Search Results (229)

Translation of mRNAs is a tightly regulated process in gene expression. In mRNA, the 5′ untranslated region (5′ UTR) controls ribosome recruitment and frequently contains structured elements that modulate translation efficacy. This study investigates stable structural motifs within the 5′ UTR of DDX23 mRNA, encoding a protein relevant for anticancer therapy, as potential regulators and targets for antisense oligonucleotides (ASOs). Despite bioinformatic predictions and transcriptomic validations suggesting RNA G-quadruplex (rG4) formation, comprehensive structural analysis using a light-up assay and CD, UV, and NMR spectroscopy revealed that most putative rG4-forming sequences do not fold into stable rG4 structures, although one of them exists in an equilibrium between rG4 and an alternative, likely hairpin, conformation. Reporter assays using a robust G4 stabilizer also argue against the significant regulatory role of rG4s in DDX23 mRNA translation. Instead, we identified and characterized a stable hairpin structure with potential regulatory function. Based on these findings, we designed fully locked nucleic acid (LNA)-modified ASOs to target this hairpin and regions flanking the upstream open reading frame (uORF) and start codon of the coding sequence. A reporter assay demonstrated that cap-proximal targeting achieved robust translation inhibition up to 80%. In contrast, targeting the efficiently translated uORF was ineffective, presumably due to steric hindrances from the ribosomal complex. The study yields crucial design principles for translation-regulating ASOs: avoid targeting regions shielded by efficient uORF translation and carefully tune ASO-RNA duplex stability to surpass endogenous structures without disrupting regulatory mechanisms. These findings provide insights into the regulation of DDX23 expression and establish a framework for developing ASO-based therapeutics with broad implications for mRNA targeting in anticancer applications. Full article

EsBax (bcl-2 Associated X protein), a member of the bcl-2 family involved in the mitochondrial apoptosis pathway, plays a crucial role in immune response and defense in invertebrates. In this study, we successfully cloned the full-length cDNA of EsBax from the Chinese mitten crab (Eriocheir sinensis) and investigated its immune-related functions. The EsBax cDNA is 3374 bp in length, including a 1563 bp open reading frame (ORF) encoding 521 amino acids, a 142 bp 5′ untranslated region (UTR), and a 1699 bp 3′ UTR. The predicted EsBax protein has a molecular weight of 58.0786 kD, a theoretical isoelectric point of 7.28, and contains three conserved BH domains (BH1-BH3), and a transmembrane domain (TM). Amino acid sequence analysis revealed the highest sequence identity (99.42%) with E. sinensis. For the expression analysis, three biological replicates were performed for each tissue and treatment group. Real-time quantitative PCR showed that EsBax mRNA was ubiquitously expressed in all examined tissues, with the highest expression in the hepatopancreas, followed by hemocytes, intestine, gill, and the lowest in muscle. Upon stimulation with lipopolysaccharide (LPS), Aeromonas hydrophila (AH), or cycloheximide (CHX), EsBax expression increased and peaked at 24 h (LPS and CHX) or 48 h (A. hydrophila), then decreased. These results suggest that EsBax expression is dynamically responsive to exogenous stimulants (LPS, A. hydrophila, and CHX) in E. sinensis, implying a potential role of EsBax in the molecular events associated with pathogen-induced apoptosis in this species. Full article

The tumor microenvironment creates strong stress conditions, including hypoxia and nutrient depletion, which cause the blocking of cap-dependent translation. Under stressful conditions, cancer cells exploit the cap-independent translation mechanism mediated by internal ribosome entry site (IRES), which ensures continued protein synthesis. IRES elements located in the 5′ untranslated regions of specific mRNAs allow selective translation of key anti-apoptotic and adaptive proteins. These proteins promote cellular processes that sustain cell survival, among them metabolic reprogramming, redox balance, and epithelial-to-mesenchymal transition, thus facilitating tumor progression and therapy resistance. IRES activity is dynamically regulated by IRES trans-acting factors, such as YB-1, PTB, and hnRNPA1, which respond to cellular stress by enhancing translation of crucial mRNAs. Emerging therapeutic strategies include pharmacological IRES inhibitors, RNA-based approaches targeting ITAF interactions, and IRES-containing vectors for controlled therapeutic gene expression. A deeper understanding of translational reprogramming, IRES structural diversity, and ITAF function is essential to develop targeted interventions to overcome therapeutic resistance and eliminate persistent tumor cell populations. Full article

