Search

Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study provides a comprehensive map of m1A in dinoflagellate mRNA and shows that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetric distribution along mature transcripts. In Amphidinium carterae, we identify 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many of which are involved in regulating carbon and nitrogen metabolism. Enriched within 3′UTRs, dinoflagellate mRNA m1A levels negatively correlate with translation efficiency. Nitrogen depletion further decreases mRNA m1A levels. Our data suggest that distinctive patterns of m1A modification might influence the expression of metabolism-related genes through translational control. Synopsis: Unlike the scarce presence in typical eukaryotes, m1A is prevalent in dinoflagellate mRNA, m1A levels correlate with the expression of metabolism-related genes and respond to nitrogen starvation. m1A, but not m6A, is the most abundant internal mRNA modification in various dinoflagellates. With major localization in the 3'UTR, m1A in dinoflagellate mRNA negatively correlates with the translation rates of mature transcripts. m1A methylation of mRNA in dinoflagellates responds to nitrogen starvation and might regulate the translation of mRNAs related to metabolism. Unlike the scarce presence in typical eukaryotes, m1A is prevalent in dinoflagellate mRNA, m1A levels correlate with the expression of metabolism-related genes and respond to nitrogen starvation. [ABSTRACT FROM AUTHOR]

Angiogenesis, the process of formation of new blood vessels from pre-existing vasculature, is essential for tumor growth and metastasis. Anti-angiogenic treatment targeting vascular endothelial growth factor (VEGF) signaling is a powerful tool to combat tumor growth; however, anti-tumor angiogenesis therapy has shown limited efficacy, with survival benefits ranging from only a few weeks to months. Compensation by upregulation of complementary growth factors and switches to different modes of vascularization have made these types of therapies less effective. Recent evidence suggests that targeting specific players in endothelial metabolism is a valuable therapeutic strategy against tumor angiogenesis. Although it is clear that metabolism can modulate the translational machinery, the reciprocal relationship between metabolism and mRNA translational control during tumor angiogenesis is not fully understood. In this review, we explore emerging examples of how endothelial cell metabolism affects mRNA translation during the formation of blood vessels. A deeper comprehension of these mechanisms could lead to the development of innovative therapeutic strategies for both physiological and pathological angiogenesis. [ABSTRACT FROM AUTHOR]

Metabolism and mRNA translation represent critical steps involved in modulating gene expression and cellular physiology. Being the most energy-consuming process in the cell, mRNA translation is strictly linked to cellular metabolism and in synchrony with it. Indeed, several mRNAs for metabolic pathways are regulated at the translational level, resulting in translation being a coordinator of metabolism. On the other hand, there is a growing appreciation for how metabolism impacts several aspects of RNA biology. For example, metabolic pathways and metabolites directly control the selectivity and efficiency of the translational machinery, as well as post-transcriptional modifications of RNA to fine-tune protein synthesis. Consistently, alterations in the intricate interplay between translational control and cellular metabolism have emerged as a critical axis underlying human diseases. A better understanding of such events will foresee innovative therapeutic strategies in human disease states. This review covers different aspects of translation and metabolism cross-regulation, ranging from how cells receive instructions to reprogram metabolism through translational control to how metabolism and metabolic states influence the protein synthesis machinery and translation control. [ABSTRACT FROM AUTHOR]

We consider a class of bilinear weakly contractive systems that entrain to periodic excitations. Entrainment is important in many natural and artificial processes. For example, in order to function properly synchronous generators must entrain to the frequency of the electrical grid, and biological organisms must entrain to the 24h solar day. A dynamical system has a positive gain of entrainment (GOE) if entrainment also yields a larger output, on average. This property is important in many applications from the periodic operation of bioreactors to the periodic production of proteins during the cell cycle division process. We derive a closed-form formula for the GOE to first-order in the control perturbation. This is used to show that in the class of systems that we consider the GOE is always a higher-order phenomenon. We demonstrate the theoretical results using two applications: the master equation and a model from systems biology called the ribosome flow model, both with time-varying and periodic transition rates. [ABSTRACT FROM AUTHOR]

Abstract Background N4‐acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N‐acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10‐mediated ac4C in HCC progression and provide a promising therapeutic approach. Methods The ac4C levels were evaluated by dot blot and ultra‐performance liquid chromatography‐tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real‐time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10‐ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull‐down, mass spectrometry, and site‐specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10‐ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. Results Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high‐throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10‐mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. Conclusions Our study identified a novel oncogenic epi‐transcriptome axis involving NAT10‐ac4C/eEF2‐HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Dysregulation of protein synthesis is one of the key mechanisms underlying autism spectrum disorder (ASD). However, the role of a major pathway controlling protein synthesis, the integrated stress response (ISR), in ASD remains poorly understood. Here, we demonstrate that the main arm of the ISR, eIF2α phosphorylation (p-eIF2α), is suppressed in excitatory, but not inhibitory, neurons in a mouse model of fragile X syndrome (FXS; Fmr1-/y). We further show that the decrease in p-eIF2α is mediated via activation of mTORC1. Genetic reduction of p-eIF2α only in excitatory neurons is sufficient to increase general protein synthesis and cause autism-like behavior. In Fmr1-/y mice, restoration of p-eIF2α solely in excitatory neurons reverses elevated protein synthesis and rescues autism-related phenotypes. Thus, we reveal a previously unknown causal relationship between excitatory neuron-specific translational control via the ISR pathway, general protein synthesis, and core phenotypes reminiscent of autism in a mouse model of FXS.

