Interference Pathway Research Articles

Regulation of MicroRNA Expression in Scleroderma and Idiopathic Pulmonary Fibrosis: A Research Study

Introduction: Scleroderma (SSc) is an autoimmune disorder with the hallmark of fibrosis of the skin, vasculature and internal organs. Patients with SSc and undifferentiated connective tissue disease (UCTD) are susceptible to interstitial lung disease (ILD), leading to decreased lung function and death. Idiopathic pulmonary fibrosis (IPF) is a form of ILD that is not associated with extrapulmonary manifestations. In this study, lung involvement of SSc was studied by observing how disease progression and pathogenesis differ among patients with SSc, UCTD, and ILD compared to healthy controls and patients with IPF. Our group has previously identified disease targets through microRNA sequencing, including the DICER enzyme, which works closely with the protein DGCR8 and the enzyme DROSHA in the RNA interference pathway. The canonical pathway stipulates that DICER processes microRNAs in the cytosol while DGCR8 and DROSHA process microRNAs in the nucleus. DICER, DROSHA, and DGCR8 are hypothesized to contribute to ILD progression. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from voluntary participants, including healthy controls. PBMCs were subsequently lysed with subcellular fractionation buffer. Western blotting was done on the resulting cytosolic and nucleic fractions for DICER, DROSHA, and DGCR8 protein expression. The cytosolic fractions were normalized to GAPDH, while the nucleic fractions were normalized to B2M. Nonparametric Kruskal‐Wallis tests were used for statistical analysis. Results: The medians were significantly higher for healthy controls for DICER in the nucleus with a p-value of 0.0302, and DROSHA in the cytosol with a p-value of 0.0406 compared to patients with SSc, UCTD, and IPF. Discussion: Differences in expression were found for DROSHA in the cytosol and DICER in the nucleus, suggesting dysregulation of the non-canonical RNA interference pathways in SSc, UCTD, and IPF patients. Variability of disease progression within the groups could lead to variable enzyme and protein levels within the same disease status. With larger sample sizes, statistically insignificant differences would become significant. Lipid nanoparticle technology could be used to deliver deficient microRNAs to silence mRNA in patients. Conclusion: Due to dysregulation of the RNA interference pathway, microRNAs may be inadequately processed in the patient groups.

Engineered gut symbiont inhibits microsporidian parasite and improves honey bee survival

Honey bees (Apis mellifera) are critical agricultural pollinators as well as model organisms for research on development, behavior, memory, and learning. The parasite Nosema ceranae, a common cause of honey bee colony collapse, has developed resistance to small-molecule therapeutics. An alternative long-term strategy to combat Nosema infection is therefore urgently needed, with synthetic biology offering a potential solution. Honey bees harbor specialized bacterial gut symbionts that are transmitted within hives. Previously, these have been engineered to inhibit ectoparasitic mites by expressing double-stranded RNA (dsRNA) targeting essential mite genes, via activation of the mite RNA interference (RNAi) pathway. In this study, we engineered a honey bee gut symbiont to express dsRNA targeting essential genes of N. ceranae via the parasite's own RNAi machinery. The engineered symbiont sharply reduced Nosema proliferation and improved bee survival following the parasite challenge. This protection was observed in both newly emerged and older forager bees. Furthermore, engineered symbionts were transmitted among cohoused bees, suggesting that introducing engineered symbionts to hives could result in colony-level protection.

Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory element analyses in sunflower (Helianthus annuus).

