Translational Regulatory Mechanisms Research Articles

Multi-omics integration analysis reveals the role of N6-methyladenosine in lncRNA translation during glioma stem cell differentiation.

Glioblastoma is one of the most lethal brain diseases in humans. Although recent studies have shown reciprocal interactions between N6-methyladenosine (m6A) modifications and long noncoding RNAs (lncRNAs) in gliomagenesis and malignant progression, the mechanism of m6A-mediated lncRNA translational regulation in glioblastoma remains unclear. Herein, we profiled the transcriptomes, translatomes, and epitranscriptomics of glioma stem cells and differentiated glioma cells to investigate the role of m6A in lncRNA translation comprehensively. We found that lncRNAs with numerous m6A peaks exhibit reduced translation efficiency. Transcript-level expression analysis demonstrates an enrichment of m6A around short open reading frames (sORFs) of translatable lncRNA transcripts. Further comparison analysis of m6A modifications in different RNA regions indicates that m6A peaks downstream of sORFs inhibit lncRNA translation more than those upstream. Observations in glioma-associated lncRNAs H19, LINC00467, and GAS5 further confirm the negative effect of m6A methylation on lncRNA translation. Overall, these findings elucidate the dynamic profiles of the m6A methylome and enhance the understanding of the complexity of lncRNA translational regulation.

Differential expression of small RNAs in biofilm-producing clinical methicillin-susceptible Staphylococcus aureus recovered from human urine

Bacterial small RNAs (sRNAs) play crucial roles in coordinating gene regulatory networks in various physiological processes, including biofilm formation. In this study, RNA sequencing was performed on biofilm (n = 4) and planktonic (n = 4) cells harvested at 10 h (pre-stationary phase of biofilm development) to identify biofilm-associated sRNAs in human methicillin-susceptible Staphylococcus aureus (MSSA) recovered from urine isolate. A total of 56 highly expressed sRNAs were identified with 15 overlapping sRNA genes (srn_9348, sprD, sRNA205, sRNA288, srn_2467, Sau-25, srn_2468, sRNA260, sRNA200, RsaE, sRNA397, Teg55, Teg60, RsaX05 and Teg140). Further validation through RT-qPCR analysis of nine sRNAs revealed that srn_9348 and sRNA260 were significantly expressed in the biofilm cells of urine sample. Both sRNAs were predicted to interact with mRNA genes including intracellular adhesin A (icaA) and host factor protein (hfq) involved in biofilm formation via cis-acting and trans-acting using CopraRNA analysis. Therefore, both sRNAs merit further investigations via reverse genetic approaches to elucidate their mechanism of translational regulation. In summary, the transcriptomic analysis conducted in this study offers new insights into the potential regulatory roles of sRNAs in MSSA biofilm development within the urinary environment.

Repression of mRNA translation initiation by GIGYF1 via disrupting the eIF3-eIF4G1 interaction.

Viruses can selectively repress the translation of mRNAs involved in the antiviral response. RNA viruses exploit the Grb10-interacting GYF (glycine-tyrosine-phenylalanine) proteins 2 (GIGYF2) and eukaryotic translation initiation factor 4E (eIF4E) homologous protein 4EHP to selectively repress the translation of transcripts such as Ifnb1, which encodes the antiviral cytokine interferon-β (IFN-β). Herein, we reveal that GIGYF1, a paralog of GIGYF2, robustly represses cellular mRNA translation through a distinct 4EHP-independent mechanism. Upon recruitment to a target mRNA, GIGYF1 binds to subunits of eukaryotic translation initiation factor 3 (eIF3) at the eIF3-eIF4G1 interaction interface. This interaction disrupts the eIF3 binding to eIF4G1, resulting in transcript-specific translational repression. Depletion of GIGYF1 induces a robust immune response by derepressing IFN-β production. Our study highlights a unique mechanism of translational regulation by GIGYF1 that involves sequestering eIF3 and abrogating its binding to eIF4G1. This mechanism has profound implications for the host response to viral infections.

