mRNA Translation Research Articles

Translation Complex Profile Sequencing Allows Discrimination of Leaky Scanning and Reinitiation in Upstream Open Reading Frame-controlled Translation

Upstream open reading frames (uORFs) are a class of translated regions (translons) in mRNA 5′ leaders. uORFs are believed to be pervasive regulators of the translation of mammalian mRNAs. Some uORFs are highly repressive but others have little or no impact on downstream mRNA translation either due to inefficient recognition of their start codon(s) or/and due to efficient reinitiation after uORF translation. While experiments with uORF reporter constructs proved to be instrumental in the investigation of uORF-mediated mechanisms of translation control, they can have serious limitations as manipulations with uORF sequences can yield various artefacts. Here we propose a general approach for using translation complex profiling (TCP-seq) data for exploring uORF regulatory characteristics. Using several examples, we show how TCP-seq could be used to estimate both repressiveness and modes of action of individual uORFs. We demonstrate how this approach could be used to assess the mechanisms of uORF-mediated translation control in the mRNA of several human genes, including EIF5, IFRD1, MDM2, MIEF1, PPP1R15B, TAF7, and UCP2.

Selective translation of nuclear mitochondrial respiratory proteins reprograms succinate metabolism in AML development and chemoresistance.

Mitochondrial adaptations dynamically reprogram cellular bioenergetics and metabolism and confer key properties for human cancers. However, the selective regulation of these mitochondrial responses remains largely elusive. Here, inspired by a genetic screening in acute myeloid leukemia (AML), we identify RAS effector RREB1 as a translational regulator and uncover a unique translation control system for nuclear-encoded mitochondrial proteins in human cancers. RREB1 deletion reduces mitochondrial activities and succinate metabolism, thereby damaging leukemia stem cell (LSC) function and AML development. Replenishing complex II subunit SDHD rectifies these deficiencies. Notably, inhibition of complex II re-sensitizes AML cells to venetoclax treatment. Mechanistically, a short RREB1 variant binds to a conserved motif in the 3' UTRs and cooperates with elongation factor eEF1A1 to enhance protein translation of nuclear-encoded mitochondrial mRNAs. Overall, our findings reveal a unique translation control mechanism for mitochondrial adaptations in AML pathogenesis and provide a potential strategy for targeting this vulnerability of LSCs.

Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43 ribonucleoprotein condensates disrupting mRNA transport and local translation in neurons.

Altered RNA metabolism and misregulation of transactive response DNA-binding protein of 43kDa (TDP-43), an essential RNA-binding protein (RBP), define amyotrophic lateral sclerosis (ALS). Intermediate-length polyglutamine (polyQ) expansions of Ataxin-2, a like-Sm (LSm) RBP, are associated with increased risk for ALS, but the underlying biological mechanisms remain unknown. Here, we studied the spatiotemporal dynamics and mRNA regulatory functions of TDP-43 and Ataxin-2 ribonucleoprotein (RNP) condensates in rodent (rat) primary cortical neurons and mouse motor neuron axons invivo. We report that Ataxin-2 polyQ expansions aberrantly sequester TDP-43 within RNP condensates and disrupt both its motility along the axon and liquid-like properties. We provide evidence that Ataxin-2 governs motility and translation of neuronal RNP condensates and that Ataxin-2 polyQ expansions fundamentally perturb spatial localization of mRNA and suppress local translation. Overall, our results support a model in which Ataxin-2 polyQ expansions disrupt stability, localization, and/or translation of critical axonal and cytoskeletal mRNAs, particularly important for motor neuron integrity.

The Drosophila ribonucleoprotein Clueless is required for ribosome biogenesis in vivo

As hubs of metabolism, mitochondria contribute critical processes to coordinate and optimize energy and intermediate metabolites. Drosophila Clueless (Clu) and vertebrate CLUH are ribonucleoproteins critical for supporting mitochondrial function yet do so in multiple ways. Clu/CLUH bind mRNAs and CLUH regulates mRNA localization and translation of mRNAs encoding proteins destined for mitochondrial import. In addition, Clu associates with ribosomal proteins and translation factors, yet whether it is required for fundamental ribosome function in vivo is not clear. In this study, we examine the Clu interactome and probe Clu’s requirement in ribosome biogenesis. We previously showed that Clu associates with ribosomal proteins. In this study, we extend these observations to show that clu null mutants display a significant decrease in overall protein synthesis. In addition, Clu associates with ribosomal proteins in an mRNA-independent manner, suggesting Clu’s core ribosomal function may be separate from its role in localizing and translating specific mRNAs. We find that Clu is present in the nucleus and associates with the ribosomal RNA (rRNA) processing protein fibrillarin but, surprisingly, that processed rRNA products are normal in the absence of Clu. Furthermore, Clu loss does not affect ribosomal protein levels, but does result in a decrease in 40S and 60S ribosomal subunits abundance. Together, these results demonstrate that Clu is present in the nucleus and required for 40S and 60S biogenesis and global translation in vivo. These results highlight the multifaceted role of Clu in supporting cell function through regulation of mRNA encoding mitochondrial proteins and ribosome biogenesis.

