Search Results (249)

Eukaryotic initiation factor 2 (EIF2) signaling plays a crucial role in regulating mRNA translation and initiating eukaryotic protein synthesis. Computational molecular network pathway analysis of the canonical pathways of the coronaviral infection revealed that EIF2 signaling is inactivated when the coronavirus pathogenesis pathway is activated and vice versa. Our computational analyses indicated that the coronavirus pathogenesis pathway and EIF2 signaling had inverse activation states. Computational investigation of upstream or downstream microRNA (miRNA) revealed that EIF2 signaling directly interacted with miRNAs, including let-7, miR-1292-3p (miRNAs with the seed CGCGCCC), miR-15, miR-34, miR-378, miR-493, miR-497, miR-7, miR-8, and MIRLET7. A total of 36 nodes, including 8 molecules (ATF4, BCL2, CCND1, DDIT3, EIF2A, EIF2AK3, EIF4E, and ERK1/2), 1 complex (the ribosomal 40s subunit), and 1 function (apoptosis) in the coronavirus pathogenesis pathway, overlapped with EIF2 signaling. Alterations in EIF2 signaling may play a role in the pathogenesis of coronavirus. Full article

It has been known for decades that eukaryotic cellular mRNAs are frequently translated by multiple ribosomes organized into polysomes of diverse topology, including circular arrangements. The closed-loop model, in which the 5′ cap and 3′ poly(A) tail are bridged by initiation factors, provided a mechanistic basis for mRNA circularization and suggested that the spatial proximity of termini facilitates ribosome recycling. Various biochemical, structural, and imaging approaches—including electron microscopy, atomic force microscopy, cryo-electron tomography, and single-molecule fluorescence—have since demonstrated that polysomes indeed adopt compact and heterogeneous conformations, with circular assemblies representing a significant fraction. Although direct visualization of ribosome recycling remains technically challenging, ribosome turnover experiments, kinetic analyses and modeling support the concept of closed-loop-assisted reinitiation (CLAR), whereby terminating ribosomes are re-utilized to sustain translation efficiency. Together, the findings suggest that mRNA circularization is a dynamic and regulated state that enhances protein synthesis under specific conditions, while linear or modular polysome architectures may dominate in others. Understanding the balance between these modes of translation remains central to elucidating the interplay between mRNA topology, ribosome dynamics, and translational control. Full article

Background/Objectives: The selection and validation of species-specific housekeeping genes (HKGs) have become increasingly common in functional genomics, with application of quantitative Polymerase Chain Reaction (qPCR) or cDNA-based qPCR (RT-qPCR). Despite the Macrobrachium amazonicum having RNA-seq studies available, there are still no data on the most stable and consistent HKGs for use in relative gene expression analyses. Therefore, the present study aimed to identify and validate seven HKGs in M. amazonicum: Eukaryotic Translation Initiation Factor (EIF), 18S ribosomal RNA (18S), Ribosomal Protein L18 (RPL18), β-actin, α-tubulin (α-tub), Elongation Factor 1-α (EF-1α), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). Methods: The HKGs were identified in the M. amazonicum transcriptome, characterized for identity confirmation, and compared against public databases. Subsequently, RT-qPCR assays were prepared using muscle, hepatopancreas, gills, testis, androgenic gland, and ovary to assess the stability of the HKG markers, employing the comparative ∆Ct, BestKeeper, NormFinder, and GeNorm methods. Results: All candidate HKGs identified showed high similarity with other decapods. Reactions performed with these markers demonstrated high specificity, PCR efficiency, and elevated coefficients of determination. The comprehensive ranking, indicated that no single HKG was stable across all tissues, with HKGs showing the best stability being tissue-specific. The most stable HKGs were RPL18 and 18S. GAPDH, historically used as an HKG, showed the poorest performance in stability ranking for most tissues tested, whereas β-actin was most suitable only for ovarian. Conclusions: These data reinforce the need for species-specific HKG validation and provide an appropriate panel of reference markers for gene expression studies in the M. amazonicum. Full article

