Small Non-Coding RNAs and Their Role in Locoregional Metastasis and Outcomes in Early-Stage Breast Cancer Patients.

Deregulation of small non-coding RNAs (sncRNAs) has been associated with the onset of metastasis. We evaluated the expression of sncRNAs in patients with early-stage breast cancer, performing RNA sequencing in 60 patients for whom tumor and sentinel lymph node (SLN) samples were available, and conducting differential expression, gene ontology, enrichment and survival analyses. Sequencing annotation classified most of the sncRNAs into small nucleolar RNA (snoRNAs, 70%) and small nuclear RNA (snRNA, 13%). Our results showed no significant differences in sncRNA expression between tumor or SLNs obtained from the same patient. Differential expression analysis showed down-regulation (n = 21) sncRNAs and up-regulation (n = 2) sncRNAs in patients with locoregional metastasis. The expression of SNHG5, SNORD90, SCARNA2 and SNORD78 differentiated luminal A from luminal B tumors, whereas SNORD124 up-regulation was associated with luminal B HER2+ tumors. Discriminating analysis and receiver-operating curve analysis revealed a signature of six snoRNAs (SNORD93, SNORA16A, SNORD113-6, SNORA7A, SNORA57 and SNORA18A) that distinguished patients with locoregional metastasis and predicted patient outcome. Gene ontology and Reactome pathway analysis showed an enrichment of biological processes associated with translation initiation, protein targeting to specific cell locations, and positive regulation of Wnt and NOTCH signaling pathways, commonly involved in the promotion of metastases. Our results point to the potential of several sncRNAs as surrogate markers of lymph node metastases and patient outcome in early-stage breast cancer patients. Further preclinical and clinical studies are required to understand the biological significance of the most significant sncRNAs and to validate our results in a larger cohort of patients.

Investigating the impact of insertion sequences and transposons in the genomes of the most significant phytopathogenic bacteria.

Genetic variability in phytopathogens is one of the main problems encountered for effective plant disease control. This fact may be related to the presence of transposable elements (TEs), but little is known about their role in host genomes. Here, we performed the most comprehensive analysis of insertion sequences (ISs) and transposons (Tns) in the genomes of the most important bacterial plant pathogens. A total of 35 692 ISs and 71 transposons were identified in 270 complete genomes. The level of pathogen-host specialization was found to be a significant determinant of the element distribution among the species. Some Tns were identified as carrying virulence factors, such as genes encoding effector proteins of the type III secretion system and resistance genes for the antimicrobial streptomycin. Evidence for IS-mediated ectopic recombination was identified in Xanthomonas genomes. Moreover, we found that IS elements tend to be inserted in regions near virulence and fitness genes, such ISs disrupting avirulence genes in X. oryzae genomes. In addition, transcriptome analysis under different stress conditions revealed differences in the expression of genes encoding transposases in the Ralstonia solanacearum, X. oryzae, and P. syringae species. Lastly, we also investigated the role of Tns in regulation via small noncoding regulatory RNAs and found these elements may target plant-cell transcriptional activators. Taken together, the results indicate that TEs may have a fundamental role in variability and virulence in plant pathogenic bacteria.

SARS-CoV-2 remodels the landscape of small non-coding RNAs with infection time and symptom severity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides. Host-derived sncRNAs, including microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), and small nucleolar RNA-derived small RNAs (sdRNAs) exhibited significant differential expression in infected patients compared to controls. Importantly, miRNA expression was predominantly down-regulated in response to SARS-CoV-2 infection, especially in patients with severe symptoms. Furthermore, we identified specific tsRNAs derived from Glu- and Gly-tRNAs as major altered elements upon infection, with 5' tRNA halves being the most abundant species and suggesting their potential as biomarkers for viral presence and disease severity prediction. Additionally, down-regulation of C/D-box sdRNAs and altered expression of tinyRNAs (tyRNAs) were observed in infected patients. These findings provide valuable insights into the host sncRNA response to SARS-CoV-2 infection and may contribute to the development of further diagnostic and therapeutic strategies in the clinic.

Development of synthetic small regulatory RNA for Rhodococcus erythropolis.