The expanding role of cap-adjacent modifications in animals

Eukaryotic mRNA cap structures directly influence mRNA stability, translation, and immune recognition. While the significance of the mRNA cap itself has been well-established, recent research has revealed the intricate modifications to the nucleotides immediately adjacent to the cap. These include 2′-O-methylation (Cap1, Cap2) and N6-methyladenosine (m6Am), which recent work suggests may be dynamically regulated, interdependent, and vital for gene-regulation. This perspective explores the expanding role of cap-adjacent modifications. Cap1 is found on metazoan mRNA, viruses which infect metazoans, and the COVID-19 mRNA vaccines. We combined basic local alignment and 2D-TLC to track cap-adjacent modifying proteins and the modifications themselves to profile their prevalence throughout eukaryotes. We confirm that Cap1 is broadly metazoan specific, but surprisingly also in a brown algae. Additionally, we find genomic presence of the eraser, FTO more predictive of the occurrence of m6Am than the m6Am-writer, PCIF1. We then outline future directions to understand and exploit these enigmatic modifications.

YAP promotes global mRNA translation to fuel oncogenic growth despite starvation.

Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play fundamental roles in stem/progenitor cell expansion during homeostasis, and their dysregulation often leads to tissue overgrowth. Here, we show that YAP activation is sufficient to overcome the restriction of global protein synthesis induced by serum starvation, enabling cells to sustain proliferation and survival despite an unfavorable environment. Mechanistically, YAP/TAZ selectively promoted the mTORC1-dependent translation of mRNAs containing 5' terminal oligopyrimidine (5'TOP) motifs, ultimately increasing the cellular polysome content. Interestingly, DNA damage-inducible transcript 4 (DDIT4), a negative regulator of mTORC1, was upregulated by serum starvation but repressed by YAP/TAZ. DDIT4 was sufficient to suppress the translation and transformative potential of uveal melanoma cells, which are often serum unresponsive due to G protein mutations. Our findings reveal a vital role for protein synthesis as a key modality of YAP/TAZ-induced oncogenic transformation and indicate the potential for targeting mTORC1 or translation to treat YAP/TAZ-driven malignancies.

Defective regulation of the eIF2-eIF2B translational axis underlies depressive-like behavior in mice and correlates with major depressive disorder in humans

Major depressive disorder (MDD) is a significant cause of disability in adults worldwide. However, the underlying causes and mechanisms of MDD are not fully understood, and many patients are refractory to available therapeutic options. Impaired control of brain mRNA translation underlies several neurodevelopmental and neurodegenerative conditions, including autism spectrum disorders and Alzheimer’s disease (AD). Nonetheless, a potential role for mechanisms associated with impaired translational control in depressive-like behavior remains elusive. A key pathway controlling translation initiation relies on the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P) which, in turn, blocks the guanine exchange factor activity of eIF2B, thereby reducing global translation rates. Here we report that the expression of EIF2B5 (which codes for eIF2Bε, the catalytic subunit of eIF2B) is reduced in postmortem MDD prefrontal cortex from two distinct human cohorts and in the frontal cortex of social isolation-induced depressive-like behavior model mice. Further, pharmacological treatment with anisomycin or with salubrinal, an inhibitor of the eIF2α phosphatase GADD34, induces depressive-like behavior in adult C57BL/6J mice. Salubrinal-induced depressive-like behavior is blocked by ISRIB, a compound that directly activates eIF2B regardless of the phosphorylation status of eIF2α, suggesting that increased eIF2α-P promotes depressive-like states. Taken together, our results suggest that impaired eIF2-associated translational control may participate in the pathophysiology of MDD, and underscore eIF2-eIF2B translational axis as a potential target for the development of novel approaches for MDD and related mood disorders.

Acute Exercise-induced Endothelial Shear Stress Activates Nos3 Mrna Translation In Vitro

Acute Exercise-induced Endothelial Shear Stress Activates Nos3 Mrna Translation In Vitro

Ribosome Quality Control mitigates the cytotoxicity of ribosome collisions induced by 5-Fluorouracil.

