Search Results (389)

We recently reported that phospholipase A2 (PLA2)-mediated production of prostaglandins within the ventromedial hypothalamus (VMH) plays a critical role in systemic glucose homeostasis. However, the role of PLA2 in the VMH in regulating food intake is still unclear. Here, we attempted to investigate the role of PLA2 in regulating food intake and body weight in male mice. We injected an adeno-associated virus encoding short hairpin RNA (AAV-shRNA) targeting cytosolic phospholipase A2 (shPla2g4a) into the VMH. We assessed food intake, body weight, oxygen consumption, glucose tolerance, and insulin sensitivity. Three weeks after the AAV injection, the shPla2g4a group exhibited increased food intake and body weight gain compared to controls (shSCRM). Energy expenditure, oxygen consumption, and respiratory quotient (RQ) were comparable between groups. Our findings suggest that the cPLA2-mediated pathway in the VMH is critical for feeding behavior and maintaining energy homeostasis. Further investigation is needed to elucidate the underlying mechanisms. Full article

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil and a single novel zinc finger with four cysteines and two histidines, from which the protein obtains its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using synthetic peptide fragments that the zinc finger of ZC4H2 is genuine and folds upon binding a zinc ion with picomolar affinity. NMR pH titration of histidines and UV–Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc, while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger are likely to perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy, it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative functions. Full article

Plant Parasitic Nematodes (PPNs) are a major problem in agriculture. Damage caused by PPNs has been estimated at USD 80–157 billion annually. The estimates could be even worse in the future in the context of a growing world population in a climate change scenario and with the removal/reduction in the use of some nematodicides due to the strong ecological impact. Biocontrol Agents (BCAs) currently constitute only 8.8% of the general pesticide market. With a view to an ecological transition, the transition from pesticides to biopesticides represents an important challenge that appears necessary not only for organic farming, but also in so-called integrated agriculture. Among the possible BCAs, microorganisms, and in particular yeast, which enjoys the GRAS (Generally Recognized As Safe) status, have the advantage of being able to be produced on a large scale by fermentation on waste substrates at low cost. In this paper, as proof of concept we constructed yeast strains expressing short hairpin RNAs (shRNAs) targeting the daf-16 gene in C. elegans. We demonstrate that oral ingestion of yeast cells expressing DAF16 shRNA is efficient in lowering daf-16 expression and lifespan, suggesting a sustainable RNA interference-based strategy to inhibit the development of PPNs. Full article

Hepatitis B virus (HBV) specifically infects hepatocytes and has a complex life cycle owing to the stabilization and pooling of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. We previously reported that the suppression of dedicator of cytokinesis 11 (DOCK11) decreases cccDNA and HBV-DNA levels and identified it as a new HBV therapeutic target. The DOCK11-associated gene, Wnt/β-catenin signaling regulator tankyrase (TNKS), was identified using in vitro methods; however, its function in the HBV life cycle remains unknown. Here, we used various inhibitors, antagonists, and short-hairpin RNA treatments related to TNKS signaling in HBV-infected hepatocytes. The role of TNKS-related Wnt/β-catenin signaling in the HBV life cycle was evaluated using immunoprecipitation assays with DOCK11 and bulk RNA sequencing methods. TNKS and Wnt/β-catenin signaling inhibitors significantly repressed cccDNA and HBV-DNA levels. Conversely, certain Wnt/β-catenin signaling agonists enhanced the HBV life cycle. DOCK11 directly binds to β-catenin to regulate HBV using its nuclear transport system. SKL2001, normally used as a Wnt/β-catenin signaling agonist, strongly reduced cccDNA in HBV-infected hepatocytes and in combination with entecavir predominantly eradicated HBV without cytotoxicity. Therefore, DOCK11 and other Wnt/β-catenin signaling molecules may be therapeutic targets to prevent persistent HBV infection. Full article