Background and Clinical Significance: Inherited thrombocytopenia (IT) is a heterogeneous group of disorders caused by mutations in over 45 genes. Among these, ANKRD26-related thrombocytopenia (ANKRD26-RT) accounts for a notable subset and is associated with variable bleeding tendencies and an increased risk of myeloid malignancies. However, the extent of this oncogenic risk appears to vary between specific gene variants. Understanding the genotype–phenotype relationship is essential for patient counseling and management. This report presents a multigenerational family carrying the rare c.−118C > G variant in the 5′ untranslated region of ANKRD26, contributing to the discussion on variant-specific cancer predisposition. Case Presentation: Two sisters aged 57 and 60 presented with lifelong bleeding diathesis and moderate thrombocytopenia. Their symptoms included easy bruising, menorrhagia, and excessive postoperative bleeding. Genetic testing confirmed heterozygosity for the ANKRD26 c.−118C > G variant. Bone marrow analysis revealed abnormal megakaryopoiesis without evidence of dysplasia or somatic mutations. One sister underwent major surgery without complications when managed with prophylactic hemostatic therapy. Their family history included multiple female relatives with similar symptoms, although formal testing was limited. Notably, none of the affected individuals developed hematologic malignancy, and only one developed esophageal cancer, with no current evidence linking this variant to solid tumors. Conclusions: This case underscores the importance of distinguishing between ANKRD26 variants when assessing malignancy risk. While ANKRD26-RT is associated with myeloid neoplasms, the c.−118C > G variant may confer a lower oncogenic potential. Variant-specific risk stratification and genetic counseling are crucial for optimizing surveillance and avoiding unnecessary interventions in low-risk individuals. Full article

In this study, we investigated the functional role of Peroxiredoxin 5 (SmPrx5) in the cuttlefish Sepiella maindroni. The full-length SmPrx5 cDNA is 934 base pairs (bp) in length, comprising a 31 bp 5′ untranslated region (UTR), a 330 bp 3′ UTR, and an open reading frame (ORF) of 573 bp that encodes a polypeptide consisting of 190 amino acids. Sequence analysis revealed the presence of a conserved peroxidase catalytic motif VPGAFTPGCSQTHLPG and the signature domain DGTGLTCSL, indicating that SmPrx5 belongs to the 2-Cys Prx subfamily. Quantitative real-time PCR (RT-qPCR) analysis demonstrated that SmPrx5 is broadly expressed across various tissues in S. maindroni, with particularly high expression levels observed in the testes, hemocytes, liver, and ovaries. Upon challenge with Vibrio alginolyticus, SmPrx5 expression was significantly upregulated in both the liver and hemocytes, peaking at 24 h post-infection and gradually returning to baseline levels within 48 h. Furthermore, the recombinant SmPrx5 protein exhibited notable antioxidant activity in vitro, suggesting its involvement in the oxidative stress response. These findings enhance our understanding of the molecular mechanisms underlying immune defense in marine cephalopods and highlight the potential role of Prx5 in host immunity. Full article

Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which can then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3′ UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here, we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted in their 5′ UTRs. Among the five different Cas RNases tested, Csy4 was found to be the most efficient in this context, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, both in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5′ end and thereby boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner. Full article

WD40 repeat and FYVE containing protein 1 (WDFY1) functions in membrane trafficking and protein complex scaffolding. WDFY1 has been studied in the immune system and in different oncogenic conditions. Therefore, comprehensive understanding of WDFY1 regulation mechanisms is much desired. In this study, we analyzed the promoter and 5′- and 3′-untranslated regions (UTRs) of wdfy1 and identified critical sequence elements, transcription factors (TFs), and miRNAs that collaboratively regulate wdfy1 gene expression. A 3.5 kb segment of the mouse wdfy1 promoter and 5′-UTR was cloned into a luciferase expression vector and transfected into HeLa cells. Luciferase assays of promoter deletion mutants revealed approximately four-fold increased activity attributed by a 500 bp distal fragment upstream of the wdfy1 5′-UTR. Four TFs (Sp1, Ap-1, Hes1, and TCF7) were found to be critical for wdfy1 expression with binding sites spread throughout the promoter and 5′-UTR regions. Cloning of a 3.2 kb fragment of wdfy1 3′-UTR into the luciferase expression vector led to an ~3.5-fold decrease in luciferase activity. Complementary siRNA and luciferase assays mutually confirmed our findings. Most importantly, IL-6, a critical cytokine in organ inflammation, was found to promote WDFY1 expression through the upregulation of Sp1 in primary renal mesangial cells. We, therefore, identified a potential inflammation-driven WDFY1 upregulation in mice. Full article

Long interspersed element-1 (LINE-1) is the only active autonomous transposon comprising about 17% of human genomes. LINE-1 transposition can cause the mutation and rearrangement of the host’s genomic DNA. The host has, therefore, developed multiple mechanisms to restrict LINE-1 mobility. Here, we report that SLFN11, a member of the Schlafen family, can restrict LINE-1 retrotransposition, and the inhibitory activity requires its helicase domain. Mechanistically, SLFN11 specifically binds to the LINE-1 5′ untranslated region (5′UTR) and blocks RNA polymerase II recruitment, thereby suppressing its transcription. Furthermore, SLFN11 promotes heterochromatinization, suggesting an epigenetic inhibition pathway. Full article

