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Summary Whereas about 70 small non-coding RNAs have been found in the Escherichia coli genome, relatively little is known about regulatory RNAs from Gram-positive bacteria. Here, we demonstrate that the recently iden- tified small untranslated RNA SR1 from the Bacillus subtilis genome is a regulatory RNA involved in fine- tuning of arginine catabolism. 2D protein gel electro- phoresis indicated three possible SR1 targets that are regulated by the transcriptional activator AhrC, which was shown to be the primary target of SR1. In vitro pairing studies and an in vivo reporter gene test dem- onstrated a specific interaction between SR1 and ahrC mRNA. This interaction did not lead to degrada- tion of ahrC mRNA, but inhibited translation at a post- initiation stage. Our data show that the Hfq chaperone was not required for the stabilization of SR1 in vivo. The amount of SR1 was increased upon addition of L-arginine and L-ornithine, but not L-citrulline or L-proline.

The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo. Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system. A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity. However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic-binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium. The altered regulation of oppA and dppA was also demonstrated using oppA-phoA and dppA-lacZ gene fusions. Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

Numerous small untranslated RNAs (sRNAs) have been identified in Escherichia coli in recent years, and their roles are gradually being defined. However, few of these sRNAs appear to be conserved in Vibrio cholerae , and both identification and characterization of sRNAs in V. cholerae remain at a preliminary stage. We have characterized one of the few sRNAs conserved between E. coli and V. cholerae : RyhB. Sequence conservation is limited to the central region of the gene, and RyhB in V. cholerae is significantly larger than in E. coli . As in E. coli , V. cholerae RyhB is regulated by the iron-dependent repressor Fur, and it interacts with the RNA-binding protein Hfq. The regulons controlled by RyhB in V. cholerae and E. coli appear to differ, although some overlap is evident. Analysis of gene expression in V. cholerae in the absence of RyhB suggests that the role of this sRNA is not limited to control of iron utilization. Quantitation of RyhB expression in the suckling mouse intestine suggests that iron availability is not limiting in this environment, and RyhB is not required for colonization of this mammalian host by V. cholerae .

Toxin-antitoxin (TA) modules are pairs of genes in which one member encodes a toxin that is neutralized or whose synthesis is prevented by the action of the product of the second gene, an antitoxin, which is either protein or RNA. We now report the identification of a TA module in the chromosome of Bacillus subtilis in which the antitoxin is an antisense RNA. The antitoxin, which is called RatA (for RNA antitoxin A), is a small (222 nucleotides), untranslated RNA that blocks the accumulation of the mRNA for a toxic peptide TxpA (for toxic peptide A; formerly YqdB). The txpA and ratA genes are in convergent orientation and overlap by ca. 75 nucleotides, such that the 3′ region of ratA is complementary to the 3′ region of txpA . Deletion of ratA led to increased levels of txpA mRNA and lysis of the cells. Overexpression of txpA also caused cell lysis and death, a phenotype that was prevented by simultaneous overexpression of ratA . We propose that the ratA transcript is an antisense RNA that anneals to the 3′ end of the txpA mRNA, thereby triggering its degradation.

The tsRNAs (tRNA-derived small RNAs) are new types of small noncoding RNAs derived from tRNAs. Gliomas are well-known malignant brain tumors. The study focused on tsRNA characterizations within gliomas. Datasets processing, bioinformatics analyses, and visualizations were performed with the packages of Python and R. Cell proliferations were demonstrated via CCK8 assays and colony formation assays, and in vivo xenograft experiments. Dual-luciferase reporter assay was performed to confirm the binding of tsRNA with its targets. Via using bioinformatics approaches, the hundreds of tsRNAs with available expression abundance were identified in gliomas dataset, most of them derived from D-loop or T-loop fragments of tRNAs. Among tsRNAs derived from tRNA-Cys-GCA, tRFdb-3003a and tRFdb-3003b (tRFdb-3003a/b) were remarkably down-regulated in gliomas. The survival outcome of gliomas patients with low tRFdb-3003a/b expressions was notably worse than that of high-expression patients. In glioma cells, tRFdb-3003a could suppress cells proliferation and colony formation ability. In vivo, tRFdb-3003a suppressed the tumor growth of xenograft gliomas. Enrichment analyses displayed the tRFdb-3003a-related mRNAs were enriched in the specific GO terms, spliceosome and autophagy pathways, and three GSEA molecular signatures. Mechanically, 3’-UTR regions of VAV2 mRNA were predicted to contain the binding positions of tRFdb-3003a/b, tRFdb-3003a and tRFdb-3003b was effective to reduce the relative luciferase activity of cells with VAV2 wild-type reporter. Overexpression of tRFdb-3003a/b could down-regulated the expression levels of VAV2 protein and mRNA in glioma cells. The tRNA-Cys-GCA derived tRFdb-3003a and tRFdb-3003b might act as key player in tumor progressions of gliomas; tRFdb-3003a/b might directly bind to VAV2 and regulate VAV2 expressions in gliomas.

The prevalence of a high-energy diet and a sedentary lifestyle has increased the incidence of type 2 diabetes (T2D). T2D is a chronic disease characterized by high blood glucose levels and insulin resistance in peripheral tissues. The pathological mechanism of this disease is not fully clear. Accumulated evidence has shown that noncoding RNAs have an essential regulatory role in the progression of diabetes and its complications. The roles of small noncoding RNAs, such as miRNAs, in T2D, have been extensively investigated, while the function of long noncoding RNAs (lncRNAs) in T2D has been unstudied. It has been reported that lncRNAs in T2D play roles in the regulation of pancreatic function, peripheral glucose homeostasis and vascular inflammation. In addition, lncRNAs carried by small extracellular vesicles (sEV) were shown to mediate communication between organs and participate in diabetes progression. Some sEV lncRNAs derived from stem cells are being developed as potential therapeutic agents for diabetic complications. In this review, we summarize the current knowledge relating to lncRNA biogenesis, the mechanisms of lncRNA sorting into sEV and the regulatory roles of lncRNAs and sEV lncRNAs in diabetes. Knowledge of lncRNAs and sEV lncRNAs in diabetes will aid in the development of new therapeutic drugs for T2D in the future.

