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The Csr (Carbon Storage Regulator) system is pivotal in controlling various cellular functions in most bacteria, primarily through the CsrA protein and its antagonistic sRNAs. However, riboregulatory networks are less explored in non-model organisms, particularly those in extreme environments. In this study, we discovered two new sRNAs of the Csr system, Pf2 and Pf3, in the Arctic bacterium Pseudoalteromonas fuliginea BSW20308, along with the previously known Pf1. By studying the impact of these Pf sRNAs on CsrA targetomes and physiological processes, we found a significant influence on various cellular functions and a collective effect on the interaction dynamics between CsrA and RNAs. Furthermore, we identified additional sRNAs that can interact with CsrA and mRNAs. Overall, our results emphasize the growing influence of the Csr system on cellular physiology through intricate sRNA regulation of CsrA, revealing riboregulatory network complexity and significance in non-model organisms., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Escherichia coli synthesizes aromatic amino acids (AAAs) through the common pathway to produce the precursor, chorismate, and the three terminal pathways to convert chorismate into Phe, Tyr, and Trp. E. coli also imports exogenous AAAs through five transporters. GcvB small RNA post-transcriptionally regulates more than 50 genes involved in amino acid uptake and biosynthesis in E. coli , but the full extent of GcvB regulon is still underestimated. This study examined all genes involved in AAA biosynthesis and transport using translation reporter assay and qRT-PCR analysis. In addition to previously verified targets, aroC , aroP , and trpE , we identified new target genes that were significantly repressed by GcvB primarily via the R1 seed region. Exceptionally, GcvB strongly inhibits the expression of aroG , which encodes the major isozyme of the first reaction in the common pathway, through direct base pairing between the aroG translation initiation region and the GcvB R3 seed sequence. RNase E mediates the degradation of target mRNAs except aroC and aroP via its C-terminal domain. GcvB overexpression prolongs the lag phase and reduces the growth rate in minimal media supplemented with AAAs and confers resistance to an antibiotic compound, azaserine, by repressing AAA transporters.IMPORTANCE E. coli strains have been genetically modified in relevant transcription factors and biosynthetic enzymes for industrial use in the fermentative production of aromatic amino acids (AAAs) and their derivative compounds. This study focuses on GcvB small RNA, a global regulator of amino acid metabolism in E. coli , and identifies new GcvB targets involved in AAA biosynthesis and uptake. GcvB represses the expression of the first and last enzymes of the common pathway and the first enzymes of Trp and Phe terminal pathways. GcvB also limits import of AAAs. This paper documents the impact of RNA-mediated regulation on AAA metabolism in E. coli ., Competing Interests: The authors declare no conflict of interest.

Gene regulation at the post-transcriptional level is prevalent in all domains of life. In bacteria, ProQ-like proteins have emerged as important RNA chaperones facilitating RNA stability and RNA duplex formation. In the major human pathogen Vibrio cholerae, post-transcriptional gene regulation is key for virulence, biofilm formation, and antibiotic resistance, yet the role of ProQ has not been studied. Here, we show that ProQ interacts with hundreds of transcripts in V. cholerae, including the highly abundant FlaX small RNA (sRNA). Global analyses of RNA duplex formation using RIL-Seq (RNA interaction by ligation and sequencing) revealed a vast network of ProQ-assisted interactions and identified a role for FlaX in motility regulation. Specifically, FlaX base-pairs with multiple sites on the flaB flagellin mRNA, preventing 30S ribosome binding and translation initiation. V. cholerae cells lacking flaX display impaired motility gene expression, altered flagella composition and reduced swimming in liquid environments. Our results provide a global view on ProQ-associated RNA duplex formation and pinpoint the mechanistic and phenotypic consequences associated with ProQ-associated sRNAs in V. cholerae., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The exact mechanisms and key functional molecules involved in skin ageing remain largely unknown. Studies linking the expression of messenger RNAs (mRNAs) and small noncoding RNAs (sncRNAs) to skin ageing are limited. In this study, we performed RNA sequencing to assess the effects of ageing on the expression of mRNAs and sncRNAs in rat skin. Our results revealed that 241 mRNAs, 109 microRNAs (miRNAs), 20 piwi-interacting RNAs (piRNAs), 45 small nucleolar RNAs (snoRNAs), and 7 small nuclear RNAs (snRNAs) were significantly differentially expressed in the skin of aged rats compared to their younger counterparts. Histological validation using RT-qPCR further verified the significant differential expression of 13 mRNAs, 7 miRNAs, 2 piRNAs, 15 snoRNAs, and 1 snRNA. Additionally, several sncRNAs showed differential expression across various tissues, suggesting that they may have broad correlations with ageing. After establishing cellular senescence in skin fibroblasts, we identified 4 mRNAs, 4 miRNAs, and 10 snoRNAs that may mediate skin ageing by modulating fibroblast senescence. Notably, overexpression or knockdown of some differentially expressed RNAs in fibroblasts influenced cellular senescence, indicating that these RNAs could play an important role in the skin ageing process. These findings highlight their potential significance for future treatments of age-related skin disorders., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Post-transcriptional gene regulation by non-coding small RNAs (sRNAs) is critical for colonization and survival of enteric pathogens, including the zoonotic pathogen Campylobacter jejuni . In this study, we utilized C. jejuni IA3902 (a representative isolate of the sheep abortion clone) and C. jejuni W7 (a highly motile variant of NCTC 11168, a human gastroenteritis strain) to further investigate regulation by sRNA CjNC110. Both motility and autoagglutination ability were confirmed to be phenotypes of conserved regulation by CjNC110. However, we demonstrated that W7∆CjNC110 does not change chicken colonization levels compared to W7 wild type, directly contrasting IA3902∆CjNC110, which had decreased colonization ability. Subsequently, we determined strain-specific phenotype variation between W7∆CjNC110 and IA3902∆CjNC110 when examining intracellular L-methionine (L-met) levels controlled by the activated methyl cycle (AMC). We hypothesized that the presence of a secondary system for L-met production conferred by MetAB in W7 but not IA3902 might explain the difference in both chicken colonization and L-met availability. Insertion of metAB within IA3902∆CjNC110 (naturally absent) restored intracellular L-met levels in IA3902∆CjNC110::metAB and overcame the colonization defect that resulted from mutagenesis of CjNC110 in IA3902. Deletion of metAB in W7∆CjNC110 (naturally present) led to a decrease in L-met in W7∆CjNC110∆metAB and a colonization defect which was otherwise masked in W7∆CjNC110. Our results indicate that regulation of the AMC leading to altered L-met availability is a conserved regulatory function of CjNC110 in C. jejuni and confirm that L-met generation via the AMC as activated by CjNC110 is critical for optimal host colonization.IMPORTANCEDuring this study, the regulatory action and conservation of function of CjNC110 between two different zoonotically important Campylobacter jejuni strains were examined. Critically, this work for the first time reveals regulation of L-methionine (L-met) production within the activated methyl cycle (AMC) by small RNA (sRNA) CjNC110 as a key factor driving C. jejuni optimal chicken colonization. As a growing body of evidence suggests that maintenance of L-met homeostasis appears to be critical for C. jejuni colonization, interventions targeting the AMC could provide a critical control point for therapeutic drug options to combat this zoonotic pathogen. Our results also indicate that even for conserved sRNAs such as CjNC110, strain-specific differences in phenotypes regulated by sRNAs may exist, independent of conserved regulatory action. Depending on the strain examined and accessory genomic content present, conserved regulatory actions might be masked, thus investigation in multiple strains may be warranted., Competing Interests: The authors declare no conflict of interest.

