Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

Plasmid-mediated conjugation is a common mechanism for most bacteria to transfer antibiotic resistance genes (ARGs). The conjugative transfer of ARGs is emerging as a major threat to human beings. Although several transfer-related factors are known to regulate this process, small RNAs (sRNAs)-based regulatory roles remain to be clarified. Here, the Hfq-binding sRNA GadY in donor strain Escherichia coli (E. coli) SM10λπ was identified as a new regulator for bacterial conjugation. Two conjugation models established in our previous studies were used, which SM10λπ carrying a chromosomally integrated IncP-1α plasmid RP4 and a mobilizable plasmid pUCP24T served as donor cells, and P. aeruginosa PAO1 or E. coli EC600 as the recipients. GadY was found to promote SM10λπ-PAO1 conjugation by base-pairing with its target mRNA SdiA, an orphan LuxR-type receptor that responds to exogenous N-acylated homoserine lactones (AHLs). However, SM10λπ-EC600 conjugation was not affected due to EC600 lacking AHLs synthase. It indicates that the effects of GadY on conjugation depended on AHLs-SdiA signalling. Further study found GadY bound SdiA to negatively regulate the global RP4 repressors KorA and KorB. When under ciprofloxacin or levofloxacin treatment, GadY expression in donor strain was enhanced, and it positively regulated quinolone-induced SM10λπ-PAO1 conjugation. Thus, our study provides a novel role for sRNA GadY in regulating plasmid-mediated conjugation, which helps us better understand bacterial conjugation to counter antibiotic resistance.

Age is an independent risk factor for atrial fibrillation (AF), and curcumin can delay aging related disease through reducing oxidative stress and inflammation. However, its target in aging-related AF remains unclear. Transfer RNA-derived small RNA (tsRNA) is a novel short non-coding RNA (sncRNA), and exerts a potential regulatory function in aging. This study was to explore the therapeutic targets of curcumin in atrium of aged mice by PANDORA-seq. Aged mice (18 month) were treated with curcumin (100 mg/kg). Rapid transjugular atrial pacing was performed to observe AF inducibility. SA-β-gal staining, reactive oxygen species (ROS) detection and qRT-PCR were used to assess the degree of aging and oxidative stress/inflammation levels. PANDORA-seq was performed to reveal the differentially expressed sncRNAs in the atrium of mice. The results showed that curcumin reduced the susceptibility AF of aged mice by improving aging-related atrial fibrosis. Compared to young mice (5 month) group, aged mice yielded 473 significantly altered tsRNA sequences, while 947 tsRNA sequences were significantly altered after treated with curcumin. Enrichment analysis revealed that the target genes were mainly related to DNA damage and protein modification. Compared with the 5 month group, the expression levels of mature-mt_tRNA-Val-TAC_CCA_end, mature-mt_tRNA-Glu-TTC_CCA_end, and mature-tRNA-Asp-GTC_CCA_end were up-regulated in the 18 month group, while the expression of mature-mt_tRNA-Thr-TGT_5_end was down-regulated. This trend was reversed in the 18 month + curcumin group. Increased cellular ROS levels, inflammation expression and senescence in aged mice atrium were improved by the down-regulation of mature-mt_tRNA-Val-TAC_CCA_end. In conclusion, our findings identified mature-mt_tRNA-Val-TAC_CCA_end participated in the mechanism of aging-related atrial fibrosis, providing new intervention target of aging-related AF.

