Regulatory RNAs Research Articles

Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation

RNA interactome studies have revealed that hundreds of zinc-finger proteins (ZFPs) are candidate RNA-binding proteins (RBPs), yet their RNA substrates and functional significance remain largely uncharacterized. Here, we present a systematic multi-omics analysis of the DNA- and RNA-binding targets and regulatory roles of more than 100 ZFPs representing 37 zinc-finger families. We show that multiple ZFPs are previously unknown regulators of RNA splicing, alternative polyadenylation, stability, or translation. The examined ZFPs show widespread sequence-specific RNA binding and preferentially bind proximal to transcription start sites. Additionally, several ZFPs associate with their targets at both the DNA and RNA levels. We highlight ZNF277, a C2H2 ZFP that binds thousands of RNA targets and acts as a multi-functional RBP. We also show that ZNF473 is a DNA/RNA-associated protein that regulates the expression and splicing of cell cycle genes. Our results reveal diverse roles for ZFPs in transcriptional and post-transcriptional gene regulation.

PARN Maintains RNA Stability to Regulate Insulin Maturation and GSIS in Pancreatic β Cells

AbstractDiabetes, a metabolic disorder characterized by hyperglycemia, underscores the importance of normal pancreatic β‐cell development and function in maintaining glucose homeostasis. Poly(A)‐specific ribonuclease (PARN) serves as the principal regulator of messenger RNA (mRNA) stability, yet its specific role in pancreatic β cells remains unclear. This study utilizes mice with targeted PARN deficiency in β cells to elucidate this role. Notably, Parn conditional knockout mice present unaltered β‐cell development and insulin sensitivity but reduced glucose‐stimulated insulin secretion (GSIS). The observed outcomes are corroborated in NIT‐1 cells. Furthermore, transcriptomic analyses reveal aberrant mRNA expression of genes crucial for insulin secretion in PARN‐deficient β cells. Insights from linear amplification of complementary DNA ends and sequencing and coimmunoprecipitation experiments reveal an interaction between PARN and polypyrimidine tract‐binding protein 1 (PTBP1), regulating the RNA stability of solute carrier family 30, member 8 (Slc30a8) and carbohydrate sulfotransferase 3 (Chst3). Interference with either PARN or PTBP1 disrupts this stability. These data indicate that PARN deficiency hampers GSIS and insulin maturation by destabilizing Slc30a8 and Chst3 RNAs. These findings provide compelling evidence indicating that PARN is a potential therapeutic target for enhancing insulin maturation and secretion.

Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48

BackgroundN6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2′-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity.ResultsHere, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2.ConclusionsOur findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

Modulation of Vibrio cholerae gene expression through conjugative delivery of engineered regulatory small RNAs.

Given the prevalence of antibiotic resistance, there is an increasing need to develop alternative approaches to managing pathogenic bacteria. In this work, we explore the feasibility of modulating the expression of various cellular systems in Vibrio cholerae using engineered regulatory sRNAs delivered into cells via DNA conjugation. These sRNAs are based on regulatory sRNAs found in V. cholerae and exploit its native regulatory machinery. By delivering these sRNAs conjugatively along with a real-time marker for DNA transfer, we found that complete knockdown of a targeted cellular system could be achieved within one cell division cycle after sRNA gene delivery. These results indicate that conjugative delivery of engineered regulatory sRNAs is a rapid and robust way of precisely targeting V. cholerae.

Rational Design of High-Efficiency Synthetic Small Regulatory RNAs and Their Application in Robust Genetic Circuit Performance Through Tight Control of Leaky Gene Expression.

