Targeting endothelial glycolytic reprogramming by tsRNA-1599 for ocular anti-angiogenesis therapy.

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

Interplay of two small RNAs fine-tunes hierarchical flagella gene expression in Campylobacter jejuni.

Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.

Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

The anti-apoptotic function of HSV-1 LAT in neuronal cell cultures but not its function during reactivation correlates with expression of two small non-coding RNAs, sncRNA1&2.

Multiple functions are associated with HSV-1 latency associated transcript (LAT), including establishment of latency, virus reactivation, and antiapoptotic activity. LAT encodes two sncRNAs that are not miRNAs and previously it was shown that they have antiapoptotic activity in vitro. To determine if we can separate the antiapoptotic function of LAT from its latency-reactivation function, we deleted sncRNA1 and sncRNA2 sequences in HSV-1 strain McKrae, creating ΔsncRNA1&2 recombinant virus. Deletion of the sncRNA1&2 in ΔsncRNA1&2 virus was confirmed by complete sequencing of ΔsncRNA1&2 virus and its parental virus. Replication of ΔsncRNA1&2 virus in tissue culture or in the eyes of WT infected mice was similar to that of HSV-1 strain McKrae (LAT-plus) and dLAT2903 (LAT-minus) viruses. The levels of gB DNA in trigeminal ganglia (TG) of mice latently infected with ΔsncRNA1&2 virus was intermediate to that of dLAT2903 and McKrae infected mice, while levels of LAT in TG of latently infected ΔsncRNA1&2 mice was significantly higher than in McKrae infected mice. Similarly, the levels of LAT expression in Neuro-2A cells infected with ΔsncRNA1&2 virus was significantly higher than in McKrae infected cells. Reactivation in TG of ΔsncRNA1&2 infected mice was similar to that of McKrae and time of reactivation in both groups were significantly faster than dLAT2903 infected mice. However, levels of apoptosis in Neuro-2A cells infected with ΔsncRNA1&2 virus was similar to that of dLAT2903 and significantly higher than that of McKrae infected cells. Our results suggest that the antiapoptotic function of LAT resides within the two sncRNAs, which works independently of its latency-reactivation function and it has suppressive effect on LAT expression in vivo and in vitro.

Untacking small RNA profiling and RNA fragment footprinting: Approaches and challenges in library construction.

Small RNAs (sRNAs) with sizes ranging from 15 to 50 nucleotides (nt) are critical regulators of gene expression control. Prior studies have shown that sRNAs are involved in a broad range of biological processes, such as organ development, tumorigenesis, and epigenomic regulation; however, emerging evidence unveils a hidden layer of diversity and complexity of endogenously encoded sRNAs profile in eukaryotic organisms, including novel types of sRNAs and the previously unknown post-transcriptional RNA modifications. This underscores the importance for accurate, unbiased detection of sRNAs in various cellular contexts. A multitude of high-throughput methods based on next-generation sequencing (NGS) are developed to decipher the sRNA expression and their modifications. Nonetheless, distinct from mRNA sequencing, the data from sRNA sequencing suffer frequent inconsistencies and high variations emanating from the adapter contaminations and RNA modifications, which overall skew the sRNA libraries. Here, we summarize the sRNA-sequencing approaches, and discuss the considerations and challenges for the strategies and methods of sRNA library construction. The pros and cons of sRNA sequencing have significant implications for implementing RNA fragment footprinting approaches, including CLIP-seq and Ribo-seq. We envision that this review can inspire novel improvements in small RNA sequencing and RNA fragment footprinting in future. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

tRNA-derived small RNA (tsr007330) regulates myocardial fibrosis after myocardial infarction through NAT10-mediated ac4C acetylation of EGR3 mRNA.