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Teg49 is a Staphylococcus aureus trans-acting regulatory sRNA derived from cleavage of the sarA P3 transcript. We showed by RNA-Seq here that the 5' trident-like structure in Teg49 regulates transcriptionally (direct and indirect) 22 genes distinct from sarA. Among these, Teg49 was noted to repress spn, encoding a 102 residue preprotein which yields the mature 73 residue peptide which inhibits the catalytic activity of myeloperoxidase in human neutrophils. Teg49 was found to regulate spn mRNA post-transcriptionally in strain SH1000 through 9-nt base-pairing between hairpin loop 2 of Teg49 and an exposed bulge of the spn mRNA. Mutations of the Teg49 binding site disrupted the repression of spn, leading to reduced degradation, and increased half-life of spn mRNA in the Teg49 mutant. The spn-Teg49 interaction was also confirmed with a synonymous spn mutation to yield enhanced spn expression in the mutant vs. the parent. The Teg49 mutant with increased spn expression exhibited enhanced resistance to MPO activity in vitro. Killing assays with human neutrophils showed that the Teg49 mutant was more resistant to killing after phagocytosis. Altogether, this study shows that Teg49 in S. aureus has a distinct and important regulatory profile whereby this sRNA modulates resistance to myeloperoxidase-mediated killing by human neutrophils.

Staphylococcus aureus is a Gram-positive commensal that can also cause a variety of infections in humans. S. aureus virulence factor gene expression is under tight control by a complex regulatory network, which includes, sigma factors, sRNAs, and two-component systems (TCS). Previous work in our laboratory demonstrated that overexpression of the sRNA tsr37 leads to an increase in bacterial aggregation. Here, we demonstrate that the clumping phenotype is dependent on a previously unannotated 88 amino acid protein encoded within the tsr37 sRNA transcript (which we named ScrA for S. aureus clumping regulator A). To investigate the mechanism of action of ScrA we performed proteomics and transcriptomics in a ScrA overexpressing strain and show that a number of surface adhesins are upregulated, while secreted proteases are downregulated. Results also showed upregulation of the SaeRS TCS, suggesting that ScrA is influencing SaeRS activity. Overexpression of ScrA in a saeR mutant abrogates the clumping phenotype confirming that ScrA functions via the Sae system. Finally, we identified the ArlRS TCS as a positive regulator of scrA expression. Collectively, our results show that ScrA is an activator of the SaeRS system and suggests that ScrA may act as an intermediary between the ArlRS and SaeRS systems.

Staphylococcus aureus RsaG is a 3′-untranslated region (3′UTR) derived sRNA from the conserved uhpT gene encoding a glucose-6-phosphate (G6P) transporter expressed in response to extracellular G6P. The transcript uhpT-RsaG undergoes degradation from 5′- to 3′-end by the action of the exoribonucleases J1/J2, which are blocked by a stable hairpin structure at the 5′-end of RsaG, leading to its accumulation. RsaG together with uhpT is induced when bacteria are internalized into host cells or in the presence of mucus-secreting cells. Using MS2-affinity purification coupled with RNA sequencing, several RNAs were identified as targets including mRNAs encoding the transcriptional factors Rex, CcpA, SarA, and the sRNA RsaI. Our data suggested that RsaG contributes to the control of redox homeostasis and adjusts metabolism to changing environmental conditions. RsaG uses different molecular mechanisms to stabilize, degrade, or repress the translation of its mRNA targets. Although RsaG is conserved only in closely related species, the uhpT 3′UTR of the ape pathogen S. simiae harbors an sRNA, whose sequence is highly different, and which does not respond to G6P levels. Our results hypothesized that the 3′UTRs from UhpT transporter encoding mRNAs could have rapidly evolved to enable adaptation to host niches., This work was supported by the Centre National de la Recherche Scientifique (CNRS), by the French National Research Agency ANR (ANR-18-CE12-0025-04 CoNoCo to P.R.). This work of the Interdisciplinary Thematic Institute IMCBio, as part of the ITI 2021–2028 program of the University of Strasbourg, CNRS, and Inserm was supported by IdEx Unistra (ANR-10-IDEX-0002), SFRI-STRAT'US (ANR 20-SFRI-0012), and by EUR IMCBio (IMCBio ANR-17-EURE-0023) under the framework of the French Investments for the Future Program. ED and LB were supported by the “Fondation pour la Recherche Médicale” (FDT201904007957 and ECO202006011534)

In many bacteria, the stabilities and functions of small regulatory RNAs (sRNAs) that act by base pairing with target RNAs most often are dependent on Hfq or ProQ/FinO-domain proteins, two classes of RNA chaperone proteins. However, while all bacteria appear to have sRNAs, many have neither Hfq nor ProQ/FinO-domain proteins raising the question of whether another factor might act as an sRNA chaperone in these organisms. Several recent studies have reported that KH domain proteins, such as KhpA and KhpB, bind sRNAs. Here we describe what is known about the distribution, structures, RNA-binding properties, and physiologic roles of KhpA and KhpB and discuss evidence for and against these proteins serving as sRNAs chaperones.

