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The coordinated action of transcriptional and post-transcriptional machineries shapes gene expression programs at steady state and determines their concerted response to perturbations. We have developed Nanodynamo, an experimental and computational workflow for quantifying the kinetic rates of nuclear and cytoplasmic steps of the RNA life cycle. Nanodynamo is based on mathematical modelling following sequencing of native RNA from cellular fractions and polysomes. We have applied this workflow to triple-negative breast cancer cells, revealing widespread post-transcriptional RNA processing that is mutually exclusive with its co-transcriptional counterpart. We used Nanodynamo to unravel the coupling between transcription, processing, export, decay and translation machineries. We have identified a number of coupling interactions within and between the nucleus and cytoplasm that largely contribute to coordinating how cells respond to perturbations that affect gene expression programs. Nanodynamo will be instrumental in unravelling the determinants and regulatory processes involved in the coordination of gene expression responses., (© 2024. The Author(s).)

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction., (© 2024 Wiley Periodicals LLC.)

The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis., (© 2024 Roučová et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs., Competing Interests: Declaration of interests R.I. is a founder, board member, and/or shareholder of Cellforma, unrelated to the present work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Translational control is important in all life, but it remains a challenge to accurately quantify. When ribosomes translate messenger (m)RNA into proteins, they attach to the mRNA in series, forming poly(ribo)somes, and can co-localize. Here, we computationally model new types of co-localized ribosomal complexes on mRNA and identify them using enhanced translation complex profile sequencing (eTCP-seq) based on rapid in vivo crosslinking. We detect long disome footprints outside regions of non-random elongation stalls and show these are linked to translation initiation and protein biosynthesis rates. We subject footprints of disomes and other translation complexes to artificial intelligence (AI) analysis and construct a new, accurate and self-normalized measure of translation, termed stochastic translation efficiency (STE). We then apply STE to investigate rapid changes to mRNA translation in yeast undergoing glucose depletion. Importantly, we show that, well beyond tagging elongation stalls, footprints of co-localized ribosomes provide rich insight into translational mechanisms, polysome dynamics and topology. STE AI ranks cellular mRNAs by absolute translation rates under given conditions, can assist in identifying its control elements and will facilitate the development of next-generation synthetic biology designs and mRNA-based therapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

mRNA therapeutics are revolutionizing the pharmaceutical industry, but methods to optimize the primary sequence for increased expression are still lacking. Here, we design 5'UTRs for efficient mRNA translation using deep learning. We perform polysome profiling of fully or partially randomized 5'UTR libraries in three cell types and find that UTR performance is highly correlated across cell types. We train models on our datasets and use them to guide the design of high-performing 5'UTRs using gradient descent and generative neural networks. We experimentally test designed 5'UTRs with mRNA encoding megaTAL TM gene editing enzymes for two different gene targets and in two different cell lines. We find that the designed 5'UTRs support strong gene editing activity. Editing efficiency is correlated between cell types and gene targets, although the best performing UTR was specific to one cargo and cell type. Our results highlight the potential of model-based sequence design for mRNA therapeutics., (© 2024. The Author(s).)

A defining feature of the bacterial cytosolic interior is a distinct membrane-less organelle, the nucleoid, that contains the chromosomal DNA. Although increasing experimental evidence indicates that macromolecular crowding is the dominant mechanism for nucleoid formation, it has remained unclear which crowders control nucleoid volume. It is commonly assumed that polyribosomes play a dominant role, yet the volume fraction of soluble proteins in the cytosol is comparable with that of polyribosomes. Here, we develop a free energy-based model for the cytosolic interior of a bacterial cell to distinguish contributions arising from polyribosomes and cytosolic proteins in nucleoid volume control. The parameters of the model are determined from the existing experimental data. We show that, while the polysomes establish the existence of the nucleoid as a distinct phase, the proteins control the nucleoid volume in physiologically relevant conditions. Our model explains experimental findings in Escherichia coli that the nucleoid compaction curves in osmotic shock measurements do not depend on cell growth rate and that dissociation of polysomes in slow growth rates does not lead to significant nucleoid expansion, while the nucleoid phase disappears in fastest growth rates. Furthermore, the model predicts a cross-over in the exclusion of crowders by their linear dimensions from the nucleoid phase: below the cross-over of 30-50 nm, the concentration of crowders in the nucleoid phase decreases linearly as a function of the crowder diameter, while decreasing exponentially above the cross-over size. Our work points to the possibility that bacterial cells maintain nucleoid size and protein concentration homeostasis via feedback in which protein concentration controls nucleoid dimensions and the nucleoid dimensions control protein synthesis rate., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

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.