Summary: Multiple myeloma (MM) is an incurable plasma cell cancer in the bone marrow. Immunomodulatory drugs, such as lenalidomide (LEN) and pomalidomide, are backbone agents in MM treatment, and LEN resistance is commonly seen in the MM clinic. In this study, we presented that heterogeneous nuclear ribonucleoprotein U (hnRNPU) affected MM resistance to LEN via the regulation of target mRNA translation. hnRNPULow MM cells exhibited upregulated CRBN and IKZF1 proteins, stringent IKZF1/3 protein degradation upon LEN addition and increased sensitivity to LEN. RNA pulldown assays and RNA electrophoretic mobility shift assays revealed that hnRNPU bound to the 3′‐untranslated region of CRBN and IKZF1 mRNA. A sucrose gradient assay suggested that hnRNPU specifically regulated CRBN and IKZF1 mRNA translation. The competition of hnRNPU binding to its target mRNAs by small RNAs with hnRNPU‐binding sites restored MM sensitivity to LEN. hnRNPU function in vivo was confirmed in an immunocompetent MM mouse model constructed by the inoculation of Crbn‐humanized murine 5TGM1 cells into CrbnI391V/+ mice. Overall, this study suggests a novel mechanism of LEN sensitivity in which hnRNPU represses CRBN and IKZF1 mRNA translation. [ABSTRACT FROM AUTHOR]

INTRODUCTION: Impaired brain protein synthesis, synaptic plasticity, and memory are major hallmarks of Alzheimer's disease (AD). The ketamine metabolite (2R,6R)‐hydroxynorketamine (HNK) has been shown to modulate protein synthesis, but its effects on memory in AD models remain elusive. METHODS: We investigated the effects of HNK on hippocampal protein synthesis, long‐term potentiation (LTP), and memory in AD mouse models. RESULTS: HNK activated extracellular signal‐regulated kinase 1/2 (ERK1/2), mechanistic target of rapamycin (mTOR), and p70S6 kinase 1 (S6K1)/ribosomal protein S6 signaling pathways. Treatment with HNK rescued hippocampal LTP and memory deficits in amyloid‐β oligomers (AβO)‐infused mice in an ERK1/2‐dependent manner. Treatment with HNK further corrected aberrant transcription, LTP and memory in aged APP/PS1 mice. DISCUSSION: Our findings demonstrate that HNK induces signaling and transcriptional responses that correct synaptic and memory deficits in AD mice. These results raise the prospect that HNK could serve as a therapeutic approach in AD. Highlights: The ketamine metabolite HNK activates hippocampal ERK/mTOR/S6 signaling pathways.HNK corrects hippocampal synaptic and memory defects in two mouse models of AD.Rescue of synaptic and memory impairments by HNK depends on ERK signaling.HNK corrects aberrant transcriptional signatures in APP/PS1 mice. [ABSTRACT FROM AUTHOR]

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2a kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell. [ABSTRACT FROM AUTHOR]

Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.

FAM86A is a class I lysine methyltransferase (KMT) that generates trimethylation on the eukaryotic translation elongation factor 2 (EEF2) at Lys525. Publicly available data from The Cancer Dependency Map project indicate high dependence of hundreds of human cancer cell lines on FAM86A expression. This classifies FAM86A among numerous other KMTs as potential targets for future anticancer therapies. However, selective inhibition of KMTs by small molecules can be challenging due to high conservation within the S-adenosyl methionine (SAM) cofactor binding domain among KMT subfamilies. Therefore, understanding the unique interactions within each KMT-substrate pair can facilitate developing highly specific inhibitors. The FAM86A gene encodes an N-terminal FAM86 domain of unknown function in addition to its C-terminal methyltransferase domain. Here, we used a combination of X-ray crystallography, the AlphaFold algorithms, and experimental biochemistry to identify an essential role of the FAM86 domain in mediating EEF2 methylation by FAM86A. To facilitate our studies, we also generated a selective EEF2K525 methyl antibody. Overall, this is the first report of a biological function for the FAM86 structural domain in any species and an example of a noncatalytic domain participating in protein lysine methylation. The interaction between the FAM86 domain and EEF2 provides a new strategy for developing a specific FAM86A small molecule inhibitor, and our results provide an example in which modeling a protein-protein interaction with AlphaFold expedites experimental biology.