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored.We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons invivo. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5'-tRHs compose the great majority of tRNA-derived fragments observed invitro, a shift to 3'-tRNAs is observed in mature axons invivo.The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication. ispartof: RNA BIOLOGY vol:18 issue:sup2 pages:832-855 ispartof: location:United States status: published

Biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled by intricate post-transcriptional networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthtase in Escherichia coli, but when bound by RapZ protein, the sRNA becomes inactivated through cleavage by the endoribonuclease RNase E. Here, we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the RapZ tetrameric quaternary structure to structural repeats in the sRNA. The nucleic acid is contacted by RapZ mostly through a highly conserved domain that shares an evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a precleavage intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised by the enzyme. The structures provide a framework for understanding how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors may be recognised for processing by RNase E., Wellcome Trust

The group A Streptococcus (GAS; Streptococcus pyogenes) causes an elaborate array of human diseases. In part, such variability in disease potential is a consequence of GAS manipulating the expression of a catalogue of virulence factors, with regulation occurring at both the transcriptional and posttranscriptional levels. The GAS small regulatory RNA (sRNA) FasX contributes to this regulatory activity, enhancing expression of the thrombolytic agent streptokinase, and reducing expression of collagen (pili) and fibronectin (PrtF1 and PrtF2) -binding adhesins. Here, we expand insight into the regulatory targets of FasX by identifying the M-related protein (Mrp), a fibrinogen-binding adhesin with anti-phagocytic activity, as a negatively-regulated target of FasX. Importantly, investigation of the consequences of FasX-mediated regulation led to the discovery that FasX is a major positive regulator of GAS survival and proliferation in non-immune whole human blood, with a 30-fold difference in GAS cell numbers between a fasX mutant strain and isogenic parental and complemented mutant strains. No difference in cell numbers were observed when these strains were grown in human serum, consistent with the protective phenotype associated with FasX occurring due to the inhibition of cell (e.g., neutrophil) – mediated GAS killing. The FasX-regulated factor/s responsible for the blood survival phenotype remain to be defined. In summary, we expand the known FasX regulon and identify a new phenotype associated with the regulatory activity of this key GAS sRNA. IMPORTANCE Small regulatory RNAs (sRNAs) represent a major class of regulatory molecule that promotes the ability of the group A Streptococcus (GAS) and other pathogens to regulate virulence factor expression. Despite FasX being the best-described sRNA in GAS, there remains much to be learned. Here, we highlight the importance of FasX, identifying for the first time that the loss of this sRNA results in a major reduction in the ability of GAS to survive in human blood, a phenotype critical to the ability of this human-specific pathogen to cause severe invasive infections. We also identified a novel regulatory target of FasX, thereby expanding the known regulon of this key sRNA.

Enterohemorrhagic Escherichia coli (EHEC) causes severe human diseases worldwide. The type three secretion system and effector proteins are essential for EHEC infection, and are encoded by the locus of enterocyte effacement (LEE). RNA-binding protein Hfq is essential for small regulatory RNA (sRNA)-mediated regulation at a post-transcriptional level and full virulence of many pathogenic bacteria. Although two early studies indicated that Hfq represses LEE expression by post-transcriptionally controlling the expression of genes grlRA and/or ler, both of which encode LEE regulators mediating a positive regulatory loop, the detailed molecular mechanism and biological significance remain unclear. Herein, we show that LEE overexpression was caused by defective RNA-binding activity of the Hfq distal face, which post-transcriptionally represses grlA and ler expression. In vitro analyses revealed that the Hfq distal face directly binds near the translational initiation site of grlA and ler mRNAs, and inhibits their translation. Taken together, we conclude that Hfq inhibits grlA and ler translation by binding their mRNAs through the distal face in an sRNA-independent manner. Additionally, we show that Hfq-mediated repression of LEE is critical for normal EHEC growth because all suppressor mutations that restored the growth defect in the hfq mutant abolished hfq deletion-induced overexpression of LEE.

Bacteria employ noncoding RNA molecules for a wide range of biological processes, including scaffolding large molecular complexes, catalyzing chemical reactions, defending against phages, and controlling gene expression. Secondary structures, binding partners, and molecular mechanisms have been determined for numerous small noncoding RNAs (sRNAs) in model aerobic bacteria. However, technical hurdles have largely prevented analogous analyses in the anaerobic gut microbiota. While experimental techniques are being developed to investigate the sRNAs of gut commensals, computational tools and comparative genomics can provide immediate functional insight. Here, using Bacteroides thetaiotaomicron as a representative microbiota member, we illustrate how comparative genomics improves our understanding of RNA biology in an understudied gut bacterium. We investigate putative RNA-binding proteins and predict a Bacteroides cold-shock protein homolog to have an RNA-related function. We apply an in silico protocol incorporating both sequence and structural analysis to determine the consensus structures and conservation of nine Bacteroides noncoding RNA families. Using structure probing, we validate and refine these predictions and deposit them in the Rfam database. Through synteny analyses, we illustrate how genomic coconservation can serve as a predictor of sRNA function. Altogether, this work showcases the power of RNA informatics for investigating the RNA biology of anaerobic microbiota members.

The carbon storage regulator system and base-pairing small RNAs (sRNAs) represent two predominant modes of bacterial post-transcriptional regulation, which globally influence gene expression. Binding of CsrA protein to the 5' UTR or initial mRNA coding sequences can affect translation, RNA stability, and/or transcript elongation. Base-pairing sRNAs also regulate these processes, often requiring assistance from the RNA chaperone Hfq. Transcriptomics studies in Escherichia coli have identified many new CsrA targets, including Spot 42 and other base-pairing sRNAs. Spot 42 synthesis is repressed by cAMP-CRP, induced by the presence of glucose, and Spot 42 post-transcriptionally represses operons that facilitate metabolism of nonpreferred carbon sources. CsrA activity is also increased by glucose via effects on CsrA sRNA antagonists, CsrB/C. Here, we elucidate a mechanism wherein CsrA binds to and protects Spot 42 sRNA from RNase E-mediated cleavage. This protection leads to enhanced repression of srlA by Spot 42, a gene required for sorbitol uptake. A second, independent mechanism by which CsrA represses srlA is by binding to and inhibiting translation of srlM mRNA, encoding a transcriptional activator of srlA. Our findings demonstrate a novel means of regulation, by CsrA binding to a sRNA, and indicate that such interactions can help to shape complex bacterial regulatory circuitry.