Synthetic sRNAs show promise as tools for targeted and programmable gene expression manipulation. However, the design of high-efficiency synthetic sRNAs is a challenging task that necessitates careful consideration of multiple factors. Therefore, this study aims to investigate rational design strategies that significantly and robustly enhance the efficiency of synthetic sRNAs. This is achieved by optimizing the following parameters: the sRNA scaffold, mRNA binding affinity, Hfq protein expression level, and mRNA secondary structure. By utilizing optimized synthetic sRNAs within a positive feedback circuit, we effectively addressed the issue of gene expression leakage─an enduring challenge in synthetic biology that undermines the reliability of genetic circuits in bacteria. Our designed synthetic sRNAs successfully prevented gene expression leakage, thus averting unintended circuit activation caused by initial expression noise, even in the absence of signal molecules. This result shows that high-efficiency synthetic sRNAs not only enable precise gene knockdown for metabolic engineering but also ensure the robust performance of synthetic circuits. The strategies developed here hold significant promise for broad applications across diverse biotechnological fields, establishing synthetic sRNAs as pivotal tools in advancing synthetic biology and gene regulation.

Mechanisms underlying sex differences in autoimmunity.

Autoimmune diseases are a leading cause of disability worldwide. Most autoimmune diseases occur more often in women than men, with rheumatic autoimmune diseases being among those most highly expressed in women. Several key factors, identified mainly in animal models and cell culture experiments, are important in increasing autoimmune disease in females. These include sex hormones, immune genes including those found on the X chromosome, sex-specific epigenetic effects on genes by estrogen and the environment, and regulation of genes and messenger RNA by microRNAs found in extracellular vesicles. Evidence is also emerging that viruses as well as drugs or toxins that damage mitochondria may contribute to increased levels of autoantibodies against nuclear and mitochondrial antigens, which are common in many autoimmune diseases. The purpose of this Review is to summarize our current understanding of mechanisms that may determine sex differences in autoimmune disease.

G3BP2 promotes tumor progression and gemcitabine resistance in PDAC via regulating PDIA3-DKC1-hENT in a stress granules-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is distinguished by its aggressive malignancy, limited treatment avenues and a tendency towards chemotherapy resistance, underscoring the critical need for advanced research to uncover new therapeutic approaches. Stress granules (SGs) that is implicated in cellular self-protection mechanism, along with its associated family molecules have shown pro-cancer effects and are closely related to tumor chemotherapy resistance. In this study we investigated the relationship between Ras GTPase-activating protein-binding proteins 2 (G3BP2), a core component of SGs, and the malignancy of PDAC as well as its resistance to the chemotherapy drug gemcitabine. Analyzing TCGA dataset revealed that the expression of G3BP1 and G3BP2 was significantly upregulated in PDAC compared with adjacent normal pancreatic tissues, and the high expression of G3BP2 rather than G3BP1 was significantly associated with poorer overall survival (OS) in PDAC patients. We demonstrated that knockdown of G3BP2 inhibited the proliferation and invasion of PANC-1 and CFPAC-1 cells in vitro and in vivo. By analyzing the differentially expressed genes in G3BP2 knockdown and overexpressed PANC-1 cells, we identified DKC1 that was associated with RNA stability and regulation as the target of G3BP2. We demonstrated that G3BP2 bound to PDIA3 mRNA and recruited them into SGs, increasing the stability of PDIA3 mRNA and attenuating its translation efficiency, thereby promoting DKC1 expression. Furthermore, DKC1 could bind to hENT mRNA and inhibited its expression, which enhanced gemcitabine resistance of PDAC. Therefore, we propose a novel mechanism wherein G3BP2 facilitates PDAC's resistance to chemotherapy by modulating PDIA3-DKC1-hENT in a SGs-dependent way, suggesting G3BP2 SGs a protentional therapeutic target for the treatment in PDAC.

Small Regulatory RNAs of the Rsm Clan in Pseudomonas.