Prokaryotic RNA N1-Methyladenosine Erasers Maintain tRNA m1A Modification Levels in Streptomyces venezuelae.

tRNA modifications help maintain tRNA structure and facilitate translation and stress response. Found in all three kingdoms of life, m1A tRNA modification occurs in the T loop of many tRNAs, stabilizes tertiary tRNA structure, and impacts translation. M1A in the T loop is reversible by three mammalian demethylase enzymes, which bypasses the need of turning over the tRNA molecule to adjust its m1A levels in cells. However, no prokaryotic tRNA demethylase enzyme has been identified that acts on endogenous RNA modifications. Using Streptomyces venezuelae as a model organism, we confirmed the presence and quantitative m1A tRNA signatures using mass spectrometry and high-throughput tRNA sequencing. We identified two RNA demethylases that can remove m1A in tRNA and validated the activity of a previously annotated tRNA m1A writer. Using single-gene knockouts of these erasers and the m1A writer, we found dynamic changes of m1A levels in many tRNAs under stress conditions. Phenotypic characterization highlighted changes in their growth and altered antibiotic production. Our identification of the first prokaryotic tRNA demethylase enzyme paves the way for investigating new mechanisms of translational regulation in bacteria.

Characterised intron retention profiles in muscle tissue of idiopathic inflammatory myopathy subtypes

ObjectivesIdiopathic inflammatory myopathies (IIMs) are a group of heterogeneous autoimmune diseases. Intron retention (IR) serves as an important post-transcriptional and translational regulatory mechanism. This study aims to identify changes in...

Context-Aware Poly(A) Signal Prediction Model via Deep Spatial-Temporal Neural Networks.

Polyadenylation [Poly(A)] is an essential process during messenger RNA (mRNA) maturation in biological eukaryote systems. Identifying Poly(A) signals (PASs) from the genome level is the key to understanding the mechanism of translation regulation and mRNA metabolism. In this work, we propose a deep dual-dynamic context-aware Poly(A) signal prediction model, called multiscale convolution with self-attention networks (MCANet), to adaptively uncover the spatial-temporal contextual dependence information. Specifically, the model automatically learns and strengthens informative features from the temporalwise and the spatialwise dimension. The identity connectivity performs contextual feature maps of Poly(A) data by direct connections from previous layers to subsequent layers. Then, a fully parametric rectified linear unit (FP-RELU) with dual-dynamic coefficients is devised to make the training of the model easier and enhance the generalization ability. A cross-entropy loss (CL) function is designed to make the model focus on samples that are easy to misclassify. Experiments on different Poly(A) signals demonstrate the superior performance of the proposed MCANet, and an ablation study shows the effectiveness of the network design for the feature learning and prediction of Poly(A) signals.

Abstract 2172 Identifying a Novel Mechanism of Translation Regulation: The ADP-dependent Activation of eEF-2K

Abstract 2172 Identifying a Novel Mechanism of Translation Regulation: The ADP-dependent Activation of eEF-2K

Decryption of sequence, structure, and functional features of SINE repeat elements in SINEUP non-coding RNA-mediated post-transcriptional gene regulation

RNA structure folding largely influences RNA regulation by providing flexibility and functional diversity. In silico and in vitro analyses are limited in their ability to capture the intricate relationships between dynamic RNA structure and RNA functional diversity present in the cell. Here, we investigate sequence, structure and functional features of mouse and human SINE-transcribed retrotransposons embedded in SINEUPs long non-coding RNAs, which positively regulate target gene expression post-transcriptionally. In-cell secondary structure probing reveals that functional SINEs-derived RNAs contain conserved short structure motifs essential for SINEUP-induced translation enhancement. We show that SINE RNA structure dynamically changes between the nucleus and cytoplasm and is associated with compartment-specific binding to RBP and related functions. Moreover, RNA–RNA interaction analysis shows that the SINE-derived RNAs interact directly with ribosomal RNAs, suggesting a mechanism of translation regulation. We further predict the architecture of 18 SINE RNAs in three dimensions guided by experimental secondary structure data. Overall, we demonstrate that the conservation of short key features involved in interactions with RBPs and ribosomal RNA drives the convergent function of evolutionarily distant SINE-transcribed RNAs.

The Role of tRNA-Centered Translational Regulatory Mechanisms in Cancer.

Cancer is a leading cause of morbidity and mortality worldwide. While numerous factors have been identified as contributing to the development of malignancy, our understanding of the mechanisms involved remains limited. Early cancer detection and the development of effective treatments are therefore critical areas of research. One class of molecules that play a crucial role in the transmission of genetic information are transfer RNAs (tRNAs), which are the most abundant RNA molecules in the human transcriptome. Dysregulated synthesis of tRNAs directly results in translation disorders and diseases, including cancer. Moreover, various types of tRNA modifications and the enzymes responsible for these modifications have been implicated in tumor biology. Furthermore, alterations in tRNA modification can impact tRNA stability, and impaired stability can prompt the cleavage of tRNAs into smaller fragments known as tRNA fragments (tRFs). Initially believed to be random byproducts lacking any physiological function, tRFs have now been redefined as non-coding RNA molecules with distinct roles in regulating RNA stability, translation, target gene expression, and other biological processes. In this review, we present recent findings on translational regulatory models centered around tRNAs in tumors, providing a deeper understanding of tumorigenesis and suggesting new directions for cancer treatment.