Background During epididymal transit, spermatozoa go through several functional maturation steps, resulting from interactions with epididymal secretomes specific to each region. In particular, the sperm membrane is under constant remodeling, with sequential attachment and shedding of various molecules provided by the epididymal lumen fluid and epididymosomes, which also deliver sncRNA cargo to sperm. As a result, the payload of sperm sncRNAs changes during the transit from the epididymis caput to the cauda. This work was designed to study the dynamics of cattle sperm sncRNAs from spermatogenesis to final maturation. Results Comprehensive catalogues of sperm sncRNAs were obtained from testicular parenchyma, epididymal caput, corpus and cauda, as well as ejaculated semen from three Holstein bulls. The primary cattle sncRNA sperm content is markedly remodeled as sperm mature along the epididymis. Expression of piRNAs, which are abundant in testis parenchyma, decreases dramatically at epididymis. Conversely, sperm progressively acquires miRNAs, rsRNAs, and tsRNAs along epididymis, with regional specificities. For instance, miRNAs and tsRNAs are enriched in epididymis cauda and ejaculated sperm, while rsRNA expression peaks at epididymis corpus. In addition, epididymis corpus contains mainly 20 nt long piRNAs, instead of 30 nt in all other locations. Beyond the bulk differences in abundance of sncRNAs classes, K-means clustering was performed to study their spatiotemporal expression profile, highlighting differences in specific sncRNAs and providing insights into their putative biological role at each maturation stage. For instance, Gene Ontology analyses using miRNA targets highlighted enriched processes such as cell cycle regulation, response to stress and ubiquitination processes in testicular parenchyma, protein metabolism in epididymal sperm, and embryonic morphogenesis in ejaculated sperm. Conclusions Our findings confirm that the sperm sncRNAome does not simply reflect a legacy of spermatogenesis. Instead, sperm sncRNA expression shows a remarkable level of plasticity resulting probably from the combination of multiple factors such as loss of the cytoplasmic droplet, interaction with epididymosomes, and more surprisingly, the putative in situ production and/or modification of sncRNAs by sperm. Given the suggested role of sncRNA in epigenetic trans-generational inheritance, our detailed spatiotemporal analysis may pave the way for a study of sperm sncRNAs role in embryo development.

Motivation Single-cell/nuclei RNA-sequencing (scRNA-seq) technologies can simultaneously quantify gene expression in thousands of cells across the genome. However, the majority of the noncoding RNAs, such as microRNAs (miRNAs), cannot currently be profiled at the same scale. MiRNAs are a class of small noncoding RNAs and play an important role in gene regulation. MiRNAs originate from the processing of primary transcripts, known as primary-microRNAs (pri-miRNAs). The pri-miRNA transcripts, independent of their cognate miRNAs, can also function as long noncoding RNAs, code for micropeptides or even interact with DNA, acting like enhancers. Therefore, it is apparent that the significance of scRNA-seq pri-miRNA profiling expands beyond using pri-miRNA as proxies of mature miRNAs. However, there are no computational methods that allow profiling and quantification of pri-miRNAs at the single-cell-type resolution. Results We have developed a simple yet effective computational framework to profile pri-MiRNAs from single-cell RNA-sequencing datasets (PPMS). Based on user input, PPMS can profile pri-miRNAs at cell-type resolution. PPMS can be applied to both newly produced and publicly available datasets obtained via single cell or single-nuclei RNA-seq. It allows users to (i) investigate the distribution of pri-miRNAs across cell types and cell states and (ii) establish a relationship between the number of cells/reads sequenced and the detection of pri-miRNAs. Here, to demonstrate its efficacy, we have applied PPMS to publicly available scRNA-seq data generated from (i) individual chambers (ventricles and atria) of the human heart, (ii) human pluripotent stem cells during their differentiation into cardiomyocytes (the heart beating cells) and (iii) hiPSCs-derived cardiomyocytes infected with severe acute respiratory syndrome coronavirus 2.

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3′ ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depended on interactions with Hfq’s flexible C-terminal domain (CTD), being more rapid when the CTD was deleted, and slower when OxyS was bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.

Small regulatory RNAs (sRNAs) are present in almost all investigated microbes, regarded as modulators and regulators of gene expression and also known to play their regulatory role in the environmentally significant process. It has been estimated that less than 1% of the microbes in nature are culturable in the laboratory, hindering our understanding of their physiology, and living strategies. However, recent big advancing of DNA sequencing and omics-related data analysis makes the understanding of the genetics, metabolic potentials, even ecological roles of uncultivated microbes possible. In this study, we used a metagenome and metatranscriptome based integrated approach to identifying small RNAs in the microbiome of Guaymas Basin sediments. Hundreds of environmental sRNAs comprising of 228 groups were identified based on their homology, 82% of which displayed high similarity with previously known small RNAs in Rfam database, whereas, “18%” are putative novel sRNA motifs. A putative cis-acting sRNA potentially binding to methyl coenzyme M reductase, a key enzyme in methanogenesis or anaerobic oxidation of methane (AOM), was discovered in the genome of ANaerobic MEthane oxidizing archaea group 1 (ANME-1), which were the dominate microbe in the sample. These sRNAs were actively expressed in local Guaymas Basin hydrothermal environment, suggesting important roles of sRNAs in regulating microbial activity in natural environments.