Ribosome quality control (RQC) resolves collided ribosomes, thus preventing their cytotoxic effects. The chemotherapeutic agent 5-Fluorouracil (5FU) is best known for its misincorporation into DNA and inhibition of thymidylate synthase. However, while a major determinant of 5FU's anticancer activity is its misincorporation into RNAs, the mechanisms by which cancer cells overcome the RNA-dependent 5FU toxicity remain ill-defined. Here, we report a role for RQC in mitigating the cytotoxic effects of 5FU. We show that 5FU treatment results in rapid induction of the mTOR signalling pathway, enhanced rate of mRNA translation initiation, and increased ribosome collisions. Consistently, a defective RQC exacerbates the 5FU-induced cell death, which is mitigated by blocking mTOR pathway or mRNA translation initiation. Furthermore, 5FU treatment enhances the expression of the key RQC factors ZNF598 and GIGYF2 via an mTOR-dependent post-translational mechanism. This adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment.

Developing messenger RNA biomarkers: A workflow to characterise and identify transcript target sequences unaffected by alternative splicing for reproducible gene transcript quantification by reverse transcriptase quantitative polymerase chain reaction

AbstractMost eukaryotic genes generate multiple messenger RNA (mRNA) transcript variants by alternative splicing. The incomplete annotation of gene transcripts in genomic databases can result in improper primer design, adversely affecting the reliability of gene expression measurements by reverse transcriptase quantitative polymerase chain reaction (RT‐qPCR). Hence, we present a workflow combining bioinformatics analyses, to select two to three evolutionarily conserved constitutive exons in rats, mice and humans as target sequences for PCR primer design, with experimental RT‐PCR amplification and amplicon sequencing to confirm the expression and identity of gene transcript targets. The application of this workflow to the characterization of neurodevelopmental biomarker genes identified an unannotated exon in the rat Map2 gene, illustrating the importance of target sequence validation for the development of translational mRNA biomarkers for toxicological and biomedical studies.

CEP112 coordinates translational regulation of essential fertility genes during spermiogenesis through phase separation in humans and mice

Spermiogenesis, the complex transformation of haploid spermatids into mature spermatozoa, relies on precise spatiotemporal regulation of gene expression at the post-transcriptional level. The mechanisms underlying this critical process remain incompletely understood. Here, we identify centrosomal protein 112 (CEP112) as an essential regulator of mRNA translation during this critical developmental process. Mutations in CEP112 are discovered in oligoasthenoteratospermic patients, and Cep112-deficient male mice recapitulate key phenotypes of human asthenoteratozoospermia. CEP112 localizes to the neck and atypical centrioles of mature sperm and forms RNA granules during spermiogenesis, enriching target mRNAs such as Fsip2, Cfap61, and Cfap74. Through multi-omics analyses and the TRICK reporter assay, we demonstrate that CEP112 orchestrates the translation of target mRNAs. Co-immunoprecipitation and mass spectrometry identify CEP112’s interactions with translation-related proteins, including hnRNPA2B1, EEF1A1, and EIF4A1. In vitro, CEP112 undergoes liquid-liquid phase separation, forming condensates that recruit essential proteins and mRNAs. Moreover, variants in patient-derived CEP112 disrupt phase separation and impair translation efficiency. Our results suggest that CEP112 mediates the assembly of RNA granules through liquid-liquid phase separation to control the post-transcriptional expression of fertility-related genes. This study not only clarifies CEP112’s role in spermatogenesis but also highlights the role of phase separation in translational regulation, providing insights into male infertility and suggesting potential therapeutic targets.

Peptide-Based Nanoparticles Suppress Hepatic Inflammation via Blockage of Human Antigen R.

Human antigen R (HuR), which is a mRNA-binding protein that stabilizes and regulates mRNA translation, is found to have increased expression in inflammation, cancer and other diseases, making HuR to be a promising drug target. This study reports a peptide-based nanoparticle (NP) system exhibits potent anti-inflammatory activity to ameliorate acute liver injury via the ability of peptides to inhibit the mRNA binding site of HuR and block downstream signaling. Molecular modeling provided structural evidence indicating that the peptides interact with the RNA-binding site of HuR, mainly via hydrogen-bonding and hydrophobic interactions. These peptide-based NPs can act as nanocarriers to deliver peptides into cells to compete with the mRNA binding site of HuR, evidenced by the reduction of antibody recognition to the native protein and the exhibition of anti-inflammatory activity against activated macrophage cells, with no adverse effect in vitro and in vivo. In LPS/D-GalN-induced hepatic sepsis with high dosage of LPS/GalN, administration of the NPs significantly attenuated necrosis and HuR expression, resulting in the significant improvement of animal survival rate, suggesting their therapeutic potential for hepatic inflammation and a broad range of HuR-overexpressed diseases.