Understanding the mechanisms of sexual development would pave the way for producing mono-sex populations to aid the aquaculture industry. This study investigates the functions of the Y-linked iDmrt1 paralogue (Po-iDMY) and insulin-like androgenic gland hormone (Po-IAG) in the process of sexual development in the tropical rock lobster, Panulirus ornatus (TRL). Previously, we identified that Po-iDMY, a male-specific heterogametic (Y-linked) paralogue of the autosomal Po-iDmrt1 found in TRL, is a second sex-linked iDmrt gene identified in invertebrates. Using 5′ and 3′ rapid amplification of cDNA ends and data from a draft male genome (with an assembly genome size of approximately 2.446 Gbp and 87% BUSCO completeness), we obtained the full-length Po-iDMY gene (encoding a protein of 312 amino acids). A 411 bp male-specific sequence located at the 3′ untranslated region of Po-iDMY mRNA was used as a sex marker, which was reported for the first time in our draft genome. However, Po-iDMY is not a master sex-determining factor since it was not expressed across developmental stages of embryos, juveniles and adults. Instead, we silenced Po-IAG at an early juvenile stage, generating two potential neo-females, implying that sexual manipulation could be a promising technique in TRL. Full article

As a globally important leguminous crop, soybean (Glycine max L.) serves as a vital source of edible oils and proteins for humans and livestock. Oils in leaves can help crops combat fungal infections, adapt to temperature changes via fatty acid modulation, and support resource recycling during leaf senescence. However, accumulating oils in leaves is a fundamental challenge due to the need to balance the inherently competing photosynthesis and fatty acid biosynthesis processes within chloroplasts. RETICULATA-RELATED (RER), known to regulate chloroplast function and plastid metabolism in Arabidopsis, plays an essential role in leaf development. Here, 14 non-redundant GmRER genes were identified in soybean and phylogenetically classified into four subclades. Most Arabidopsis RER genes were evolutionarily preserved as gene duplicates in soybean, except for GmRER5 and GmRER6. RNA secondary structures spanning the coding sequences (CDSs), the 5′- and 3′- untranslated regions (UTRs) of GmRERs, displayed exceptional structural plasticity in CDSs, while exhibiting limited conservation in UTRs. In contrast, protein structures retained conserved folds, underscoring evolutionary constraints on functional domains despite transcriptional plasticity. Notably, GmRER4a and GmRER4b represented an exceptional case of high similarity in both protein and RNA structures. Expression profiling across fourteen tissues and three abiotic stress conditions revealed a dynamic shift in expression levels between leaf-predominant and root-enriched GmRER paralogs after stress treatments. A comparative transcriptome analysis of six soybean landraces further revealed transcriptional polymorphism in the GmRER family, which was associated with the expression patterns of lipid biosynthesis regulators. Our comprehensive characterization of GmRERs may offer potential targets for soybean breeding optimization in overall plant oil production. Full article

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5′ and 3′ UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines. Full article

Nipah virus (NiV), a highly lethal zoonotic pathogen causing encephalitis and respiratory diseases with mortality rates up to 40–70%, faces research limitations due to its strict biosafety level 4 (BSL-4) containment requirements, hindering antiviral development. To address this, we generated two NiV minigenome replicons (Fluc- and EGFP-based) expressed via helper plasmids encoding viral N, P, and L proteins, enabling replication studies under BSL-2 conditions. The minigenome replicon recapitulated the cytoplasmic inclusion body (IB) formation observed in live NiV infections. We further demonstrated that IB assembly is driven by liquid–liquid phase separation (LLPS), with biochemical analyses identifying the C-terminal N core domain of the N protein, as well as N₀ and XD domains and the intrinsically disordered region (IDR) of the P protein, as essential structural determinants for LLPS-mediated IB biogenesis. The targeted siRNA silencing of the 5′ and 3′ untranslated regions (UTRs) significantly reduced replicon-derived mRNA levels, validating the regulatory roles of these regions. Importantly, the minigenome replicon demonstrated sensitivity to type I/II/III interferons and antivirals (remdesivir, azvudine, molnupiravir), establishing its utility for drug screening. This study provides a safe and efficient platform for investigating NiV replication mechanisms and accelerating therapeutic development, circumventing the constraints of BSL-4 facilities while preserving key virological features. Full article