The RNA chaperone Hfq plays crucial roles in bacterial gene expression and is a major facilitator of small regulatory RNA (sRNA) action. The toroidal architecture of the Hfq hexamer presents three well-characterized surfaces that allow it to bind sRNAs to stabilize them and engage target transcripts. Hfq-interacting sRNAs are categorized into two classes based on the surfaces they use to bind Hfq. By characterizing a systematic alanine mutant library of Hfq to identify amino acid residues that impact survival of Escherichia coli experiencing nitrogen (N) starvation, we corroborated the important role of the three RNA-binding surfaces for Hfq function. We uncovered two, previously uncharacterized, conserved residues, V22 and G34, in the hydrophobic core of Hfq, to have a profound impact on Hfq's RNA-binding activity in vivo. Transcriptome-scale analysis revealed that V22A and G34A Hfq mutants cause widespread destabilization of both sRNA classes, to the same extent as seen in bacteria devoid of Hfq. However, the alanine substitutions at these residues resulted in only modest alteration in stability and structure of Hfq. We propose that V22 and G34 have impact on Hfq function, especially critical under cellular conditions when there is an increased demand for Hfq, such as N starvation., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small RNAs (sRNAs) play a crucial role in modulating target gene expression through short base-pairing interactions and serve as integral components of many stress response pathways and regulatory circuits in bacteria. Transcriptome analyses have facilitated the annotation of dozens of sRNA candidates in the ubiquitous environmental model bacterium Caulobacter crescentus, but their physiological functions have not been systematically investigated so far. To address this gap, we have established CauloSOEP, a multi-copy plasmid library of C. crescentus sRNAs, which can be studied in a chosen genetic background and under select conditions. Demonstrating the power of CauloSOEP, we identified sRNA AbnZ to impair cell viability and morphology. AbnZ is processed from the 3' end of the polycistronic abn mRNA encoding the tripartite envelope-spanning efflux pump AcrAB-NodT. A combinatorial approach revealed the essential membrane translocation module TamAB as a target of AbnZ, implying that growth inhibition by AbnZ is linked to repression of this system., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The timings of several cellular events like cell lysis, cell division, or pore formation in endosomes are regulated by the time taken for the relevant proteins to cross a threshold in number or concentration. Since protein synthesis is stochastic, the threshold crossing time is a first passage problem. The exact distributions of these first passage processes have been obtained recently for unregulated and autoregulated genes. Many proteins are however regulated by post-transcriptional regulation, controlled by small noncoding RNAs (sRNAs). Certain mathematical models of gene expression with post-transcriptional sRNA regulation have been recently exactly mapped to models without sRNA regulation. Utilizing this mapping and the exact distributions, we calculate exact results on fluctuations (full distribution, all cumulants, and characteristic times) of protein threshold crossing times in the presence of sRNA regulation. We derive two interesting predictions from these exact results. We show that the size of the fluctuation of the threshold crossing times have a nonmonotonic U-shaped behavior as a function of the rates of binding and unbinding of the sRNA-mRNA complex. Thus there are optimal parameters that minimize noise. Furthermore, the fluctuations in models with sRNA regulation may be higher or lower compared to the model without regulation, depending on the mean protein burst size.

Bacteria reside in constantly changing environments and require rapid and precise adjustments of gene expression to ensure survival. Small regulatory RNAs (sRNAs) are a crucial element that bacteria utilize to achieve this. sRNAs are short RNA molecules that modulate gene expression usually through base-pairing interactions with target RNAs, primarily mRNAs. These interactions can lead to either negative outcomes such as mRNA degradation or translational repression or positive outcomes such as mRNA stabilization or translation enhancement. In recent years, high-throughput approaches such as RIL-seq (RNA interaction by ligation and sequencing) revolutionized the sRNA field by enabling the identification of sRNA targets on a global scale, unveiling intricate sRNA-RNA networks. In this review, we discuss the insights gained from investigating sRNA-RNA networks in well-studied bacterial species as well as in understudied bacterial species. Having a complete understanding of sRNA-mediated regulation is critical for the development of new strategies for controlling bacterial growth and combating bacterial infections., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Recently, members of a unique species of non-coding RNA, known as transfer RNA-derived small RNAs (tsRNAs) have been reported to serve multiple molecular functions, including in cells that mediate immunity. Because of their low molecular weights, tsRNAs were previously difficult to detect and were thus overlooked, until now. In this review, we delve into the biogenesis of tsRNAs and their diverse biological functions, ranging from transcriptional regulation to modulation of mRNA translation. We highlight the current evidence demonstrating their involvement in the immune response, as well as how tsRNAs modulate immunity to influence tumor growth and spread, autoimmune disease pathology and infection by pathogens. We surmise that tsRNAs are likely informative as diagnostic markers of cellular homeostasis and disease, and that therapeutic targeting of tsRNAs could be beneficial for a range of human diseases. Improved knowledge on the functions for tsRNAs in the mammalian immune system will enable us to leverage tsRNAs for their effective clinical use as treatments for human health challenges., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Offspring health outcomes are often linked with epigenetic alterations triggered by maternal nutrition and intrauterine environment. Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of paternal effects. Here, we characterised the baseline sncRNA landscape of human sperm and the effect of a 6-week dietary intervention on their expression profile. This study involves sncRNAseq profiling, that was performed on a subset (n = 17) of the participants enrolled in the PREPARE trial: 9 from the control group and 8 from the intervention group. 5'tRFs, miRNAs and piRNAs were the most abundant sncRNA subtypes identified; their expression was associated with age, BMI, and sperm quality. Nutritional intervention with olive oil, vitamin D and omega-3 fatty acids altered expression of 3 tRFs, 15 miRNAs and 112 piRNAs, targeting genes involved in fatty acid metabolism and transposable elements in the sperm genome. PREPARE Trial registration number: ISRCTN50956936, Trial registration date: 10/02/2014., Competing Interests: Declarations. Competing interests: N.K. (Agency for Science, Technology and Research, Singapore), K.M.G. (University of Southampton), K.A.L. (University of Southampton), and Y.S.C. (National University of Singapore) are part of an academic consortium that has received research funding from Abbott Nutrition, Nestec, BenevolentAI Bio Ltd. and Danone. P.C.C. serves on the Scientific Advisory Board of Smartfish, the producer of the drinks used in the study. P.C.C. (University of Southampton,) has received advisory and/or speaking honoraria from Fresenius-Kabi, B. Braun, Baxter Healthcare, Abbott Nutrition, and Danone/Nutricia, sellers of parenteral and enteral feeds, and from DSM, Pronova BioPharma/BASF AS and Smartfish, sellers of products containing omega-3 fatty acids. K.M.G. (University of Southampton,) has received reimbursement for speaking at conferences sponsored by companies selling nutritional products. The remaining authors declare no competing interests. The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Ethics approval: Ethical approval for the PREPARE trial was granted by an NHS Research Ethics Committee (13/SC/0544). All participants provided written informed consent., (© 2025. The Author(s).)