Vibrio cholerae CsrA controls ToxR levels by increasing the stability and translation of toxR mRNA.

Vibrio cholerae is uniquely adapted to life in marine environments as well as in the human intestinal tract. Global regulators such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates synthesis of ToxR, a key virulence factor of V. cholerae . Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization, the nutrient-rich small intestinal mucosa.

Genome-wide impact of codon usage bias on translation optimization in Drosophila melanogaster

Accuracy and efficiency are fundamental to mRNA translation. Codon usage bias is widespread across species. Despite the long-standing association between optimized codon usage and improved translation, our understanding of its evolutionary basis and functional effects remains limited. Drosophila is widely used to study codon usage bias, but genome-scale experimental data are scarce. Using high-resolution mass spectrometry data from Drosophila melanogaster, we show that optimal codons have lower translation errors than nonoptimal codons after accounting for these biases. Genomic-scale analysis of ribosome profiling data shows that optimal codons are translated more rapidly than nonoptimal codons. Although we find no long-term selection favoring synonymous mutations in D. melanogaster after diverging from D. simulans, we identify signatures of positive selection driving codon optimization in the D. melanogaster population. These findings expand our understanding of the functional consequences of codon optimization and serve as a foundation for future investigations.

Deoxyhypusine synthase deficiency syndrome zebrafish model: aberrant morphology, epileptiform activity, and reduced arborization of inhibitory interneurons

DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase (DHPS). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5AHYP-dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome.

Circadian regulation of translation.

Most, if not all organisms exhibit robust rhythmicity of their biological functions, allowing a perpetual adaptation to external clues within the daily 24 hours-cycle. Studies on circadian rhythm regulation primarily focused on transcriptional level, considering mRNA levels to represent the primary determinant of oscillations of intracellular protein levels. However, a plethora of emerging evidence suggests that post-transcriptional regulation, particularly rhythmic mRNA translation, is not solely reliant on the oscillation of transcription. Instead, the circadian regulation of mRNA translation plays a critical role as well. A comprehensive understanding of these mechanisms underlying rhythmic translation and its regulation should bridge the gap in rhythm regulation beyond RNA fluctuations in research, and greatly enhance our comprehension of rhythm generation and maintenance. In this review, we summarize the major mechanisms of circadian regulation of translation, including regulation of translation initiation, elongation, and the alteration in rhythmic translation to external stresses, such as endoplasmic reticulum (ER) stress and ageing. We also illuminate the complex interplay between phase separation and mRNA translation. Together, we have summarized various facets of mRNA translation in circadian regulation, to set on forthcoming studies into the intricate regulatory mechanisms underpinning circadian rhythms and their implications for associated disorders.

Proteomic Characterization of Corneal Epithelial and Stromal Cell-Derived Extracellular Vesicles.

Communication between the different layers of the cornea (epithelium and stroma) is a complex, yet crucial element in the corneal healing process. Upon corneal injury, it has been reported that the bi-directional cross talk between the epithelium and stroma via the vesicular secretome, namely, extracellular vesicles (EVs), can lead to accelerated wound closure upon injury. However, the distinct protein markers of EVs derived from human corneal epithelial (HCE) cells, keratocytes (HCKs), fibroblasts (HCFs), and myofibroblasts (HCMs) remain poorly understood. All EVs were enriched for CD81 and showed increased expression levels of ITGAV and FN1 in HCM-EVs compared to HCE- and HCF-EVs. All EVs were negative for GM130 and showed minimal differences in biophysical properties (particle concentration, median particle size, and zeta potential). At the proteomic level, we show that HCM-EVs are enriched with proteins associated with fibrosis pathways, such as COL6A1, COL6A2, MMP1, MMP2, TIMP1, and TIMP2, compared to HCE-, HCK-, and HCF-EVs. Interestingly, HCE-EVs express proteins involved with the EIF-2 signaling pathway (stress-induced signals to regulate mRNA translation), such as RPS21, RALB, EIF3H, RALA, and others, compared to HCK-, HCF-, and HCM-EVs. In this study, we isolated EVs from cell-conditioned media from HCE, HCKs, HCFs, and HCMs and characterized their biophysical and protein composition by Western blot, nanoparticle tracking analysis, and proteomics. This study supports the view that EVs from the corneal epithelium and stroma have a distinct molecular composition and may provide novel protein markers to distinguish the difference between HCE-, HCK-, HCF-, and HCM-EVs.