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 N 6 -Methyladenosine (m 6 A) 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., Competing Interests: Declaration of competing interest Authors declare no conflict of interests., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: The authors declare no conflict of interest.

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.

GEMIN5 is a multifunctional protein involved in various aspects of RNA biology, including biogenesis of snRNPs and translation control. Reduced levels of GEMIN5 confer a differential translation to selective groups of mRNAs, and biallelic variants reducing protein stability or inducing structural conformational changes are associated with neurological disorders. Here, we show that upregulation of GEMIN5 can be detrimental as it modifies the steady state of mRNAs and enhances alternative splicing (AS) events of genes involved in a broad range of cellular processes. RNA-Seq identification of the mRNAs associated with polysomes in cells with high levels of GEMIN5 revealed that a significant fraction of the differential AS events undergo translation. The association of mRNAs with polysomes was dependent on the type of AS event, being more frequent in the case of exon skipping. However, there were no major differences in the percentage of genes showing open-reading frame disruption. Importantly, differential AS events in mRNAs engaged in polysomes, eventually rendering non-functional proteins, encode factors controlling cell growth. The broad range of mRNAs comprising AS events engaged in polysomes upon GEMIN5 upregulation supports the notion that this multifunctional protein has evolved as a gene expression balancer, consistent with its dual role as a member of the SMN complex and as a modulator of protein synthesis, ultimately impinging on cell homoeostasis.

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., (© 2023. Springer Nature Limited.)

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot 1 . 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 p54 nrb and PSPC1 as well as nucleolin and RPS2, two p54 nrb -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., Competing Interests: AG, ER, FD, FH, FM, JA, FP, IA, EB, OB, CF, AH, PV, EL, FT, BG, AP No competing interests declared, (© 2022, Godet, Roussel et al.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [ 35 S]methionine labeling, and RiboPuromycylation. [ 35 S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that HBA (α-globin) and HBB (β-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and β-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells 1 . Here we use advances in cryo-electron tomography and sub-tomogram analysis 2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes 4 . By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells., (© 2022. The Author(s).)

Eukaryotic translation is a complex process that involves the interplay of various translation factors to convert genetic information into a specific amino acid chain. According to an elegant model of eukaryotic translation initiation, the 3' poly(A) tail of an mRNA, which is occupied by poly(A)-binding proteins (PABPs), communicates with the 5'-cap bound by eIF4E to enhance translation. Although the circularization of mRNA resulting from the communication is widely understood, it has yet to be directly observed. To explore mRNA circularization in translation, we analyzed the level of colocalization of eIF4E, eIF4G, and PABP on individual mRNAs in polysomal and subpolysomal fractions using single polysome analysis. Our results show that the three tested proteins barely coexist in mRNA in either polysomal or subpolysomal fractions, implying that the closed-loop structure generated by the communication between eIF4E, eIF4G, and PAPB may be transient during translation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jong-Bong Lee reports financial support was provided by Korea Ministry of Science and ICT. Yoon Ki Kim reports financial support was provided by Korea Ministry of Science and ICT. Jong-Bong Lee reports a relationship with Korea Ministry of Science and ICT that includes: funding grants. Yoon Ki Kim reports a relationship with Korea Ministry of Science and ICT that includes: funding grants., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Selective translation allows to orchestrate the expression of specific proteins in response to different signals through the concerted action of cis-acting elements and RNA-binding proteins (RBPs). Gemin5 is a ubiquitous RBP involved in snRNP assembly. In addition, Gemin5 regulates translation of different mRNAs through apparently opposite mechanisms of action. Here, we investigated the differential function of Gemin5 in translation by identifying at a genome-wide scale the mRNAs associated with polysomes. Among the mRNAs showing Gemin5-dependent enrichment in polysomal fractions, we identified a selective enhancement of specific transcripts. Comparison of the targets previously identified by CLIP methodologies with the polysome-associated transcripts revealed that only a fraction of the targets was enriched in polysomes. Two different subsets of these mRNAs carry unique cis-acting regulatory elements, the 5' terminal oligopyrimidine tracts (5'TOP) and the histone stem-loop (hSL) structure at the 3' end, respectively, encoding ribosomal proteins and histones. RNA-immunoprecipitation (RIP) showed that ribosomal and histone mRNAs coprecipitate with Gemin5. Furthermore, disruption of the TOP motif impaired Gemin5-RNA interaction, and functional analysis showed that Gemin5 stimulates translation of mRNA reporters bearing an intact TOP motif. Likewise, Gemin5 enhanced hSL-dependent mRNA translation. Thus, Gemin5  promotes polysome association of only a subset of its targets, and as a consequence, it favors translation of the ribosomal and the histone mRNAs. Together, the results presented here unveil Gemin5 as a novel translation regulator of mRNA subsets encoding proteins involved in fundamental cellular processes., (© 2022. The Author(s).)