Exogenous RNA application, also known as spray-induced gene silencing (SIGS), is a new approach in plant biotechnology that utilizes RNA interference (RNAi) to modify plant traits. This technique involves applying RNA solutions of double-stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), or microRNA (miRNA) directly onto plant surfaces. This triggers RNAi-mediated silencing of specific genes within the plant or invading pathogens. While extensively studied for enhancing resistance to pathogens, the application of exogenous RNA to regulate plant endogenous genes remains less explored, creating a rich area for further research. This review summarizes and analyzes the studies reporting on the exogenously induced silencing of plant endogenes and transgenes using various RNA types. We also discuss the RNA production and delivery approaches, analyze the uptake and transport of exogenous RNAs, and the mechanism of action. The analysis revealed that SIGS/exoRNAi affects the expression of plant genes, which may contribute to crop improvement and plant gene functional studies. Full article

Mitochondria maintain cellular homeostasis through the dynamic balance of fusion and fission, which relies on nuclear-encoded mitochondrial fusion proteins, mitofusins 1 and 2 (Mfn1, Mfn2). Changes in Mfn1 and Mfn2 expression significantly affect mitochondrial fusion and fission, thereby affecting cellular metabolism. This study investigated the effect of Mfn1 expression on cell proliferation, apoptosis, and mitochondrial function by overexpressing Mfn1 (in OE-Mfn1 cells) and silencing Mfn1 using short hairpin RNA (shRNA) (in shMfn1 cells). Cell proliferation capacity, mitochondrial membrane potential, and mitochondrial ATP content were measured. To investigate the effects of Mfn1 on cellular metabolism and epigenetic modifications, the levels of metabolites α-KG, A-CoA, and SAM, as well as the levels of cellular methylation and acetylation, were detected by ELISA. Differentially expressed genes and metabolites were assessed by RNA-seq and LC-MS. This study demonstrates that alterations in Mfn1 gene expression can significantly affect mitochondrial metabolism and cell proliferation and apoptosis. In addition, Mfn1 affects the expression of genes encoding enzymes that are responsible for histone methylation and acetylation, thereby regulating these modifications. These findings provide a theoretical basis for further elucidation of the mechanisms by which Mfn1 affects cell proliferation, regulates metabolites, and modulates chromatin epigenetic modification. Full article

The biological significance of sodium–glucose cotransporter 2 (SGLT2) in clear cell renal cell carcinoma (ccRCC) has yet to be elucidated. In this study, we aimed to determine the role of SGLT2 in ccRCC tumor progression. The human ccRCC line KMRC-1, which contains a von Hippel–Lindau (VHL) gene mutation, was used to assess the effects of the SGLT2 inhibitor (SGLT2i) dapagliflozin on proliferation and migration in media containing different glucose concentrations (25, 12.5, or 5 mM). Dapagliflozin significantly reduced cell proliferation and migration in 25 mM glucose medium. Similarly, SGLT2 knockdown involving short hairpin RNA lentiviral transfection significantly decreased cell viability, migration, and colony formation compared with the control subline in 25 mM glucose medium. Moreover, tumor progression was inhibited in the media with low glucose concentrations. Remarkably, 2 µM dapagliflozin inhibited the progression of ccRCC at concentrations as low as 5 mM (normoglycemic model) glucose medium as well as 25 mM (severe glycemia model) glucose medium. In addition, dapagliflozin treatment significantly enhanced the apoptosis of ccRCC cells. Our findings demonstrate that SGLT2 impacts the progression of ccRCC with the VHL mutation. In light of the above findings, SGLT2is, which exert the dual effects of SGLT2 blockade and glycemic control, may represent a novel therapeutic agent, particularly in patients with ccRCC who suffer from concurrent diabetes mellitus. To the best of our knowledge, this is the first preclinical study demonstrating the impact of SGLT2 inhibition on the progression of ccRCC with the VHL mutation. Full article

Grafting significantly enhances plant quality, including stress resistance and fruit quality. We previously found that grafting watermelon onto pumpkin can alter the metabolite content, but the involvement of mobile RNA was unclear. Here, we established and comprehensively analyzed mobile mRNA (mb-mRNA) profiles, transcriptomes, and metabolomes between the rootstock (pumpkin) and scion (watermelon). A total of 834 mobile RNAs were identified in the pulp and stem of pumpkin-grafted watermelon. GO (Gene Ontology) and KO (Kyoto Encyclopedia of Genes and Genomes Orthology) analyses revealed photosynthesis- and carbon fixation-related mobile RNAs (e.g., Photosystem II D2, P700 chlorophyll a apoprotein) in the watermelon pulp and cell division-related mobile RNAs in the stem. Additionally, transcription factors like MADS and DNAJ exhibited mobility. The secondary structure prediction of the MADS-box transcription factor (CmoCh20G002790) showed multiple loop structures (e.g., internal and hairpin loops) related to its mobility. An integrated analysis of transcript and metabolite profiles indicated that photosynthesis-related products are regulated not only by the scion’s own RNA but also by mb-mRNA synthesized by the rootstock. This research advances our understanding of grafting’s molecular mechanisms and provides insights for improving crop quality and sustainability in agriculture. Full article