RNA interference (RNAi) regulates a variety of eukaryotic gene expressions that are engaged in response to stress, growth, and the conservation of genomic stability during developmental phases. It is also intimately connected to the post-transcriptional gene silencing (PTGS) process and chromatin modification levels. The entire process of RNA interference (RNAi) pathway gene families mediates RNA silencing. The main factors of RNA silencing are the Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) gene families. To the best of our knowledge, genome-wide identification of RNAi gene families like DCL, AGO, and RDR in sunflower (Helianthus annuus) has not yet been studied despite being discovered in some species. So, the goal of this study is to find the RNAi gene families like DCL, AGO, and RDR in sunflower based on bioinformatics approaches. Therefore, we accomplished an inclusive in silico investigation for genome-wide identification of RNAi pathway gene families DCL, AGO, and RDR through bioinformatics approaches such as (sequence homogeneity, phylogenetic relationship, gene structure, chromosomal localization, PPIs, GO, sub-cellular localization). In this study, we have identified five DCL (HaDCLs), fifteen AGO (HaAGOs), and ten RDR (HaRDRs) in the sunflower genome database corresponding to the RNAi genes of model plant Arabidopsis thaliana based on genome-wide analysis and a phylogenetic method. The analysis of the gene structure that contains exon-intron numbers, conserved domain, and motif composition analyses for all HaDCL, HaAGO, and HaRDR gene families indicated almost homogeneity among the same gene family. The protein-protein interaction (PPI) network analysis illustrated that there exists interconnection among identified three gene families. The analysis of the Gene Ontology (GO) enrichment showed that the detected genes directly contribute to the RNA gene-silencing and were involved in crucial pathways. It was observed that the cis-acting regulatory components connected to the identified genes were shown to be responsive to hormone, light, stress, and other functions. That was found in HaDCL, HaAGO, and HaRDR genes associated with the development and growth of plants. Finally, we are able to provide some essential information about the components of sunflower RNA silencing through our genome-wide comparison and integrated bioinformatics analysis, which open the door for further research into the functional mechanisms of the identified genes and their regulatory elements.

The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.

Seasonality influences key physiological components contributing to Culex pipiens vector competence.

Mosquitoes are the most important animal vector of disease on the planet, transmitting a variety of pathogens of both medical and veterinary importance. Mosquito-borne diseases display distinct seasonal patterns driven by both environmental and biological variables. However, an important, yet unexplored component of these patterns is the potential for seasonal influences on mosquito physiology that may ultimately influence vector competence. To address this question, we selected Culex pipiens, a primary vector of the West Nile virus (WNV) in the temperate United States, to examine the seasonal impacts on mosquito physiology by examining known immune and bacterial components implicated in mosquito arbovirus infection. Semi-field experiments were performed under spring, summer, and late-summer conditions, corresponding to historically low-, medium-, and high-intensity periods of WNV transmission, respectively. Through these experiments, we observed differences in the expression of immune genes and RNA interference (RNAi) pathway components, as well as changes in the distribution and abundance of Wolbachia in the mosquitoes across seasonal cohorts. Together, these findings support the conclusion that seasonal changes significantly influence mosquito physiology and components of the mosquito microbiome, suggesting that seasonality may impact mosquito susceptibility to pathogen infection, which could account for the temporal patterns in mosquito-borne disease transmission.

SiRNA and apoptosis in breast cancer cells: A minireview

Small interfering RNA (siRNA), is a class of double-stranded RNA at first non-coding RNA molecules, operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. siRNAs have been widely used to study gene function and extensively exploited for their potential therapeutic applications. siRNAs have gained attention as a potential therapeutic reagent due to their ability to inhibit specific genes in cancer cells. Increased resistance to apoptosis is a challenging issue for treatment of many cancers, including breast cancer. It has been recently reported that siRNAs and RNAi technology can be used to increase the apoptotic susceptibility of cancer cells. It has been shown that apoptosis is induced in cancer cells by siRNA-mediated silencing of the livin/ML-IAP/KIAP gene. Association of siRNA with apoptosis via mitochondrial depolarization and caspase-3 activation has been highlighted in cancer cells. Although many aspects of siRNA actions in cancer cells have been revealed through in vivo and in vitro studies, the biological mechanisms underlying siRNA mediated knockdown of gene expression and apoptosis induction in cancer cells are not yet fully understood. The main aim of this review is to investigate the effects of siRNAs on apoptosis induction in breast cancer cells.

Argonaute 1 and 5 proteins play crucial roles in the defence against cucumber green mottle mosaic virus in watermelon.