The duck represents an important reservoir of influenza viruses for transmission to other avian and mammalian hosts, including humans. The increased pathogenicity of the recently emerging clades of highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype in ducks features systemic viral spread and organ-to-organ variation in viral transcription and tissue damage. We previously reported that experimental infection of Sudani ducks (Cairina moschata) with an Egyptian HPAI (H5N1) virus (clade 2.2.1.2) features high viral replication and severe tissue damage in lung, but lower viral replication and only mild histological changes in brain. Little is known about the involvement of miRNA in organ-specific responses to H5N1 viruses in ducks, and involvement of the other classes of small noncoding RNA (sncRNA) has not been investigated so far. Following RNA sequencing, we have annotated the duck sncRNome and compared global expression changes of the four major sncRNA classes (miRNAs, piRNAs, snoRNAs, snRNAs) between duck lung and brain during a 120 h time course of infection with this HPAI strain. We find major organ-specific differences in miRNA, piRNA and snoRNA populations even before infection and substantial reprogramming of all sncRNA classes throughout infection, which was less pronounced in brain. Pathway prediction analysis of miRNA targets revealed enrichment of inflammation-, infection- and apoptosis-related pathways in lung, but enrichment of metabolism-related pathways (including tryptophan metabolism) in brain. Thus, organ-specific differences in sncRNA responses may contribute to differences in viral replication and organ damage in ducks infected with isolates from this emerging HPAI clade, and likely other strains.

Small nucleolar RNA SNORD50A and SNORD50B (SNORD50A/B) has been reported to be recurrently deleted and function as a putative tumor suppressor in different types of cancer by binding to and suppressing the activity of the KRAS oncoproteins. Its deletion correlates with poorer patient survival. However, in this study, we surprisingly found that SNORD50A/B loss predicted a better survival in breast cancer patients carrying wild-type p53. Functional studies showed that SNORD50A/B deletion strongly inhibited the proliferation, migration, invasion and tumorigenic potential, and induced cell cycle arrest and apoptosis in p53 wild-type breast cancer cells, while exerted the opposite effects in p53 mutated breast cancer cells. This was also supported by ectopically expressing SNORD50A/B in both p53 wild-type and mutated breast cancer cells. Mechanistically, SNORD50A/B clearly enhances the interaction between E3 ubiquitin ligase TRIM21 and its substrate GMPS by forming a complex among them, thereby promoting GMPS ubiquitination and its subsequent cytoplasmic sequestration. SNORD50A/B deletion in p53 wild-type breast cancer cells will release GMPS and induce the translocation of GMPS into the nucleus, where GMPS can recruit USP7 and form a complex with p53, thereby decreasing p53 ubiquitination, stabilizing p53 proteins, and inhibiting malignant phenotypes of cancer cells. Altogether, the present study first reports that SNORD50A/B plays an oncogenic role in p53 wild-type breast cancers by mediating TRIM21-GMPS interaction.

Noncoding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA–chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq–sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA–protein interactions can be more reliably detected. We find that the concentration of the RNA–DNA adapter is an important parameter in determining the signal in the system and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq–RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein–RNA interactions with the B3H system and will facilitate exploration of the binding mechanisms of these interactions.

Small non-coding RNAs (sncRNAs) play critical roles in multiple regulatory processes, including transcription, post-transcription, and translation. Emerging evidence reveals the critical roles of sncRNAs in cancer development and their potential role as biomarkers and/or therapeutic targets. In this paper, we review recent research on four sncRNA species with functional significance in cancer: small nucleolar RNAs, transfer RNA, small nuclear RNAs, and piwi-interacting RNAs. We introduce their functional roles in tumorigenesis and discuss the potential utility of sncRNAs as prognostic and diagnostic biomarkers and therapeutic targets. We further summarize approaches to characterize sncRNAs in a high-throughput manner, including the specific library construction and computational framework. Our review provides a perspective of the functions, clinical utility, and characterization of sncRNAs in cancer.

DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.

The ten-eleven translocation 2 (TET2) protein, which oxidizes 5-methylcytosine in DNA, can also bind RNA; however, the targets and function of TET2-RNA interactions in vivo are not fully understood. Using stringent affinity tags introduced at the Tet2 locus, we purified and sequenced TET2-crosslinked RNAs from mouse embryonic stem cells (mESCs) and found a high enrichment for tRNAs. RNA immunoprecipitation with an antibody against 5-hydroxymethylcytosine (hm5C) recovered tRNAs that overlapped with those bound to TET2 in cells. Mass spectrometry (MS) analyses revealed that TET2 is necessary and sufficient for the deposition of the hm5C modification on tRNA. Tet2 knockout in mESCs affected the levels of several small noncoding RNAs originating from TET2-bound tRNAs that were enriched by hm5C immunoprecipitation. Thus, our results suggest a new function of TET2 in promoting the conversion of 5-methylcytosine to hm5C on tRNA and regulating the processing or stability of different classes of tRNA fragments.