The transformation of normal to malignant cells is accompanied by substantial changes in gene expression programs through diverse mechanisms. Here, we examined the changes in the landscape of transcription start sites and alternative promoter (AP) usage and their impact on the translatome in TCL1-driven chronic lymphocytic leukemia (CLL). Our findings revealed a marked elevation of APs in CLL B cells from Eµ-Tcl1 transgenic mice, which are particularly enriched with intra-genic promoters that generate N-terminally truncated or modified proteins. Intra-genic promoter activation is mediated by (1) loss of function of 'closed chromatin' epigenetic regulators due to the generation of inactive N-terminally modified isoforms or reduced expression; (2) upregulation of transcription factors, including c-Myc , targeting the intra-genic promoters and their associated enhancers. Exogenous expression of Tcl1 in MEFs is sufficient to induce intra-genic promoters of epigenetic regulators and promote c-Myc expression. We further found a dramatic translation downregulation of transcripts bearing CNY cap-proximal trinucleotides, reminiscent of cells undergoing metabolic stress. These findings uncovered the role of Tcl1 oncogenic function in altering promoter usage and mRNA translation in leukemogenesis., Competing Interests: AO, TH, SB, KD, IS, RD No competing interests declared, (© 2022, Ogran et al.)

Intronic polyadenylation (IPA) isoforms, which contain alternative last exons, are widely regulated in various biological processes and by many factors. However, little is known about their cytoplasmic regulation and translational status. In this study, we provide the first evidence that the genome-wide patterns of IPA isoform regulation during a biological process can be very distinct between the transcriptome and translatome, and between the nucleus and cytosol. Indeed, by 3'-seq analyses on breast cancer cells, we show that the genotoxic anticancer drug, doxorubicin, preferentially down-regulates the IPA to the last-exon (IPA:LE) isoform ratio in whole cells (as previously reported) but preferentially up-regulates it in polysomes. We further show that in nuclei, doxorubicin almost exclusively down-regulates the IPA:LE ratio, whereas in the cytosol, it preferentially up-regulates the isoform ratio, as in polysomes. Then, focusing on IPA isoforms that are up-regulated by doxorubicin in the cytosol and highly translated (up-regulated and/or abundant in polysomes), we identify several IPA isoforms that promote cell survival to doxorubicin. Mechanistically, by using an original approach of condition- and compartment-specific CLIP-seq (CCS-iCLIP) to analyze ELAVL1-RNA interactions in the nucleus and cytosol in the presence and absence of doxorubicin, as well as 3'-seq analyses upon ELAVL1 depletion, we show that the RNA-binding protein ELAVL1 mediates both nuclear down-regulation and cytosolic up-regulation of the IPA:LE isoform ratio in distinct sets of genes in response to doxorubicin. Altogether, these findings reveal differential regulation of the IPA:LE isoform ratio across subcellular compartments during drug response and its coordination by an RNA-binding protein., (© 2022 Chakraborty et al.; Published by Cold Spring Harbor Laboratory Press.)

Many glycine-rich RNA-binding proteins (GR-RBPs) have critical functions in RNA processing and metabolism. Here, we describe a role for the tomato (Solanum lycopersicum) GR-RBP SlRBP1 in regulating mRNA translation. We found that SlRBP1 knockdown mutants (slrbp1) displayed reduced accumulation of total chlorophyll and impaired chloroplast ultrastructure. These phenotypes were accompanied by deregulation of the levels of numerous key transcripts associated with chloroplast functions in slrbp1. Furthermore, native RNA immunoprecipitation-sequencing (nRIP-seq) recovered 61 SlRBP1-associated RNAs, most of which are involved in photosynthesis. SlRBP1 binding to selected target RNAs was validated by nRIP-qPCR. Intriguingly, the accumulation of proteins encoded by SlRBP1-bound transcripts, but not the mRNAs themselves, was reduced in slrbp1 mutants. Polysome profiling followed by RT-qPCR assays indicated that the polysome occupancy of target RNAs was lower in slrbp1 plants than in wild-type. Furthermore, SlRBP1 interacted with the eukaryotic translation initiation factor SleIF4A2. Silencing of SlRBP1 significantly reduced SleIF4A2 binding to SlRBP1-target RNAs. Taking these observations together, we propose that SlRBP1 binds to and channels RNAs onto the SleIF4A2 translation initiation complex and promotes the translation of its target RNAs to regulate chloroplast functions., (� American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