The intricate regulatory roles of small non-coding RNAs (sncRNAs), including PIWI-interacting RNAs (piRNAs) and microRNAs (miRNAs), have been increasingly recognized in the modulation of cellular functions and are associated with the pathogenesis of various diseases, notably cancer. However, the specific dysregulation patterns of sncRNAs in non-muscle-invasive bladder cancer (NMIBC) have yet to be fully delineated, highlighting a significant gap in our current understanding. To elucidate the expressional dynamics of sncRNAs for patients with NMIBC, we characterized the profile of piRNAs and miRNAs by next-generation sequencing. We identified the differentially expressed sncRNAs between tumor and paracancerous tissues and characterized their distribution along the genome. We further revealed a set of immune-related piRNAs and dysregulated miRNAs that might be associated with NMIBC pathogenesis. Differentially expressed piRNAs were predominantly localized at the long arms of chromosomes 13, 1, and 6. Notably, the targets of specific piRNAs, including piR-hsa-2215234, piR-hsa-105306, piR-hsa-102066, and piR-hsa-236465, show significant associated with antigen processing and presentation pathway. Additionally, differentially expressed miRNAs are mainly located on chromosome 14 and their target genes tend to be involved in cancer-related pathways, suggesting their potential regulatory roles in NMIBC. Collectively, this study revealed the global sncRNA dysregulation in NMIBC, and the identified sncRNAs are implicated in the modulation of both immune and cancer pathways, suggesting their contribution to the pathogenesis and potential targets for immunotherapy., Competing Interests: Declarations. Ethics approval and consent to participate: We ensure the authenticity of research data and the absence of unethical research activities. All patients signed informed consent forms. Our study adhered to the Declaration of Helsinki. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

We performed next-generation sequencing (NGS) on RNA from 19 paired leiomyoma (Lyo) and myometrium (Myo) specimens, stratified by race/ethnicity (White: n = 7; Black: n = 12) and mediator complex subunit 12 (MED12) mutation status (mutated: n = 10; non-mutated: n = 9). Analysis identified 2,189 small non-coding RNAs (sncRNAs) with altered expression in Lyo compared to paired Myo (≥1.5-fold change), including small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), and PIWI-interacting RNAs (piRNAs). Among these, 17 sncRNAs showed differential expression in the MED12-mutated group versus Myo, while minimal changes were observed in the non-mutated group. Additionally, 31 sncRNAs displayed differential expression in Black women compared to White women. For validation, five novel miRNAs (miR-19a-3p, miR-99a-5p, miR-3196, miR-499a-5p, and miR-30d-3p) and five piRNAs (piR-009295, piR-020326, piR-020365, piR-006426, and piR-020485) were analyzed in 51 paired Lyo samples using qRT-PCR. Reduced expression of the selected sncRNAs was confirmed in Lyo versus Myo, with miR-19a-3p, miR-3196, miR-30d-3p, piR-006426, and piR-020485 linked to MED12 status, while miR-499a-5p and miR-30d-3p were associated with race/ethnicity. These findings suggest that sncRNA dysregulation contributes to altered gene expression in Lyo, influenced by MED12 mutation and racial background.

Background: Infection and inflammation of the genital tract are major potentially treatable factors contributing to male infertility. The profile of small non-coding RNA (sncRNAs) in spermatozoa can be altered by environmental exposures and inflammatory conditions., Objectives: Experimental autoimmune epididymo-orchitis (EAEO) is a well-established model of autoimmune-induced chronic testicular and epididymal inflammation. This model investigates the effect of chronic inflammation on sperm sncRNA profiles and offspring phenotypes., Materials and Methods: Regarding the EAEO model, mice were immunized with testis homogenates thrice. Subsequently, flow cytometry and histological analyses were conducted on EAEO mice. Next-generation sequencing was used to profile small RNA of spermatozoa from the caput, corpus, and cauda epididymis. We performed a comprehensive integrative analysis of sperm sncRNAs and chronic epididymitis and identified their molecular signatures. The metabolic functions of the first-generation (F1) offspring were evaluated using a glucose tolerance test (GTT)., Results: Body weight and metabolic function were significantly altered in F1 offspring from EAEO sperm donors. The analysis of cauda sperm sncRNA profiles revealed that the proportions of miRNAs and tsRNAs increased and decreased, respectively, after autoimmunization. Three differentially expressed miRNAs and seven differentially expressed tsRNAs were significantly correlated with F1 metabolic dysfunction. The expression patterns of miRNAs and tsRNAs in mice partially overlapped with those observed in the spermatozoa from human patients with chronic epididymitis., Discussion and Conclusions: We revealed that autoimmune epididymo-orchitis alters sncRNA profiles in mouse spermatozoa. Offspring from mice with autoimmune orchitis develop metabolic disorders. A comprehensive analysis of human and mouse inflammation data revealed an association between alterations in the miRNA and tsRNA profiles of epididymal spermatozoa and offspring phenotypes., (© 2023 American Society of Andrology and European Academy of Andrology.)