The integrated stress response protects against ER stress but is not required for altered translation and lifespan from dietary restriction in Caenorhabditis elegans.

The highly conserved integrated stress response (ISR) reduces and redirects mRNA translation in response to certain forms of stress and nutrient limitation. It is activated when kinases phosphorylate a key residue in the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). General Control Nonderepressible-2 (GCN2) is activated to phosphorylate eIF2α by the presence of uncharged tRNA associated with nutrient scarcity, while protein kinase R-like ER kinase-1 (PERK) is activated during the ER unfolded protein response (UPRER). Here, we investigated the role of the ISR during nutrient limitation and ER stress with respect to changes in protein synthesis, translationally driven mRNA turnover, and survival in Caenorhabditis elegans. We found that, while GCN2 phosphorylates eIF2α when nutrients are restricted, the ability to phosphorylate eIF2α is not required for changes in translation, nonsense-mediated decay, or lifespan associated with dietary restriction (DR). Interestingly, loss of both GCN2 and PERK abolishes increased lifespan associated with dietary restriction, indicating the possibility of other substrates for these kinases. The ISR was not dispensable under ER stress conditions, as demonstrated by the requirement for PERK and eIF2α phosphorylation for decreased translation and wild type-like survival. Taken together, results indicate that the ISR is critical for ER stress and that other translation regulatory mechanisms are sufficient for increased lifespan under dietary restriction.

A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates β-Cell Identity and Function.

As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of beta cell identity and function. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.

Transcriptomic and Translatomic Analyses Reveal Insights into the Signaling Pathways of the Innate Immune Response in the Spleens of SPF Chickens Infected with Avian Reovirus.

Avian reovirus (ARV) infection is prevalent in farmed poultry and causes viral arthritis and severe immunosuppression. The spleen plays a very important part in protecting hosts against infectious pathogens. In this research, transcriptome and translatome sequencing technology were combined to investigate the mechanisms of transcriptional and translational regulation in the spleen after ARV infection. On a genome-wide scale, ARV infection can significantly reduce the translation efficiency (TE) of splenic genes. Differentially expressed translational efficiency genes (DTEGs) were identified, including 15 upregulated DTEGs and 396 downregulated DTEGs. These DTEGs were mainly enriched in immune regulation signaling pathways, which indicates that ARV infection reduces the innate immune response in the spleen. In addition, combined analyses revealed that the innate immune response involves the effects of transcriptional and translational regulation. Moreover, we discovered the key gene IL4I1, the most significantly upregulated gene at both the transcriptional and translational levels. Further studies in DF1 cells showed that overexpression of IL4I1 could inhibit the replication of ARV, while inhibiting the expression of endogenous IL4I1 with siRNA promoted the replication of ARV. Overexpression of IL4I1 significantly downregulated the mRNA expression of IFN-β, LGP2, TBK1 and NF-κB; however, the expression of these genes was significantly upregulated after inhibition of IL4I1, suggesting that IL4I1 may be a negative feedback effect of innate immune signaling pathways. In addition, there may be an interaction between IL4I1 and ARV σA protein, and we speculate that the IL4I1 protein plays a regulatory role by interacting with the σA protein. This study not only provides a new perspective on the regulatory mechanisms of the innate immune response after ARV infection but also enriches the knowledge of the host defense mechanisms against ARV invasion and the outcome of ARV evasion of the host's innate immune response.

Metabolic flux phenotyping of secondary metabolism in cyanobacteria.

Cyanobacteria generate energy from photosynthesis and produce various secondary metabolites with diverse commercial and pharmaceutical applications. Unique metabolic and regulatory pathways in cyanobacteria present new challenges for researchers to enhance their product yields, titers, and rates. Therefore, further advancements are critically needed to establish cyanobacteria as a preferred bioproduction platform. Metabolic flux analysis (MFA) quantitatively determines the intracellular flows of carbon within complex biochemical networks, which elucidate the control of metabolic pathways by transcriptional, translational, and allosteric regulatory mechanisms. The emerging field of systems metabolic engineering (SME) involves the use of MFA and other omics technologies to guide the rational development of microbial production strains. This review highlights the potential of MFA and SME to optimize the production of cyanobacterial secondary metabolites and discusses the technical challenges that lie ahead.

