Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

Polysome-bound mRNAs and translational mechanisms regulate drought tolerance in rice.

Plants evolved several acquired tolerance traits for drought stress adaptation to maintain the cellular homeostasis. Drought stress at the anthesis stage in rice affects productivity due to the inefficiency of protein synthesis machinery. The effect of translational mechanisms on different pathways involved in cellular tolerance plays an important role. We report differential responses of translation-associated mechanisms in rice using polysome bound mRNA sequencing at anthesis stage drought stress in resistant Apo and sensitive IR64 genotypes. Apo maintained higher polysomes with 60 S-to-40 S and polysome-to-monosome ratios which directly correlate with protein levels under stress. IR64 has less protein levels under stress due to defective translation machinery and reduced water potential. Many polysome-bound long non-coding RNAs (lncRNA) were identified in both genotypes under drought, influencing translation. Apo had higher levels of N6-Methyladenosine (m6A) mRNA modifications that contributed for sustained translation. Translation machinery in Apo could maintain higher levels of photosynthetic machinery-associated proteins in drought stress, which maintain gas exchange, photosynthesis and yield under stress. The protein stability and ribosome biogenesis mechanisms favoured improved translation in Apo. The phytohormone signalling and transcriptional responses were severely affected in IR64. Our results demonstrate that, the higher translation ability of Apo favours maintenance of photosynthesis and physiological responses that are required for drought stress adaptation.

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells.

Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.

Unveiling Polysomal Long Non-Coding RNA Expression on the First Day of Adipogenesis and Osteogenesis in Human Adipose-Derived Stem Cells.

Understanding the intricate molecular mechanisms governing the fate of human adipose-derived stem cells (hASCs) is essential for elucidating the delicate balance between adipogenic and osteogenic differentiation in both healthy and pathological conditions. Long non-coding RNAs (lncRNAs) have emerged as key regulators involved in lineage commitment and differentiation of stem cells, operating at various levels of gene regulation, including transcriptional, post-transcriptional, and post-translational processes. To gain deeper insights into the role of lncRNAs' in hASCs' differentiation, we conducted a comprehensive analysis of the lncRNA transcriptome (RNA-seq) and translatome (polysomal-RNA-seq) during a 24 h period of adipogenesis and osteogenesis. Our findings revealed distinct expression patterns between the transcriptome and translatome during both differentiation processes, highlighting 90 lncRNAs that are exclusively regulated in the polysomal fraction. These findings underscore the significance of investigating lncRNAs associated with ribosomes, considering their unique expression patterns and potential mechanisms of action, such as translational regulation and potential coding capacity for microproteins. Additionally, we identified specific lncRNA gene expression programs associated with adipogenesis and osteogenesis during the early stages of cell differentiation. By shedding light on the expression and potential functions of these polysome-associated lncRNAs, we aim to deepen our understanding of their involvement in the regulation of adipogenic and osteogenic differentiation, ultimately paving the way for novel therapeutic strategies and insights into regenerative medicine.

Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.

Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.

Human cytomegalovirus modulates mTORC1 to redirect mRNA translation within quiescently infected monocytes.

Human cytomegalovirus (HCMV) utilizes peripheral blood monocytes as a means to systemically disseminate throughout the host. Following viral entry, HCMV stimulates non-canonical Akt signaling leading to the activation of mTORC1 and the subsequent translation of select antiapoptotic proteins within infected monocytes. However, the full extent to which the HCMV-initiated Akt/mTORC1 signaling axis reshapes the monocyte translatome is unclear. We found HCMV entry alone was able to stimulate widescale changes to mRNA translation levels and that inhibition of mTOR, a component of mTORC1, dramatically attenuated HCMV-induced protein synthesis. Although monocytes treated with normal myeloid growth factors also exhibited increased levels of translation, mTOR inhibition had no effect, suggesting HCMV activation of mTOR stimulates the acquisition of a unique translatome within infected monocytes. Indeed, polyribosomal profiling of HCMV-infected monocytes identified distinct prosurvival transcripts that were preferentially loaded with ribosomes when compared to growth factor-treated cells. Sirtuin 1 (SIRT1), a deacetylase that exerts prosurvival effects through regulation of the PI3K/Akt pathway, was found to be highly enriched following HCMV infection in an mTOR-dependent manner. Importantly, SIRT1 inhibition led to the death of HCMV-infected monocytes while having minimal effect on uninfected cells. SIRT1 also supported a positive feedback loop to sustain Akt/mTORC1 signaling following viral entry. Taken together, HCMV profoundly reshapes mRNA translation in an mTOR-dependent manner to enhance the synthesis of select factors necessary for the survival of infected monocytes.IMPORTANCEHuman cytomegalovirus (HCMV) infection is a significant cause of morbidity and mortality among the immunonaïve and immunocompromised. Peripheral blood monocytes are a major cell type responsible for disseminating the virus from the initial site of infection. In order for monocytes to mediate viral spread within the host, HCMV must subvert the naturally short lifespan of these cells. In this study, we performed polysomal profiling analysis, which demonstrated HCMV to globally redirect mRNA translation toward the synthesis of cellular prosurvival factors within infected monocytes. Specifically, HCMV entry into monocytes induced the translation of cellular SIRT1 to generate an antiapoptotic state. Defining the precise mechanisms through which HCMV stimulates survival will provide insight into novel anti-HCMV drugs able to target infected monocytes.