Rhodococci have been regarded as ideal chassis for biotransformation, biodegradation, and biosynthesis for their unique environmental persistence and robustness. However, most species of Rhodococcus are still difficult to metabolically engineer due to the lack of genetic tools and techniques. In this study, synthetic sRNA strategy was exploited for gene repression in R. erythropolis XP. The synthetic sRNA based on the RhlS scaffold from Pseudomonas aeruginosa functions better in repressing sfgfp expression than those based on E. coli MicC, SgrS, and P. aeruginosa PrrF1-2 scaffold. The RhlS-based sRNAs were applied to study the influence of sulfur metabolism on biodesulfurization (BDS) efficiency in R. erythropolis XP and successfully identified two genes involved in sulfur metabolism that affect the BDS efficiency significantly. The RhlS-based synthetic sRNAs show promise in the metabolic engineering of Rhodococcus and promote the industrial applications of Rhodococcus in environmental remediation and biosynthesis.

In Silico Design, In Vitro Construction, and In Vivo Application of Synthetic Small Regulatory RNAs in Bacteria.

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described.

Assessing small RNA profiles in potato diploid hybrid and its resynthesized allopolyploid reveals conserved abundance with distinct genomic distribution.

KEY MESSAGE: The shock produced by the allopolyploidization process on a potato interspecific diploid hybrid displays a non-random remobilization of the small RNAs profile on a variety of genomic features. Allopolyploidy, a complex process involving interspecific hybridization and whole genome duplication, significantly impacts plant evolution, leading to the emergence of novel phenotypes. Polyploids often present phenotypic nuances that enhance adaptability, enabling them to compete better and occasionally to colonize new habitats. Whole-genome duplication represents a genomic "shock" that can trigger genetic and epigenetic changes that yield novel expression patterns. In this work, we investigate the polyploidization effect on a diploid interspecific hybrid obtained through the cross between the cultivated potato Solanum tuberosum and the wild potato Solanum kurtzianum, by assessing the small RNAs (sRNAs) profile of the parental diploid hybrid and its derived allopolyploid. Small RNAs are key components of the epigenetic mechanisms involved in silencing by RNA-directed DNA Methylation (RdDM). A sRNA sequencing (sRNA-Seq) analysis was performed to individually profile the 21 to 22 nucleotide (21 to 22-nt) and 24-nt sRNA size classes due to their unique mechanism of biogenesis and mode of function. The composition and distribution of different genomic features and differentially accumulated (DA) sRNAs were evaluated throughout the potato genome. We selected a subset of genes associated with DA sRNAs for messenger RNA (mRNA) expression analysis to assess potential impacts on the transcriptome. Interestingly, we noted that 24-nt DA sRNAs that exclusively mapped to exons were correlated with differentially expressed mRNAs between genotypes, while this behavior was not observed when 24-nt DA sRNAs were mapped to intronic regions. These findings collectively emphasize the nonstochastic nature of sRNA remobilization in response to the genomic shock induced by allopolyploidization.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σE-dependent small RNA with unusual mRNA activation activity.

The oral commensal Fusobacterium nucleatum can spread to extra-oral sites, where it is associated with diverse pathologies, including pre-term birth and cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of the RNA regulatory networks that allow this bacterium to adapt to its various niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor, σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped the 5' and 3' ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156-nucleotide sRNA previously misannotated as the 5' untranslated region (UTR) of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as is FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5' and 3' UTRs in F. nucleatum and expands our understanding of the σE response in this evolutionarily distant bacterium.IMPORTANCEThe oral microbe Fusobacterium nucleatum can colonize secondary sites, including cancer tissue, and likely deploys complex regulatory systems to adapt to these new environments. These systems are largely unknown, partly due to the phylogenetic distance of F. nucleatum to other model organisms. Previously, we identified a global stress response mediated by σE that displays functional conservation with the envelope stress response in Proteobacteria, comprising a coding and noncoding regulatory arm. Through global identification of transcriptional start and stop sites, we uncovered the small RNA (sRNA) FoxJ as a novel component of the noncoding arm of the σE response in F. nucleatum. Together with its companion sRNA FoxI, FoxJ post-transcriptionally modulates the synthesis of envelope proteins, revealing a conserved function for σE-dependent sRNAs between Fusobacteriota and Proteobacteria. Moreover, FoxJ activates the gene expression for several targets, which is a mode of regulation previously unseen in the noncoding arm of the σE response.

Epigenetic weapons of plants against fungal pathogens.