Small non-coding ribonucleic acids (sncRNAs) play an important role in cell regulation and are closely related to the pathogenesis of heart diseases. However, the role and molecular mechanism of transfer RNA-derived small RNAs (tsRNAs) in myocardial fibrosis after myocardial infarction (MI) remain unknown. In this study, we identified and validated sncRNAs (mainly miRNA and tsRNA) associated with myocardial fibrosis after MI through PANDORA sequencing of rat myocardial tissue. As a key enzyme of N4-acetylcytidine (ac4C) acetylation modification, N-acetyltransferase 10 (NAT10) plays an important role in regulating messenger RNA (mRNA) stability and translation efficiency. We found that NAT10 is highly expressed in infarcted myocardial tissue, and the results of acetylated RNA immunoprecipitation sequencing (acRIP-seq) analysis suggest that early growth response 3 (EGR3) may be an important molecule in the pathogenesis of NAT10-mediated myocardial fibrosis. Both in vivo and in vitro experiments have shown that inhibition of NAT10 can reduce the expression of EGR3 and alleviate myocardial fibrosis after MI. tsRNA can participate in gene regulation by inhibiting target genes. The expression of tsr007330 was decreased in myocardial infarction tissue. We found that overexpression of tsr007330 in rat myocardial tissue could antagonize NAT10, improve myocardial function in MI and alleviate myocardial fibrosis. In conclusion, tsRNAs (rno-tsr007330) may regulate the occurrence of myocardial fibrosis by regulating NAT10-mediated EGR3 mRNA acetylation. This study provides new insights into the improvement of myocardial fibrosis after MI by targeting tsRNA therapy.

Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

Identifying quantitative sncRNAs signature using global sequencing as a potential biomarker for tuberculosis diagnosis and their role in regulating host response.

OBJECTIVES: The study aimed to identify a quantitative signature of circulating small non-coding RNAs (sncRNAs) as a biomarker for pulmonary tuberculosis disease (active-TB/ATB) and explore their regulatory roles in host-pathogen interactions and disease progression. METHODS: We conducted a cross-sectional study recruiting subjects diagnosed with active-TB (drug-sensitive and drug-resistant) and healthy controls. Sera samples were collected and utilized for preparing small RNA libraries. Quantitative patterns of circulating sncRNAs (miRNAs, piRNAs and tRFs) were identified via high-throughput sequencing and DeSeq2 analysis and validated in independent active-TB cohorts. Functional knockdown for two selected miRNAs were also performed. RESULTS: A diagnostic signature of four sncRNAs for both drug-sensitive and drug-resistant active-TB cases was validated, exhibiting an AUC of 0.96 (95% CI: 0.937-0.996, p < 0.001) with 86.7% sensitivity (95% CI: 0.775-0.932) and 91.7% specificity (95% CI: 0.730-0.990) in ROC analysis. Functional knockdown demonstrated regulatory roles of hsa-miR-223-5p and hsa-miR-10b-5p in Mycobacterium tuberculosis (Mtb) growth and pro-inflammatory cytokine expression (IL-6 and IL-8). CONCLUSION: The study identified a diagnostic tool utilizing a signature of four sncRNAs with high specificity and sensitivity, enhancing our understanding of sncRNAs as ATB diagnostic biomarker. Additionally, hsa-miR-223-5p and hsa-miR-10b-5p demonstrated potential roles in Mtb pathogenesis and host-response to infection.

sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

Slings and arrows: sRNAs mediate intragenomic competition.

Bacterial genomes are littered with exogenous: competing DNA elements. Here, Sprenger et al. demonstrate that the Vibrio cholerae prophage VP882 modulates host functions via production of regulatory sRNAs to promote phage development. Alternatively, host sRNAs inhibit the VP882 lytic phase by specifically regulating phage genes.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Novel non-invasive molecular signatures for oral cavity cancer, by whole transcriptome and small non-coding RNA sequencing analyses: Predicted association with PI3K/AKT/mTOR pathway.