Profiling mitochondria-polyribosome lncRNAs associated with pluripotency.

Pluripotent stem cells (PSCs) provide unlimited resources for regenerative medicine because of their potential for self-renewal and differentiation into many different cell types. The pluripotency of these PSCs is dynamically regulated at multiple cellular organelle levels. To delineate the factors that coordinate this inter-organelle crosstalk, we profiled those long non-coding RNAs (lncRNAs) that may participate in the regulation of multiple cellular organelles in PSCs. We have developed a unique strand-specific RNA-seq dataset of lncRNAs that may interact with mitochondria (mtlncRNAs) and polyribosomes (prlncRNAs). Among the lncRNAs differentially expressed between induced pluripotent stem cells (iPSCs), fibroblasts, and positive control H9 human embryonic stem cells, we identified 11 prlncRNAs related to stem cell reprogramming and exit from pluripotency. In conjunction with the total RNA-seq data, this dataset provides a valuable resource to examine the role of lncRNAs in pluripotency, particularly for studies investigating the inter-organelle crosstalk network involved in germ cell development and human reproduction.

m6A modification negatively regulates translation by switching mRNA from polysome to P-body via IGF2BP3.

In the cytoplasm, mRNAs are dynamically partitioned into translating and non-translating pools, but the mechanism for this regulation has largely remained elusive. Here, we report that m6A regulates mRNA partitioning between polysome and P-body where a pool of non-translating mRNAs resides. By quantifying the m6A level of polysomal and cytoplasmic mRNAs with m6A-LAIC-seq and m6A-LC-MS/MS in HeLa cells, we observed that polysome-associated mRNAs are hypo-m6A-methylated, whereas those enriched in P-body are hyper-m6A-methylated. Downregulation of the m6A writer METTL14 enhances translation by switching originally hyper-m6A-modified mRNAs from P-body to polysome. Conversely, by proteomic analysis, we identify a specific m6A reader IGF2BP3 enriched in P-body, and via knockdown and molecular tethering assays, we demonstrate that IGF2BP3 is both necessary and sufficient to switch target mRNAs from polysome to P-body. These findings suggest a model for the dynamic regulation of mRNA partitioning between the translating and non-translating pools in an m6A-dependent manner.

Extensive Angular Sampling Enables the Sensitive Localization of Macromolecules in Electron Tomograms.

Cryo-electron tomography provides 3D images of macromolecules in their cellular context. To detect macromolecules in tomograms, template matching (TM) is often used, which uses 3D models that are often reliable for substantial parts of the macromolecules. However, the extent of rotational searches in particle detection has not been investigated due to computational limitations. Here, we provide a GPU implementation of TM as part of the PyTOM software package, which drastically speeds up the orientational search and allows for sampling beyond the Crowther criterion within a feasible timeframe. We quantify the improvements in sensitivity and false-discovery rate for the examples of ribosome identification and detection. Sampling at the Crowther criterion, which was effectively impossible with CPU implementations due to the extensive computation times, allows for automated extraction with high sensitivity. Consequently, we also show that an extensive angular sample renders 3D TM sensitive to the local alignment of tilt series and damage induced by focused ion beam milling. With this new release of PyTOM, we focused on integration with other software packages that support more refined subtomogram-averaging workflows. The automated classification of ribosomes by TM with appropriate angular sampling on locally corrected tomograms has a sufficiently low false-discovery rate, allowing for it to be directly used for high-resolution averaging and adequate sensitivity to reveal polysome organization.