The development of high-throughput technologies has enhanced our understanding of small non-coding RNAs (sncRNAs) and their crucial roles in various diseases, including atrial fibrillation (AF). This study aimed to systematically delineate sncRNA profiles in AF patients. PANDORA-sequencing was used to examine the sncRNA profiles of atrial appendage tissues from AF and non-AF patients. Differentially expressed sncRNAs were identified using the R package DEGseq 2 with a fold change >2 and p < 0.05. The target genes of the differentially expressed sncRNAs were predicted using MiRanda and RNAhybrid. Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. In AF patients, the most abundant sncRNAs were ribosomal RNA-derived small RNAs (rsRNAs), followed by transfer RNA-derived small RNAs (tsRNAs), and microRNAs (miRNAs). Compared with non-AF patients, 656 rsRNAs, 45 miRNAs, 191 tsRNAs and 51 small nucleolar RNAs (snoRNAs) were differentially expressed in AF patients, whereas no significantly differentially expressed piwi-interacting RNAs were identified. Two out of three tsRNAs were confirmed to be upregulated in AF patients by quantitative reverse transcriptase polymerase chain reaction, and higher plasma levels of tsRNA 5006c-LysCTT were associated with a 2.55-fold increased risk of all-cause death in AF patients (hazard ratio: 2.55; 95% confidence interval, 1.56-4.17; p < 0.001). Combined with our previous transcriptome sequencing results, 32 miRNA, 31 snoRNA, 110 nucleus-encoded tsRNA, and 33 mitochondria-encoded tsRNA target genes were dysregulated in AF patients. GO and KEGG analyses revealed enrichment of differentially expressed sncRNA target genes in AF-related pathways, including the \'calcium signaling pathway\' and \'adrenergic signaling in cardiomyocytes.\' The dysregulated sncRNA profiles in AF patients suggest their potential regulatory roles in AF pathogenesis. Further research is needed to investigate the specific mechanisms of sncRNAs in the development of AF and to explore potential biomarkers for AF treatment and prognosis.

Infertility, a worldwide reproductive health concern, impacts approximately one in five couples. Male infertility, stemming from spermatogenic dysfunction and reduced sperm quality, stands as a primary factor contributing to infertility. Given the global decrease in male fertility linked to environmental factors like the greenhouse effect, it is crucial to develop a comprehensive understanding of how increased temperatures impact both the quantity and quality of sperm. In this study, we utilized Pandora-seq technology to detect the small non-coding RNAs (sncRNAs) expression profile in the testicular tissue of heat-stressed mice. The investigation explores the dynamic shifts in sncRNAs within the mouse testis under heat stress, including miRNAs, tsRNAs, piRNAs, rsRNAs and other sncRNAs. Furthermore, we successfully identified differentially expressed sncRNAs in testicular tissues before and after heat stress. Subsequently, we conducted functional enrichment analysis on the potential predicted target genes of differentially expressed miRNAs and tsRNAs. These datasets will constitute a valuable foundational resource for further investigations into the decline in male reproductive capacity triggered by heat stress.

In recent years, polymyxin has been used as a last-resort therapy for carbapenem-resistant bacterial infections. The emergence of heteroresistance (HR) to polymyxin hampers the efficacy of polymyxin treatment by amplifying resistant subpopulation. However, the mechanisms behind polymyxin HR remain unclear. Small noncoding RNAs (sRNAs) play an important role in regulating drug resistance. The purpose of this study was to investigate the effects and mechanisms of sRNA on polymyxin B (PB)-HR in carbapenem-resistant Klebsiella pneumoniae. In this study, a novel sRNA PhaS was identified by transcriptome sequencing. PhaS expression was elevated in the PB heteroresistant subpopulation. Overexpression and deletion of PhaS were constructed in three carbapenem-resistant K. pneumoniae strains. Population analysis profiling, growth curve, and time-killing curve analysis showed that PhaS enhanced PB-HR. In addition, we verified that PhaS directly targeted phoP through the green fluorescent protein reporter system. PhaS promoted the expression of phoP, thereby encouraging the expression of downstream genes pmrD and arnT. This upregulation of arnT promoted the 4-amino-4-deoxyL-arabinosaccharide (L-Ara4N) modification of lipid A in PhaS overexpressing strains, thus enhancing PB-HR. Further, within the promoter region of PhaS, specific PhoP recognition sites were identified. ONPG assays and RT-qPCR analysis confirmed that PhaS expression was positively modulated by PhoP and thus up-regulated by PB stimulation. To sum up, a novel sRNA enhancing PB-HR was identified and a positive feedback regulatory pathway of sRNA-PhoP/Q was demonstrated in the study. This helps to provide a more comprehensive and clear understanding of the underlying mechanisms behind polymyxin HR in carbapenem-resistant K. pneumoniae.

Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.