Enterovirus A71 (EV-A711) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host’s translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1–4) from the EV-A71 5′UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

Although bacterial small noncoding RNAs (sRNAs) are known to play a critical role in various cellular processes, including pathogenesis, the identity and action of such sRNAs are still poorly understood in many organisms. Here we have performed a genome‐wide screen and functional analysis of the sRNAs in Xanthomonas campestris pv. campestris (Xcc), an important phytopathogen. The 50–500‐nt RNA fragments isolated from the wild‐type strain grown in a virulence gene‐inducing condition were sequenced and a total of 612 sRNA candidates (SRCs) were identified. The majority (82%) of the SRCs were derived from mRNA, rather than specific sRNA genes. A representative panel of 121 SRCs were analysed by northern blotting; 117 SRCs were detected, supporting the contention that the overwhelming majority of the 612 SRCs identified are indeed sRNAs. Phenotypic analysis of strains overexpressing different candidates showed that a particular sRNA, RsmU, acts as a negative regulator of virulence, the hypersensitive response, and cell motility in Xcc. In vitro electrophoretic mobility shift assay and in vivo coimmunoprecipitation analyses indicated that RsmU interacted with the global posttranscriptional regulator RsmA, although sequence analysis displayed that RsmU is not a member of the sRNAs families known to antagonize RsmA. Northern blotting analyses demonstrated that RsmU has two isoforms that are processed from the 3′‐untranslated region of the mRNA of XC1332 predicted to encode ComEA, a periplasmic protein required for DNA uptake in bacteria. This work uncovers an unexpected major sRNA biogenesis strategy in bacteria and a hidden layer of sRNA‐mediated virulence regulation in Xcc., The RsmA‐sequestering noncoding RNA RsmU derived from the 3′‐UTR of XC1332 mRNA negatively regulates virulence, the hypersensitive response, and cell motility in Xanthomonas campestris.

Polyploidy promotes morphological, physiological, and reproductive diversity in plants. The imminent effect of chromosome doubling in plants is the enlargement of organs such as flowers and fruits, which increases the commercial value of crops. Flowering plays a vital role in the growth and development of angiosperms. Here, we prepared an isolated microspore culture of 'FT', a doubled haploid (DH) line of Chinese cabbage (Brassica rapa L. ssp. pekinensis), and obtained diploid and autotetraploid plants with the same genetic background. Compared with diploids, the autotetraploids were characterized by large floral organs, dark petals, delayed flowering, and reduced fertility. The indole-3-acetic acid (IAA) and jasmonic acid (JA) levels in autotetraploid petals were significantly higher and the abscisic acid (ABA) level was significantly lower than those in the diploid petals. The lutein level in autotetraploid petals was nearly two times higher than that in the diploid petals. A comparative transcriptome analysis revealed 14,412 differentially expressed genes (DEGs) between the diploids and autotetraploids, and they were enriched in 117 Gene Ontology terms and 110 Kyoto Encyclopedia of Genes and Genomes pathways. We detected 231 DEGs related to phytohormone signal transduction and 29 DEGs involved in carotenoid biosynthesis. An miRNA-target mRNA analysis showed that 32 DEGs regulated by 16 DEMs were associated with flowering timing (BraA03000336, BraA09004319, and BraA09000515), petal development (BraA05002408, BraA01004006, BraA09004069, and BraA04000966), flower opening (BraA07000350), and pollen development (BraA01000720, BraA09005727, and BraA01000253). This study provides information to help elucidate the molecular mechanisms underlying phenotypic variations induced by autopolyploidy in Chinese cabbage.

Helicobacter pylori (Hp) infection is a major cause of gastrointestinal disease. However, the pathogenesis of gastric mucosa injury by Hp has remained elusive. Small non‑coding RNA (sRNA) is a type of widespread RNA in prokaryotic organisms and regulates bacterial growth, reproduction and virulence. In the present study, Hp sRNA profiles were generated to reveal the sequences and possible functions of sRNA by bioinformatics analysis. The role of sRNA in tinidazole (TNZ) treatment was also explored. Total sRNAs of HP26695 were sequenced using an Illumina HiSeq2000. Detected Tags were then compared with a known sRNA database to build an sRNA profile. Reverse transcription‑quantitative (RT‑q)PCR products were sequenced directly and agarose gel electrophoresis was used to identify NAT‑67 and 5'ureB‑sRNA in HP. Furthermore, HP was treated with TNZ for 6, 12 and 24 h. The bacterial concentration was measured, the expression of NAT‑67, 5'ureB‑sRNA and ceuE was determined by RT‑qPCR and superoxide dismutase (SOD) activity and reactive oxygen species (ROS) production were detected. A total of 163 sRNA tags were predicted in Hp through bioinformatics analysis. Among them, 35 tags were evolutionarily aconserved in different Hp strains. By target prediction, it was indicated that certain candidate sRNAs were associated with bacterial oxidative stress, virulence and chemotaxis. It was also observed that NAT‑67 and 5'ureB‑sRNA were downregulated in TNZ‑treated HP. TNZ treatment inhibited the growth of Hp, which was accompanied by downregulation of ceuE and SOD activity, as well as upregulation of ROS. RNA sequencing and bioinformatics are valuable in predicting the expression profile and function of sRNA in HP. sRNA‑targeted genes may be associated with virulence, oxidative stress and chemokines. Downregulation of NAT‑67 by TNZ may be involved in Hp oxidative stress regulation, which may comprise one of the mechanisms of the antibacterial effects of TNZ.

Background Emerging evidence has shown the involvement of dysregulated transfer RNAs (tRNAs) and small RNAs derived from transfer RNAs (tsRNAs) in the pathophysiology of human diseases. The role of tRNAs and tsRNAs in systemic lupus erythematosus (SLE) remains unclear. Therefore, this study aims to investigate the possible regulatory roles of tRNAs and tsRNAs in the pathological mechanism of SLE. Methods Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 20 SLE patients and 20 normal controls (NCs) to obtain tRNAs and tsRNAs, followed by tRNA and tsRNA expression profiling by the NextSeq system. Target genes were predicted by informatics analysis. Subsequently, to explore the function of messenger RNA (mRNA) in these target genes, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the Cytoscape plug-in BinGo, the DAVID database, and Cytoscape software. Results A total of 101 tRNAs and 355 tsRNAs were found to be differentially expressed in SLE patients versus NCs by RNA microarray. GO analysis revealed that the altered target genes of the selected tRNAs and tsRNAs were most enriched similarly in immune response and the immune system process. Moreover, KEGG pathway analysis demonstrated that altered target genes of tRNAs were most enriched in systemic lupus erythematosus, while the altered target genes of tsRNAs were most enriched in the T cell receptor signalling pathway, Th1 and Th2 cell differentiation and primary immunodeficiency. These pathways may be related to the initiation of SLE. Conclusion Our results provide a novel perspective for studying the tRNA-related and tsRNA-related pathogenesis of SLE.