The timely and accurate detection of cancer is crucial for preventing disease progression and for the early treatment of confirmed cases. MiRNAs are cancer markers. In this study, a simple miRNA detection method is proposed. Three hairpins were designed based on gold nanoparticles combined with catalytic hairpin assembly nucleic acid amplification technology. The low-pH method was used for rapid coupling, and hairpin H1 was opened by miR-378, triggering the cycle reaction and signal amplification and finally forming a Y-shaped structure, thereby narrowing the distance between gold nanoparticles and achieving colorimetric detection. The absorbance change (A₆₂₀/A₅₂₀) was proportional to the concentration of miR-378 (0.05–5 nM), with a detection limit of 0.05 nM. This method also has an evident detection effect on real samples. HeLa and L-02 cell extracts were analyzed using this method. The former showed no obvious color change, whereas the maximum absorption peak of the latter showed a red shift, and the color changed from red to purple. The minimum number of cells that could be detected using HeLa cells was 500 cells/mL. Full article

(1) Background: Previous theoretical studies have provided arguments for the existence of a circular or hairpin RNA that could have served as a primitive informational and functional molecule at the origin of life. The present article consists of searching in current genomes for RNAs closest to this primitive RNA in terms of the occurrence of similar nucleotide motifs. (2) Methods: In searching for the smallest possible RNA capable of interacting with amino acids in the construction of the peptides of the primitive living world, we found a circular docosamer RNA molecule (length 22), which we called AL (for ALpha or Archetypal Loop). Then, we started to systematically track AL relics in current genomes in the form of motifs like pentamers or pairs of consecutive codons in common with AL. (3) Results: The sequence correspondence between AL and RNA sequences of organisms from different kingdoms of life (Archaea, Bacteria, and Eukarya) was found with high statistical significance, with a frequency gradient depending on both the antiquity of the species and the functional necessity of the genes. (4) Conclusions: Considering the suitability of AL as a candidate for being a primitive sequence, and the evolution of the different species considered, we can consider the AL RNA as a possible actor that favored the appearance of life on Earth. Full article

Background/Objectives: Small cell lung cancer (SCLC) is one of the malignancies with the worst prognosis, and there have been no major breakthroughs in its treatment for a long time. The majority of patients are diagnosed at the extensive stage, where the only option is chemotherapy, and even the addition of immune checkpoint inhibitors results in only modest benefits. The characterization of the molecular mechanisms behind therapy resistance has relevance in finding novel therapeutic approaches. Previous studies showed the possibility of annexin A1’s (ANXA1) involvement in the immunosuppressive tumor microenvironment in SCLC, and there are studies showing the direct effects of ANXA1 modulation on cancer cell aggressiveness. Methods: We aimed to characterize the roles of ANXA1 expression using publicly available transcriptomic data, the RNA-seq-based predictive algorithms EPIC and ESTIMATE, and immunohistochemistry on patient samples. For the in vitro studies, we silenced ANXA1 expression with short hairpin RNA in three SCLC cell lines, measured the growth rate with the trypan blue exclusion assay, assessed the chemosensitivity to cisplatin and etoposide with the Presto Blue^TM viability assay, and performed Western blots to assess changes in the levels of metabolic and mesenchymal markers and transcriptional drivers. Results: ANXA1-high tumors are associated with significantly increased immune infiltrates, stromality, and tumor-associated macrophages (TAMs). The ANXA1 protein is expressed on tumor cells and TAMs at the tissue level. ANXA1 silencing in H841 cells did not affect the growth rate; in SW1271 cells, shANXA1 cells grew significantly slower than shCTRL cells. Meanwhile, in H1048 cells, proliferation was significantly faster. Despite the different growth rates of the tested cell lines, ANXA1 silencing decreased the chemosensitivity to both cisplatin and etoposide in all three cell lines. Gene expression changes in mesenchymal markers, metabolic markers, dominant transcriptional drivers, and immune-relevant molecules were also characterized. Conclusions: This is the first comprehensive characterization of ANXA1 in SCLC to reveal its role in the tumor’s cell biology and the TME, aiming to boost further research in the field. Full article