In addition to their central functions in translation, ribosomes can adopt inactive structures that are fully assembled yet devoid of mRNA. We describe how the abundance of idle eukaryotic ribosomes is influenced by a broad range of biological conditions spanning viral infection, nutrient deprivation, and developmental cues. Vacant ribosomes may provide a means to exclude ribosomes from translation while also shielding them from degradation, and the variable identity of factors that occlude ribosomes may impart distinct functionality. We propose that regulated changes in the balance of idle and active ribosomes provides a means to fine-tune translation. We provide an overview of idle ribosomes, describe what is known regarding their function, and highlight questions that may clarify their biological roles., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors, and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of PE in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2α phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity., Competing Interests: RC, MA, SH, GP No competing interests declared, (© 2022, Crawford et al.)

Regulated loading of eIF3-bound 40S ribosomes on capped mRNA is generally dependent upon the translation initiation factor eIF4E; however, mRNA translation often proceeds during physiological stress, such as virus infection, when eIF4E availability and activity are limiting. It remains poorly understood how translation of virus and host mRNAs are regulated during infection stress. While initially sensitive to mTOR inhibition, which limits eIF4E-dependent translation, we show that protein synthesis in human cytomegalovirus (HCMV)-infected cells unexpectedly becomes progressively reliant upon eIF3d. Targeting eIF3d selectively inhibits HCMV replication, reduces polyribosome abundance, and interferes with expression of essential virus genes and a host gene expression signature indicative of chronic ER stress that fosters HCMV reproduction. This reveals a strategy whereby cellular eIF3d-dependent protein production is hijacked to exploit virus-induced ER stress. Moreover, it establishes how switching between eIF4E and eIF3d-responsive cap-dependent translation can differentially tune virus and host gene expression in infected cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages anti-rRNA ribosome immunoprecipitation on whole polysomes to measure ribosome association (RA). We compare RA to its analogue in ribosome profiling and RNA sequencing, translation efficiency, and demonstrate both the performance of riboPLATE-seq and its utility in detecting translational alterations induced by specific inhibitors of protein kinases., (© 2022. The Author(s).)

Phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) and subsequent inhibition of protein synthesis is a major survival response to different stresses in animal and yeast cells. However, the role of this regulatory mechanism in plants is not unambiguously established to date. Here we describe a slight reduction of polysome abundance in Nicotiana benthamiana after the transient expression of a cDNA, AteIF2α(S56D), encoding a phosphomimetic form of Arabidopsis thaliana eIF2α. In contrast, the expression of a cDNA, AteIF2α(S56A), that encodes a non-phosphorylatable form of AteIF2α caused slightly elevated polysome formation compared to the control. Recombinant AteIF2α(S56A) was detected in association with 40S ribosomal subunit-containing complexes and also in the polysomal fraction, while recombinant AteIF2α(S56D) was detected mainly in complex with 40S subunits. Intentional phosphorylation of TaeIF2α induced by L-histidinol in a wheat germ (Triticum aestivum) cell-free extract did not reduce the abundance of polysomes. Interestingly, the phosphorylated TaeIF2(αP) was not detected in the polysomal fraction, similar to AteIF2α(S56D) in the in vivo experiment. Using mRNAs with a 'Strepto-tag' in the 3' untranslated region, the 48S pre-initiation complexes isolated from histidinol-treated wheat germ extracts were shown to contain phosphorylated TaeIF2(αP). Thus, the phosphorylation of plant eIF2 does not greatly affect its ability to participate in the initiation of mRNA translation, in contrast to animals and yeast, in which eIF2α phosphorylation results in profound suppression of protein synthesis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3∆ mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57-associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32 P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. IMPORTANCE As an RNA-binding protein, KSHV ORF57 regulates RNA splicing, stability, and translation and inhibits host innate immunity by blocking the formation of RNA granules in virus-infected cells. In this study, ORF57 was found to interact with many host noncoding RNAs, including lncRNAs, snoRNAs, and rRNAs, to carry out additional unknown functions. ORF57 binds a group of lncRNAs via the RNA motifs identified by ORF57 CLIP-seq to regulate their expression. ORF57 associates with snoRNAs independently of fibrillarin and NOP58 proteins and with rRNA in the regions that commonly bind snoRNAs. Knockdown of fibrillarin expression decreases the expression of snoRNAs and CDK4 but does not affect viral gene expression. More importantly, we found that ORF57 binds translationally active polysomes and enhances PABPC1 but prevents Ago2 association with polysomes. Data provide compelling evidence on how ORF57 in KSHV-infected cells might regulate protein synthesis by blocking Ago2's hostile activities on translation.