The formation of paralogs through gene duplication is a core evolutionary process. For paralogs that encode components of protein complexes such as the ribosome, a central question is whether they encode functionally distinct proteins or whether they exist to maintain appropriate total expression of equivalent proteins. Here, we systematically tested evolutionary models of paralog function using the ribosomal protein paralogs Rps27 (eS27) and Rps27l (eS27L) as a case study. Evolutionary analysis suggests that Rps27 and Rps27l likely arose during whole-genome duplication(s) in a common vertebrate ancestor. We show that Rps27 and Rps27l have inversely correlated mRNA abundance across mouse cell types, with the highest Rps27 in lymphocytes and the highest Rps27l in mammary alveolar cells and hepatocytes. By endogenously tagging the Rps27 and Rps27l proteins, we demonstrate that Rps27- and Rps27l-ribosomes associate preferentially with different transcripts. Furthermore, murine Rps27 and Rps27l loss-of-function alleles are homozygous lethal at different developmental stages. However, strikingly, expressing Rps27 protein from the endogenous Rps27l locus or vice versa completely rescues loss-of-function lethality and yields mice with no detectable deficits. Together, these findings suggest that Rps27 and Rps27l are evolutionarily retained because their subfunctionalized expression patterns render both genes necessary to achieve the requisite total expression of two equivalent proteins across cell types. Our work represents the most in-depth characterization of a mammalian ribosomal protein paralog to date and highlights the importance of considering both protein function and expression when investigating paralogs.

Background Information: The dual‐specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC‐ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. Results: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans‐acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr‐phosphorylated HNRNPC preferentially binds to IRES‐containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c‐MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated‐HNRNPC/RNA interaction reduces HNRNPC‐p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA‐dependent manner. Conclusions and Significance: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC‐ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM‐domain proteins. [ABSTRACT FROM AUTHOR]

Cellular stress is an intrinsic part of cell physiology that underlines cell survival or death. The ability of mammalian cells to regulate global protein synthesis (aka translational control) represents a critical, yet underappreciated, layer of regulation during the stress response. Various cellular stress response pathways monitor conditions of cell growth and subsequently reshape the cellular translatome to optimize translational outputs. On the molecular level, such translational reprogramming involves an intricate network of interactions between translation machinery, RNA-binding proteins, mRNAs, and non-protein coding RNAs. In this review, we will discuss molecular mechanisms, signaling pathways, and targets of translational control that contribute to cellular adaptation to stress and to cell survival or death. [ABSTRACT FROM AUTHOR]

In recent years, research into Parkinson's disease and similar neurodegenerative disorders has increasingly suggested that these conditions are synonymous with failures in proteostasis. However, the spotlight of this research has remained firmly focused on the tail end of proteostasis, primarily aggregation, misfolding, and degradation, with protein translation being comparatively overlooked. Now, there is an increasing body of evidence supporting a potential role for translation in the pathogenesis of PD, and its dysregulation is already established in other similar neurodegenerative conditions. In this paper, we consider how altered protein translation fits into the broader picture of PD pathogenesis, working hand in hand to compound the stress placed on neurons, until this becomes irrecoverable. We will also consider molecular players of interest, recent evidence that suggests that aggregates may directly influence translation in PD progression, and the implications for the role of protein translation in our development of clinically useful diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Diphthamide is a conserved post-translational modification of the essential eukaryotic translation elongation factor 2 (eEF2). It assures translational frame accuracy, and its absence increases translational frameshifts. The enzymes and pathways for diphthamide synthesis are conserved from yeast to man, in eukaryotes and archaea, yet absent in bacteria. Diphthamide is the molecular target of ADP-ribosylating toxins from bacterial pathogens including Corynebacterium diphtheriae and Pseudomonas aeruginosa. Deletions or promoter mutations of diphthamide synthesis genes are associated with cancer. Homozygous or compound heterozygous mutations that compromise diphthamide synthesis enzymes can cause diphthamide deficiency syndrome. Diphthamide-dependent reading frame maintenance limits –1 programmed ribosomal frameshifts (−1PRFs). −1PRF is necessary to generate proteins that are needed for propagation of HIV and SARS-CoV-2. Some −1PRF-dependent viruses target diphthamide synthesis enzymes for degradation. Diphthamide, a complex modification on eukaryotic translation elongation factor 2 (eEF2), assures reading-frame fidelity during translation. Diphthamide and enzymes for its synthesis are conserved in eukaryotes and archaea. Originally identified as target for diphtheria toxin (DT) in humans, its clinical relevance now proves to be broader than the link to pathogenic bacteria. Diphthamide synthesis enzymes (DPH1 and DPH3) are associated with cancer, and DPH gene mutations can cause diphthamide deficiency syndrome (DDS). Finally, new analyses provide evidence that diphthamide may restrict propagation of viruses including SARS-CoV-2 and HIV-1, and that DPH enzymes are targeted by viruses for degradation to overcome this restriction. This review describes how diphthamide is synthesized and functions in translation, and covers its clinical relevance in human development, cancer, and infectious diseases. [ABSTRACT FROM AUTHOR]