In the natural environment, plants face constant exposure to biotic stress caused by fungal attacks. The plant's response to various biotic stresses relies heavily on its ability to rapidly adjust the transcriptome. External signals are transmitted to the nucleus, leading to activation of transcription factors that subsequently enhance the expression of specific defense-related genes. Epigenetic mechanisms, including histone modifications and DNA methylation, which are closely linked to chromatin states, regulate gene expression associated with defense against biotic stress. Additionally, chromatin remodelers and non-coding RNA play a significant role in plant defense against stressors. These molecular modifications enable plants to exhibit enhanced resistance and productivity under diverse environmental conditions. Epigenetic mechanisms also contribute to stress-induced environmental epigenetic memory and priming in plants, enabling them to recall past molecular experiences and utilize this stored information for adaptation to new conditions. In the arms race between fungi and plants, a significant aspect is the cross-kingdom RNAi mechanism, whereby sRNAs can traverse organismal boundaries. Fungi utilize sRNA as an effector molecule to silence plant resistance genes, while plants transport sRNA, primarily through extracellular vesicles, to pathogens in order to suppress virulence-related genes. In this review, we summarize contemporary knowledge on epigenetic mechanisms of plant defense against attack by pathogenic fungi. The role of epigenetic mechanisms during plant-fungus symbiotic interactions is also considered.

RNA compaction and iterative scanning for small RNA targets by the Hfq chaperone.

RNA-guided enzymes must quickly search a vast sequence space for their targets. This search is aided by chaperones such as Hfq, a protein that mediates regulation by bacterial small RNAs (sRNAs). How RNA binding proteins enhance this search is little known. Using single-molecule Förster resonance energy transfer, we show that E. coli Hfq performs a one-dimensional scan in which compaction of the target RNA delivers sRNAs to sites distant from the location of Hfq recruitment. We also show that Hfq can transfer an sRNA between different target sites in a single mRNA, favoring the most stable duplex. We propose that compaction and segmental transfer, combined with repeated cycles of base pairing, enable the kinetic selection of optimal sRNA targets. Finally, we show that RNA compaction and sRNA transfer require conserved arginine patches. We suggest that arginine patches are a widespread strategy for enabling the movement of RNA across protein surfaces.

Microarray analysis of tRNA-derived small RNA (tsRNA) in LPS-challenged macrophages treated with metformin.

Metformin, a widely used anti-diabetic drug, has demonstrated its efficacy in addressing various inflammatory conditions. tRNA-derived small RNA (tsRNA), a novel type of small non-coding RNA, exhibits diverse regulatory functions and holds promise as both a diagnostic biomarker and a therapeutic target for various diseases. The purpose of this study is to investigate whether the abundance of tsRNAs changed in LPS versus LPS + metformin-treated cells, utilizing microarray technology. Firstly, we established an in vitro lipopolysaccharide (LPS)-induced inflammation model using RAW264.7 macrophages and assessed the protective effects of metformin against inflammatory damage. Subsequently, we extracted total RNA from both LPS-treated and metformin + LPS-treated cell samples for microarray analysis to identify differentially abundant tsRNAs (DA-tsRNAs). Furthermore, we conducted bioinformatics analysis to predict target genes for validated DA-tsRNAs and explore the biological functions and signaling pathways associated with DA-tsRNAs. Notably, metformin was found to inhibit the inflammatory response in RAW264.7 macrophages. The microarray results revealed a total of 247 DA-tsRNAs, with 58 upregulated and 189 downregulated tsRNAs in the Met + LPS group compared to the LPS group. The tsRNA-mRNA network was visualized, shedding light on potential interactions. The results of bioinformatics analysis suggested that these potential targets of specific tsRNAs were mainly related to inflammation and immunity. Our study provides compelling evidence that metformin exerts anti-inflammatory effects and modulates the abundance of tsRNAs in LPS-treated RAW264.7 macrophages. These findings establish a valuable foundation for using tsRNAs as potential biomarkers for metformin in the treatment of inflammatory conditions.

Small RNAs from mitochondrial genome recombination sites are incorporated into T. gondii mitoribosomes.

The mitochondrial genomes of apicomplexans comprise merely three protein-coding genes, alongside a set of thirty to forty genes encoding small RNAs (sRNAs), many of which exhibit homologies to rRNA from E. coli. The expression status and integration of these short RNAs into ribosomes remains unclear and direct evidence for active ribosomes within apicomplexan mitochondria is still lacking. In this study, we conducted small RNA sequencing on the apicomplexan Toxoplasma gondii to investigate the occurrence and function of mitochondrial sRNAs. To enhance the analysis of sRNA sequencing outcomes, we also re-sequenced the T. gondii mitochondrial genome using an improved organelle enrichment protocol and Nanopore sequencing. It has been established previously that the T. gondii genome comprises 21 sequence blocks that undergo recombination among themselves but that their order is not entirely random. The enhanced coverage of the mitochondrial genome allowed us to characterize block combinations at increased resolution. Employing this refined genome for sRNA mapping, we find that many small RNAs originated from the junction sites between protein-coding blocks and rRNA sequence blocks. Surprisingly, such block border sRNAs were incorporated into polysomes together with canonical rRNA fragments and mRNAs. In conclusion, apicomplexan ribosomes are active within polysomes and are indeed assembled through the integration of sRNAs, including previously undetected sRNAs with merged mRNA-rRNA sequences. Our findings lead to the hypothesis that T. gondii's block-based genome organization enables the dual utilization of mitochondrial sequences as both messenger RNAs and ribosomal RNAs, potentially establishing a link between the regulation of rRNA and mRNA expression.