INTRODUCTION: Identification of molecular biomarkers in the saliva and serum of oral cavity cancer patients represents a first step in the development of essential and efficient clinical tools for early detection and post-treatment monitoring. We hypothesized that molecular analyses of paired saliva and serum samples from an individual would likely yield better results than analyses of either serum or saliva alone. MATERIALS AND METHODS: We performed whole-transcriptome and small non-coding RNA sequencing analyses on 32 samples of saliva and serum collected from the same patients with oral squamous cell carcinoma (OSCC) and healthy controls (HC). RESULTS: We identified 12 novel saliva and serum miRNAs and a panel of unique miRNA and mRNA signatures, significantly differentially expressed in OSCC patients relative to HC (log2 fold change: 2.6-26.8; DE: 0.02-0.000001). We utilized a combined panel of the 10 top-deregulated miRNAs and mRNAs and evaluated their putative diagnostic potential (>87% sensitivity; 100% specificity), recommending seven of them for further validation. We also identified unique saliva and serum miRNAs associated with OSCC and smoking history (OSCC smokers vs. never-smokers or HC: log2 fold change: 22-23; DE: 0.00003-0.000000001). Functional and pathway analyses indicated interactions between the discovered OSCC-related non-invasive miRNAs and mRNAs and their targets, through PI3K/AKT/mTOR signaling. CONCLUSION: Our data support our hypothesis that using paired saliva and serum from the same individuals and deep sequencing analyses can provide unique combined mRNA and miRNA signatures associated with canonical pathways that may have a diagnostic advantage relative to saliva or serum alone and may be useful for clinical testing. We believe this data will contribute to effective preventive care by post-treatment monitoring of patients, as well as suggesting potential targets for therapeutic approaches.

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.

Transcriptional activation of the Mycobacterium tuberculosis virulence-associated small RNA MTS1338 by the response regulators DosR and PhoP.

MTS1338, a distinctive small RNA in pathogenic mycobacteria, plays a crucial role in host-pathogen interactions during infection. Mycobacterial cells encounter heterogeneous stresses in macrophages, which highly upregulate MTS1338. A dormancy regulatory factor DosR regulates the intracellular abundance of MTS1338. Herein, we investigated the interplay of DosR and a low pH-inducible gene regulator PhoP binding to the MTS1338 promoter. We identified that DosR strongly binds to two regions upstream of the MTS1338 gene. The proximal region possesses a threefold higher affinity than the distal site, but the presence of both regions increased the affinity for DosR by > 10-fold. PhoP did not bind to the MTS1338 gene but binds to the DosR-bound MTS1338 gene, suggesting a concerted mechanism for MTS1338 expression.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Small RNAs direct attack and defense mechanisms in a quorum sensing phage and its host.

Many, if not all, bacteria use quorum sensing (QS) to control collective behaviors, and more recently, QS has also been discovered in bacteriophages (phages). Phages can produce communication molecules of their own, or "listen in" on the host's communication processes, to switch between lytic and lysogenic modes of infection. Here, we study the interaction of Vibrio cholerae with the lysogenic phage VP882, which is activated by the QS molecule DPO. We discover that induction of VP882 results in the binding of phage transcripts to the major RNA chaperone Hfq, which in turn outcompetes and downregulates host-encoded small RNAs (sRNAs). VP882 itself also encodes Hfq-binding sRNAs, and we demonstrate that one of these sRNAs, named VpdS, promotes phage replication by regulating host and phage mRNA levels. We further show that host-encoded sRNAs can antagonize phage replication by downregulating phage mRNA expression and thus might be part of the host's phage defense arsenal.

A novel ionizing radiation-induced small RNA, DrsS, promotes the detoxification of reactive oxygen species in Deinococcus radiodurans.