Transfer RNA-derived small RNAs (tsRNAs), a recently identified subclass of small non-coding RNAs (sncRNAs), emerge through the cleavage of mature transfer RNA (tRNA) or tRNA precursors mediated by specific enzymes. The tumor necrosis factor (TNF) protein, a signaling molecule produced by activated macrophages, plays a pivotal role in systemic inflammation. Its multifaceted functions include the capacity to eliminate or hinder tumor cells, enhance the phagocytic capabilities of neutrophils, confer resistance against infections, induce fever, and prompt the production of acute phase proteins. Notably, four TNF-related tsRNAs have been conclusively linked to distinct diseases. Examples include 5\'tiRNA-Gly in skeletal muscle injury, tsRNA-21109 in systemic lupus erythematosus (SLE), tRF-Leu-AAG-001 in endometriosis (EMs), and tsRNA-04002 in intervertebral disk degeneration (IDD). These tsRNAs exhibit the ability to suppress the expression of TNF-α. Additionally, KEGG analysis has identified seven tsRNAs potentially involved in modulating the TNF pathway, exerting their influence across a spectrum of non-cancerous diseases. Noteworthy instances include aberrant tiRNA-Ser-TGA-001 and tRF-Val-AAC-034 in intrauterine growth restriction (IUGR), irregular tRF-Ala-AGC-052 and tRF-Ala-TGC-027 in obesity, and deviant tiRNA-His-GTG-001, tRF-Ser-GCT-113, and tRF-Gln-TTG-035 in irritable bowel syndrome with diarrhea (IBS-D). This comprehensive review explores the biological functions and mechanisms of tsRNAs associated with the TNF signaling pathway in both cancer and other diseases, offering novel insights for future translational medical research.

Pancreatic adenocarcinoma (PAAD) is a life-threatening cancer. Exploring new diagnosis and treatment targets helps improve its prognosis. tRNA-derived small non-coding RNAs (tsRNAs) are a novel type of gene expression regulators and their dysregulation is closely related to many human cancers. Yet the expression and functions of tsRNAs in PAAD are not well understood. Our study used RNA sequencing to identify tsRNA expression profiles in PAAD cells cultured in no or high glucose media and found tRF-18-8R6546D2 was an uncharacterized tsRNA, which has significantly high expression in PAAD cells and tissues. Clinically, tRF-18-8R6546D2 is linked to poor prognosis in PAAD patients and can be used to distinguish them from healthy populations. Functionally, in vitro and vivo, tRF-18-8R6546D2 over-expression promoted PAAD cell proliferation, migration and invasion, inhibited apoptosis, whereas tRF-18-8R6546D2 knock-down showed opposite effects. Mechanistically, tRF-18-8R6546D2 promoted PAAD malignancy partly by directly silencing ASCL2 and further regulating its downstream genes such as MYC and CASP3. These findings show that tRF-18-8R6546D2 is a novel oncogenic factor and can be a promising diagnostic or prognostic biomarker and therapeutic target for PAAD.

Rationale: Current treatments for ocular angiogenesis primarily focus on blocking the activity of vascular endothelial growth factor (VEGF), but unfavorable side effects and unsatisfactory efficacy remain issues. The identification of novel targets for anti-angiogenic treatment is still needed. Methods: We investigated the role of tsRNA-1599 in ocular angiogenesis using endothelial cells, a streptozotocin (STZ)-induced diabetic model, a laser-induced choroidal neovascularization model, and an oxygen-induced retinopathy model. CCK-8 assays, EdU assays, transwell assays, and matrigel assays were performed to assess the role of tsRNA-1599 in endothelial cells. Retinal digestion assays, Isolectin B4 (IB4) staining, and choroidal sprouting assays were conducted to evaluate the role of tsRNA-1599 in ocular angiogenesis. Transcriptomic analysis, metabolic analysis, RNA pull-down assays, and mass spectrometry were utilized to elucidate the mechanism underlying angiogenic effects mediated by tsRNA-1599. Results: tsRNA-1599 expression was up-regulated in experimental ocular angiogenesis models and endothelial cells in response to angiogenic stress. Silencing of tsRNA-1599 suppressed angiogenic effects in endothelial cells in vitro and inhibited pathological ocular angiogenesis in vivo. Mechanistically, tsRNA-1599 exhibited little effect on VEGF signaling but could cause reduced glycolysis and NAD+/NADH production in endothelial cells by regulating the expression of HK2 gene through interacting with YBX1, thus affecting endothelial effects. Conclusions: Targeting glycolytic reprogramming of endothelial cells by a tRNA-derived small RNA represents an exploitable therapeutic approach for ocular neovascular diseases.