MiR-218 Promotes Dopaminergic Differentiation and Controls Neuron Excitability and Neurotransmitter Release through the Regulation of a Synaptic-Related Genes Network.

In the brain, microRNAs (miRNAs) are believed to play a role in orchestrating synaptic plasticity at a higher level by acting as an additional mechanism of translational regulation, alongside the mRNA/polysome system. Despite extensive research, our understanding of the specific contribution of individual miRNA to the function of dopaminergic neurons (DAn) remains limited. By performing a dopaminergic-specific miRNA screening, we have identified miR-218 as a critical regulator of DAn activity in male and female mice. We have found that miR-218 is specifically expressed in mesencephalic DAn and is able to promote dopaminergic differentiation of embryonic stem cells and functional maturation of transdifferentiated induced DA neurons. Midbrain-specific deletion of both genes encoding for miR-218 (referred to as miR-218-1 and mir218-2) affects the expression of a cluster of synaptic-related mRNAs and alters the intrinsic excitability of DAn, as it increases instantaneous frequencies of evoked action potentials, reduces rheobase current, affects the ionic current underlying the action potential after hyperpolarization phase, and reduces dopamine efflux in response to a single electrical stimulus. Our findings provide a comprehensive understanding of the involvement of miR-218 in the dopaminergic system and highlight its role as a modulator of dopaminergic transmission.SIGNIFICANCE STATEMENT In the past decade, several miRNAs have emerged as potential regulators of synapse activity through the modulation of specific gene expression. Among these, we have identified a dopaminergic-specific miRNA, miR-218, which is able to promote dopaminergic differentiation and regulates the translation of an entire cluster of synapse related mRNAs. Deletion of miR-218 has notable effects on dopamine release and alters the intrinsic excitability of dopaminergic neurons, indicating a direct control of dopaminergic activity by miR-218.

AGEs induce high expression of Dll4 via endoplasmic reticulum stress PERK signaling-mediated internal ribosomal entry site mechanism in macrophages

Background and aimAdvanced glycation end products (AGEs)- exposed macrophages was characterized by Delta-like ligand 4 (Dll4) high expressed and has been shown to participate in diabetes-related atherosclerosis. This study was aimed to investigate the translational regulatory mechanism of Dll4 high expression in macrophages exposed to AGEs. MethodsHuman Dll4 5′ untranslated region (5′UTR) sequence was cloned and inserted into a bicistronic reporter plasmid. Human THP-1 macrophages transfected with the bicistronic reporter plasmids were exposed to AGEs. Dual-luciferase assay was used to detect internal ribosome entry site (IRES) activity contained in Dll4 5′UTR. Small interference RNA transfection was used to knock-down specific gene expression. Localization of protein was analyzed. ResultsAGEs exposure significantly induced IRES activity in Dll4 5′ UTR in human macrophages. Internal potential promoter and ribosome read-through mechanisms were excluded. Inhibition of endoplasmic reticulum stress and specific silencing of protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) signaling pathway activation reduced IRES activity in Dll4 5′ UTR in human macrophages. Dll4 5′ UTR IRES activity was also inhibited by targeted silencing of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Moreover, specific inhibition of PERK/eIF2α signaling pathway led to deactivation of hnRNPA1, resulting to reduction of AGEs- induced Dll4 5’ UTR IRES activity in human macrophages. ConclusionsAGEs induced Dll4 5′ UTR IRES activity in human macrophages which was dependent on endoplasmic reticulum stress PERK/eIF2α signaling pathway. hnRNPA1 acted the role as an ITAF was also indispensable for AGEs-induced Dll4 5′UTR IRES activity in human macrophages.

Comprehensive quantitative modeling of translation efficiency in a genome-reduced bacterium.

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

Synergistic, long-term effects of glutamate dehydrogenase 1 deficiency and mild stress on cognitive function and mPFC gene and miRNA expression