RNA silencing, a core part of plants' antiviral defence, requires the ARGONAUTE, DICER-like, and RNA-dependent RNA polymerase proteins. However, how these proteins contribute to watermelon's RNA interference (RNAi) pathway response to cucumber green mottle mosaic virus (CGMMV) has not been characterized. Here, we identifyseven ClAGO, four ClDCL, and 11 ClRDR genes in watermelon and analyse their expression profiles when infected with CGMMV. ClAGO1 and ClAGO5 expression levels were highly induced by CGMMV infection. The results of ClAGO1 and ClAGO5 overexpression and silencing experiments suggest that these genes play central roles in watermelon's antiviral defence. Furthermore, co-immunoprecipitation and bimolecular fluorescence complementation experiments showed that ClAGO1 interacts with ClAGO5 in vivo, suggesting that ClAGO1 and ClAGO5 co-regulate watermelon defence against CGMMV infection. We also identified the ethylene response factor (ERF) binding site in the promoters of the ClAGO1 and ClAGO5 genes, and ethylene (ETH) treatment significantly increased ClAGO5 expression. Two ERF genes (Cla97C08G147180 and Cla97C06G122830) closely related to ClAGO5 expression were identified using co-expression analysis. Subcellular localization revealed that two ERFs and ClAGO5 predominantly localize at the nucleus, suggesting that enhancement of resistance to CGMMV by ETH is probably achieved through ClAGO5 but not ClAGO1. Our findings reveal aspects of the mechanisms underlying RNA silencing in watermelon against CGMMV.

OPA1 Dominant Optic Atrophy: Pathogenesis and Therapeutic Targets.

OPA1 Dominant Optic Atrophy: Pathogenesis and Therapeutic Targets.

Impact of stereopure chimeric backbone chemistries on the potency and durability of gene silencing by RNA interference.

Herein, we report the systematic investigation of stereopure phosphorothioate (PS) and phosphoryl guanidine (PN) linkages on siRNA-mediated silencing. The incorporation of appropriately positioned and configured stereopure PS and PN linkages to N-acetylgalactosamine (GalNAc)-conjugated siRNAs based on multiple targets (Ttr and HSD17B13) increased potency and durability of mRNA silencing in mouse hepatocytes in vivo compared with reference molecules based on clinically proven formats. The observation that the same modification pattern had beneficial effects on unrelated transcripts suggests that it may be generalizable. The effect of stereopure PN modification on silencing is modulated by 2'-ribose modifications in the vicinity, particularly on the nucleoside 3' to the linkage. These benefits corresponded with both an increase in thermal instability at the 5'-end of the antisense strand and improved Argonaute 2 (Ago2)loading. Application of one of our most effective designs to generate a GalNAc-siRNA targeting human HSD17B13 led to ∼80% silencing that persisted for at least 14 weeks after administration of a single 3 mg/kg subcutaneous dose in transgenic mice. The judicious use of stereopure PN linkages improved the silencing profile of GalNAc-siRNAs without disrupting endogenous RNA interference pathways and without elevating serum biomarkers for liver dysfunction, suggesting they may be suitable for therapeutic application.

Rift Valley Fever Virus Primes Immune Responses in Aedes aegypti Cells.

The ongoing global emergence of arthropod-borne (arbo) viruses has accelerated research into the interactions of these viruses with the immune systems of their vectors. Only limited information exists on how bunyaviruses, such as Rift Valley fever virus (RVFV), are sensed by mosquito immunity or escape detection. RVFV is a zoonotic phlebovirus (Bunyavirales; Phenuiviridae) of veterinary and human public health and economic importance. We have shown that the infection of mosquitoes with RVFV triggers the activation of RNA interference pathways, which moderately restrict viral replication. Here, we aimed to better understand the interactions between RVFV and other vector immune signaling pathways that might influence RVFV replication and transmission. For this, we used the immunocompetent Aedes aegypti Aag2 cell line as a model. We found that bacteria-induced immune responses restricted RVFV replication. However, virus infection alone did not alter the gene expression levels of immune effectors. Instead, it resulted in the marked enhancement of immune responses to subsequent bacterial stimulation. The gene expression levels of several mosquito immune pattern recognition receptors were altered by RVFV infection, which may contribute to this immune priming. Our findings imply that there is a complex interplay between RVFV and mosquito immunity that could be targeted in disease prevention strategies.

Disruption of the Aspergillus fumigatus RNA interference machinery alters the conidial transcriptome.