Agnodice: indexing experimentally supported bacterial sRNA-RNA interactions.

In the last decade, the immense growth in the field of bacterial small RNAs (sRNAs), along with the biotechnological breakthroughs in Deep Sequencing permitted the deeper understanding of sRNA-RNA interactions. However, microbiology is currently lacking a thoroughly curated collection of this rapidly expanding universe. We present Agnodice (https://dianalab.e-ce.uth.gr/agnodice), our effort to systematically catalog and annotate experimentally supported bacterial sRNA-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Deep Sequencing interactome identification techniques. It comprises 39,600 entries which are annotated at strain-level resolution and pertain to 399 sRNAs and 12,137 target RNAs identified in 71 bacterial strains. The database content is exclusively experimentally supported, incorporating interactions derived via low yield as well as state-of-the-art high-throughput methods. The entire content of the database is freely accessible and can be directly downloaded for further analysis. Agnodice will serve as a valuable source, enabling microbiologists to form novel hypotheses, design/identify novel sRNA-based drug targets, and explore the therapeutic potential of microbiomes from the perspective of small regulatory RNAs.IMPORTANCEAgnodice (https://dianalab.e-ce.uth.gr/agnodice) is an effort to systematically catalog and annotate experimentally supported bacterial small RNA (sRNA)-RNA interactions. Agnodice, for the first time, incorporates thousands of bacterial sRNA-RNA interactions derived from a diverse set of experimental methodologies including state-of-the-art Next Generation Sequencing interactome identification techniques.

The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division.

The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress resistance; however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation, and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of RNA-RNA pairs, our study revealed an Hfq-dependent small regulatory RNA (sRNA), DinR, which is processed from the 3' terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled division inhibition during the SOS response.

The Amyloid Assembly of the Bacterial Hfq Is Lipid-Driven and Lipid-Specific.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as "functional amyloids". In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

Short noncoding RNAs as predictive biomarkers for the development from inflammatory bowel disease unclassified to Crohn's disease or ulcerative colitis.

Numerous pathogenic processes are mediated by short noncoding RNAs (sncRNA). Twenty percent of inflammatory bowel disease (IBD) patients are labelled as IBD unclassified (IBDU) at disease onset. Most IBDU patients are reclassified as Crohn's disease (CD) or ulcerative colitis (UC) within few years. Since the therapeutic methods for CD and UC differ, biomarkers that can forecast the categorization of IBDU into CD or UC are highly desired. Here, we investigated whether sncRNAs can predict CD or UC among IBDU patients. 35 IBDU patients who were initially diagnosed with IBDU were included in this retrospective investigation; of them, 12, 15, and 8 were reclassified into CD (IBDU-CD), UC (IBDU-UC), or remained as IBDU (IBDU-IBDU), respectively. Eight IBD patients, were included as references. SncRNA profiling on RNA from mucosal biopsies were performed using Affymetrix miRNA 4.0 array. Selected probe sets were validated using RT-qPCR. Among all patients and only adults, 306 and 499 probe sets respectively were differentially expressed between IBDU-CD and IBDU-UC. Six of the probe sets were evaluated by RT-qPCR, of which miR-182-5p, miR-451a and ENSG00000239080 (snoU13) together with age and sex resulted in an AUC of 78.6% (95% CI: 60-97) in discriminating IBDU-CD from IBDU-UC. Based on the three sncRNAs profile it is possible to predict if IBDU patients within 3 years will be reclassified as CD or UC. We showed that the expression profile of IBDU patients differ from that of definite CD or UC, suggesting that a subgroup of IBDU patients may compose a third unique IBD subtype.

Suppression of host plant defense by bacterial small RNAs packaged in outer membrane vesicles.