Structure and Dynamics of the CCCH-Type Tandem Zinc Finger Domain of POS-1 and Implications for RNA Binding Specificity.

CCCH-type tandem zinc finger (TZF) motifs are found in many RNA-binding proteins involved in regulating mRNA stability, translation, and splicing. In Caenorhabditis elegans, several RNA-binding proteins that regulate embryonic development and cell fate determination contain CCCH TZF domains, including POS-1. Previous biochemical studies have shown that despite high levels of sequence conservation, POS-1 recognizes a broader set of RNA sequences compared to the human homologue tristetraprolin. However, the molecular basis of these differences remains unknown. In this study, we refined the consensus RNA sequence and determined the differing binding specificities of the two zinc fingers of POS-1. We also determined the solution structure and characterized the internal dynamics of the TZF domain of POS-1. From the structure, we identified unique features that define the RNA binding specificity of POS-1. We also observed that the TZF domain of POS-1 is in equilibrium between interconverting conformations. Transitions between these conformations require internal motions involving many residues with correlated dynamics in each ZF. We propose that the correlated dynamics are necessary to allow allosteric communication between the nucleotide-binding pockets observed in the N-terminal ZF. Our study shows that both the structure and conformational plasticity of POS-1 are important in ensuring recognition of its RNA binding targets.

MiRNA-mediated gene silencing in Drosophila larval development involves GW182-dependent and independent mechanisms.

MicroRNAs (miRNAs) regulate a wide variety of biological processes by silencing their target genes. Argonaute (AGO) proteins load miRNAs to form an RNA-induced silencing complex (RISC), which mediates translational repression and/or mRNA decay of the targets. A scaffold protein called GW182 directly binds AGO and the CCR4-NOT deadenylase complex, initiating the mRNA decay reaction. Although previous studies have demonstrated the critical role of GW182 in cultured cells as well as in cell-free systems, its biological significance in living organisms remains poorly explored, especially in Drosophila melanogaster. Here, we generated gw182-null flies using the CRISPR/Cas9 system and found that, unexpectedly, they can survive until an early second-instar larval stage. Moreover, in vivo miRNA reporters can be effectively repressed in gw182-null first-instar larvae. Nevertheless, gw182-null flies have defects in the expression of chitin-related genes and the formation of the larval trachea system, preventing them from completing larval development. Our results highlight the importance of both GW182-dependent and -independent silencing mechanisms in vivo.

IGF2BP1-mediated the stability and protein translation of FGFR1 mRNA regulates myogenesis through the ERK signaling pathway

N6-methyladenosine (m6A) is the most prevalent post-transcriptional modification of RNAs and plays a key regulatory role in various biological processes. As a member of the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) family, IGF2BP1 has recently demonstrated its ability to specifically bind m6A-modified sites within mRNAs and effectively regulate their mRNA stability. However, the precise roles of IGF2BP1 in mammalian skeletal muscle development, along with its downstream mRNA targets during myogenesis, have yet to be fully elucidated. Here, we observed that IGF2BP1 expression significantly decreased during myogenic differentiation. Knockdown of IGF2BP1 significantly inhibited myoblast proliferation while promoted myogenic differentiation. In contrast, IGF2BP1 overexpression robustly stimulated myoblast proliferation but suppressed their differentiation. Combined analysis of high-throughput sequencing and RNA stability assays revealed that IGF2BP1 can enhance fibroblast growth factor receptor 1 (FGFR1) mRNA stability and promote its translation in an m6A-dependent manner, thereby regulating its expression level and the Extracellular Signal-Regulated Kinase (ERK) pathway. Additionally, knockdown of FGFR1 rescued the phenotypic changes (namely increased cell proliferation and suppressed differentiation) induced by IGF2BP1 overexpression via attenuating ERK signaling. Taken together, our findings suggest that IGF2BP1 maintains the stability and translation of FGFR1 mRNA in an m6A-dependent manner, thereby inhibiting skeletal myogenesis through activation of the ERK signaling pathway. This study further enriches the understanding of the molecular mechanisms by which RNA methylation regulates myogenesis, providing valuable insights into the role of IGF2BP1-mediated post-transcriptional regulation in muscle development.