Celf4 controls mRNA translation underlying synaptic development in the prenatal mammalian neocortex.

Abnormalities in neocortical and synaptic development are linked to neurodevelopmental disorders. However, the molecular and cellular mechanisms governing initial synapse formation in the prenatal neocortex remain poorly understood. Using polysome profiling coupled with snRNAseq on human cortical samples at various fetal phases, we identify human mRNAs, including those encoding synaptic proteins, with finely controlled translation in distinct cell populations of developing frontal neocortices. Examination of murine and human neocortex reveals that the RNA binding protein and translational regulator, CELF4, is expressed in compartments enriched in initial synaptogenesis: the marginal zone and the subplate. We also find that Celf4/CELF4-target mRNAs are encoded by risk genes for adverse neurodevelopmental outcomes translating into synaptic proteins. Surprisingly, deleting Celf4 in the forebrain disrupts the balance of subplate synapses in a sex-specific fashion. This highlights the significance of RNA binding proteins and mRNA translation in evolutionarily advanced synaptic development, potentially contributing to sex differences.

Translational control by Trypanosoma brucei DRBD18 contributes to the maintenance of the procyclic state.

Trypanosoma brucei occupies distinct niches throughout its life cycle, within both the mammalian and tsetse fly hosts. The immunological and biochemical complexity and variability of each of these environments require a reshaping of the protein landscape of the parasite both to evade surveillance and face changing metabolic demands. In kinetoplastid protozoa, including T. brucei, posttranscriptional control mechanisms are the primary means of gene regulation, and these are often mediated by RNA-binding proteins. DRBD18 is a T. brucei RNA-binding protein that reportedly interacts with ribosomal proteins and translation factors. Here, we tested a role for DRBD18 in translational control. We validate the DRBD18 interaction with translating ribosomes and the translation initiation factor, eIF3a. We further show that DRBD18 depletion by RNA interference leads to altered polysomal profiles with a specific depletion of heavy polysomes. Ribosome profiling analysis reveals that 101 transcripts change in translational efficiency (TE) upon DRBD18 depletion: 41 exhibit decreased TE and 60 exhibit increased TE. A further 66 transcripts are buffered, that is, changes in transcript abundance are compensated by changes in TE such that the total translational output is expected not to change. In DRBD18-depleted cells, a set of transcripts that codes for procyclic form-specific proteins is translationally repressed while, conversely, transcripts that code for bloodstream form- and metacyclic form-specific proteins are translationally enhanced. RNA immunoprecipitation/qRT-PCR indicates that DRBD18 associates with members of both repressed and enhanced cohorts. These data suggest that DRBD18 contributes to the maintenance of the procyclic state through both positive and negative translational control of specific mRNAs.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h-1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Evolutionarily divergent mTOR remodels translatome for tissue regeneration.

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation.

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

The translational landscape of bread wheat during grain development.

The dynamics of gene expression in crop grains has typically been investigated at the transcriptional level. However, this approach neglects translational regulation, a widespread mechanism that rapidly modulates gene expression to increase the plasticity of organisms. Here, we performed ribosome profiling and polysome profiling to obtain a comprehensive translatome data set of developing bread wheat (Triticum aestivum) grains. We further investigated the genome-wide translational dynamics during grain development, revealing that the translation of many functional genes is modulated in a stage-specific manner. The unbalanced translation between subgenomes is pervasive, which increases the expression flexibility of allohexaploid wheat. In addition, we uncovered widespread previously unannotated translation events, including upstream open reading frames (uORFs), downstream open reading frames (dORFs), and open reading frames (ORFs) in long noncoding RNAs, and characterized the temporal expression dynamics of small ORFs. We demonstrated that uORFs act as cis-regulatory elements that can repress or even enhance the translation of mRNAs. Gene translation may be combinatorially modulated by uORFs, dORFs, and microRNAs. In summary, our study presents a translatomic resource that provides a comprehensive and detailed overview of the translational regulation in developing bread wheat grains. This resource will facilitate future crop improvements for optimal yield and quality.

Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes.

Regulation of mRNA translation in astrocytes gains a growing interest. However, until now, successful ribosome profiling of primary astrocytes has not been reported. Here, we optimized the standard 'polysome profiling' method and generated an effective protocol for polyribosome extraction, which enabled genome-wide assessment of mRNA translation dynamics along the process of astrocyte activation. Transcriptome (RNAseq) and translatome (Riboseq) data generated at 0, 24 and 48 h after cytokines treatment, revealed dynamic genome-wide changes in the expression level of ∼12 000 genes. The data clarify whether a change in protein synthesis rate results from a change in mRNA level or translation efficiency per se. It exhibit different expression strategies, based on changes in mRNA abundance and/or translation efficiency, which are specifically assigned to gene subsets depending on their function. Moreover, the study raises an important take-home message related to the possible presence of 'difficult to extract' polyribosome sub-groups, in all cell types, thus illuminating the impact of ribosomes extraction methodology on experiments addressing translation regulation.

Nanopore Direct RNA Sequencing of Monosome- and Polysome-Bound RNA.

Polysome fractionation makes use of density gradients and ultracentrifugation to separate transcripts based on their specific number of bound ribosomes, and can be combined with downstream analysis such as cDNA-seq (commonly known as RNA-seq), microarray analysis, RT-qPCR, or Northern blotting. Here, we describe the application of Nanopore direct RNA sequencing to quantify monosome- and polysome-bound full-length transcripts after polysome fractionation, RNA cleanup, and size selection, using the yeast glucose stress response as an example use case.

Ribosomes in RNA Granules Are Stalled on mRNA Sequences That Are Consensus Sites for FMRP Association.

Local translation in neurons is partly mediated by the reactivation of stalled polysomes. Stalled polysomes may be enriched within the granule fraction, defined as the pellet of sucrose gradients used to separate polysomes from monosomes. The mechanism of how elongating ribosomes are reversibly stalled and unstalled on mRNAs is still unclear. In the present study, we characterize the ribosomes in the granule fraction using immunoblotting, cryogenic electron microscopy (cryo-EM), and ribosome profiling. We find that this fraction, isolated from 5-d-old rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homologue. Cryo-EM analysis of ribosomes in this fraction indicates they are stalled, mainly in the hybrid state. Ribosome profiling of this fraction reveals (1) an enrichment for footprint reads of mRNAs that interact with FMRPs and are associated with stalled polysomes, (2) an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development, and (3) increased ribosome occupancy on mRNAs encoding RNA binding proteins. Compared with those usually found in ribosome profiling studies, the footprint reads were longer and were mapped to reproducible peaks in the mRNAs. These peaks were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the granule fraction to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall ribosomes during translation elongation in neurons.SIGNIFICANCE STATEMENT Neurons send mRNAs to synapses in RNA granules, where they are not translated until an appropriate stimulus is given. Here, we characterize a granule fraction obtained from sucrose gradients and show that polysomes in this fraction are stalled on consensus sequences in a specific state of translational arrest with extended ribosome-protected fragments. This finding greatly increases our understanding of how neurons use specialized mechanisms to regulate translation and suggests that many studies on neuronal translation may need to be re-evaluated to include the large fraction of neuronal polysomes found in the pellet of sucrose gradients used to isolate polysomes.

Polyribosomes of circular topology are prevalent in mammalian cells.

Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been shown to possess various spatial conformations, including a ring-shaped configuration which was considered to be functionally important. However, a recent in situ cryo-ET analysis of predominant regular inter-ribosome contacts did not confirm the abundance of ring-shaped polyribosomes in a cell cytoplasm. To address this discrepancy, here we analyzed the cryo-ET structure of polyribosomes in diluted lysates of HeLa cells. It was shown that the vast majority of the ribosomes were combined into polysomes and were proven to be translationally active. Tomogram analysis revealed that circular polyribosomes are indeed very common in the cytoplasm, but they mostly possess pseudo-regular structures without specific inter-ribosomal contacts. Although the size of polyribosomes varied widely, most circular polysomes were relatively small in size (4-8 ribosomes). Our results confirm the recent data that it is cellular mRNAs with short ORF that most commonly form circular structures providing an enhancement of translation.

Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and RPS2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.