Upon transcription, most mRNAs associate with the small ribosomal subunit, after which a fully translating ribosome assembles. Under starvation or stress, however, mRNA–ribosome associations are blocked and many mRNAs are instead sequestered with specific RNA-binding proteins into stress granules or other subcellular condensates, a process that has been extensively studied. In contrast, much less attention has been paid to the fate of ribosomes under these same conditions. Ribosomes can remain fully assembled but unbound to mRNA, entering an inactive, dormant state. Dormancy is often supported by specific protein factors which protect ribosomes from degradation and facilitate reactivation once growth conditions improve. In this review, we highlight that dormant ribosome states are well defined in prokaryotes, in part because they possess distinct and experimentally tractable features, such as stable vacant 100S dimers. In eukaryotes, by contrast, analogous disomes are largely absent, making their discovery more indirect and method-dependent. We therefore focus on how evidence for eukaryotic dormant ribosomes has been assembled through multiple independent findings and how their interpretation depends critically on the experimental approaches used to study them. Finally, we consider atypical ribosomal states, such as translationally inactive polysomes in neurons, which underscore the context-dependent nature of ribosome activity. Full article

N6-methyladenosine (m⁶A) represents the most abundant and tightly controlled modification within eukaryotic mRNA, critically influencing RNA metabolism and function. The m⁶A methyltransferase Like-3 (METTL3), responsible for the complex’s catalytic function, has emerged as a central epitranscriptomic regulator governing mRNA stability, alternative splicing, nuclear export, and the efficiency of mRNA translation. Converging research shows that METTL3 is involved in the pathogenesis of numerous disorders via m⁶A-dependent, post-transcriptional regulation of gene programs controlling cell growth, migration, and immune pathways. Regarding pulmonary pathophysiology, METTL3-mediated m⁶A is tied to disease initiation and progression in conditions such as asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), lung infections, acute respiratory distress syndrome (ARDS). This review summarizes the contemporary evidence for METTL3’s roles and regulatory network in diverse pulmonary pathologies. We further highlight emerging strategies for targeting METTL3 as a potential therapeutic approach, underscoring its promise as a novel epitranscriptomic target. Beyond inflammatory and fibrotic disorders, we also summarize emerging evidence linking METTL3 to lung cancer and briefly outline other respiratory conditions (e.g., ILD, bronchiectasis, and secondary pulmonary hypertension), highlighting common translational themes and remaining gaps. Further studies are required to clarify the disease-specific and context-dependent actions of METTL3 and to advance the clinical translation of m⁶A-based therapies. Full article

Background/Objectives: Selectively expressible RNA (seRNA) molecules represent a promising new platform for the induction of cell type-specific protein expression. Based on the sense–antisense interaction of the seRNA antisense domain with target cell-specific RNA molecules, the partial degradation of the seRNA molecule induces the activation of an internal ribosomal entry site to initiate translation. The selective expression of seRNA encoded proteins exclusively in target cells works both in vitro and in vivo but is associated with a lower expression intensity compared with classical mRNAs. Furthermore, seRNAs have so far been transfected into cells by plasmid-encoded seRNA expression systems, which is limiting their broad medical applicability. Here, we focus on the characterization of plasmid-based seRNA uptake and activation as well as on options to transfer the seRNA technology to additional vector systems to increase target cell-specific effector expression. Methods: seRNA constructs were generated as expression plasmids, AAV, DNA minicircles and IVT-RNA and delivered into different eukaryotic cell lines by transfection/transduction. Analyses were performed using fluorescence microscopy and, for quantitative analyses, flow cytometry. RNA stability and expression analyses were performed using qRT-PCR. Results: We show that seRNA-based plasmid systems are efficiently transfected into cells but that reduced RNA steady-state levels are present compared with control expression plasmids. This effect is most likely based on reduced transcription efficiency rather than seRNA stability. Furthermore, seRNA transcription from viral vectors or circular DNA significantly increased the effector expression of seRNAs and enabled linear expression regulation while maintaining target cell-specific activation and inactivation in non-target cells. Optimal results were achieved by adapting the technology to in vitro transcribed seRNA. Conclusions: Our data show that seRNA technology develops its full functionality regardless of the type of transfer vector used. Furthermore, expression strength can be regulated within a wide range while maintaining consistent functionality which will enable broad applicability in medicine in the future. Full article