Bacteria of the genus Pseudomonas are ubiquitous on Earth due to their great metabolic versatility and adaptation to fluctuating environments and different hosts. Some groups are important animal/human and plant pathogens, whereas others are studied for their biotechnological applications, including bioremediation, biological control of phytopathogens and plant growth promotion. Notably, their adaptability is mediated by various signal transduction systems, with the post-transcriptional Gac-Rsm cascade playing a key role. This pervasive Pseudomonas pathway controls major transitions at the population level, such as motile/sessile lifestyle, primary/secondary metabolism or replicative/infective behaviour. A hallmark of the Gac-Rsm cascade is the participation of small, regulatory, non-coding RNAs of the Rsm clan. These RNAs are synthetised in response to cell-density-dependent autoinducer signals channelled through the GacS/GacA two-component system, and they counteract, by molecular mimicry, the translational control that RNA-binding proteins of the RsmA family exert over hundreds of mRNAs. Rsm RNAs have been investigated in a few Pseudomonas model species, evidencing the presence of a variable number and families of genes depending on the taxonomic clade. However, the global picture of the distribution of these riboregulators at the genus level was unknown until now. We have undertaken a comprehensive survey and annotation of the vast array of gene sequences encoding members of the Rsm RNA clan in 245 complete genomes that cover 28 phylogenomic clades across the entire genus. The properties of the different families of rsm genes, their phylogenetic radiation, as well as the features of their promoters and adjacent regions, are discussed. The novel insights presented in our manuscript will significantly boost research on the biology of these prevalent RNAs in understudied species of the genus Pseudomonas and closely related genera.

Evaluation of clinically significant miRNAs level by machine learning approaches utilizing total transcriptome data

Analysis of the mechanisms underlying the occurrence and progression of cancer represents a key objective in contemporary clinical bioinformatics and molecular biology. Utilizing omics data, particularly transcriptomes, enables a detailed characterization of expression patterns and post-transcriptional regulation across various RNA types relative to the entire transcriptome. Here, we assembled a dataset comprising transcriptomic data from approximately 16.000 patients encompassing over 160 types of cancer. We employed state-of-the-art gradient boosting algorithms to discern intricate correlations in the expression levels of four clinically significant microRNAs, specifically hsa-mir-21, hsa-let-7a-1, hsa-let-7b, and hsa-let-7i, with the expression levels of the remaining 60.660 unique RNAs. Our analysis revealed a dependence of the expression levels of the studied microRNAs on the concentrations of several small nucleolar RNAs and regulatory long non-coding RNAs. Notably, the roles of these RNAs in the development of specific cancer types had been previously established through experimental evidence. Subsequent evaluation of the created database will facilitate the identification of a broader spectrum of overarching dependencies related to changes in the expression levels of various RNA classes in diverse cancers. In future it will make possible discovery of unique alterations specific to certain types of malignant transformations.

Subcellular Level Spatial Transcriptomics with PHOTON.

The subcellular localization of RNA is closely linked to its function. Many RNA species are partitioned into organelles and other subcellular compartments for storage, processing, translation, or degradation. Thus, capturing the subcellular spatial distribution of RNA would directly contribute to the understanding of RNA functions and regulation. Here, we present PHOTON (Photoselection of Transcriptome over Nanoscale), a method which combines high resolution imaging with high throughput sequencing to achieve spatial transcriptome profiling at subcellular resolution. We demonstrate PHOTON as a versatile tool to accurately capture the transcriptome of target cell types in situ at the tissue level such as granulosa cells in the ovary, as well as RNA content within subcellular compartments such as the nucleolus and the stress granule. Using PHOTON, we also reveal the functional role of m6A modification on mRNA partitioning into stress granules. These results collectively demonstrate that PHOTON is a flexible and generalizable platform for understanding subcellular molecular dynamics through the transcriptomic lens.