WhiB6 in pathogenic mycobacteria is highly upregulated during NO stress, hypoxia, and macrophage infection. Its expression primarily results from transcriptional control by a two-component response regulator PhoP in response to the various stresses exerted on mycobacterial cells inside phagosomes. Herein, we investigated the transcriptional and posttranscriptional regulatory mechanism of whiB6 expression. We found that PhoP binds to the PhoP-signature sequences located upstream to the core promoter region of whiB6 gene and controls its expression at the transcriptional level. Phosphorylation of PhoP is obligatory for binding to whiB6 promoter. A dormancy regulatory factor DosR, although doesn't bind to whiB6 gene, can bind to the PhoP-bound whiB6 gene implicating its potential role in whiB6 expression. A virulence-associated sRNA MTS1338 directly binds to the coding region of whiB6 gene presumably protecting it from cellular ribonucleases. Induction of MTS1338 in response to low pH and oxidative stress increases whiB6 accumulation likely through the stabilization of whiB6 transcript at the posttranscriptional level. This study substantially increases our knowledge of the regulation of whiB6 expression in M. tuberculosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Small noncoding RNAs (sncRNAs) can regulate gene expression by guiding the RNA-induced silencing complex (RISC) to targeted transcripts for translational repression and/or target destabilization. Here, we present a robust benchtop protocol, termed CIMERA-seq, for the unambiguous profiling of sncRNA:target RNA interactions in a genome-wide and cell-type-selective manner. We describe steps for in vivo crosslinking and harvesting tissue, immunoprecipitation and covalent ligation of sncRNAs to target RNAs within the RISC, and sequencing of the resulting chimeric sncRNA:target RNA interactions. For complete details on the use and execution of this protocol, please refer to Li et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Small non-coding RNAs play a pivotal role in regulating various metabolic processes in both prokaryotic and eukaryotic organisms. However, knowledge about small RNAs (sRNAs) in Streptomyces clavuligerus ( S. clavuligerus ) is scarce. This study aimed to use cutting-edge bioinformatics tools and a compendium of RNA-seq data to predict the potential coding of sRNAs that might be present in the genome of S. clavuligerus ATCC 27064. In the genome of S. clavuligerus , 606 intergenic regions (IGRs) are conserved, and 272 possess a highly thermodynamically stable and conserved secondary structure, indicating the presence of non-coding RNA in these regions. The transcriptome assembly of S. clavuligerus showed that the genome is completely functional, as all the annotated genes are expressed under the conditions analyzed. From this assembly, transcripts originating from IGRs were labeled as putative sRNAs, and their differential expression during the growth curve of S. clavuligerus for clavulanic acid (CA) production was established. The interactome of these differentially expressed (DE) RNAs displayed the sRNAs as global regulators, as they can have multiple mRNA targets. The functional annotation of the target genes of DE sRNAs demonstrated that they are directly involved in secondary metabolite production. Specifically, two sRNA have the genes of the biosynthetic gene cluster of CA as targets. Thus, these molecules add an additional layer to the regulatory cascade for CA biosynthesis, and we propose them as targets for metabolic engineering to increase CA production.

Small RNAs (sRNAs) controlling gene expression by imperfect base-pairing with mRNA(s) are widespread in bacteria. They regulate multiple genes, including genes involved in iron homeostasis, through a wide variety of mechanisms. We previously showed that OmrA and OmrB sRNAs repress the synthesis of the Escherichia coli FepA receptor for iron-enterobactin complexes. We now report that five additional sRNAs, namely RprA, RybB, ArrS, RseX and SdsR, responding to different environmental cues, also repress fepA, independently of one another. While RprA follows the canonical mechanism of pairing with the translation initiation region, repression by ArrS or RseX requires a secondary structure far upstream within the long fepA 5' untranslated region. We also demonstrate a dual action of SdsR, whose 5'-part pairs with the fepA translation initiation region while its 3'-end behaves like ArrS or RseX. Strikingly, mutation analysis shows a key role for the loops of these sRNAs' intrinsic terminators in the regulation. Furthermore, regulation depends on both the Hfq chaperone and the RNase E endonuclease. Overall, our data strongly suggest that FepA levels must be tightly controlled under a variety of conditions and highlight the diversity of mechanisms that underly the regulation of gene expression by sRNAs in bacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Pseudomonas aeruginosa, an opportunistic bacterial pathogen of public health concern, is known for its metabolic versatility, adaptability in harsh environment, and pathogenic aggressiveness. P. aeruginosa relies on various regulatory networks modulated by small non-coding RNAs, which in turn influence different physiological traits such as metabolism, stress response, and pathogenesis. In this study, srbA sRNA has been shown to play a diverse role in regulating cellular metabolism and the production of different virulence factors in P. aeruginosa. srbA was found to control the TCA cycle, a key regulatory pathway for cellular metabolism and energy production, by regulating three main enzymes: citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-ketoglutarate dehydrogenase E1 subunit (sucA) at both the transcriptional and translational levels. By modulating the TCA cycle, srbA could help the bacteria to adapt nutritional stress by lowering energy consumption. Additionally, srbA has been found to differentially regulate production of various virulence factors such as rhamnolipid, elastase, LasA protease, and pyocyanin under both nutrient-rich and nutrient-limiting conditions. It could also influence motilities in P. aeruginosa, linked to biofilm formation and pathogenicity. Thus, srbA might hold a promise in the research area for identifying virulence pathways and developing novel therapeutic targets to combat the global pathogenic threat of P. aeruginosa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vibrio alginolyticus, the causative agent of aquatic vertebrates and invertebrates, can cause severe infections (e.g. septicemia, gill necrosis, and surface ulcers) and high mortality in aquatic organisms, leading to serious economic losses in global aquaculture. Small non-coding RNAs (sRNAs), emerging modulators of gene expression, played vital regulatory roles in virulence, pathogenicity, and physiological metabolism of bacteria. In this work, the modulation of physiological functions and metabolome of V. alginolyticus by the quorum-regulatory sRNA, Qrr1, was figured out. We found that the deletion of qrr1 induced significant cell shape elongation. Meanwhile, Qrr1 could inhibit the production of alkaline serine protease by weakening the expression of main regulator LuxR in the quorum sensing (QS) system. Moreover, the untargeted metabolomics and lipidomics approaches showed that most of nucleotides, organic acids, carbohydrates, and lipidome (both lipid content and category) were significantly altered in response to the qrr1 deletion. Spearman correlation analysis demonstrated that most of the intermediates involved in glutamate metabolism, sphingolipid metabolism, and glycerolipid metabolism displayed high correlations with cell virulence factors. These findings illuminate the mechanism of bacterial virulence regulation and further exploit potential therapeutic targets for virulence prevention in V. alginolyticus., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Pseudomonas aeruginosa is an important gram-negative pathogen that often causes skin, respiratory, and bloodstream infections. The virulence of P. aeruginosa is regulated by sRNAs through a complex network, which is still incompletely understood. This study employed transcriptome sequencing (RNA-Seq) to screen sRNA 111, and real-time quantitative PCR was conducted for validation. In addition, an overexpression strain of sRNA111 was constructed and the effects of overexpression of sRNA111 on virulence factors such as biofilm formation, pyocyanin, swarming motility, inflammatory factors, and biofilm-associated factors were analyzed. The results revealed that sRNA111 might be closely related to the formation of P. aeruginosa biofilm. Compared to the vector, sRNA111 was found to enhance P. aeruginosa biofilm formation while also suppressing swarming motility, invasive ability, and adhesion capability. Meanwhile, sRNA111 up-regulated the expression of biofilm-related genes, such as Anr, pel family, polB, recA, etc., more significantly in the biofilm environment compared with the wild strain. In addition, sRNA111 effectively promoted the expression of inflammatory factors in the biofilm environment compared with the planktonic environment. These results implied that sRNA111 regulates the formation of P. aeruginosa biofilm and other virulence factors and further modulates the process of infection immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Small non-coding RNAs (ncRNAs) have recently emerged as potential biomarkers of sarcopenia. However, previous studies have rarely explored the association of small ncRNAs with sarcopenic components, especially muscle strength and physical performance. We aimed to examine circulating small ncRNA profiles to detect low muscle strength and physical performance in older adults., Methods: Ninety-eight older adults were randomly selected from Korean Frailty and Aging Cohort Study and classified into the "Normal," "Low muscle strength (MS) only," "Low physical performance (PP) only," and "Low MS and PP" groups by Asian Working Group for Sarcopenia 2019 criteria. We used high-throughput sequencing to delineate small ncRNA profiles in plasma. Differentially expressed small ncRNAs were analyzed to reveal distinct patterns based on muscle strength and physical performance status., Results: In "Low MS and PP" group, 119 miRNAs, 86 piRNAs, 92 snoRNAs, 106 snRNAs, and 15 tRNAs were differentially expressed compared to "Normal" group (p < 0.05). After Benjamini-Hochberg adjustment, 39 miRNAs, 2 piRNAs, 75 snoRNAs, 48 snRNAs, and 15 tRNAs showed differential expression in "Low MS and PP" group compared to than "Normal" group (adjusted p < 0.05). No significant differences were observed in comparisons between the other groups (adjusted p > 0.05)., Conclusion: The expression of circulating small ncRNAs were comprehensively characterized, revealing distinct signatures in older adults with both low muscle strength and physical performance compared to normal individuals. Although preliminary, this characterization can advance small ncRNA research on age-related declines in muscle strength and physical performance by providing foundational data for further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Vault RNAs (vtRNAs) are evolutionarily conserved small noncoding RNAs transcribed by RNA polymerase III. Vault RNAs were initially described as components of the vault particle, but have since been assigned multiple vault-independent functions, including regulation of PKR activity, apoptosis, autophagy, lysosome biogenesis, and viral particle trafficking. The full-length transcript has also been described as a noncanonical source of miRNAs, which are processed in a DICER-dependent manner. As central molecules in vault-dependent and independent processes, vtRNAs have been attributed numerous biological roles, including regulation of cell proliferation and survival, response to viral infections, drug resistance, and animal development. Yet, their impact to mammalian physiology remains largely unexplored. To study vault RNAs in vivo , we generated a mouse line with a conditional Vaultrc5 loss-of-function allele. Because Vaultrc5 is the sole murine vtRNA, this allele enables the characterization of the physiological requirements of this conserved class of small regulatory RNAs in mammals. Using this strain, we show that mice constitutively null for Vaultrc5 are viable and histologically normal but have a slight reduction in platelet counts, pointing to a potential role for vtRNAs in hematopoiesis. This work paves the way for further in vivo characterizations of this abundant but mysterious RNA molecule. Specifically, it enables the study of the biological consequences of constitutive or lineage-specific Vaultrc5 deletion and of the physiological requirements for an intact Vaultrc5 during normal hematopoiesis or in response to cellular stresses such as oncogene expression, viral infection, or drug treatment., (Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Terminal nucleotidyltransferases add nucleotides to the 3' end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, the expression of PUP-2, PUP-3, and especially PUP-4 limits uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that the addition of U-tails to nonadenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as 'master regulators' of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology., (© 2024. The Author(s).)