Background: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Methods: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. Results: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Conclusions: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency. [ABSTRACT FROM AUTHOR]

In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications. [ABSTRACT FROM AUTHOR]

ABSTRACTBacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage–host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host’s translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host–phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling.IMPORTANCEThe ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host–phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Multi-RNA co-transfection is starting to be employed to stimulate immune responses to SARS-CoV-2 viral infection. While there are good reasons to utilize such an approach, there is little background on whether there are synergistic RNA-dependent cellular effects. To address this issue, we use transcriptome-induced phenotype remodeling (TIPeR) via phototransfection to assess whether mRNAs encoding the Spike and Nucleocapsid proteins of SARS-CoV-2 virus into single human astrocytes (an endogenous human cell host for the virus) and mouse 3T3 cells (often used in high-throughput therapeutic screens) synergistically impact host cell biologies. An RNA concentration-dependent expression was observed where an increase of RNA by less than 2-fold results in reduced expression of each individual RNAs. Further, a dominant inhibitory effect of Nucleocapsid RNA upon Spike RNA translation was detected that is distinct from codon-mediated epistasis. Knowledge of the cellular consequences of multi-RNA transfection will aid in selecting RNA concentrations that will maximize antigen presentation on host cell surface with the goal of eliciting a robust immune response. Further, application of this single cell stoichiometrically tunable RNA functional genomics approach to the study of SARS-CoV-2 biology promises to provide details of the cellular sequalae that arise upon infection in anticipation of providing novel targets for inhibition of viral replication and propagation for therapeutic intervention.

Advanced prostate malignancies are a leading cause of cancer-related deaths in men, in large part due to our incomplete understanding of cellular drivers of disease progression. We investigate prostate cancer cell dynamics at single-cell resolution from disease onset to the development of androgen independence in an in vivo murine model. We observe an expansion of a castration-resistant intermediate luminal cell type that correlates with treatment resistance and poor prognosis in human patients. Moreover, transformed epithelial cells and associated fibroblasts create a microenvironment conducive to pro-tumorigenic immune infiltration, which is partially androgen responsive. Androgen-independent prostate cancer leads to significant diversification of intermediate luminal cell populations characterized by a range of androgen signaling activity, which is inversely correlated with proliferation and mRNA translation. Accordingly, distinct epithelial populations are exquisitely sensitive to translation inhibition, which leads to epithelial cell death, loss of pro-tumorigenic signaling, and decreased tumor heterogeneity. Our findings reveal a complex tumor environment largely dominated by castration-resistant luminal cells and immunosuppressive infiltrates.

ABSTRACT Human cytomegalovirus (HCMV) requires the robust expression of two immediate early proteins, IE1 and IE2, immediately upon infection to suppress the antiviral response and promote viral gene expression. While transcriptional control of IE1 and IE2 has been extensively studied, the role of post-transcriptional regulation of IE1 and IE2 expression is relatively unexplored. We previously found that the shared major immediate early 5′ untranslated region (MIE 5′ UTR) of the mature IE1 and IE2 transcripts plays a critical role in facilitating the translation of the IE1 and IE2 mRNAs. As RNA secondary structure in 5′ UTRs can regulate mRNA translation efficiency, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to identify RNA structures in the shared MIE 5′ UTR. We found that the MIE 5′ UTR contains three stable stem loop structures. Using a series of recombinant viruses to investigate the role of each stem loop in IE1 and IE2 protein synthesis, we found that the stem loop closest to the 5′ end of the MIE 5′ UTR (SL1) is both necessary and sufficient for efficient IE1 and IE2 mRNA translation and HCMV replication. The positive effect of SL1 on mRNA translation and virus replication was dependent on its location within the 5′ UTR. Surprisingly, a synthetic stem loop with the same free energy as SL1 in its native location also supported wild type levels of IE1 and IE2 mRNA translation and virus replication, suggesting that the presence of RNA structure at a specific location in the 5′ UTR, rather than the primary sequence of the RNA, is critical for efficient IE1 and IE2 protein synthesis. These data reveal a novel post-transcriptional regulatory mechanism controlling IE1 and IE2 expression and reinforce the critical role of RNA structure in regulating HCMV protein synthesis and replication.IMPORTANCEThese results reveal a new aspect of immediate early gene regulation controlled by non-coding RNA structures in viral mRNAs. Previous studies have largely focused on understanding viral gene expression at the level of transcriptional control. Our results show that a complete understanding of the control of viral gene expression must include an understanding of viral mRNA translation, which is driven in part by RNA structure(s) in the 5′ UTR of viral mRNAs. Our results illustrate the importance of these additional layers of regulation by defining specific 5′ UTR RNA structures regulating immediate early gene expression in the context of infection and identify important features of RNA structure that govern viral mRNA translation efficiency. These results may therefore broadly impact current thinking on how viral gene expression is regulated for human cytomegalovirus and other DNA viruses.

