Regulation Of mRNA Translation Research Articles

Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner.

Stress response regulation of mRNA translation: Implications for antioxidant enzyme expression in cancer

From tumorigenesis to advanced metastatic stages, tumor cells encounter stress, ranging from limited nutrient and oxygen supply within the tumor microenvironment to extrinsic and intrinsic oxidative stress. Thus, tumor cells seize regulatory pathways to rapidly adapt to distinct physiologic conditions to promote cellular survival, including manipulation of mRNA translation. While it is now well established that metastatic tumor cells must up-regulate their antioxidant capacity to effectively spread and that regulation of antioxidant enzymes is imperative to disease progression, relatively few studies have assessed how translation and the hijacking of RNA systems contribute to antioxidant responses of tumors. Here, we review the major stress signaling pathways involved in translational regulation and discuss how these are affected by oxidative stress to promote prosurvival changes that manipulate antioxidant enzyme expression. We describe how tumors elicit these adaptive responses and detail how stress-induced translation can be regulated by kinases, RNA-binding proteins, RNA species, and RNA modification systems. We also highlight opportunities for further studies focused on the role of mRNA translation and RNA systems in the regulation of antioxidant enzyme expression, which may be of particular importance in the context of metastatic progression and therapeutic resistance.

Investigating the Activities of CAF20 and ECM32 in the Regulation of PGM2 mRNA Translation

Translation is a fundamental process in biology, and understanding its mechanisms is crucial to comprehending cellular functions and diseases. The regulation of this process is closely linked to the structure of mRNA, as these regions prove vital to modulating translation efficiency and control. Thus, identifying and investigating these fundamental factors that influence the processing and unwinding of structured mRNAs would be of interest due to the widespread impact in various fields of biology. To this end, we employed a computational approach and identified genes that may be involved in the translation of structured mRNAs. The approach is based on the enrichment of interactions and co-expression of genes with those that are known to influence translation and helicase activity. The in silico prediction found CAF20 and ECM32 to be highly ranked candidates that may play a role in unwinding mRNA. The activities of neither CAF20 nor ECM32 have previously been linked to the translation of PGM2 mRNA or other structured mRNAs. Our follow-up investigations with these two genes provided evidence of their participation in the translation of PGM2 mRNA and several other synthetic structured mRNAs.

Activation of platelet-derived growth factor receptors regulate connective tissue growth factor protein levels via the AKT pathway in malignant mesothelioma cells.

The incidence of malignant mesothelioma (MM), a disease linked to refractory asbestos exposure, continues to increase globally, and remains largely resistant to various treatments. Our previous studies have identified a strong correlation between connective tissue growth factor (CTGF) protein expression and MM malignancy, underscoring the importance of understanding CTGF regulation in MM cells. In this study, we demonstrate for the first time that stimulation with platelet-derived growth factor receptor (PDGFR) ligand, PDGF-BB, increases CTGF protein expression levels without affecting CTGF mRNA levels. Inhibition of PDGFR resulted in a reduction of CTGF protein expression, indicating that PDGFR activation is essential in regulating CTGF protein expression in MM cells. PDGF-BB also activated the protein kinase B (AKT) pathway, and inhibition of AKT phosphorylation abolished the PDGFR-induced CTGF protein expression, suggesting that PDGFR acts upstream of CTGF via the AKT pathway. This reinforces the role of CTGF protein as a key regulator of MM malignancy. Additionally, PDGFR activation led to the phosphorylation of mTOR and 4E-BP1, critical regulators of protein synthesis downstream of AKT, suggesting that PDGFR controls CTGF protein expression through the regulation of CTGF mRNA translation.

AMBRA1 controls the translation of immune-specific genes in T lymphocytes

T cell receptor (TCR) engagement causes a global cellular response that entrains signaling pathways, cell cycle regulation, and cell death. The molecular regulation of mRNA translation in these processes is poorly understood. Using a whole-genome CRISPR screen for regulators of CD95 (FAS/APO-1)-mediated T cell death, we identified AMBRA1, a protein previously studied for its roles in autophagy, E3 ubiquitin ligase activity, and cyclin regulation. T cells lacking AMBRA1 resisted FAS-mediated cell death by down-regulating FAS expression at the translational level. We show that AMBRA1 is a vital regulator of ribosome protein biosynthesis and ribosome loading on select mRNAs, whereby it plays a key role in balancing TCR signaling with cell cycle regulation pathways. We also found that AMBRA1 itself is translationally controlled by TCR stimulation via the CD28-PI3K-mTORC1-EIF4F pathway. Together, these findings shed light on the molecular control of translation after T cell activation and implicate AMBRA1 as a translational regulator governing TCR signaling, cell cycle progression, and T cell death.