Major depressive disorder (MDD) is a complex, common, and moderately heritable illness, and accumulating evidence suggests that psychological stress may affect male fertility. Mounting evidence indicates sncRNAs in sperm are sensitive to environmental changes and mediate the inheritance of paternally acquired metabolic and mental traits. In order to know the impact of psychological stress on reproduction and alterations in sperm sncRNAs, in this study, depressive-like mice induced by chronic unpredictable mild stimulation (CUMS) were used to investigate the impact of psychological stress on reproduction and alterations in sperm sncRNAs. The results showed that CUMS treatments for 4 weeks induced depressive behavior in male mice and significantly affected sperm quality. The results obtained from small RNA sequencing indicated that alterations occurred in the distribution and composition of small non-coding RNAs (sncRNAs), encompassing PIWI-interacting RNAs (piRNAs), rRNA-derived small RNAs (rsRNAs), and tRNA-derived small RNAs (tsRNAs). Furthermore, the offspring of male mice with depressive-like behavior have a significant reduction in survival rate at 21 days after birth, and those that did survive displayed an increased susceptibility to depression. This study provides some theoretical support for understanding the effects of psychological stress on reproduction, as well as information exchange from psychological stimulation to germ cells, and then from germ cells to next generation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Small noncoding RNAs (sncRNAs) play important regulatory roles in bacterial physiological processes and host-pathogen interactions. Meanwhile, bacterial outer membrane vesicles (OMVs), as naturally secreted outer membrane structures, play a vital role in the interaction between bacteria and their living environment, including the host environment. However, most current studies focus on the biological functions of sncRNAs in bacteria or hosts, while neglecting the roles and regulatory mechanisms of the OMVs that encapsulate these sncRNAs. Therefore, this review aims to summarize the intracellular regulatory roles of bacterial sncRNAs in promoting pathogen survival by regulating virulence, modulating bacterial drug resistance, and regulating iron metabolism, and their extracellular regulatory function for influencing host immunity through host-pathogen interactions. Additionally, we introduce the key role played by OMVs, which serve as important cargoes in bacterial sncRNA-host interactions. We propose emerging pathways of sncRNA action to further discuss the mode of host-pathogen interactions, highlighting that the inhibition of sncRNA delivery by OMVs may prevent the occurrence of infection to some extent. Hence, this review lays the foundation for future prophylactic treatments against bacterial infections and strategies for addressing bacterial drug resistance. KEY POINTS: •sncRNAs have intracellular and extracellular regulatory functions in bacterial physiological processes and host-pathogen interactions. •OMVs are potential mediators between bacterial sncRNAs and host cells. •OMVs encapsulating sncRNAs have more potential biological functions., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 .