During retrovirus assembly, Gag packages unspliced viral RNA as the virion genome. Genome packaging is usually specific with occasional exceptions of cross-packaging RNA from distantly related retroviruses. For example, HIV-1 Gag can efficiently package HIV-2 RNA. To better understand how HIV-1 Gag selects packaging substrates, we defined elements in the HIV-2 5′ untranslated region (UTR) that are important for this process. Although sharing little homology, both HIV-1 and HIV-2 5′ UTRs have unpaired guanosines essential for packaging by their own Gag. Simultaneously substituting guanosines of nine sites in the HIV-2 5′ UTR caused severe defects in HIV-1 Gag-mediated packaging. Two of the nine sites are particularly important, mutating each one impaired HIV-1 Gag-mediated packaging, whereas the other sites required mutations in multiple sites to produce similar effects. Additionally, we identified one site that impacts HIV-1 Gag but is dispensable for HIV-2 Gag selective packaging. Furthermore, combining mutations has an additive effect on packaging defects for HIV-1 Gag, in contrast to the previously reported synergistic effects for HIV-2 Gag. Our study demonstrates that Gag proteins from two different retroviruses recognize and use mostly the same set of cis-acting elements to mediate RNA packaging and provide the mechanistic basis for genome cross-packaging. Full article

Background/Objectives: Modified messenger RNA (modRNA) is a promising gene delivery method used to upregulate genes in cardiac tissue, with applications in both clinical and preclinical settings to prevent cardiac remodeling after ischemic injury. The 5′ untranslated region (5′UTR) plays a crucial role in regulating the translation efficiency of mRNA into functional proteins. Due to the high production cost and short half-life of modRNA, it is essential to identify novel 5′UTR designs that enhance modRNA translation in the heart. Methods: Here, we present an artificial 5′UTR, termed “Top Heart 5′UTR”, designed based on ribonucleotide frequency analyses of 1000 genes highly expressed in the heart. This novel artificial 5′UTR contains a unique 20-nucleotide sequence, consisting of 11 previously uncharacterized nucleotides (CCCCCGCCCCC) and 9 well-described nucleotides from the Kozak sequence upstream of the start codon (ATG). Results: This design significantly improves modRNA translation efficiency in cardiomyocytes (CMs) and heart cells both in vitro and in vivo. Specifically, the Top Heart 5′UTR increases translation efficiency by approximately 30–60% in both mouse and human CMs compared to a standard 5′UTR control. Moreover, the artificial 5′UTR induces a 2–2.5 times higher translation of modRNA in the mouse heart 24 and 48 h post-delivery. Conclusions: Our findings may contribute to the development of a superior modRNA platform for use in preclinical and clinical studies, potentially allowing reduced dosages or increased gene expression at the same dosage level. This approach can be extended to identify optimized 5′UTRs for various cell types or organs, including applications in cancer therapies. Full article

Porcine sapelovirus (PSV), porcine kobuvirus (PKV), porcine teschovirus (PTV), and porcine enterovirus G (EV-G) are all important viruses in the swine industry. These viruses play important roles in the establishment of similar clinical signs of diseases in pigs, including diarrhea, encephalitis, and reproductive and respiratory disorders. The early accurate detection of these viruses is crucial for dealing with these diseases. In order for the differential detection of these four viruses, specific primers and TaqMan probes were designed for the conserved regions in the 5′ untranslated region (UTR) of these four viruses, and one-step quadruplex reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PSV, PKV, PTV, and EV-G was developed. The results showed that this assay had the advantages of high sensitivity, strong specificity, excellent repeatability, and simple operation. Probit regression analysis showed that the assay obtained low limits of detection (LODs) for PSV, PKV, PTV, and EV-G, with 146.02, 143.83, 141.92, and 139.79 copies/reaction, respectively. The assay showed a strong specificity of detecting only PSV, PKV, PTV, and EV-G, and had no cross-reactivity with other control viruses. The assay exhibited excellent repeatability of the intra-assay coefficient of variation (CV) of 0.28–1.58% and the inter-assay CV of 0.20–1.40%. Finally, the developed quadruplex RT-qPCR was used to detect 1823 fecal samples collected in Guangxi Province, China between January 2024 and December 2024. The results indicated that the positivity rates of PSV, PKV, PTV, and EV-G were 15.25% (278/1823), 21.72% (396/1823), 18.82% (343/1823), and 27.10% (494/1823), respectively, and there existed phenomena of mixed infections. Compared with the reference RT-qPCR/RT-PCR established for these four viruses, the coincidence rates for the detection results of PSV, PKV, PTV, and EV-G reached 99.51%, 99.40%, 99.51%, and 99.01%, respectively. In conclusions, the developed quadruplex RT-qPCR could simultaneously detect PSV, PKV, PTV, and EV-G, and provided an efficient and convenient detection method to monitor the epidemic status and variation of these viruses. Full article