Thermospermine plays a critical role in negatively regulating xylem development in angiosperms. A mutant of Arabidopsis thaliana that is defective in thermospermine biosynthesis, acaulis5 (acl5), exhibits a dwarf phenotype with excessive xylem formation. Mechanistically thermospermine acts in attenuating the inhibitory effect of an evolutionarily conserved upstream open reading frame (uORF) on the main ORF of SAC51, which encodes a basic helix-loop-helix protein involved in xylem repression. Here, we revealed that a semidominant suppressor of acl5, sac503, which partially restores the acl5 phenotype, has a point mutation in the conserved uORF of SAC51 with no amino acid substitution in the deduced peptide sequence. In transgenic lines carrying the β-glucuronidase (GUS) reporter gene fused with the SAC51 5ʹ region containing the uORF, the mutant construct was shown to confer higher GUS activity than does the wild-type SAC51 construct. We confirmed that sac503 mRNA was more stable than SAC51 mRNA in acl5. These results suggest that the single-base change in sac503 positively affects the translation of its main ORF instead of thermospermine. We further found that the uORF-GUS fusion protein could be synthesized in planta from the wild-type and sac503 translational fusion constructs. [ABSTRACT FROM AUTHOR]

The eIF4E family of translation initiation factors bind 5' methylated caps and act as the limiting step for mRNA translation. The canonical eIF4E1A is required for cell viability, yet other related eIF4E families exist and are utilized in specific contexts or tissues. Here, we describe a family called Eif4e1c, for which we find roles during heart development and regeneration in zebrafish. The Eif4e1c family is present in all aquatic vertebrates but is lost in all terrestrial species. A core group of amino acids shared over 500 million years of evolution forms an interface along the protein surface, suggesting that Eif4e1c functions in a novel pathway. Deletion of eif4e1c in zebrafish caused growth deficits and impaired survival in juveniles. Mutants surviving to adulthood had fewer cardiomyocytes and reduced proliferative responses to cardiac injury. Ribosome profiling of mutant hearts demonstrated changes in translation efficiency of mRNA for genes known to regulate cardiomyocyte proliferation. Although eif4e1c is broadly expressed, its disruption had most notable impact on the heart and at juvenile stages. Our findings reveal context-dependent requirements for translation initiation regulators during heart regeneration. [ABSTRACT FROM AUTHOR]

Recent advances in mRNA therapeutics demand efficient toolkits for the incorporation of nucleoside analogues into mRNA suitable for downstream applications. Herein, we report the application of a versatile enzyme cascade for the triphosphorylation of a broad range of nucleoside analogues, including unprotected nucleobases containing chemically labile moieties. Our biomimetic system was suitable for the preparation of nucleoside triphosphates containing adenosine, cytidine, guanosine, uridine and non‐canonical core structures, as determined by capillary electrophoresis coupled to mass spectrometry. This enabled us to establish an efficient workflow for transcribing and purifying functional mRNA containing these nucleoside analogues, combined with mass spectrometric verification of analogue incorporation. Our combined methodology allows for analyses of how incorporation of nucleoside analogues that are commercially unavailable as triphosphates affect mRNA properties: The translational fidelity of the produced mRNA was demonstrated in analyses of how incorporated adenosine analogues impact translational recoding. For the SARS‐CoV‐2 frameshifting site, analyses of the mRNA pseudoknot structure using circular dichroism spectroscopy allowed insight into how the pharmacologically active 7‐deazaadenosine destabilises RNA secondary structure, consistent with observed changes in recoding efficiency. [ABSTRACT FROM AUTHOR]

Aims Mitochondria play a vital role in cellular metabolism and energetics and support normal cardiac function. Disrupted mitochondrial function and homeostasis cause a variety of heart diseases. Fam210a (family with sequence similarity 210 member A), a novel mitochondrial gene, is identified as a hub gene in mouse cardiac remodelling by multi-omics studies. Human FAM210A mutations are associated with sarcopenia. However, the physiological role and molecular function of FAM210A remain elusive in the heart. We aim to determine the biological role and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health in vivo. Methods and results Tamoxifen-induced αMHC MCM-driven conditional knockout of Fam210a in the mouse cardiomyocytes induced progressive dilated cardiomyopathy and heart failure, ultimately causing mortality. Fam210a deficient cardiomyocytes exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Furthermore, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in cardiomyocytes at the early stage before contractile dysfunction and heart failure. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response, resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of heart failure. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial-encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischaemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodelling and damage in ischaemia-induced heart failure. Conclusion These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal cardiomyocyte contractile function. This study also offers a new therapeutic target for treating ischaemic heart disease. [ABSTRACT FROM AUTHOR]