Glutamate abnormalities in the medial prefrontal cortex (mPFC) are associated with cognitive deficits. We previously showed that homozygous deletion of CNS glutamate dehydrogenase 1 (Glud1), a metabolic enzyme critical for glutamate metabolism, leads to schizophrenia-like behavioral abnormalities and increased mPFC glutamate; mice heterozygous for CNS Glud1 deletion (C-Glud1+/− mice) showed no cognitive or molecular abnormalities. Here, we examined the protracted behavioral and molecular effects of mild injection stress on C-Glud1+/− mice. We found spatial and reversal learning deficits, as well as large-scale mPFC transcriptional changes in pathways associated with glutamate and GABA signaling, in stress-exposed C-Glud1+/− mice, but not in their stress-naïve or C-Glud1+/+ littermates. These effects were observed several weeks following stress exposure, and the expression levels of specific glutamatergic and GABAergic genes differentiated between high and low reversal learning performance. An increase in miR203-5p expression immediately following stress may provide a translational regulatory mechanism to account for the delayed effect of stress exposure on cognitive function. Our findings show that chronic glutamate abnormalities interact with acute stress to induce cognitive deficits, and resonate with gene x environment theories of schizophrenia. Stress-exposed C-Glud1+/− mice may model a schizophrenia high-risk population, which is uniquely sensitive to stress-related ‘trigger’ events.

Preprint Highlight: mRNA Location and Translation Rate Determine Protein Targeting to Dual Destinations

NET1 is a guanine nucleotide exchange factor for RhoA GTPase that plays different roles in the nucleus and cytoplasm. While perinuclear and peripheral mRNA translation sites have been identified, the mechanisms of spatial translation regulation and impacts on protein function are not generally understood. Here, the authors use genetic tools to manipulate NET1 mRNA targeting sequences, as well as proximity ligation, mass spectrometry, and imaging. They show that mRNA localization, translation rate, and the order of appearance of targeting domains during translation determine the extent of protein synthesis in each location, promoting site-specific NET1 function. This novel regulation of protein targeting by RNA translation rate and location uncovers the potential to engineer mRNA localization to optimize protein function and will likely impact research on cell biology, immunology, and beyond. This preprint has been assigned the following badges: New Hypothesis, New Methods, Open Software, Cross-Validation. Read the preprint on bioRxiv ( Gasparski et al., 2023 ): https://doi.org/10.1101/2023.04.24.538105 .

Cytosolic and mitochondrial ribosomal proteins mediate the locust phase transition via divergence of translational profiles

The phase transition from solitary to gregarious locusts is crucial in outbreaks of locust plague, which threaten agricultural yield and food security. Research on the regulatory mechanisms of phase transition in locusts has focused primarily on the transcriptional or posttranslational level. However, the translational regulation of phase transition is unexplored. Here, we show a phase-dependent pattern at the translation level, which exhibits different polysome profiles between gregarious and solitary locusts. The gregarious locusts exhibit significant increases in 60S and polyribosomes, while solitary locusts possess higher peaks of the monoribosome and a specific "halfmer." The polysome profiles, a molecular phenotype, respond to changes in population density. In gregarious locusts, ten genes involved in the cytosolic ribosome pathway exhibited increased translational efficiency (TE). In solitary locusts, five genes from the mitochondrial ribosome pathway displayed increased TE. The high expression of large ribosomal protein 7 at the translational level promotes accumulation of the free 60S ribosomal subunit in gregarious locusts, while solitary locusts employ mitochondrial small ribosomal protein 18cto induce the assembly of mitochondrial ribosomes, causing divergence of the translational profiles and behavioral transition. This study reveals the translational regulatory mechanism of locust phase transition, in which the locusts employ divergent ribosome pathways to cope with changes in population density.

Exploring structural determinants and the role of nucleolin in formation of the long-range interactions between untranslated regions of p53 mRNA.

p53 protein is a key regulator of cellular homeostasis by coordinating the framework of antiproliferative pathways as a response to various stress factors. Although the main mechanism of stress-dependent induction of p53 protein relies on post-translational modifications influencing its stability and activity, a growing amount of evidence suggests that complex regulation of p53 expression occurs also at the mRNA level. This study explores structural determinants of long-range RNA-RNA interactions in p53 mRNA, crucial for stress-dependent regulation of p53 protein translation. We demonstrate that the 8-nt bulge motif plays a key structural role in base-pairing of complementary sequences from the 5' and 3' untranslated regions of p53 mRNA. We also show that one of the p53 translation regulators, nucleolin, displays an RNA chaperone activity and facilitates the association of sequences involved in the formation of long-range interactions in p53 mRNA. Nucleolin promotes base-pairing of complementary sequences through the bulge motif, because mutations of this region reduce or inhibit pairing while compensatory mutations restore this interaction. Mutational analysis of nucleolin reveals that all four RNA recognition motifs are indispensable for optimal RNA chaperone activity of nucleolin. These observations help to decipher the unique mechanism of p53 protein translation regulation pointing to bulge motif and nucleolin as the critical factors during intramolecular RNA-RNA recognition in p53 mRNA.