Stress granules (SGs) are cytoplasmic biomolecular condensates that are formed against a variety of stress conditions when translation initiation is perturbed. SGs form through the weak protein-protein, protein-RNA, and RNA-RNA interactions, as well as through the intrinsically disordered domains and post-translation modifications within RNA binding proteins (RBPs). SGs are known to contribute to cell survivability by minimizing the stress-induced damage to the cells by delaying the activation of apoptosis. Here, we find that dihydrocapsaicin (DHC), an analogue of capsaicin, is a SG inducer that promotes polysome disassembly and reduces global protein translation via phosphorylation of eIF2α. DHC-mediated SG assembly is controlled by the phosphorylation of eIF2α at serine 51 position and is controlled by all four eIF2α stress kinases (i.e., HRI, PKR, PERK, and GCN2) with HRI showing maximal effect. We demonstrate that DHC is a bonafide compound that induces SG assembly, disassembles polysome, phosphorylates eIF2α in an HRI dependent manner, and thereby arrest global translation. Together, our results suggest that DHC is a novel SG inducer and an alternate to sodium arsenite to study SG dynamics., Competing Interests: Declaration of competing interest The Authors declare no competing or financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Protein synthesis and degradation determine the relationship between mRNA and corresponding protein amounts. This relationship can change in a dynamic and selective fashion when translational efficiencies of transcript subsets are altered downstream of, for example, translation factors and/or RNA binding proteins. Notably, even transcription factors such as estrogen receptor alpha (ERα) can modulate mRNA translation in a transcript-selective manner. Yet, despite ample evidence suggesting a key role for mRNA translation in shaping the proteome in health and disease, it remains largely unexplored. Here, we present a guide for the extraction of mRNA engaged in translation using polysome fractionation with linear and optimized sucrose gradients. The isolated polysome-associated RNA is then quantified, in parallel with total mRNA from the same conditions, using methods such as RNA sequencing; and the resulting data set is analyzed to derive transcriptome-wide insights into how mRNA translation is modulated. The methods we describe are applicable to cultured cells, small numbers of FACS-isolated primary cells, and small tissue samples from biobanks or animal studies. Accordingly, this approach can be applied to study in detail how ERα and other factors control gene expression by selectively modulating mRNA translation both in vitro and in vivo., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated Stress Response (ISR) is the immediate shutdown of most translation through phosphorylation of the alpha subunit of translation initiation factor eIF2 and activation of eIF4E binding proteins. While these posttranslational modifications largely inhibit cap-dependent translation, many mRNA resist this inhibition by alternative translation mechanisms involving cis-regulatory sequences and structures in 5' transcript leaders, including upstream Open Reading Frames (uORFs), Internal Ribosome Entry Sites (IRESes), and Cap-Independent Translation Elements (CITEs). Studies of uORF and IRES activity are often performed on a gene-by-gene basis; however, high-throughput methods have recently emerged. Here, we describe a protocol for Polysome Library Sequencing (PoLib-Seq; Fig. 1), a multiplexed assay of reporter gene translation that can be used during the ISR. A designer library of reporter RNAs are transfected into tissue-culture cells, and their translation is assayed via sucrose gradient fractionation followed by high-throughput sequencing. As an example, we include PoLib-seq results simultaneously assaying translation of wildtype and uORF mutant human ATF4 reporter RNAs, recapitulating the known function of uORF1 in resisting translational inhibition during the ISR., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