A plethora of gene regulatory mechanisms with eccentric attributes in Deinoccocus radiodurans confer it to possess a distinctive ability to survive under ionizing radiation. Among the many regulatory processes, small RNA (sRNA)-mediated regulation of gene expression is prevalent in bacteria but barely investigated in D. radiodurans. In the current study, we identified a novel sRNA, DrsS, through RNA-seq analysis in D. radiodurans cells while exposed to ionizing radiation. Initial sequence analysis for promoter identification revealed that drsS is potentially co-transcribed with sodA and dr_1280 from a single operon. Elimination of the drsS allele in D. radiodurans chromosome resulted in an impaired growth phenotype under γ-radiation. DrsS has also been found to be upregulated under oxidative and genotoxic stresses. Deletion of the drsS gene resulted in the depletion of intracellular concentration of both Mn2+ and Fe2+ by ~70% and 40%, respectively, with a concomitant increase in carbonylation of intracellular protein. Complementation of drsS gene in ΔdrsS cells helped revert its intracellular Mn2+ and Fe2+ concentration and alleviated carbonylation of intracellular proteins. Cells with deleted drsS gene exhibited higher sensitivity to oxidative stress than wild-type cells. Extrachromosomally expressed drsS in ΔdrsS cells retrieved its oxidative stress resistance properties by catalase-mediated detoxification of reactive oxygen species (ROS). In vitro binding assays indicated that DsrS directly interacts with the coding region of the katA transcript, thus possibly protecting it from cellular endonucleases in vivo. This study identified a novel small RNA DrsS and investigated its function under oxidative stress in D. radiodurans. IMPORTANCE: Deinococcus radiodurans possesses an idiosyncratic quality to survive under extreme ionizing radiation and, thus, has evolved with diverse mechanisms which promote the mending of intracellular damages caused by ionizing radiation. As sRNAs play a pivotal role in modulating gene expression to adapt to altered conditions and have been delineated to participate in almost all physiological processes, understanding the regulatory mechanism of sRNAs will unearth many pathways that lead to radioresistance in D. radiodurans. In that direction, DrsS has been identified to be a γ-radiation-induced sRNA, which is also induced by oxidative and genotoxic stresses. DrsS appeared to activate catalase under oxidative stress and detoxify intracellular ROS. This sRNA has also been shown to balance intracellular Mn(II) and Fe concentrations protecting intracellular proteins from carbonylation. This novel mechanism of DrsS identified in D. radiodurans adds substantially to our knowledge of how this bacterium exploits sRNA for its survival under stresses.

Iron starvation increases the production of the Pseudomonas aeruginosa RsmY and RsmZ sRNAs in static conditions.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that induces virulence gene expression in response to host-mediated iron starvation. Recently, our laboratory showed that some virulence factors are responsive to iron limitation in static but not shaking growth conditions. One of these is the HSI-2-type six secretion system (T6SS), which is also induced during chronic infection. Iron regulation of T6SS was partially impacted by the iron-responsive PrrF sRNA and completely dependent upon the Pseudomonas quinolone signal (PQS) biosynthetic gene pqsA. Here, we analyzed the impact of iron on the expression of two small regulatory RNAs (sRNAs), RsmY and RsmZ, that activate the expression of T6SS by sequestering the RsmA translation inhibitor. Our results demonstrate that iron starvation induces the expression of RsmY and RsmZ in static but not shaking cultures. We further show that this induction occurs through the rsmY and rsmZ promoters and is dependent upon PqsA. Disruption of either the pqsR gene also eliminated iron-dependent regulation of rsmY and rsmZ promoter activity. Taken together, our results show novel targets of iron regulation that are specific to static growth, highlighting the importance of studying regulatory mechanisms in static communities that may be more representative of growth during chronic infection.IMPORTANCEIron is a central component of various bacterial metabolic pathways making it an important host-acquired nutrient for pathogens to establish infection. Previous iron regulatory studies primarily relied on shaking bacterial cultures; while these ensure cultural homogeneity, they do not reflect growth conditions during infection. We recently showed that static growth of Pseudomonas aeruginosa promotes iron-dependent regulation of a type six secretion system (T6SS), a virulence factor that is induced during chronic infections. In the current study, we found that static growth also promotes iron-dependent regulation of the RsmY and RsmZ sRNAs, which are global regulators that affect T6SS during chronic P. aeruginosa lung infection. Hence, our work demonstrates the Rsm sRNAs as potential effectors of iron regulation during static growth that may also be relevant in chronic infection.

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.