M6A reader YTHDF2 governs the onset of atrial fibrillation by modulating Cacna1c translation.

Atrial fibrillation (AF) is the most common arrhythmia, which is tightly associated with the abnormal expression and function of ion channels in the atrial cardiomyocytes. N6-methyladenosine (m6A), a widespread chemical modification in eukaryotic mRNA, is known to play a significant regulatory role in the pathogenesis of heart disease. However, the significance of m6A regulatory proteins in the onset of AF remains unclear. Here, we demonstrate that the m6A reader protein YTHDF2 regulates atrial electrical remodeling and AF onset by modulating the Cav1.2 expression. Firstly, YTHDF2 expression was selectively upregulated in rat atrial cardiomyocytes with AF. Secondly, YTHDF2 knockout reduced AF susceptibility in mice. Thirdly, the knockout of YTHDF2 increased Cav1.2 protein levels in an m6A-in-dependent manner, ultimately prolonging the atrial myocardial refractory period, a critical electrophysiological substrate for the onset of AF. Fourthly, the N-terminal domain of YTHDF2 was identified as critical for Cacna1c mRNA translation regulation. Overall, our findings unveil that YTHDF2 can alter Cav1.2 protein expression in an m6A-independent manner, thereby facilitating the onset of AF. Our study suggests that YTHDF2 may be a potential intervention target for AF.

Transient Inhibition of Translation Improves Cardiac Function After Ischemia/Reperfusion by Attenuating the Inflammatory Response.

The myocardium adapts to ischemia/reperfusion (I/R) by changes in gene expression, determining the cardiac response to reperfusion. mRNA translation is a key component of gene expression. It is largely unknown how regulation of mRNA translation contributes to cardiac gene expression and inflammation in response to reperfusion and whether it can be targeted to mitigate I/R injury. To examine translation and its impact on gene expression in response to I/R, we measured protein synthesis after reperfusion in vitro and in vivo. Underlying mechanisms of translational control were examined by pharmacological and genetic targeting of translation initiation in mice. Cell type-specific ribosome profiling was performed in mice that had been subjected to I/R to determine the impact of mRNA translation on the regulation of gene expression in cardiomyocytes. Translational regulation of inflammation was studied by quantification of immune cell infiltration, inflammatory gene expression, and cardiac function after short-term inhibition of translation initiation. Reperfusion induced a rapid recovery of translational activity that exceeds baseline levels in the infarct and border zone and is mediated by translation initiation through the mTORC1 (mechanistic target of rapamycin complex 1)-4EBP1 (eIF4E-binding protein 1)-eIF (eukaryotic initiation factor) 4F axis. Cardiomyocyte-specific ribosome profiling identified that I/R increased translation of mRNA networks associated with cardiac inflammation and cell infiltration. Short-term inhibition of the mTORC1-4EBP1-eIF4F axis decreased the expression of proinflammatory cytokines such as Ccl2 (C-C motif chemokine ligand 2) of border zone cardiomyocytes, thereby attenuating Ly6Chi monocyte infiltration and myocardial inflammation. In addition, we identified a systemic immunosuppressive effect of eIF4F translation inhibitors on circulating monocytes, directly inhibiting monocyte infiltration. Short-term pharmacological inhibition of eIF4F complex formation by 4EGI-1 or rapamycin attenuated translation, reduced infarct size, and improved cardiac function after myocardial infarction. Global protein synthesis is inhibited during ischemia and shortly after reperfusion, followed by a recovery of protein synthesis that exceeds baseline levels in the border and infarct zones. Activation of mRNA translation after reperfusion is driven by mTORC1/eIF4F-mediated regulation of initiation and mediates an mRNA network that controls inflammation and monocyte infiltration to the myocardium. Transient inhibition of the mTORC1-/eIF4F axis inhibits translation and attenuates Ly6Chi monocyte infiltration by inhibiting a proinflammatory response at the site of injury and of circulating monocytes.