The RNA interference (RNAi) pathway has evolved numerous functionalities in eukaryotes, with many on display in Kingdom Fungi. RNAi can regulate gene expression, facilitate drug resistance, or even be altogether lost to improve growth potential in some fungal pathogens. In the WHO fungal priority pathogen, Aspergillus fumigatus, the RNAi system is known to be intact and functional. To extend our limited understanding of A. fumigatus RNAi, we first investigated the genetic variation in RNAi-associated genes in a collection of 217 environmental and 83 clinical genomes, where we found that RNAi components are conserved even in clinical strains. Using endogenously expressed inverted-repeat transgenes complementary to a conditionally essential gene (pabA) or a nonessential gene (pksP), we determined that a subset of the RNAi componentry is active in inverted-repeat transgene silencing in conidia and mycelium. Analysis of mRNA-seq data from RNAi double-knockout strains linked the A. fumigatus dicer-like enzymes (DclA/B) and RNA-dependent RNA polymerases (RrpA/B) to regulation of conidial ribosome biogenesis genes; however, surprisingly few endogenous small RNAs were identified in conidia that could explain this broad change. Although RNAi was not clearly linked to growth or stress response defects in the RNAi knockouts, serial passaging of RNAi knockout strains for six generations resulted in lineages with diminished spore production over time, indicating that loss of RNAi can exert a fitness cost on the fungus. Cumulatively, A. fumigatus RNAi appears to play an active role in defense against double-stranded RNA species alongside a previously unappreciated housekeeping function in regulation of conidial ribosomal biogenesis genes.

Switching Quantum Interference in Single‐Molecule Junctions by Mechanical Tuning

AbstractThe charge transport through single‐molecule electronic devices can be controlled mechanically by changing the molecular geometrical configuration in situ, but the tunable conductance range is typically less than two orders of magnitude. Herein, we proposed a new mechanical tuning strategy to control the charge transport through the single‐molecule junctions via switching quantum interference patterns. By designing molecules with multiple anchoring groups, we switched the electron transport between the constructive quantum interference (CQI) pathway and the destructive quantum interference (DQI) pathway, and more than four orders of magnitude conductance variation can be achieved by shifting the electrodes in a range of about 0.6 nm, which is the highest conductance range ever achieved using mechanical tuning.

Switching Quantum Interference in Single-Molecule Junctions by Mechanical Tuning.

The charge transport through single-molecule electronic devices can be controlled mechanically by changing the molecular geometrical configuration in situ, but the tunable conductance range is typically less than two orders of magnitude. Herein, we proposed a new mechanical tuning strategy to control the charge transport through the single-molecule junctions via switching quantum interference patterns. By designing molecules with multiple anchoring groups, we switched the electron transport between the constructive quantum interference (CQI) pathway and the destructive quantum interference (DQI) pathway, and more than four orders of magnitude conductance variation can be achieved by shifting the electrodes in a range of about 0.6 nm, which is the highest conductance range ever achieved using mechanical tuning.

Signature of quantum interference effect in inter-layer Coulomb drag in graphene-based electronic double-layer systems

The distinguishing feature of a quantum system is interference arising from the wave mechanical nature of particles which is clearly central to macroscopic electronic properties. Here, we report the signature of quantum interference effect in inter-layer transport process. Via systematic magneto-drag experiments on graphene-based electronic double-layer systems, we observe low-field correction to the Coulomb-scattering-dominated inter-layer drag resistance in a wide range of temperature and carrier density, with its characteristics sensitive to the band topology of graphene layers. These observations can be attributed to a new type of quantum interference between drag processes, with the interference pathway comprising different carrier diffusion paths in the two constituent conductors. The emergence of such effect relies on the formation of superimposing planar diffusion paths, among which the impurity potentials from intermediate insulating spacer play an essential role. Our findings establish an ideal platform where the interplay between quantum interference and many-body interaction is essential.

Control of Raman Scattering Quantum Interference Pathways in Graphene.

Graphene is an ideal platform to study the coherence of quantum interference pathways by tuning doping or laser excitation energy. The latter produces a Raman excitation profile that provides direct insight into the lifetimes of intermediate electronic excitations and, therefore, on quantum interference, which has so far remained elusive. Here, we control the Raman scattering pathways by tuning the laser excitation energy in graphene doped up to 1.05 eV. The Raman excitation profile of the G mode indicates its position and full width at half-maximum are linearly dependent on doping. Doping-enhanced electron-electron interactions dominate the lifetimes of Raman scattering pathways and reduce Raman interference. This will provide guidance for engineering quantum pathways for doped graphene, nanotubes, and topological insulators.

RNA interference is essential to modulating the pathogenesis of mosquito-borne viruses in the yellow fever mosquito Aedes aegypti

While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects' antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the Aedes aegypti Dicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.