514 Roles of core regulatory noncoding RNAs in bovine Staphylococcus aureus subclinical mastitis

Abstract Staphylococcus aureus (S. aureus) is an opportunistic pathogen frequently associated with subclinical mastitis and accounts for a large proportion of the economic losses due to mastitis on Canadian dairy farms. Despite a plethora of investigations on the molecular mechanisms of mastitis, little information is available on the roles of regulatory noncoding RNAs (ncRNA). This study aimed to uncover the regulatory roles of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in the response to subclinical mastitis due to S. aureus of cows. Transcriptome sequencing (RNA-Seq and miRNA-Seq) was conducted on milk somatic cells from 13 cows with S. aureus subclinical mastitis [high somatic cell counts (SCC) of ≥350,000 cells/mL for ≥3 consecutive months and positive for S. aureus only], and 5 healthy cows (low SCC< 100,000 cells/mL for ≥3 consecutive months and negative for mastitis pathogens). RNASeq and smRNASeq nf-core bioinformatics pipelines were used to process the data. Results showed that 97 miRNAs (36 up-regulated and 61 down-regulated) and 1,565 lncRNAs (617 up-regulated and 948 down-regulated) were differentially expressed (DE; FDR< 0.05) between S. aureus subclinical mastitic cows and healthy controls. Competing endogenous networks comprising co-expressed differentially expressed mRNAs (DEGs), miRNAs (DEMs), and lncRNAs (DELs) were constructed. CytoHubba, a Cytoscape plugin, was utilized to identify the top hub regulatory ncRNAs including bta-miR-2387, bta-miR-331, bta-miR-95, bta-miR-744, bta-miR-455, bta-novel-miR-60, bta-miR-3533, bta-miR-500, bta-miR-1249 and bta-novel-miR-66). The results revealed that the main hubs (downregulated bta-miR-2387 and upregulated bta-miR-331) potentially regulate numerous DEGs during S. aureus subclinical mastitis such as NFKB1, IL10RA, IL17RA, TLR10, LOC112442665, LOC112448481 and LOC112442015, etc., and TUNAR, LOC112445429, LOC112444484 and LOC101907369, respectively. Functional analysis of bta-miR-331 target genes revealed roles in several KEGG pathways (e.g., tight junction, bacterial invasion of epithelial cells) and biological processes gene ontology (GO) terms (e.g., cell adhesion, cell locomotion and cell motility). Similarly, roles for bta-miR-2387 in KEGG pathways like JAK-STAT signaling pathway, NOD-like receptor signaling pathway and inflammatory bowel disease, and biological process GO terms such as inflammatory response, neutrophil migration, leucocyte migration and response to cytokine were revealed. MiR-331 has been found to promote cell proliferation, migration, and invasion of breast cancer. Furthermore, bta-miR-2387 has been shown to regulate several immune pathways which aid in improving the movement of spermatids across the epithelium and preleptotene spermatocytes across the blood-testis barrier during spermatogenesis. This supports our findings and suggests that bta-miR-2387 and bta-miR-331 could be pivotal regulators of immune-related genes and pathways during S. aureus subclinical mastitis. Thus, they hold potential as biomarkers for the development of therapeutic and diagnostic tools for managing subclinical mastitis.

Exploring the transcription start sites and other genomic features facilitates the accurate identification and annotation of small RNAs across multiple stress conditions in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) is a pathogen that is known for its ability to persist in harsh environments and cause chronic infections. Understanding the regulatory networks of MTB is crucial for developing effective treatments. Small regulatory RNAs (sRNAs) play important roles in gene expression regulation in all kingdoms of life, and their classification based solely on genomic location can be imprecise due to the computational-based prediction of protein-coding genes in bacteria, which often neglects segments of mRNA such as 5'UTRs, 3'UTRs, and intercistronic regions of operons. To address this issue, our study simultaneously discovered genomic features such as TSSs, UTRs, and operons together with sRNAs in the M. tuberculosis H37Rv strain (ATCC 27294) across multiple stress conditions. Our analysis identified 1,376 sRNA candidates and 8,173 TSSs in MTB, providing valuable insights into its complex regulatory landscape. TSS mapping enabled us to classify these sRNAs into more specific categories, including promoter-associated sRNAs, 5'UTR-derived sRNAs, 3'UTR-derived sRNAs, true intergenic sRNAs, and antisense sRNAs. Three of these sRNA candidates were experimentally validated using 3'-RACE-PCR: predictedRNA_0240, predictedRNA_0325, and predictedRNA_0578. Future characterization and validation are necessary to fully elucidate the functions and roles of these sRNAs in MTB. Our study is the first to simultaneously unravel TSSs and sRNAs in MTB and demonstrate that the identification of other genomic features, such as TSSs, UTRs, and operons, allows for more accurate and specific classification of sRNAs.