N6-methyladenosine (m⁶A) is the most abundant internal RNA modification in eukaryotes and plays a critical role in gene expression regulation by influencing RNA stability, splicing, nuclear export, and translation. Emerging evidence suggests that dysregulation of m⁶A contributes to neuroinflammation, neurotoxicity, and synaptic dysfunction—key features of neurodegenerative diseases. This review aims to examine the role of m6A modification in neurodegenerative diseases from a cell-type-specific perspective. We systematically reviewed recent studies investigating m⁶A modifications in neurons and glial cells. Data from transcriptomic, epitranscriptomic, and functional studies were analyzed to understand how m⁶A dynamics influence disease-related processes. Findings indicate that m⁶A modifications regulate neuroinflammation and immune responses in microglia, modulate astrocytic support functions, affect myelination through oligodendrocytes, and alter m⁶A patterns in neurons, impacting synaptic plasticity, stress responses, and neuronal survival. These cell-type-specific roles of m⁶A contribute to the progression of neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Amyotrophic lateral sclerosis (ALS). Understanding m⁶A-modulated mechanisms in specific neural cell types may facilitate the development of targeted interventions for neurodegenerative diseases. Full article

The localization and remodeling of mRNPs is inextricably linked to translational control. In recent years there has been great progress in the field of mRNA translational control due to the characterization of the proteins and small RNAs that compose mRNPs. But our initial assumptions about the physical nature and participation of germ cell granules/condensates in mRNA regulation may have been misguided. These “granules” were found to be non-membrane-bound liquid–liquid phase-separated (LLPS) condensates that form around proteins with intrinsically disordered regions (IDRs) and RNA. Their macrostructures are dynamic as germ cells differentiate into gametes and subsequently join to form embryos. In addition, they segregate translation-repressing RNA-binding proteins (RBPs), selected eIF4 initiation factors, Vasa/GLH-1 and other helicases, several Argonautes and their associated small RNAs, and frequently components of P bodies and stress granules (SGs). Condensate movement, separation, fusion, and dissolution were long conjectured to mediate the translational control of mRNAs residing in contained mRNPs. New high-resolution microscopy and tagging techniques identified order in their organization, showing the segregation of similar mRNAs and the stratification of proteins into distinct mRNPs. Functional transitions from repression to activation seem to corelate with the overt granule dynamics. Yet increasing evidence suggests that the resident mRNPs, and not the macroscopic condensates, exert the bulk of the regulation. Full article

Introduction: Bovine leukemia virus is a single-stranded RNA virus that targets B cell CD5⁺ lymphocytes in cattle. Only a tiny percentage of individuals develop malignant lymphoproliferative disorders, while most remain healthy carriers or experience persistent lymphocytosis. The exact mechanisms leading to lymphoma development are complex and not fully understood. RNA-seq analysis of cows’ peripheral blood leukocytes (PBLs) with and without Bovine leukemia virus (BLV) antibodies was conducted to gain a deeper understanding of molecular events beyond BLV infection. Method: Eighteen samples were selected, and their RNA was sequenced. For gene expression analysis and protein–protein network interactions, three groups were selected, including healthy negative samples (CT, n = 7), asymptomatic carriers (AC, n = 5), and persistent lymphocytosis (PL, n = 6), to provide the differentially expressed gene (DEG) and protein–protein interaction network (PPIN) outputs. Results: Our results demonstrated that in comparison to CT, ACs upregulated TLR7 and transcription activation factors. In the CT vs. PL group, MHC class II, transcription activation factors, and anti-inflammatory cytokines increased, while the acute-phase proteins, antiviral receptors, and inflammatory cytokines decreased. Additionally, antiviral receptors, acute-phase proteins, and inflammatory receptors were downregulated in the PL versus the AC groups. Moreover, PPINs analysis suggested that nuclear receptor corepressor 1 (NCOR1), serine/arginine repetitive matrix 2 (SRRM2), LUC7 like 3 pre-mRNA splicing factor (LUC7L3), TWIST neighbor (TWISTNB), U6 small nuclear RNA and mRNA degradation associated (LSM4), eukaryotic translation elongation factor 2 (EEF2), ubiquitin C (UBC), CD74, and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNP A2B1) are possible hub gene candidates in the PL group. Conclusions: Our results suggest that innate and cellular immune responses are more loose in severe BLV infectious conditions, while the PPINs revealed that new protein interactions are necessary for oncogenesis. Full article