ERASing endoplasmic reticulum stress: the faster, the better.

The endoplasmic reticulum (ER) has evolved multiple mechanisms to maintain homeostasis under stress conditions. A recent study by Efstathiou et al. identified a novel mechanism of silencing ER-associated RNAs by the exogenous RNA interference pathway. This adaptive response reduces protein flux in the ER under stressful conditions.

Magnon-induced absorption via quantum interference.

We propose a scheme to generate magnon-induced absorption (MIA) in a two-cavity magnonics system. By placing an yttrium iron garnet (YIG) sphere into one of two coupled microwave cavities, three different interference pathways are established by the photon coupling and magnon-photon coupling, leading to the conversion from suppression to enhancement on resonance under proper conditions. Most interestingly, the analytical results of the probe absorption are solved based on bright and dark modes in a dressed-state picture, which can be used to explain the position, width, and height of the absorption peaks accurately. Furthermore, we investigate the noise spectral density (NSD) of the microwave cavity and find out the similar MIA phenomena, which may provide a feasible way to remotely detect a magnon with an optical method.

Ingestion of Species-Specific dsRNA Alters Gene Expression and Can Cause Mortality in the Forest Pest, Ips calligraphus

Ips calligraphus (Germar) is a conifer pest that causes economically and ecologically significant tree mortality, particularly when forests are stressed. As forests become increasingly vulnerable to pest outbreaks due to habitat fragmentation, invasive species, or climate change, innovative management strategies are needed to augment traditional approaches. Manipulating the RNA interference (RNAi) pathway is emerging as a novel pest management technology that could serve as a means of managing I. calligraphus while minimizing non-target effects. Demonstrating effectiveness of exogenous double-stranded RNA (dsRNA) in inducing changes in gene expression and causing mortality is an essential step. In this study, oral ingestion of dsRNA caused significant changes in gene expression and increased mortality for two of the three target dsRNAs tested. Additionally, we sequenced 5 mRNA libraries from adult beetles to assemble a transcriptome, from which we identified sequences of target genes for dsRNAs, and 10 genes in the I. calligraphus transcriptome putatively involved in the RNAi pathway. We demonstrate that oral ingestion of exogenous dsRNA can trigger the RNAi pathway. This is the first published study to artificially trigger the RNAi pathway in an Ips spp. and the first step in evaluating the potential for pest management strategies utilizing RNAi against this pest.

Heterochromatin and RNAi act independently to ensure genome stability in Mucorales human fungal pathogens

Chromatin modifications play a fundamental role in controlling transcription and genome stability and yet despite their importance, are poorly understood in early-diverging fungi. We present a comprehensive study of histone lysine and DNA methyltransferases across the Mucoromycota, emphasizing heterochromatin formation pathways that rely on the Clr4 complex involved in H3K9-methylation, the Polycomb-repressive complex 2 driving H3K27-methylation, or DNMT1-like methyltransferases that catalyze 5mC DNA methylation. Our analysis uncovered H3K9-methylated heterochromatin as the major chromatin modification repressing transcription in these fungi, which lack both Polycomb silencing and cytosine methylation. Although small RNAs generated by RNA interference (RNAi) pathways facilitate the formation of heterochromatin in many eukaryotic organisms, we show that RNAi is not required to maintain either genomic or centromeric heterochromatin in Mucor. H3K9-methylation and RNAi act independently to control centromeric regions, suggesting a functional subspecialization. Whereas the H3K9 methyltransferase Clr4 and heterochromatin formation are essential for cell viability, RNAi is dispensable for viability yet acts as the main epigenetic, regulatory force repressing transposition of centromeric GremLINE1 elements. Mutations inactivating canonical RNAi lead to rampant transposition and insertional inactivation of targets resulting in antimicrobial drug resistance. This fine-tuned, Rdrp2-dependent RNAi activity is critical for genome stability, restricting GremLINE1 retroelements to the centromeres where they occupy long heterochromatic islands. Taken together, our results suggest that RNAi and heterochromatin formation are independent genome defense and regulatory mechanisms in the Mucorales, contributing to a paradigm shift from the cotranscriptional gene silencing observed in fission yeasts to models in which heterochromatin and RNAi operate independently in early-diverging fungi.