mRNA translation plays a critical role in determining proteome composition. In health, regulation of mRNA translation facilitates rapid gene expression responses to intra- and extracellular signals. Moreover, dysregulated mRNA translation is a common feature in disease states, including neurological disorders and cancer. Yet, most studies of gene expression focus on analysis of mRNA levels, leaving variations in translational efficiencies largely uncharacterized. Here, we outline procedures to identify mRNA-selective alterations in translational efficiencies on a transcriptome-wide scale using the anota2seq package. Anota2seq compares expression data originating from translated mRNA to data from matched total mRNA to identify changes in translated mRNA not paralleled by corresponding changes in total mRNA (interpreted as changes in translational efficiencies impacting protein levels), congruent changes in total and translated mRNA (interpreted as changes in transcription and/or mRNA stability), and changes in total mRNA not paralleled by corresponding alterations in translated mRNA (interpreted as translational buffering). To illustrate the functionality of the anota2seq analysis package, we demonstrate a detailed analysis using a polysome-profiling data set quantified by RNA sequencing, revealing that estrogen receptor α modulates gene expression via a type of translational buffering termed offsetting. Notably, this anota2seq analysis procedure is also applicable to ribosome-profiling (RiboSeq) data sets and can be adapted to a variety of other data types and experimental contexts. Finally, we provide guidance for extending anota2seq analysis to examine associations between untranslated regions and altered translational efficiencies as well as targeted cellular functions to gain insights into mechanisms and phenotypic consequences of altered mRNA translation. Thus, this step-by-step manual allows users to interrogate selective changes in mRNA translation on a transcriptome-wide scale using the Bioconductor package anota2seq., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Translational control provides a strategy for rapid optimization of gene expression and restoration of protein homeostasis in response to cellular stresses. An important mechanism for translational control involves phosphorylation of eIF2, which invokes the integrated stress response (ISR). In the ISR, initiation of bulk protein synthesis is lowered coincident with enhanced translation efficiency of select gene transcripts that serve critical functions in stress adaptation. In this chapter, we focus on polysome profiling as a tool for establishing and characterizing translation control induced by eIF2 phosphorylation during environmental stresses. We describe in detail the experimental strategies of polysome profiling for detecting bulk repression of the translational machinery and quantifying translational control of key stress-induced gene transcripts. These experimental strategies can be adjusted to measure individual gene transcripts or genome-wide analyses and can be adapted to measure changes in the levels of ribosome subunits and associated factors invoked by various cellular cues in the ISR., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

mRNA translation is a key step in gene expression that allows the cell to qualitatively and quantitatively modulate the cell's proteome according to intra- or extracellular signals. Polysome profiling is the most comprehensive technique to study both the translation state of mRNAs and the protein machinery associated with the mRNAs being translated. Here we describe the procedure commonly used in our laboratory to gain insights into the molecular mechanisms underlying translation regulation under pathophysiological conditions., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation., (© 2021. Springer Science+Business Media, LLC.)

The Pax-5 gene encodes a transcription factor that is essential for B-cell commitment and maturation. However, Pax-5 deregulation is associated with various cancer lesions, notably hematopoietic cancers. Mechanistically, studies have characterized genetic alterations within the Pax -5 locus that result in either dominant oncogenic function or haploinsufficiency-inducing mutations leading to oncogenesis. Apart from these mutations, some examples of aberrant Pax-5 expression cannot be associated with genetic alterations. In the present study, we set out to elucidate potential alterations in post-transcriptional regulation of Pax-5 expression and establish that Pax-5 transcript editing represents an important means to aberrant expression. Upon the profiling of Pax-5 mRNA in leukemic cells, we found that the 3'end of the Pax-5 transcript is submitted to alternative polyadenylation (APA) and alternative splicing events. Using rapid amplification of cDNA ends (3'RACE) from polysomal fractions, we found that Pax-5 3' untranslated region (UTR) shortening correlates with increased ribosomal occupancy for translation. These observations were also validated using reporter gene assays with truncated 3'UTR regions cloned downstream of a luciferase gene. We also showed that Pax-5 3'UTR editing has direct repercussions on regulatory elements such as miRNAs, which in turn impact Pax-5 protein expression. More importantly, we found that advanced staging of various hematopoietic cancer lesions relates to shorter Pax-5 3'UTRs. Altogether, our findings identify novel molecular mechanisms that account for aberrant expression and function of the Pax-5 oncogene in cancer cells. These findings also present new avenues for strategic intervention in Pax-5 -mediated cancers.