This review highlights the emerging functional implications of nonsense-mediated mRNA decay (NMD) in human diseases, with a focus on its therapeutic potential for cardiovascular disease. NMD, conserved from yeast to humans, is involved in apoptosis, autophagy, cellular differentiation, and gene expression regulation. NMD is a highly conserved surveillance mechanism that degrades mRNAs containing premature termination codons (PTCs) located upstream of the final exon-exon junction. NMD serves to prevent the translation of aberrant mRNA and prevents the formation of defective protein products that could result in diseases. Key players in this pathway include up-frameshift proteins (UPFs), nonsense-mediated mRNA decay associated with p13K-related kinases (SMGs), and eukaryotic release factors (eRFs), among others. Dysregulation of NMD has been linked to numerous pathological conditions such as dilated cardiomyopathy, cancer, viral infections, and various neurodevelopmental and genetic disorders. This review will examine the regulatory mechanisms by which NMD regulation or dysregulation may contribute to disease mitigation or progression and its potential for cardiovascular disease therapy. We will further explore how modulating NMD could prevent the outcomes of mutations underlying genetically induced cardiovascular conditions and its applications in personalized medicine due to its role in gene regulation. While recent advances have provided valuable insights into NMD machinery and its therapeutic potential, further studies are needed to clarify the precise roles of key NMD components in cardiovascular disease prevention and treatment. Full article

Background: Protein kinase R (PKR) inhibits general mRNA translation by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2). PKR also modulates NF-κB signaling during viral infections, but comparative studies of PKR-mediated NF-κB responses across mammalian species and their regulation by viral inhibitors remain largely unexplored. This study aimed to characterize the conserved antiviral and inflammatory roles of mammalian PKR orthologs and investigate their modulation by poxviral inhibitors. Methods: Using reporter gene assays and quantitative RT-PCR, we assessed the impact of 17 mammalian PKR orthologs on general translation inhibition, stress-responsive translation, and NF-κB-dependent induction of target genes. Congenic human and rabbit cell lines infected with a myxoma virus strain lacking PKR inhibitors were used to compare the effects of human and rabbit PKR on viral replication and inflammatory responses. Site-directed mutagenesis was employed to determine key residues responsible for differential sensitivity to the viral inhibitor M156. Results: All 17 mammalian PKR orthologs significantly inhibited general translation, strongly activated stress-responsive ATF4 translation, and robustly induced NF-κB target genes. Inhibition of these responses was specifically mediated by poxviral K3 orthologs that effectively suppressed PKR activation. Comparative analyses showed human and rabbit PKRs similarly inhibited virus replication and induced cytokine transcripts. Amino acid swaps between rabbit PKRs reversed their sensitivity to viral inhibitor M156 and NF-κB activation. Conclusions: Our data show that the tested PKR orthologs exhibit conserved dual antiviral and inflammatory regulatory roles, which can be antagonized by poxviral K3 orthologs that exploit eIF2α mimicry to modulate the PKR-NF-κB axis. Full article

Circular RNAs (circRNAs) are covalently closed RNA molecules generated through a non-canonical splicing event known as back-splicing. This particular class of non-coding RNAs has attracted growing interest due to its evolutionary conservation across eukaryotes, high expression in the central nervous system, and frequent dysregulation in various pathological conditions, including cancer. Traditionally, circRNAs have been characterised by their ability to function as microRNA (miRNA) and protein sponges. However, recent discoveries from multiple research groups have uncovered a novel and potentially transformative mechanism of action: the direct interaction of circRNAs with messenger RNAs (mRNAs) to regulate their fate. These interactions can influence mRNA stability and translation, revealing a new layer of post-transcriptional gene regulation. In this review, we present and analyse the latest evidence supporting the emerging role of circRNAs in diverse biological contexts. We highlight the growing body of research demonstrating circRNA-mRNA interactions as a functional regulatory mechanism and explore their involvement in key physiological and pathophysiological processes. Understanding this novel mechanism expands our knowledge of RNA-based regulation and opens new opportunities for therapeutic strategies targeting circRNA-mRNA networks in human disease. Full article

