alpha (TNFα) and receptor-interacting protein kinase 3 (RIPK3) levels and its phosphorylation (p-RIPK3), as well as decreased B-cell lymphoma 2 (Bcl-2) and increased Bcl-2-associated X protein (Bax) levels and c-Jun N-terminal kinase (JNK) phosphorylation, suggesting an activation of necroptosis and apoptosis, respectively. These results suggest that QUIN activates the autophagy, but on day 7, it is blocked and organelle and cellular damage, neurodegeneration, and behavior alterations could be caused by necroptosis and apoptosis activation.

Identification and functional characterization of mRNAs that exhibit stop codon readthrough in Arabidopsis thaliana

Stop codon readthrough (SCR) is the process of continuation of translation beyond the stop codon, generating protein isoforms with C-terminal extensions. SCR has been observed in viruses, fungi, and multicellular organisms, including mammals. However, SCR is largely unexplored in plants. In this study, we have analyzed ribosome profiling datasets to identify mRNAs that exhibit SCR in Arabidopsis thaliana. Analyses of the ribosome density, ribosome coverage, and three-nucleotide periodicity of the ribosome profiling reads in the mRNA region downstream of the stop codon provided strong evidence for SCR in mRNAs of 144 genes. We show that SCR generated putative evolutionarily conserved nuclear localization signals, transmembrane helices, and intrinsically disordered regions in the C-terminal extensions of several of these proteins. Furthermore, gene ontology functional enrichment analysis revealed that these 144 genes belong to three major functional groups—translation, photosynthesis, and abiotic stress tolerance. Using a luminescence-based readthrough assay, we experimentally demonstrated SCR in representative mRNAs belonging to each of these functional classes. Finally, using microscopy, we show that the SCR product of one gene that contains a nuclear localization signal at the C-terminal extension, CURT1B, localizes to the nucleus as predicted. Based on these observations, we propose that SCR plays an important role in plant physiology by regulating protein localization and function.

Extrinsic noise effects on ribosomal traffic during the translation process

Any cellular process at the microscopic level is governed by both extrinsic and intrinsic noise (IN) and same is true for mRNA translation as well. Even though the effect of stochasticity via the IN is well studied for mRNA translation, same cannot be said about extrinsic noise (EN). In this article, we incorporate EN in a model of mRNA translation and carry out stochastic simulations of the same. We then evaluate various statistics related to the residence time of the ribosome and the ribosomal traffic on the mRNA and subsequent protein production. We also study the effect of slow codons and ribosome dropoff. From our simulations, we show that noise in the translation initiation rate rather than the translation termination rate acts to significantly broaden the distribution of ribosome residence times on the mRNA lattice. This EN also has an effect on ribosomal current, density and protein production. Further, the presence of slow codons acts to increase the mean residence times. However, this increase also depends on the number and position of the slow codons on the lattice. We also show that the slow codons act to mask any effect from the EN themselves. Our results, therefore, have implications towards a better understanding of the role the individual components play during the translation process.

Preferential translation of p53 target genes

ABSTRACT The transcription factor p53 exerts its tumour suppressive effect through transcriptional activation of numerous target genes controlling cell cycle arrest, apoptosis, cellular senescence and DNA repair. In addition, there is evidence that p53 influences the translation of specific mRNAs, including translational inhibition of ribosomal protein synthesis and translational activation of MDM2. A challenge in the analysis of translational control is that changes in mRNA abundance exert a kinetic (passive) effect on ribosome densities. In order to separate these passive effects from active regulation of translation efficiency in response to p53 activation, we conducted a comprehensive analysis of translational regulation by comparative analysis of mRNA levels and ribosome densities upon DNA damage induced by neocarzinostatin in wild-type and TP53−/− HCT116 colorectal carcinoma cells. Thereby, we identified a specific group of mRNAs that are preferentially translated in response to p53 activation, many of which correspond to p53 target genes including MDM2, SESN1 and CDKN1A. By subsequent polysome profile analysis of SESN1 and CDKN1A mRNA, we could demonstrate that p53-dependent translational activation relies on a combination of inducing the expression of translationally advantageous isoforms and trans-acting mechanisms that further enhance the translation of these mRNAs.

Beginners guide to ribosome profiling

Beginners guide to ribosome profiling

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data.

Identification of open reading frames (ORFs), especially those encoding small peptides and being actively translated under specific physiological contexts, is critical forcomprehensive annotations of context-dependent translatomes. Ribosome profiling, a technique for detecting the binding locations and densities of translating ribosomes on RNA, offers an avenue to rapidly discover where translation is occurring at the genome-wide scale. However, it is not a trivial task in bioinformatics to efficiently and comprehensively identify the translating ORFs for ribosome profiling. Described here is an easy-to-use package, named RiboCode, designed to search for actively translating ORFs of any size from distorted and ambiguous signals in ribosome profiling data. Taking our previously published dataset as an example, this article provides step-by-step instructions for the entire RiboCode pipeline, from preprocessing of the raw data to interpretation of the final output result files. Furthermore, for evaluating the translation rates of the annotated ORFs, procedures for visualization and quantification of ribosome densities on each ORF are also described in detail. In summary, the present article is a useful and timely instruction for the research fields related to translation, small ORFs, and peptides.