The computational prediction of RNA three-dimensional (3D) structures remains a significant challenge, largely due to the limited understanding of RNA folding pathways. Although the scarcity of resolved native RNA structures has hindered the effectiveness of machine learning-based prediction methods, small, local structural motifs are both recurring and abundant in the available data. Precisely modeling these geometric motifs presents a promising approach to improving 3D structure prediction. In this paper, we introduce a novel mathematical model that represents RNA 3D structures as collections of interacting helices with concise geometric descriptions. By using a small set of parameters for each modeled helix, our method maps RNA strand segments onto helices within a 3D space, facilitating the effective assembly of large RNA structures. Preliminary tests on RNA sequences from the Protein Data Bank demonstrated the model’s potential in predicting key structural elements, including double helices, hairpin loops, and bulges. Full article

Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies. Full article

Mucor lusitanicus has emerged as a model organism for studying RNAi in early-diverging fungi. This fungus exhibits intricate RNAi pathways that play crucial roles in regulating gene expression, destroying invasive exogenous genetic material, and controlling the movement of transposable elements (TEs) to ensure genome stability. One of the most fascinating RNAi pathways of this fungus is the non-canonical RNAi pathway (NCRIP), which is independent of Dicer and Argonaute proteins and uses the atypical RNase III R3B2 to degrade specific target messenger RNAs (mRNAs), playing an essential role in genome stability and virulence. Despite accumulating data suggesting that this pathway is a degradation mechanism, there has been no conclusive evidence. Here, we conducted a comparative transcriptomic analysis of mRNA and small RNAs regulated by r3b2, identifying 35 direct NCRIP targets. Most of these direct NCRIP targets correspond to TEs, highlighting the significant role of this RNAi pathway in TE control. Detailed functional analysis of the NCRIP targets confirmed the crucial role of r3b2 in regulating gene expression of protein-coding genes and controlling TEs other than centromeric GremLINE1 transposons, emphasizing the important role of r3b2 in genome stability. Interestingly, the RNAs of the NCRIP targets harbor a unique motif consisting of CAG repeats which are known to form hairpin structures which are targeted by RNA interference. Additionally, the generation of transformants expressing mRNAs containing the luciferase reporter gene along direct NCRIP targets reveals that this RNAi pathway is a true degradation mechanism for specific mRNAs. These results are expected to contribute to the understanding of the regulation of the NCRIP pathway through the analysis of its direct targets identified here. Full article

MicroRNA-155-3p (miR-155-3p) is an important biomarker in various pathological conditions, including cancer, making the development of sensitive and specific detection methods crucial. Here, we present a molecular beacon (MB-G4) that underwent a conformational switch upon hybridization with miR-155-3p, enabling the formation of a G-quadruplex (G4) structure. This G4 was recognized by the fluorogenic ligand N-methyl mesoporphyrin IX (NMM), producing a fluorescence signal proportional to the target concentration, making it a new detection method. The conformational dynamics of MB-G4 were characterized through circular dichroism (CD) spectroscopy and native polyacrylamide gel electrophoresis (PAGE), confirming the transition from a hairpin structure to an RNA–DNA hybrid duplex that facilitated G4 formation. The optimization of the experimental conditions, including the potassium chloride (KCl) and NMM concentrations, ensured selective detection with minimal background signal. The detection limit (LOD) was determined to be 10.85 nM, using a linear fluorescence response curve, and the specificity studies demonstrated a clear distinction between miR-155-3p and miR-155-5p. Furthermore, MB-G4 was studied with total RNA extracted from the lung cancer cell line A549 to evaluate its detection in a more complex environment and was able to detect its target, validating its potential for biological sample analysis. Full article