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2–4EHP (or EIF4E2) translation repressor complex to the mRNA 5′-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifnb1 mRNA that encodes the cytokine interferon β, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA mediated silencing. Here, we demonstrate the pervasive augmentation of miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with argonaute 2 (AGO2), the core component of the miRNA-induced silencing complex (miRISC), via GIGYF2 and enhances the translational repression mediated by natural miRNA-binding sites in the 3′ untranslated region of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program by co-opting the host miRNA-mediated silencing machinery. [ABSTRACT FROM AUTHOR]

Aldolase A (ALDOA), a crucial glycolytic enzyme, is often aberrantly expressed in various types of cancer. Although ALDOA has been reported to play additional roles beyond its conventional enzymatic role, its nonmetabolic function and underlying mechanism in cancer progression remain elusive. Here, it is shown that ALDOA promotes liver cancer growth and metastasis by accelerating mRNA translation independent of its catalytic activity. Mechanistically, ALDOA interacted with insulin‐ like growth factor 2 mRNA‐binding protein 1 (IGF2BP1) to facilitate its binding to m6A‐modified eIF4G mRNA, thereby increasing eIF4G protein levels and subsequently enhancing overall protein biosynthesis in cells. Importantly, administration of GalNAc‐conjugated siRNA targeting ALDOA effectively slows the tumor growth of orthotopic xenografts. Collectively, these findings uncover a previously unappreciated nonmetabolic function of ALDOA in modulating mRNA translation and highlight the potential of specifically targeting ALDOA as a prospective therapeutic strategy in liver cancer. [ABSTRACT FROM AUTHOR]

Metabolic reprogramming of non-cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer-derived metabolic and non-metabolic factors, is an emerging aspect of cancer-host interaction. We show that in normal and cancer-associated fibroblasts, breast cancer-secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer-derived miR-105 and miR-204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re-feeding, 13 C-arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer-derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR-105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re-prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma-produced proteins during periodic nutrient fluctuations.

In fluctuating environmental conditions, organisms must modulate their bioenergetic production in order to maintain cellular homeostasis for optimal fitness. Mitochondria are hubs for metabolite and energy generation. Mitochondria are also highly dynamic in their function: modulating their composition, size, density, and the network-like architecture in relation to the metabolic demands of the cell. Here, we review the recent research on the post-transcriptional regulation of mitochondrial composition focusing on mRNA localization, mRNA translation, protein import, and the role that dynamic mitochondrial structure may have on these gene expression processes. As mitochondrial structure and function has been shown to be very important for age-related processes, including cancer, metabolic disorders, and neurodegeneration, understanding how mitochondrial composition can be affected in fluctuating conditions can lead to new therapeutic directions to pursue.

Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discrete steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

Abstract Aldolase A (ALDOA), a crucial glycolytic enzyme, is often aberrantly expressed in various types of cancer. Although ALDOA has been reported to play additional roles beyond its conventional enzymatic role, its nonmetabolic function and underlying mechanism in cancer progression remain elusive. Here, it is shown that ALDOA promotes liver cancer growth and metastasis by accelerating mRNA translation independent of its catalytic activity. Mechanistically, ALDOA interacted with insulin‐ like growth factor 2 mRNA‐binding protein 1 (IGF2BP1) to facilitate its binding to m6A‐modified eIF4G mRNA, thereby increasing eIF4G protein levels and subsequently enhancing overall protein biosynthesis in cells. Importantly, administration of GalNAc‐conjugated siRNA targeting ALDOA effectively slows the tumor growth of orthotopic xenografts. Collectively, these findings uncover a previously unappreciated nonmetabolic function of ALDOA in modulating mRNA translation and highlight the potential of specifically targeting ALDOA as a prospective therapeutic strategy in liver cancer.

During their quest for growth, adaptation, and survival, cancer cells create a favorable environment through the manipulation of normal cellular mechanisms. They increase anabolic processes, including protein synthesis, to facilitate uncontrolled proliferation and deplete the tumor microenvironment of resources. As a dynamic adaptation to the self-imposed oncogenic stress, cancer cells promptly hijack translational control to alter gene expression. Rewiring the cellular proteome shifts the phenotypic balance between growth and adaptation to promote therapeutic resistance and cancer cell survival. The integrated stress response (ISR) is a key translational program activated by oncogenic stress that is utilized to fine-tune protein synthesis and adjust to environmental barriers. Here, we focus on the role of ISR signaling for driving cancer progression. We highlight mechanisms of regulation for distinct mRNA translation downstream of the ISR, expand on oncogenic signaling utilizing the ISR in response to environmental stresses, and pinpoint the impact this has for cancer cell plasticity during resistance to therapy. There is an ongoing need for innovative drug targets in cancer treatment, and modulating ISR activity may provide a unique avenue for clinical benefit. [ABSTRACT FROM AUTHOR]