Influenza A virus (IAV) induces acute respiratory infections in birds and various mammals, including humans, and presents a significant global public health concern, with considerable economic consequences. Recently, researchers have shown keen interest in noncanonical small noncoding RNAs (sncRNAs) as carriers of epigenetic information, including tRNA-derived small RNAs (tsRNAs), rRNA-derived small RNA (rsRNAs), and Y RNA-derived small RNAs (ysRNAs). Particularly, tsRNAs and rsRNAs are detected in diverse species and demonstrate evolutionary conservation. We analyzed sncRNAs sequencing data in the pulmonary tissue of two genetically distinct mouse strains, C57BL/6J and DBA/2J, to explore strain-specific variations of sncRNAs in response to IAV infection. We systematically compiled information on noncanonical sncRNAs in these two strains and investigated the tsRNAs/rsRNAs/ysRNAs profiles influenced by IAV infection. Specifically, four noncanonical sncRNA families, including rsRNA-12S, GtsRNA-Arg-CCT, GtsRNA-Arg-TCT, and GtsRNA-Lys-TTT, exhibited upregulation upon IAV infection. Notably, DBA/2J mice showed earlier systemic differential expression of noncanonical sncRNAs after IAV infection compared to C57BL/6J mice. Additionally, our study revealed a strain-specific biogenesis of MtsRNAs in response to IAV infection. Also, distinct co-expression patterns of MtsRNAs were observed between C57BL/6J and DBA/2J mice, with DBA/2J mice showing broader positive co-expression of MtsRNAs with various sncRNA families compared to C57BL/6J mice. Our study provides a novel insight into noncanonical sncRNAs and their implications in IAV pathology and mouse strain specificity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Small RNA (sRNA) in bacteria serve as the key messengers in regulating genes associated with quorum sensing controlled bacterial virulence. This study was aimed to unveil the regulatory role of sRNA PA0730.1 on the expression of various traits of Pseudomonas aeruginosa linked to pathogenicity, with special emphasis on the growth, colony morphology, cell motility, biofilm formation, and the expression of diverse virulence factors. PA0730.1 sRNA was found to be upregulated both during planktonic stationary growth phase and at biofilm state of P. aeruginosa PAO1. PA0730.1 deleted strain showed significant growth retardation with increased doubling time. Overexpression of PA0730.1 led to enhanced motility and biofilm formation, while the ∆PA0730.1 strain displayed significant inhibition in motility and biofilm formation. Furthermore, PA0730.1 was found to regulate the synthesis of selected virulence factors of P. aeruginosa. These observations in PA0730.1 + and ∆PA0730.1 were found to be correlated with the PA0730.1-mediated repression of transcription regulators, mucA and rpoS, both at transcriptional and translational levels. The results suggest that PA0730.1 sRNA might be a promising target for developing new drug to counter P. aeruginosa pathogenesis, and could also help in RNA oligonucleotide based therapeutic research for formulating a novel therapeutant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: Non-coding RNAs (ncRNAs) comprise a heterogeneous cluster of RNA molecules. Emerging evidence suggests their involvement in various aspects of tumorigenesis, particularly in gynecological malignancies. Notably, ncRNAs have been implicated as mediators within tumor signaling pathways, exerting their influence through interactions with RNA or proteins. These findings further highlight the hypothesis that ncRNAs constitute therapeutic targets and point out their clinical potential as stratification biomarkers., Areas Covered: The review outlines the use of small ncRNAs, including miRNAs, tRNA-derived small RNAs, PIWI-interacting RNAs and circular RNAs, for diagnostic, prognostic, and predictive purposes in gynecological cancers. It aims to increase our knowledge of their functions in tumor biology and their translation into clinical practice., Expert Opinion: By leveraging interdisciplinary collaborations, scientists can decipher the riddle of small ncRNA biomarkers as diagnostic, prognostic and predictive biomarkers of gynecological tumors. Integrating small ncRNA-based assays into clinical practice will allow clinicians to provide cure plans for each patient, reducing the likelihood of adverse responses. Nevertheless, addressing challenges such as standardizing experimental methodologies and refining diagnostic assays is imperative for advancing small ncRNA research in gynecological cancer.

Transfer RNA-derived small RNAs (tsRNAs) - categorized as tRNA-derived fragments (tRFs), tRNA-derived stress-induced RNAs (tiRNAs) and internal tRF (itRF) - are small non-coding RNAs that participate in various cellular processes such as translation inhibition and responses to cellular stress. We here identified tsRNA profiles within susceptible tissues in animal models of amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Parkinson's disease (PD) to pinpoint disease-specific tsRNAs and those shared across neurodegenerative diseases. We performed small RNA sequencing in the SOD1G93A and TDP43A315T mouse models of ALS (spinal cord), the TauP301S model of FTD (hippocampus), and the parkin/POLG model of PD (substantia nigra). Bioinformatic analysis showed higher expression of 5' tiRNAs selectively in the two ALS models, lower expression of 3' tRFs in both the ALS and FTD mouse models, and lower expression of itRF Arg in the PD model. Experimental validation confirmed the expression of tsRNAs. Gene Ontology analysis of targets associated with validated 3' tRFs indicated functions in the regulation of synaptic and neuronal pathways. Our profiling of tsRNAs indicates disease-specific fingerprints in animal models of neurodegeneration, which require validation in human disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Bacterial small regulatory RNA (sRNA) plays a crucial role in cell metabolism and could be used as a new potential drug target in the treatment of pathogen-induced disease. However, experimental methods for identifying sRNAs still require a large investment of human and material resources., Methods: In this study, we propose a novel sRNA prediction model called sRNAdeep based on the DistilBERT feature extraction and TextCNN methods. The sRNA and non-sRNA sequences of bacteria were considered as sentences and then fed into a composite model consisting of deep learning models to evaluate classification performance., Results: By filtering sRNAs from BSRD database, we obtained a validation dataset comprised of 2438 positive and 4730 negative samples. The benchmark experiments showed that sRNAdeep displayed better performance in the various indexes compared to previous sRNA prediction tools. By applying our tool to Mycobacterium tuberculosis (MTB) genome, we have identified 21 sRNAs within the intergenic and intron regions. A set of 272 targeted genes regulated by these sRNAs were also captured in MTB. The coding proteins of two genes (lysX and icd1) are implicated in drug response, with significant active sites related to drug resistance mechanisms of MTB., Conclusion: In conclusion, our newly developed sRNAdeep can help researchers identify bacterial sRNAs more precisely and can be freely available from https://github.com/pyajagod/sRNAdeep.git ., (© 2024. The Author(s).)

tRNA-derived small RNAs (tsRNAs) are a novel class of small noncoding RNAs, precisely cleaved from tRNA, functioning as regulatory molecules. The topic of tsRNAs in injuries has not been extensively discussed, and studies on tsRNAs are entering a new era. Here, we provide a fresh perspective on this topic. We systematically reviewed the classification, generation, and biological functions of tsRNAs in response to stress, as well as their potential as biomarkers and therapeutic targets in various injuries, including lung injury, liver injury, renal injury, cardiac injury, neuronal injury, vascular injury, skeletal muscle injury, and skin injury. We also provided a fresh perspective on the association between stress-induced tsRNAs and organ injury from a clinical perspective., Competing Interests: The authors declare that they have no competing interests., (© 2024 Wang et al.)