Regulation of an RNA toxin-antitoxin system, SdsR-RyeA, by a small RNA GcvB

The toxin-antitoxin (TA) system regulates many physiological processes in free-living bacteria. One such TA system in Escherichia coli comprises an RNA toxin SdsR and an antitoxin RyeA. An overabundance of SdsR is toxic to the cells. RyeA normalizes SdsR abundance and helps the cells to adapt to altered conditions. The current study showed that a novel small RNA (sRNA) regulator GcvB directly interacts with RyeA to maintain its abundance in the cells under normal or low pH conditions. The deletion of the gcvB allele in the E. coli chromosome resulted in a ∼3-fold decrease in intrabacterial RyeA accumulation. An ectopic expression of GcvB in ΔgcvB strain reinstated RyeA abundance to its normal level. Induction of GcvB in the cells upon exposure to low pH resulted in a simultaneous increase in intracellular RyeA. While GcvB increases RyeA abundance in the cells, SdsR accumulation is divergently regulated by GcvB. The absence of the gcvB gene in E. coli leads to upregulation of SdsR and vice versa. The GcvB-mediated decrease of SdsR accumulation stems from the increased RyeA-driven normalization of SdsR. This study delineates a novel mechanism for the regulation of the expression of an RNA toxin SdsR by another sRNA regulator GcvB through a feed-forward control.

Exploration on cold adaptation of Antarctic lichen via detection of positive selection genes

Lichen as mutualistic symbiosis is the dominant organism in various extreme terrestrial environment on Earth, however, the mechanisms of their adaptation to extreme habitats have not been fully elucidated. In this study, we chose the Antarctic dominant lichen species Usnea aurantiacoatra to generate a high-quality genome, carried out phylogenetic analysis using maximum likelihood and identify genes under positive selection. We performed functional enrichment analysis on the positively selected genes (PSGs) and found that most of the PSGs focused on transmembrane transporter activity and vacuole components. This suggest that the genes related to energy storage and transport in Antarctic U. aurantiacoatra were affected by environmental pressure. Inside of the 86 PSGs screened, two protein interaction networks were identified, which were RNA helicase related proteins and regulator of G-protein signaling related proteins. The regulator of the G-protein signaling gene (UaRGS1) was chosen to perform further verification by the lichen genetic manipulation system Umbilicaria muhlenbergii. Given that the absence of UmRgs1 resulted in elevated lethality to cold shock, the role for UaRgs1 in Antarctic U. aurantiacoatra resistance to cold can be inferred. The investigation of lichen adaptation to extreme environments at the molecular level will be opened up.

Genetic and Epigenetic Interactions Involved in Senescence of Stem Cells.

Cellular senescence is a permanent condition of cell cycle arrest caused by a progressive shortening of telomeres defined as replicative senescence. Stem cells may also undergo an accelerated senescence response known as premature senescence, distinct from telomere shortening, as a response to different stress agents. Various treatment protocols have been developed based on epigenetic changes in cells throughout senescence, using different drugs and antioxidants, senolytic vaccines, or the reprogramming of somatic senescent cells using Yamanaka factors. Even with all the recent advancements, it is still unknown how different epigenetic modifications interact with genetic profiles and how other factors such as microbiota physiological conditions, psychological states, and diet influence the interaction between genetic and epigenetic pathways. The aim of this review is to highlight the new epigenetic modifications that are involved in stem cell senescence. Here, we review recent senescence-related epigenetic alterations such as DNA methylation, chromatin remodeling, histone modification, RNA modification, and non-coding RNA regulation outlining new possible targets for the therapy of aging-related diseases. The advantages and disadvantages of the animal models used in the study of cellular senescence are also briefly presented.