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.

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.

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.

MiRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity

Activity-dependent protein synthesis is crucial for long-lasting forms of synaptic plasticity. However, our understanding of translational mechanisms controlling GABAergic synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the mechanisms controlling plasticity-induced gephyrin translation remain unknown. We identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting inhibitory synaptic structure and function. iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and promoting de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Together, we delineate a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

Crosstalk between ubiquitination and translation in neurodevelopmental disorders.

Ubiquitination is one of the most conserved post-translational modifications and together with mRNA translation contributes to cellular protein homeostasis (proteostasis). Temporal and spatial regulation of proteostasis is particularly important during synaptic plasticity, when translation of specific mRNAs requires tight regulation. Mutations in genes encoding regulators of mRNA translation and in ubiquitin ligases have been associated with several neurodevelopmental disorders. RNA metabolism and translation are regulated by RNA-binding proteins, critical for the spatial and temporal control of translation in neurons. Several ubiquitin ligases also regulate RNA-dependent mechanisms in neurons, with numerous ubiquitination events described in splicing factors and ribosomal proteins. Here we will explore how ubiquitination regulates translation in neurons, from RNA biogenesis to alternative splicing and how dysregulation of ubiquitin signaling can be the underlying cause of pathology in neurodevelopmental disorders, such as Fragile X syndrome. Finally we propose that targeting ubiquitin signaling is an attractive novel therapeutic strategy for neurodevelopmental disorders where mRNA translation and ubiquitin signaling are disrupted.

Methyltransferase ATMETTL5 writes m6A on 18S ribosomal RNA to regulate translation in Arabidopsis.

Aberrant RNA modifications can lead to dysregulated gene expression and impeded growth in plants. Ribosomal RNA (rRNA) constitutes a substantial portion of total RNA, while the precise functions and molecular mechanisms underlying rRNA modifications in plants remain largely elusive. Here, we elucidated the exclusive occurrence of the canonical RNA modification N6-methyladenosine (m6A) solely 18S rRNA, but not 25S rRNA. We identified a completely uncharacterized protein, ATMETTL5, as an Arabidopsis m6A methyltransferase responsible for installing m6A methylation at the 1771 site of the 18S rRNA. ATMETTL5 is ubiquitously expressed and localized in both nucleus and cytoplasm, mediating rRNA m6A methylation. Mechanistically, the loss of ATMETTL5-mediated methylation results in attenuated translation. Furthermore, we uncovered the role of ATMETTL5-mediated methylation in coordinating blue light-mediated hypocotyl growth by regulating the translation of blue light-related messenger RNAs (mRNAs), specifically HYH and PRR9. Our findings provide mechanistic insights into how rRNA modification regulates ribosome function in mRNA translation and the response to blue light, thereby advancing our understanding of the role of epigenetic modifications in precisely regulating mRNA translation in plants.

What goes up must come down: off switches for regulatory RNAs.

Small RNAs base pair with and regulate mRNA translation and stability. For both bacterial small regulatory RNAs and eukaryotic microRNAs, association with partner proteins is critical for the stability and function of the regulatory RNAs. We review the mechanisms for degradation of these RNAs: displacement of the regulatory RNA from its protein partner (in bacteria) or destruction of the protein and its associated microRNAs (in eukaryotes). These mechanisms can allow specific destruction of a regulatory RNA via pairing with a decay trigger RNA or function as global off switches by disrupting the stability or function of the protein partner.

SMYD5 methylation of rpL40 links ribosomal output to gastric cancer.