Drought stress is a predominant abiotic constraint adversely affecting global rice (Oryza sativa) production and threatening food security. While the transcriptional and post-transcriptional regulation of drought-responsive pathways has been widely investigated, the emerging field of epitranscriptomics, particularly RNA chemical modifications such as N6-methyladenosine (m⁶A), adds a new dimension to gene regulation under stress. The most prevalent internal modification in eukaryotic messenger RNA influences RNA metabolism by interacting dynamically with enzymes that add, remove, or recognize the modification. Recent studies in rice reveal that m⁶A deposition is not static but dynamically regulated in response to water-deficit conditions, influencing transcript stability, splicing, nuclear export, and translation efficiency of key drought-responsive genes. This review critically synthesizes current findings on the distribution and functional implications of m⁶A and other epitranscriptomic marks (e.g., 5-methylcytosine [m⁵C], pseudouridine [Ψ]) in modulating rice responses to drought. We discuss the regulatory circuitry involving m⁶A effectors such as OsMTA, OsFIP37, and YTH domain proteins and their integration with known drought-signaling pathways including ABA and reactive oxygen species (ROS) cascades. We also highlight emerging high-resolution technologies such as m⁶A-seq, direct RNA sequencing, and nanopore-based detection that facilitate epitranscriptomic profiling in rice. Finally, we propose future directions for translating epitranscriptomic knowledge into crop improvement, including CRISPR/Cas-based modulation of RNA modification machinery to enhance drought tolerance. Full article

The eukaryotic translation elongation factor 1 (eEF1) family exhibits critical roles in RNA viral infection beyond its canonical function in protein synthesis. This review analyzes the structural characteristics of eEF1A and the eEF1B complex, and their regulatory mechanisms during viral infection. eEF1A impacts viral replication by stabilizing viral RNA-dependent RNA polymerase (RdRp) complexes, modulating genomic RNA synthesis, and facilitating viral assembly through cytoskeletal regulation. eEF1B subunits contribute through enhancing viral mRNA translation, regulating nuclear transport of viral components, and mediating post-translational modifications. The high conservation of eEF1 proteins across species and their involvement in multiple stages of viral replication establish them as promising broad-spectrum antiviral targets. Current eEF1-targeting compounds like plitidepsin demonstrate efficacy against diverse viral families, though therapeutic development faces challenges in balancing antiviral activity with host toxicity. This review provides a theoretical foundation for developing novel antiviral strategies targeting host–virus interaction interfaces and offers insights into addressing emerging infectious diseases. Full article

N6-methyladenosine (m⁶A) is one of the most prevalent methylated modifications of mRNA in eukaryotes. This reversible alteration can directly or indirectly influence biological functions, including RNA degradation, translation, and splicing. This study investigates the impact of chronic morphine administration and varying withdrawal durations (1 day, 1 week, 4 weeks, and 12 weeks) on the m⁶A modification levels in brain regions critical to addiction development and persistence. Our findings indicate that in the prefrontal cortex, the m⁶A levels and METTL3 expression decrease, accompanied by an increase in FTO and ALKBH5 expression, followed by fluctuating, but statistically insignificant changes in methylation-regulating enzymes over prolonged withdrawal. In the striatum, reductions in m⁶A levels and METTL3 expression are observed at 4 weeks of withdrawal, preceded by non-significant fluctuations in enzyme expression and the m⁶A modification levels. In contrast, no changes in the m⁶A modification levels or the expression of related enzymes are detected in the hippocampus and the cerebellum. Our data suggest that m⁶A modification and its regulatory enzymes undergo region-specific and time-dependent changes in response to chronic morphine exposure and subsequent withdrawal. Full article