Qki5 safeguards spinal motor neuron function by defining the motor neuron-specific transcriptome via pre-mRNA processing

Many RNA-binding proteins (RBPs) are linked to the dysregulation of RNA metabolism in motor neuron diseases (MNDs). However, the molecular mechanisms underlying MN vulnerability have yet to be elucidated. Here, we found that such an RBP, Quaking5 (Qki5), contributes to formation of the MN-specific transcriptome profile, termed "MN-ness," through the posttranscriptional network and maintenance of the mature MNs. Immunohistochemical analysis and single-cell RNA sequencing (scRNA-seq) revealed that Qki5 is predominantly expressed in MNs, but not in other neuronal populations of the spinal cord. Furthermore, comprehensive RNA sequencing (RNA-seq) analyses revealed that Qki5-dependent RNA regulation plays a pivotal role in generating the MN-specific transcriptome through pre-messenger ribonucleic acid (mRNA) splicing for the synapse-related molecules and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathways. Indeed, MN-specific ablation of the Qki5 caused neurodegeneration in postnatal mice and loss of Qki5 function resulted in the aberrant activation of stress-responsive JNK/SAPK pathway both in vitro and in vivo. These data suggested that Qki5 plays a crucial biological role in RNA regulation and safeguarding of MNs and might be associated with pathogenesis of MNDs.

CircSMAD3 represses VSMC phenotype switching and neointima formation via promoting hnRNPA1 ubiquitination degradation.

Circular RNAs (circRNAs) are novel regulatory RNAs with high evolutionary conservation and stability, which makes them effective therapeutic agents for various vascular diseases. The SMAD family is a downstream mediator of the canonical transforming growth factor beta (TGF-β) signalling pathway and has been considered as a critical regulator in vascular injury. However, the role of circRNAs derived from the SMAD family members in vascular physiology remains unclear. In this study, we initially identified potential functional circRNAs originating from the SMAD family using integrated transcriptome screening. circSMAD3, derived from the SMAD3 gene, was identified to be significantly downregulated in vascular injury and atherosclerosis. Transcriptome analysis was conducted to comprehensively illustrate the pathways modulated by circRNAs. Functionally, circSMAD3 repressed vascular smooth muscle cell (VSMC) proliferation and phenotype switching in vitro evidenced by morphological assays, and ameliorated arterial injury-induced neointima formation in vivo. Mechanistically, circSMAD3 interacted with heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) within the nucleus, enhanced its interaction with E3 ligase WD repeat domain 76 to promote hnRNPA1 ubiquitination degradation, facilitated p53 pre-RNA splicing, activated the p53γ signalling pathway, and finally suppressed VSMC proliferation and phenotype switching. Our study identifies circSMAD3 as a novel epigenetic regulator that suppresses VSMC proliferation and phenotype switching, thereby attenuating vascular remodelling and providing a new circRNA-based therapeutic strategy for cardiovascular diseases.

Spatially exploring RNA biology in archival formalin-fixed paraffin-embedded tissues.

The capability to spatially explore RNA biology in formalin-fixed paraffin-embedded (FFPE) tissues holds transformative potential for histopathology research. Here, we present pathology-compatible deterministic barcoding in tissue (Patho-DBiT) by combining in situ polyadenylation and computational innovation for spatial whole transcriptome sequencing, tailored to probe the diverse RNA species in clinically archived FFPE samples. It permits spatial co-profiling of gene expression and RNA processing, unveiling region-specific splicing isoforms, and high-sensitivity transcriptomic mapping of clinical tumor FFPE tissues stored for 5 years. Furthermore, genome-wide single-nucleotide RNA variants can be captured to distinguish malignant subclones from non-malignant cells in human lymphomas. Patho-DBiT also maps microRNA regulatory networks and RNA splicing dynamics, decoding their roles in spatial tumorigenesis. Single-cell level Patho-DBiT dissects the spatiotemporal cellular dynamics driving tumor clonal architecture and progression. Patho-DBiT stands poised as a valuable platform to unravel rich RNA biology in FFPE tissues to aid in clinical pathology evaluation.

The activity of the quorum sensing regulator HapR is modulated by the bacterial extracellular vesicle (BEV)-associated protein ObfA of Vibrio cholerae.

Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.

A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure.

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.