Dysregulated transcription due to disruption in histone lysine methylation dynamics is an established contributor to tumorigenesis1,2. However, whether analogous pathologic epigenetic mechanisms act directly on the ribosome to advance oncogenesis is unclear. Here we find that trimethylation of the core ribosomal protein L40 (rpL40) at lysine 22 (rpL40K22me3) by the lysine methyltransferase SMYD5 regulates mRNA translation output to promote malignant progression of gastric adenocarcinoma (GAC) with lethal peritoneal ascites. A biochemical-proteomics strategy identifies the monoubiquitin fusion protein partner rpL40 (ref. 3) as the principal physiological substrate of SMYD5 across diverse samples. Inhibiting the SMYD5-rpL40K22me3 axis in GAC cell lines reprogrammes protein synthesis to attenuate oncogenic gene expression signatures. SMYD5 and rpL40K22me3 are upregulated in samples from patients with GAC and negatively correlate with clinical outcomes. SMYD5 ablation in vivo in familial and sporadic mouse models of malignant GAC blocks metastatic disease, including peritoneal carcinomatosis. Suppressing SMYD5 methylation of rpL40 inhibits human cancer cell and patient-derived GAC xenograft growth and renders them hypersensitive to inhibitors of PI3K and mTOR. Finally, combining SMYD5 depletion with PI3K-mTOR inhibition and chimeric antigen receptor T cell administration cures an otherwise lethal in vivo mouse model of aggressive GAC-derived peritoneal carcinomatosis. Together, our work uncovers a ribosome-based epigenetic mechanism that facilitates the evolution of malignant GAC and proposes SMYD5 targeting as part of a potential combination therapy to treat this cancer.

The Role of Long Non-Coding RNF144A-AS1 in Cancer Progression.

RNAs transcribing more than 200 nucleotides without encoding proteins are termed long non-coding RNAs (LncRNAs). LncRNAs can be used as decoy molecules, signal molecules, scaffolds, and guide molecules. Long non-coding RNAs can interact with DNA, chromatin-modifying complexes, and transcriptional regulatory proteins, regulating gene expression in the cell nucleus. It is distributed in cytoplasm; they also participate in mRNA degradation and translational regulation via miRNAs, other transcription products, and proteins. They play a significant role in the development of various diseases, including tumors. Cancer seriously threatens human life and health. Regretfully, a great deal of newly diagnosed cancer patients found to have metastasized. RNF144A-AS1, also referred to as GRASLND, was initially recognized for its regulation of chondrogenic differentiation in MSCs. Focusing on RNF144A-AS1, this review summarizes and discusses the latest progress of RNF144A-AS1 in bladder cancer, glioblastoma, papillary renal cell carcinoma, gastric cancer, osteosarcoma, head and neck squamous cell carcinoma, and ovarian cancer. RNF144A-AS1 has good potential in tumor treatment and diagnosis.

Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

Non-Coding RNA in Tumor Cells and Tumor-Associated Myeloid Cells-Function and Therapeutic Potential.

The RNA world is wide, and besides mRNA, there is a variety of other RNA types, such as non-coding (nc)RNAs, which harbor various intracellular regulatory functions. This review focuses on small interfering (si)RNA and micro (mi)RNA, which form a complex network regulating mRNA translation and, consequently, gene expression. In fact, these RNAs are critically involved in the function and phenotype of all cells in the human body, including malignant cells. In cancer, the two main targets for therapy are dysregulated cancer cells and dysfunctional immune cells. To exploit the potential of mi- or siRNA therapeutics in cancer therapy, a profound understanding of the regulatory mechanisms of RNAs and following targeted intervention is needed to re-program cancer cells and immune cell functions in vivo. The first part focuses on the function of less well-known RNAs, including siRNA and miRNA, and presents RNA-based technologies. In the second part, the therapeutic potential of these technologies in treating cancer is discussed, with particular attention on manipulating tumor-associated immune cells, especially tumor-associated myeloid cells.

Dysregulated ribosome quality control in human diseases.

Precise regulation of mRNA translation is of fundamental importance for maintaining homeostasis. Conversely, dysregulated general or transcript-specific translation, as well as abnormal translation events, have been linked to a multitude of diseases. However, driven by the misconception that the transient nature of mRNAs renders their abnormalities inconsequential, the importance of mechanisms that monitor the quality and fidelity of the translation process has been largely overlooked. In recent years, there has been a dramatic shift in this paradigm, evidenced by several seminal discoveries on the role of a key mechanism in monitoring the quality of mRNA translation - namely, Ribosome Quality Control (RQC) - in the maintenance of homeostasis and the prevention of diseases. Here, we will review recent advances in the field and emphasize the biological significance of the RQC mechanism, particularly its implications in human diseases.