Noncoding small RNAs (sRNAs) packaged in bacterial outer membrane vesicles (OMVs) function as novel mediators of interspecies communication. While the role of bacterial sRNAs in enhancing virulence is well established, the role of sRNAs in the interaction between OMVs from phytopathogenic bacteria and their host plants remains unclear. In this study, we employ RNA sequencing to characterize differentially packaged sRNAs in OMVs of the phytopathogen Xanthomonas oryzae pv. oryzicola (Xoc). Our candidate sRNA (Xosr001) was abundant in OMVs and involved in the regulation of OsJMT1 to impair host stomatal immunity. Xoc loads Xosr001 into OMVs, which are specifically ttransferred into the mechanical tissues of rice leaves. Xosr001 suppresses OsJMT1 transcript accumulation in vivo, leading to a reduction in MeJA accumulation in rice leaves. Furthermore, the application of synthesized Xosr001 sRNA to the leaves of OsJMT1-HA-OE transgenic line results in the suppression of OsJMT1 expression by Xosr001. Notably, the OsJMT1-HA-OE transgenic line exhibited attenuated stomatal immunity and disease susceptibility upon infection with ΔXosr001 compared to Xoc. These results suggest that Xosr001 packaged in Xoc OMVs functions to suppress stomatal immunity in rice.

The serum small non-coding RNA (SncRNA) landscape as a molecular biomarker of age associated muscle dysregulation and insulin resistance in older adults.

Small noncoding RNAs (sncRNAs) are implicated in age-associated pathologies, including sarcopenia and insulin resistance (IR). As potential circulating biomarkers, most studies have focussed on microRNAs (miRNAs), one class of sncRNA. This study characterized the wider circulating sncRNA transcriptome of older individuals and associations with sarcopenia and IR. sncRNA expression including miRNAs, transfer RNAs (tRNAs), tRNA-associated fragments (tRFs), and piwi-interacting RNAs (piRNAs) was measured in serum from 21 healthy and 21 sarcopenic Hertfordshire Sarcopenia Study extension women matched for age (mean 78.9 years) and HOMA2-IR. Associations with age, sarcopenia and HOMA2-IR were examined and predicted gene targets and biological pathways characterized. Of the total sncRNA among healthy controls, piRNAs were most abundant (85.3%), followed by tRNAs (4.1%), miRNAs (2.7%), and tRFs (0.5%). Age was associated (FDR < 0.05) with 2 miRNAs, 58 tRNAs, and 14 tRFs, with chromatin organization, WNT signaling, and response to stress enriched among gene targets. Sarcopenia was nominally associated (p < .05) with 12 tRNAs, 3 tRFs, and 6 piRNAs, with target genes linked to cell proliferation and differentiation such as Notch Receptor 1 (NOTCH1), DISC1 scaffold protein (DISC1), and GLI family zinc finger-2 (GLI2). HOMA2-IR was nominally associated (p<0.05) with 6 miRNAs, 9 tRNAs, 1 tRF, and 19 piRNAs, linked with lysine degradation, circadian rhythm, and fatty acid biosynthesis pathways. These findings identify changes in circulating sncRNA expression in human serum associated with chronological age, sarcopenia, and IR. These may have clinical utility as circulating biomarkers of ageing and age-associated pathologies and provide novel targets for therapeutic intervention.

Small RNA-big impact: exosomal miRNAs in mitochondrial dysfunction in various diseases.

Mitochondria are multitasking organelles involved in maintaining the cell homoeostasis. Beyond its well-established role in cellular bioenergetics, mitochondria also function as signal organelles to propagate various cellular outcomes. However, mitochondria have a self-destructive arsenal of factors driving the development of diseases caused by mitochondrial dysfunction. Extracellular vesicles (EVs), a heterogeneous group of membranous nano-sized vesicles, are present in a variety of bodily fluids. EVs serve as mediators for intercellular interaction. Exosomes are a class of small EVs (30-100 nm) released by most cells. Exosomes carry various cargo including microRNAs (miRNAs), a class of short noncoding RNAs. Recent studies have closely associated exosomal miRNAs with various human diseases, including diseases caused by mitochondrial dysfunction, which are a group of complex multifactorial diseases and have not been comprehensively described. In this review, we first briefly introduce the characteristics of EVs. Then, we focus on possible mechanisms regarding exosome-mitochondria interaction through integrating signalling networks. Moreover, we summarize recent advances in the knowledge of the role of exosomal miRNAs in various diseases, describing how mitochondria are changed in disease status. Finally, we propose future research directions to provide a novel therapeutic strategy that could slow the disease progress mediated by mitochondrial dysfunction.