In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the carboxyl terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic amino terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.

Many bacterial small RNAs (sRNAs) efficiently inhibit translation of target mRNAs by forming a duplex that sequesters the Shine-Dalgarno (SD) sequence or start codon and prevents formation of the translation initiation complex. There are a growing number of examples of sRNA-mRNA binding interactions distant from the SD region, but how these mediate translational regulation remains unclear. Our previous work in Escherichia coli and Salmonella identified a mechanism of translational repression of manY mRNA by the sRNA SgrS through a binding interaction upstream of the manY SD. Here, we report that SgrS forms a duplex with a uridine-rich translation-enhancing element in the manY 5’ untranslated region. Notably, we show that the enhancer is ribosome-dependent and that the small ribosomal subunit protein S1 interacts with the enhancer to promote translation of manY. In collaboration with the chaperone protein Hfq, SgrS interferes with the interaction between the translation enhancer and ribosomal protein S1 to repress translation of manY mRNA. Since bacterial translation is often modulated by enhancer-like elements upstream of the SD, sRNA-mediated enhancer silencing could be a common mode of gene regulation.

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily of Herpesviridae. PRV-induced pseudorabies is a highly contagious disease that has caused huge economic losses to the global swine industry. The PRV gE/gI gene deletion vaccine strain (Fa ΔgE/gI strain) constructed from the PRV Fa wild-type strain was shown to have a protective effect against infection. However, the interaction between PRV gE/gI genes and host miRNA needs further exploration, and little is known about the regulatory mechanisms of non-coding RNAs during PRV infection. miRNAs play a key regulatory role in viral infection and immune responses, so we analyzed the differential expression of miRNAs induced by the PRV Fa ΔgE/gI strain and Fa wild-type strain in the PK15 cell line. High-throughput sequencing reads were aligned to known Sus scrofa pre-miRNAs in the miRBase database. Target genes of differentially expressed miRNAs were predicted using the miRGen 3.0 database, then filtered miRNA target genes were subjected to Gene Ontology (GO) analysis and Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis. Stem-loop quantitative real-time PCR was performed to confirm the accuracy of high-throughput sequencing data. In total, 387, 472, and 490 annotated and novel mature miRNAs were identified from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells, respectively. Five PRV-encoded miRNAs were also identified. GO analysis showed that target genes of differentially expressed miRNAs in PRV Fa ΔgE/gI strain-infected and Fa wild-type strain-infected PK-15 cells were mainly involved in biological regulation and metabolic processes. STRING analysis showed that immune-related target genes of differentially expressed miRNAs in the Toll-like receptor signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-κB signaling pathway, and transforming growth factor-β signaling pathway were interrelated. This is the first report of the small RNA transcriptome in PRV mutant wild-type strain-infected and Fa ΔgE/gI strain-infected porcine cell lines. Our findings will contribute to the prevention and treatment of PRV mutant strains. Electronic supplementary material The online version of this article (10.1007/s11262-020-01760-6) contains supplementary material, which is available to authorized users.

RyeA/SraC is a cis-encoded small RNA (sRNA), which act as an anti-toxin to RpoS-regulated RyeB toxin in Escherichia coli. Ectopic expression of RyeA was reported to diminish the RyeB accumulation by serving as a RNA trap. Lower abundance of RyeA in the early exponential growth phase turned out to be the outcome of its degradation by RNase BN/Z. In the current study, we show that RyeA is an acid stress inducible sRNA, and global stress responsive factor RpoS appeared to be inessential in RyeA induction. Although, ryeB-pphA dicistronic transcript at low pH condition was stimulated by ∼4-fold, however, RyeB population was found to be decreased by > 50% under the same condition by the decoy action of enhanced RyeA accumulation. Investigation of the mechanism of RyeA induceduction at low pH in the exponential phase, revealed that RNase BN/Z, which catabolizes RyeA in the exponential phase, appeared to be highly sensitive to low pH stress. Both mRNA and protein level of RNase BN transpired to be decreased to

Gene silencing mediated by small noncoding RNAs (sRNAs) is a fundamental gene regulation mechanism in eukaryotes that broadly governs cellular processes. It has been established that sRNAs are critical regulators of plant growth, development, and antiviral defence, while accumulating studies support positive roles of sRNAs in plant defence against bacteria and eukaryotic pathogens such as fungi and oomycetes. Emerging evidence suggests that plant sRNAs move between species and function as antimicrobial agents against nonviral parasites. Multiple plant pathosystems have been shown to involve a similar exchange of small RNAs between species. Recent analysis about extracellular sRNAs shed light on the understanding of the selection and transportation of sRNAs moving from plant to parasites. In this review, we summarize current advances regarding the function and regulatory mechanism of plant endogenous small interfering RNAs (siRNAs) in mediating plant defence against pathogen intruders including viruses, bacteria, fungi, oomycetes, and parasitic plants. Beyond that, we propose potential mechanisms behind the sorting of sRNAs moving between species and the idea that engineering siRNA-producing loci could be a useful strategy to improve disease resistance of crops.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3′ stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3′ region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. IMPORTANCE Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.