A serine/threonine-specific protein kinase B (PKB), also known as Akt, is a key factor in the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway that regulates cell survival, metabolism and proliferation. Akt phosphorylates many downstream specific substrates, which subsequently control the nuclear envelope breakdown (NEBD), centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. In vertebrates, Akt is also an important player during oogenesis and preimplantation development. In the signaling pathways regulating mRNA translation, Akt is involved in the control of mammalian target of rapamycin complex 1 (mTORC1) and thereby regulates the activity of a translational repressor, the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1). In this review, we summarize the functions of Akt in mitosis, meiosis and early embryonic development. Additionally, the role of Akt in the regulation of mRNA translation is addressed with respect to the significance of this process during early development. [ABSTRACT FROM AUTHOR]

Ketamine has shown antidepressant effects in patients with major depressive disorder (MDD) resistant to first‐line treatments and approved for use in this patient population. Ketamine induces several forms of synaptic plasticity, which are proposed to underlie its antidepressant effects. However, the molecular mechanism of action directly responsible for ketamine's antidepressant effects remains under active investigation. It was recently demonstrated that the effectors of the mammalian target of rapamycin complex 1 (mTORC1) signalling pathway, namely, eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2 (4E‐BP1 and 4E‐BP2), are central in mediating ketamine‐induced synaptic plasticity and behavioural antidepressant‐like effect. 4E‐BPs are a family of messenger ribonucleic acid (mRNA) translation repressors inactivated by mTORC1. We observed that their expression in inhibitory interneurons mediates ketamine's effects in the forced swim and novelty suppressed feeding tests and the long‐lasting inhibition of GABAergic neurotransmission in the hippocampus. In addition, another effector pathway that regulates translation elongation downstream of mTORC1, the eukaryotic elongation factor 2 kinase (eEF2K), has been implicated in ketamine's behavioural effects. We will discuss how ketamine's rapid antidepressant effect depends on the activation of neuronal mRNA translation through 4E‐BP1/2 and eEF2K. Furthermore, given that these pathways also regulate cognitive functions, we will discuss the evidence of ketamine's effect on cognitive function in MDD. Overall, the data accrued from pre‐clinical research have implicated the mRNA translation pathways in treating mood symptoms of MDD. However, it is yet unclear whether the pro‐cognitive potential of subanesthetic ketamine in rodents also engages these pathways and whether such an effect is consistently observed in the treatment‐resistant MDD population. [ABSTRACT FROM AUTHOR]

DDX41 mutation has been observed in myeloid malignancies including myelodysplastic syndromes and acute myeloid leukemia, but the underlying causative mechanisms of these diseases have not been fully elucidated. The DDX41 protein is an ATP-dependent RNA helicase with roles in RNA metabolism. We previously showed that DDX41 is involved in ribosome biogenesis by promoting the processing of newly transcribed pre-ribosomal RNA. To build on this finding, in this study, we leveraged ribosome profiling technology to investigate the involvement of DDX41 in translation. We found that DDX41 knockdown resulted in both translationally increased and decreased transcripts. Both gene set enrichment analysis and gene ontology analysis indicated that ribosome-associated genes were translationally promoted after DDX41 knockdown, in part because these transcripts had significantly shorter transcript length and higher transcriptional and translational levels. In addition, we found that transcripts with 5'-terminal oligopyrimidine motifs tended to be translationally upregulated when the DDX41 level was low. Our data suggest that a translationally regulated feedback mechanism involving DDX41 may exist for ribosome biogenesis. [ABSTRACT FROM AUTHOR]

Cancer persister cells remain a significant barrier to effective anti-cancer therapy. We found that melanoma persister cells undergo a reversible reprogramming of mRNA translation. A subset of mRNAs, harboring N6-methyladenosine in their 5'-untranslated regions, is translationally up-regulated in an eIF4A-dependent manner. Targeting eIF4A prevents the emergence of resistant clones.

Although historically research has focused on transcription as the central governor of protein expression, protein translation is now increasingly being recognized as a major factor for determining protein levels within cells. The central nervous system relies on efficient updating of the protein landscape. Thus, coordinated regulation of mRNA localization, initiation, or termination of translation is essential for proper brain function. In particular, dendritic protein synthesis plays a key role in synaptic plasticity underlying learning and memory as well as cognitive processes. Increasing evidence suggests that impaired mRNA translation is a common feature found in numerous psychiatric disorders. In this review, we describe how malfunction of translation contributes to development of psychiatric diseases, including schizophrenia, major depression, bipolar disorder, and addiction.

Abstract Background Chronic mTORC1 activation in skeletal muscle is linked with age‐associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E‐binding proteins (4EBPs). Whole‐body knockout of S6K1 or muscle‐specific over‐expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1‐mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1‐mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. Methods Mice with myofiber‐specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1‐mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt‐TSC1mKO or S6K1‐TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. Results Here, we show that 4EBP1mt‐TSC1mKO, but not S6K1‐TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P