The study of small, regulatory RNAs (sRNA) that act by base-pairing with target RNAs in bacteria has been steadily advancing, particularly with the availability of more and more transcriptome and RNA-RNA interactome datasets. While the characterization of multiple sRNAs has helped to elucidate their mechanisms of action, these studies also are providing insights into protein function, control of metabolic flux, and connections between metabolic pathways as we will discuss here. In describing several examples of the metabolic insights gained, we will summarize the different types of base-pairing sRNAs including mRNA-derived sRNAs, sponge RNAs, RNA mimics, and dual-function RNAs as well as suggest how information about sRNAs could be exploited in the future., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Ltd.)

Extracellular vesicles and particles (EVPs) play a crucial role in mediating cell-to-cell communication by transporting various molecular cargos, with small non-coding RNAs (ncRNAs) holding particular significance. A thorough investigation into the abundance and sorting mechanisms of ncRNA within EVPs is imperative for advancing their clinical applications. We have developed EVPsort, which not only provides an extensive overview of ncRNA profiling in 3,162 samples across various biofluids, cell lines, and disease contexts but also seamlessly integrates 19 external databases and tools. This integration encompasses information on associations between ncRNAs and RNA-binding proteins (RBPs), motifs, targets, pathways, diseases, and drugs. With its rich resources and powerful analysis tools, EVPsort extends its profiling capabilities to investigate ncRNA sorting, identify relevant RBPs and motifs, and assess functional implications. EVPsort stands as a pioneering database dedicated to comprehensively addressing both the abundance and sorting of ncRNA within EVPs. It is freely accessible at https://bioinfo.vanderbilt.edu/evpsort/., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Listeria pathogenicity island 1 (LIPI-1) is a genetic region containing a cluster of genes essential for virulence of the bacterial pathogen Listeria monocytogenes . Main virulence factors in LIPI-1 include long 5' untranslated regions (5'UTRs), among which is Rli51, a small RNA (sRNA) in the 5'UTR of the Zn-metalloprotease-coding mpl . So far, Rli51 function and molecular mechanisms have remained obscure. Here, we show that Rli51 exhibits a dual mechanism of regulation, functioning as a cis - and as a trans -acting sRNA. Under nutrient-rich conditions, rli51-mpl transcription is prematurely terminated, releasing a short 121-nucleotide-long sRNA. Rli51 is predicted to function as a transcription attenuator that can fold into either a terminator or a thermodynamically more stable antiterminator. We show that the sRNA Rli21/RliI binds to a single-stranded RNA loop in Rli51, which is essential to mediate premature transcription termination, suggesting that sRNA binding could stabilize the terminator fold. During intracellular infection, rli51 transcription is increased, which generates a higher abundance of the short Rli51 sRNA and allows for transcriptional read-through into mpl . Comparative intracellular bacterial transcriptomics in rli51 -null mutants and the wild-type reference strain EGD-e suggests that Rli51 upregulates iron-scavenging proteins and downregulates virulence factors from LIPI-1. MS2 affinity purification confirmed that Rli51 binds transcripts of the heme-binding protein Lmo2186 and Lmo0937 in vivo. These results prove that Rli51 functions as a trans -acting sRNA in intracellular bacteria. Our research shows a growth condition-dependent mechanism of regulation for Rli51, preventing unintended mpl transcription in extracellular bacteria and regulating genes important for virulence in intracellular bacteria.

Small RNAs (sRNAs) are major regulators of gene expression in bacteria, exerting their regulation primarily via base pairing with their target transcripts and modulating translation. Accumulating evidence suggest that sRNAs can also affect the stability of their target transcripts by altering their accessibility to endoribonucleases. Yet, the effects of sRNAs on transcript stability and the mechanisms underlying them have not been studied in wide scale. Here we employ large-scale RNA-seq-based methodologies in the model bacterium Escherichia coli to quantitatively study the functional interaction between a sRNA and an endoribonuclease in regulating gene expression, using the well-established sRNA, GcvB, and the major endoribonuclease, RNase E. Studying single and double mutants of gcvB and rne and analysing their RNA-seq results by the Double Mutant Cycle approach, we infer distinct modes of the interplay between GcvB and RNase E. Transcriptome-wide mapping of RNase E cleavage sites provides further support to the results of the RNA-seq analysis, identifying cleavage sites in targets in which the functional interaction between GcvB and RNase E is evident. Together, our results indicate that the most dominant mode of GcvB-RNase E functional interaction is GcvB enhancement of RNase E cleavage, which varies in its magnitude between different targets., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of in vivo interactions by sequencing of the resulting chimeric RNAs. Modifications are incorporated to increase the capacity for processing low-abundance samples and permit cell-type-selective profiling of sncRNA:target RNA interactions, as demonstrated in mouse brain cortex. CIMERA-seq represents a cohesive and optimized method for unambiguously characterizing the in vivo network of sncRNA:target RNA interactions in numerous biological contexts and even subcellular fractions. Genome-wide and cell-type-selective CIMERA-seq enhances researchers' ability to study gene regulation by sncRNAs in diverse model systems and tissue types., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