Extracellular small RNAs (sRNAs) are abundant in many biofluids, but little is known about their mechanisms of transport and stability in RNase-rich environments. We previously reported that high-density lipoproteins (HDLs) in mice were enriched with multiple classes of sRNAs derived from the endogenous transcriptome, but also from exogenous organisms. Here, we show that human HDL transports tRNA-derived sRNAs (tDRs) from host and nonhost species, the profiles of which were found to be altered in human atherosclerosis. We hypothesized that HDL binds to tDRs through apolipoprotein A-I (apoA-I) and that these interactions are conferred by RNA-specific features. We tested this using microscale thermophoresis and electrophoretic mobility shift assays and found that HDL binds to tDRs and other single-stranded sRNAs with strong affinity but did not bind to double-stranded RNA or DNA. Furthermore, we show that natural and synthetic RNA modifications influenced tDR binding to HDL. We demonstrate that reconstituted HDL bound to tDRs only in the presence of apoA-I, and purified apoA-I alone were able to bind sRNA. Conversely, phosphatidylcholine vesicles did not bind tDRs. In summary, we conclude that HDL binds to single-stranded sRNAs likely through nonionic interactions with apoA-I. These results highlight binding properties that likely enable extracellular RNA communication and provide a foundation for future studies to manipulate HDL-sRNA interactions for therapeutic approaches to prevent or treat disease.

Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5′-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.

Objectives. The epithelial-to-mesenchymal transition (EMT) is one key step for the invasion and metastasis of colorectal cancer (CRC). Up until now, the underlying mechanism of EMT in CRC is still unpromising. Thus, it is essential to have a better understanding of its carcinogenesis. The transfer RNA-derived small fragments (tsRNAs) are a new group of small noncoding RNAs (sncRNAs), including tRNA-derived stress-induced RNAs (tiRNAs) and tRNA-derived fragments (tRFs), which have been observed to play an important role in many cancers. However, the relationship between tRFs and EMT in CRC is still unknown. Herein, we aimed to investigate the involvement of tRFs in EMT and its contribution to CRC development. Methods. We identified the differentially expressed tsRNAs in colorectal cancer cell line HT29 treated with TGF-β compared with control cells by using high-throughput sequencing and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). QRT-PCR was conducted to validate the differentially expressed fragments in 68 CRC tumor samples (22 women and 46 men) and adjacent nontumor samples. The association of the expression of tRFs with CRC metastasis and clinical stage was analyzed. Meanwhile, the correlation between tRF expression and overall survival (OS) was also analyzed. TargetScan and miRanda and multiple bioinformatic approaches were used to predict the possible target genes of tsRNAs and analyze possible functions of the tRFs. Results. A series of differentially expressed tsRNAs were identified in TGF-β-treated HT29 cells compared with control cells. tRF-phe-GAA-031 and tRF-VAL-TCA-002 were found to be significantly upregulated in CRC tissues compared to adjacent nontumor tissues. They were significantly correlated with distant metastasis and clinical stage. We compared the differences between tumor samples and nontumor tissues from the ROC curves. The area under the ROC curve (AUC) was up to 0.7554 (95% confidence interval: 0.6739 to 0.8369, p < 0.0001 ) for tRF-Phe-GAA-031 and up to 0.7313 (95% confidence interval: 0.6474 to 0.8151, p < 0.0001 ) for tRF-VAL-TCA-002. For OS analysis, higher tRF-phe-GAA-031 and tRF-VAL-TCA-002 expressions were associated with shorter survival for CRC patients. Conclusion. A series of differentially expressed tsRNAs are identified in the EMT process of CRC. And tRF-phe-GAA-031 and tRF-VAL-TCA-002 are higher expressed in CRC tissues, and they might play an important role in the metastasis of CRC. Meanwhile, they may be potential biomarkers and intervention targets in the clinical treatment of CRC.

In all domains of life, RNA chaperones safeguard and guide the fate of the cellular RNA pool. RNA chaperones comprise structurally diverse proteins that ensure proper folding, stability, and ribonuclease resistance of RNA, and they support regulatory activities mediated by RNA. RNA chaperones constitute a topologically diverse group of proteins that often present an unstructured region and bind RNA with limited nucleotide sequence preferences. In bacteria, three main proteins - Hfq, ProQ, and CsrA - have been shown to regulate numerous complex processes, including bacterial growth, stress response and virulence. Hfq and ProQ have well-studied activities as global chaperones with pleiotropic impact, while CsrA has a chaperone-like role with more defined riboregulatory function. Here, we describe relevant novel insights into their common features, including RNA binding properties, unstructured domains, and interplay with other proteins important to RNA metabolism.

Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30–55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.

Post-transcriptional regulatory networks in Gammaproteobacteria are to a large extent built around the two globally acting RNA-binding proteins (RBPs) CsrA and Hfq. Both RBPs interact with small regulatory RNAs (sRNAs), but the functional outcomes of these interactions are generally distinct. Whereas Hfq both stabilizes sRNAs and promotes their base-pairing to target mRNAs, the sRNAs bound by CsrA act as sequestering molecules that titrate the RBP away from its mRNA targets. In this issue of Molecular Microbiology, Lai et al. reveal that CsrA interacts with the Hfq-associated and base-pairing sRNA Spot 42. In this case, CsrA increases Spot 42 stability by masking a cleavage site for endoribonuclease RNase E, thereby promoting Spot 42-dependent regulation of srlA mRNA. Interestingly, the effect of CsrA on srlA expression is two-fold. In addition to affecting Spot 42-dependent regulation, CsrA directly inhibits translation of SrlM, an activator of srlA transcription. Together, this study reveals a new function for CsrA and indicates more intricate connections between the CsrA and Hfq networks than previously anticipated. Several recent studies have identified additional RBPs that interact with sRNAs. With new RBP identification methods at hand, it will be intriguing to see how many more sRNA-binding proteins will be uncovered.

Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3'UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.