Abstract Background RNA secondary structure has broad impact on the fate of RNA metabolism. The reduced stability of secondary structures near the translation initiation site/start codon of the coding region promotes the efficiency of translation in both prokaryotic and eukaryotic species. However, the inaccuracy of in silico folding and the focus on the coding region limit our understanding of the global relationship between the whole mRNA structure and translation efficiency. Leveraging high-throughput RNA structure probing data in the transcriptome, we aim to systematically investigate the role of RNA structure in regulating translation efficiency. Results Here, we analyze the influences of hundreds of sequence and structural features on translation efficiency in the mouse embryonic stem cells (mESCs) and zebrafish developmental stages. Our findings reveal that overall in vivo RNA structure has a higher relative importance in predicting translation efficiency than in vitro RNA structure in both mESCs and zebrafish. Also, RNA structures in 3’ untranslated region (UTR) have much stronger influence on translation efficiency compared to those in coding regions or 5' UTR. Furthermore, strong alternation between in vitro and in vivo structures in 3' UTR are detected in highly translated mRNAs in mESCs but not zebrafish. Instead, moderate alteration between in vitro and in vivo RNA structures in the 5’ UTR and proximal coding regions are detected in highly translated mRNAs in zebrafish. Conclusions Our results suggest the openness of the 3’ UTR promotes the translation efficiency in both mice and zebrafish, with the in vivo structure in 3’ UTR more important in mice than in zebrafish. This reveals a novel role of RNA secondary structure on translational regulation.

Abstract Background Vertebrate CPEB proteins bind mRNAs at cytoplasmic polyadenylation elements (CPEs) in their 3′ UTRs, leading to cytoplasmic changes in their poly(A) tail lengths; this can promote translational repression or activation of the mRNA. However, neither the regulation nor the mechanisms of action of the CPEB family per se have been systematically addressed to date. Results Based on a comparative analysis of the four vertebrate CPEBs, we determine their differential regulation by phosphorylation, the composition and properties of their supramolecular assemblies, and their target mRNAs. We show that all four CPEBs are able to recruit the CCR4-NOT deadenylation complex to repress the translation. However, their regulation, mechanism of action, and target mRNAs define two subfamilies. Thus, CPEB1 forms ribonucleoprotein complexes that are remodeled upon a single phosphorylation event and are associated with mRNAs containing canonical CPEs. CPEB2–4 are regulated by multiple proline-directed phosphorylations that control their liquid–liquid phase separation. CPEB2–4 mRNA targets include CPEB1-bound transcripts, with canonical CPEs, but also a specific subset of mRNAs with non-canonical CPEs. Conclusions Altogether, these results show how, globally, the CPEB family of proteins is able to integrate cellular cues to generate a fine-tuned adaptive response in gene expression regulation through the coordinated actions of all four members.

Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA‐binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T‐cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth. Synopsis: How immune cells invading the tumor microenvironment cope with detrimental stress conditions such as hypoxia and nutrient deprivation remains poorly understood. Here, RNA‐binding protein CPEB4 is shown to mediate a novel branch of transient unfolded protein response (UTR) in CD8 T cells, supporting effector cell viability and antitumor function. Transient acute ER stress promotes CPEB4 translational upregulationCPEB4 mediates chronic ER stress adaptation in CD8 cellsT‐cell effector function requires CPEB4‐mediated chronic ER stress adaptation.CPEB4 promotes T cell‐mediated antitumor immunity in vivo. [ABSTRACT FROM AUTHOR]

Complexes of two or more proteins form many, if not most, of the intracellular "machines" that execute physical and chemical work, and transmit information. Complexes can form from stochastic post‐translational interactions of fully formed proteins, but recent attention has shifted to co‐translational interactions in which the most common mechanism involves binding of a mature constituent to an incomplete polypeptide emerging from a translating ribosome. Studies in yeast have revealed co‐translational interactions during formation of multiple major complexes, and together with recent mammalian cell studies, suggest widespread utilization of the mechanism. These translation‐dependent interactions can involve a single or multiple mRNA templates, can be uni‐ or bi‐directional, and can use multi‐protein sub‐complexes as a binding component. Here, we discuss benefits of co‐translational complex assembly including accuracy and efficiency, overcoming hidden interfaces, localized and hierarchical assembly, and reduction of orphan protein degradation, toxicity, and dominant‐negative pathogenesis, all serving to improve cell fitness. [ABSTRACT FROM AUTHOR]

Ribosomes, in general, are viewed as constitutive macromolecular machines where protein synthesis takes place; however, this view has been recently challenged, supporting the hypothesis of ribosome specialization and opening a completely new field of research. Recent studies have demonstrated that ribosomes are heterogenous in their nature and can provide another layer of gene expression control by regulating translation. Heterogeneities in ribosomal RNA and ribosomal proteins that compose them favor the selective translation of different sub-pools of mRNAs and functional specialization. In recent years, the heterogeneity and specialization of ribosomes have been widely reported in different eukaryotic study models; however, few reports on this topic have been made on protozoa and even less on protozoa parasites of medical importance. This review analyzes heterogeneities of ribosomes in protozoa parasites highlighting the specialization in their functions and their importance in parasitism, in the transition between stages in their life cycle, in the change of host and in response to environmental conditions. [ABSTRACT FROM AUTHOR]