The tRNA-derived small RNAs (tsRNAs) can be categorized into two main groups: tRNA-derived fragments (tRFs) and tRNA-derived stress-induced RNAs (tiRNAs). Each group possesses specific molecular sizes, nucleotide compositions, and distinct physiological functions. Notably, hypoxia-inducible factor-1 (HIF-1), a transcriptional activator dependent on oxygen, comprises one HIF-1β subunit and one HIF-α subunit (HIF-1α/HIF-2α/HIF-3α). The activation of HIF-1 plays a crucial role in gene transcription, influencing key aspects of cancer biology such as angiogenesis, cell survival, glucose metabolism, and invasion. The involvement of HIF-1α activation has been demonstrated in numerous human diseases, particularly cancer, making HIF-1 an attractive target for potential disease treatments. Through a series of experiments, researchers have identified two tiRNAs that interact with the HIF-1 pathway, impacting disease development: 5'tiRNA-His-GTG in colorectal cancer (CRC) and tiRNA-Val in diabetic retinopathy (DR). Specifically, 5'tiRNA-His-GTG promotes CRC development by targeting LATS2, while tiRNA-Val inhibits Sirt1, leading to HIF-1α accumulation and promoting DR development. Clinical data have further indicated that certain tsRNAs' expression levels are associated with the prognosis and pathological features of CRC patients. In CRC tumor tissues, the expression level of 5'tiRNA-His-GTG is significantly higher compared to normal tissues, and it shows a positive correlation with tumor size. Additionally, KEGG analysis has revealed multiple tRFs involved in regulating the HIF-1 pathway, including tRF-Val-AAC-016 in diabetic foot ulcers (DFU) and tRF-1001 in pathological ocular angiogenesis. This comprehensive article reviews the biological functions and mechanisms of tsRNAs related to the HIF-1 pathway in diseases, providing a promising direction for subsequent translational medicine research., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Transfer RNA-derived small RNAs (tsRNAs) are a class of small non-coding RNA (sncRNA) molecules derived from tRNA, including tRNA derived fragments (tRFs) and tRNA halfs (tiRNAs). tsRNAs can affect cell functions by participating in gene expression regulation, translation regulation, intercellular signal transduction, and immune response. They have been shown to play an important role in various human diseases, including cardiovascular diseases (CVDs). Targeted regulation of tsRNAs expression can affect the progression of CVDs. The tsRNAs induced by pathological conditions can be detected when released into the extracellular, giving them enormous potential as disease biomarkers. Here, we review the biogenesis, degradation process and related functional mechanisms of tsRNAs, and discuss the research progress and application prospects of tsRNAs in different CVDs, to provide a new perspective on the treatment of CVDs., Competing Interests: Declaration of Competing Interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: To investigate the epigenetic footprint of idiopathic inflammatory myopathies (IIM) through characterization of circulating extracellular vesicles (EVs) and the expression of EV-derived small non-coding RNAs (sncRNAs)., Methods: In this cross-sectional study, EVs were isolated by size-exclusion chromatography from plasma of patients with IIM and age- and sex-matched healthy donors (HD). EV-derived sncRNAs were sequenced and quantified using Next-Generation Sequencing (NGS). Following quality control and normalization, filtered count reads were used for differential microRNA (miRNA) and piwi-interacting RNA (piRNA) expression analyses. Putative gene targets enriched for pathways implicated in IIM were analyzed. Patients' clinical and laboratory characteristics at the time of sampling were recorded., Results: Forty-seven IIM patients and 45 HD were enrolled. MiR-486-5p (p < 0.01), miR-122-5p, miR-192-5p, and miR-32-5p were significantly upregulated (p < 0.05 for all), while miR-142-3p (p < 0.001), miR-141-3p (p < 0.01), let-7a-5p (p < 0.05) and miR-3613-5p (p < 0.05) downregulated in EVs from IIM patients versus HD. MiR-486-5p was associated with raised muscle enzymes levels. Several target genes of up/downregulated miRNAs in IIM participate in inflammation, necroptosis, interferon and immune signaling. Six piRNAs were significantly dysregulated in IIM EVs versus HD (p < 0.05). Within IIM, miR-335-5p was selectively upregulated and miR-27a-5p downregulated in dermatomyositis (n = 21, p < 0.01). Finally, plasma EV levels were significantly increased in cancer-associated myositis (CAM, n = 12) versus non-CAM IIM (n = 35, p = 0.02) and HD (p < 0.01). EVs cargo in CAM was significantly enriched of let-7f-5p and depleted of miR-143-3p., Conclusion: Through an unbiased screening of EV-derived sncRNAs, we characterize miRNAs and piRNAs in the EVs cargo as potential biomarkers and modifiers of diverse IIM phenotypes., Competing Interests: Declaration of competing interest The authors have no conflict of interest in context of this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Small non-coding RNAs (sncRNAs) present in the conditioned medium (CM) of bovine preimplantation embryos are potential noninvasive biomarkers for assessing embryo quality. Accurate quantification of sncRNA levels in the spent CM is of utmost importance in this regard. RT-qPCR is considered as the gold standard for quantifying RNA. In order to standardize RT-qPCR data in the sample type under investigation, the use of suitable stable sncRNAs is essential. Here, we selected 10 sncRNAs from small RNA sequencing of CM samples derived from both bovine blastocysts and degenerate embryos, and evaluated their expression stability together with that of cel-miR-39 as a spike and the often-used U6 small nuclear RNA at different embryo developmental stages. In CM of 2-cell embryos, rsRNA-1044 showed the most stable expression, while tDR-1:32-Gly-CCC-1 was the most stable expressed sncRNA in CM of the stages beyond the 2-cell stage. Next, tDR-1:32-Gly-CCC-1 was used for normalizing the RT-qPCR data from the CM of blastocysts and degenerate embryos. Bta-miR-155 and tDR-39:75-Arg-CCG-2 were found to be significantly up-regulated in the CM of blastocysts compared to that of the degenerated embryos (P = 0.028 and P = 0.017, respectively), suggesting their expression levels are related to embryo development stage. In conclusion, tDR-1:32-Gly-CCC-1 can serve as a suitable reference sncRNA for normalization of RT-qPCR data of the CM from bovine blastocysts., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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&#95;0240, predictedRNA&#95;0325, and predictedRNA&#95;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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This review comprehensively investigates the intricate interplay between small non-coding RNAs (sncRNAs) and pancreatic ductal adenocarcinoma (PDAC), a devastating malignancy with limited therapeutic options. Our analysis reveals the pivotal roles of sncRNAs in various facets of PDAC biology, spanning diagnosis, pathogenesis, drug resistance, and therapeutic strategies. sncRNAs have emerged as promising biomarkers for PDAC, demonstrating distinct expression profiles in diseased tissues. sncRNA differential expression patterns, often detectable in bodily fluids, hold potential for early and minimally invasive diagnostic approaches. Furthermore, sncRNAs exhibit intricate involvement in PDAC pathogenesis, regulating critical cellular processes such as proliferation, apoptosis, and metastasis. Additionally, mechanistic insights into sncRNA-mediated pathogenic pathways illuminate novel therapeutic targets and interventions. A significant focus of this review is dedicated to unraveling sncRNA mechanisms underlying drug resistance in PDAC. Understanding these mechanisms at the molecular level is imperative for devising strategies to overcome drug resistance. Exploring the therapeutic landscape, we discuss the potential of sncRNAs as therapeutic agents themselves as their ability to modulate gene expression with high specificity renders them attractive candidates for targeted therapy. In summary, this review integrates current knowledge on sncRNAs in PDAC, offering a holistic perspective on their diagnostic, pathogenic, and therapeutic relevance. By elucidating the roles of sncRNAs in PDAC biology, this review provides valuable insights for the development of novel diagnostic tools and targeted therapeutic approaches, crucial for improving the prognosis of PDAC patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Small non-coding RNAs (sncRNAs) derived from tRNAs are known as tRNA-derived small RNAs (tsRNAs). These tsRNAs are further categorized into tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), which play significant roles in the various molecular mechanisms underlying certain human diseases. However, the generation of tsRNAs and their potential roles during Dengue virus (DENV) infection is not yet known. Here, we performed small RNA sequencing to identify the generation and alterations in tsRNAs expression profiles of DENV-infected Huh7 cells. Upon DENV infection, tRNA fragmentation was found to be increased. We identified a significant number of differentially expressed tsRNAs during DENV infection. Interestingly, the 3'tRF population showed upregulation, while the i-tRF population exhibited downregulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to analyze the impact of differentially expressed tsRNAs on DENV pathogenesis. Our results suggest that differentially expressed tsRNAs are involved in transcriptional regulation via RNA polymerase II promoter and metabolic pathways. Overall, our study contributes significantly to our understanding of the roles played by tsRNAs in the complex dynamics of DENV infection., (© 2024. The Author(s).)