An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection.

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

Identification of an RNA sponge that controls the RoxS riboregulator of central metabolism in Bacillus subtilis.

sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.

A LysR-type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens.

Small RNAs (sRNAs) are universal posttranscriptional regulators of gene expression and hundreds of sRNAs are frequently found in each and every bacterium. In order to coordinate cellular processes in response to ambient conditions, many sRNAs are differentially expressed. Here, we asked how these small regulators are regulated using Agrobacterium tumefaciens as a model system. Among the best-studied sRNAs in this plant pathogen are AbcR1 regulating numerous ABC transporters and PmaR, a regulator of peptidoglycan biosynthesis, motility, and ampicillin resistance. We report that the LysR-type regulator VtlR (also known as LsrB) controls expression of AbcR1 and PmaR. A vtlR/lsrB deletion strain showed growth defects, was sensitive to antibiotics and severely compromised in plant tumor formation. Transcriptome profiling by RNA-sequencing revealed more than 1,200 genes with altered expression in the mutant. Consistent with the function of VtlR/LsrB as regulator of AbcR1, many ABC transporter genes were affected. Interestingly, the transcription factor did not only control the expression of AbcR1 and PmaR. In the mutant, 102 sRNA genes were significantly up- or downregulated. Thus, our study uncovered VtlR/LsrB as the master regulator of numerous sRNAs. Thereby, the transcriptional regulator harnesses the regulatory power of sRNAs to orchestrate the expression of distinct sub-regulons.

Identification and Quantification of Small RNAs.

RNA silencing plays a critical role in diverse biological processes in plants including growth, development, and responses to abiotic and biotic stresses. RNA silencing is guided by small non-coding RNAs (sRNAs) with the length of 21-24 nucleotides (nt) that are loaded into Argonaute (AGO) to repress expression of target loci and transcripts through transcriptional or posttranscriptional gene silencing mechanisms. Identification and quantitative characterization of sRNAs are crucial steps toward appreciation of their functions in biology. Here, we developed a step-by-step protocol to precisely illustrate the process of cloning of sRNA libraries and correspondingly computational analysis of the recovered sRNAs. This protocol can be used in all kinds of organisms, including Arabidopsis, and is compatible with various high-throughput sequence technologies such as Illumina Hiseq. Thus, we wish that this protocol represents an accurate way to identify and quantify sRNAs in vivo.

The transcriptome of Listeria monocytogenes during co-cultivation with cheese rind bacteria suggests adaptation by induction of ethanolamine and 1,2-propanediol catabolism pathway genes.

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50-90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.

Y RNAs: Biogenesis, Function and Implications for the Cardiovascular System.

In recent years, progress in the field of high-throughput sequencing technology and its application to a wide variety of biological specimens has greatly advanced the discovery and cataloging of a diverse set of non-coding RNAs (ncRNAs) that have been found to have unexpected biological functions. Y RNAs are an emerging class of highly conserved, small ncRNAs. There is a growing number of reports in the literature demonstrating that Y RNAs and their fragments are not just random degradation products but are themselves bioactive molecules. This review will outline what is currently known about Y RNA including biogenesis, structure and functional roles. In addition, we will provide an overview of studies reporting the presence and functions attributed to Y RNAs in the cardiovascular system.

RNA sequencing reveals small RNAs in Bacillus pumilus under different growth phases of the protease fermentation process.

Bacillus pumilus, an endospore-forming soil bacterium, produces a wide array of extracellular proteins, such as proteases, which are already applied in the chemical, detergent and leather industries. Small noncoding regulatory RNAs (sRNAs) in bacteria are important RNA regulators that act in response to various environmental signals. Here, an RNA-seq-based transcriptome analysis was applied to B. pumilus SCU11, a strain that produces extracellular alkaline protease, across various growth phases of the protease fermentation process. Through bioinformatics screening of the sequencing data and visual inspection, 84 putative regulatory sRNAs were identified in B. pumilus, including 21 antisense sRNAs and 63 sRNAs in intergenic regions. We experimentally validated the expression of 48 intergenic sRNAs by quantitative RT-PCR (qRT-PCR). Meanwhile, the expression of 6 novel sRNAs was confirmed by northern blotting, and the expression profiles of 5 sRNAs showed close correlation with the growth phase. We revealed that the sRNA Bpsr137 was involved in flagellum and biofilm formation in B. pumilus. The identification of a global set of sRNAs increases the inventory of regulatory sRNAs in Bacillus and implies the important regulatory roles of sRNA in B. pumilus. These findings will contribute another dimension to the optimization of crucial metabolic activities of B. pumilus during a productive fermentation process.

Regulatory RNA at the crossroads of carbon and nitrogen metabolism in photosynthetic cyanobacteria.

Cyanobacteria are photosynthetic bacteria that populate widely different habitats. Accordingly, cyanobacteria exhibit a wide spectrum of lifestyles, physiologies, and morphologies and possess genome sizes and gene numbers which may vary by up to a factor of ten within the phylum. Consequently, large differences exist between individual species in the size and complexity of their regulatory networks. Several non-coding RNAs have been identified that play crucial roles in the acclimation responses of cyanobacteria to changes in the environment. Some of these regulatory RNAs are conserved throughout the cyanobacterial phylum, while others exist only in a few taxa. Here we give an overview on characterized regulatory RNAs in cyanobacteria, with a focus on regulators of photosynthesis, carbon and nitrogen metabolism. However, chances are high that these regulators represent just the tip of the iceberg.

Human ribonuclease Dicer ­ structure and functions / Ludzka rybonukleaza Dicer ­ struktura i funkcje biologiczne.

Endoribonuclease III Dicer plays a crucial role in the biogenesis of small regulatory RNAs, such as microRNAs (miRNAs) and small inter­fering RNAs (siRNAs). However, this is not the only role that Dicer plays in cells. For example, it has been shown that Dicer is involved in processing of diverse classes of RNA, including tRNA and snoRNA, cleavage of repeat-element-derived RNAs, and maintenance of genome integrity. Dicer has also been found to participate in the chromosome fragmentation during apoptosis or in the inflammatory processes. More­over, a recent discovery of Dicer-binding passive sites in mRNAs and long non-coding RNAs, and its putative nucleic acid chaperone activity, has pointed out a novel regulatory role of the enzyme. Here we focus on human Dicer and review its structure and function including recent findings on miRNA-independent roles and their impact on cell biology.

Quorum-sensing based small RNA regulation for dynamic and tuneable gene expression.

OBJECTIVES: Developing a dynamic regulation strategy is an essential step in establishing an automatic control system for manipulating metabolic fluxes and cellular behaviors. To broaden the extent of the application, a system that can generally control any gene of interest is demanded. RESULTS: Through characterization and optimization, the strategy repressed the immediate expression incrementally from 0 to 90% during culturing. Moreover, by changing single base pair in the lux box of the Plux promoter, the degree of repression of the target genomic gene was tuned to a difference of 70%. This strategy is expected to control metabolic flux without disrupting cell growth. CONCLUSIONS: We engineered bacterial small RNA to develop a pathway-independent strategy that can dynamically repress the expression of any gene at the posttranscription level.

High-Throughput Sequencing Analysis of Small RNAs Derived from Coleus Blumei Viroids.

Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid-host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5'-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.

Neuronal Small RNAs Control Behavior Transgenerationally.

It is unknown whether the activity of the nervous system can be inherited. In Caenorhabditis elegans nematodes, parental responses can transmit heritable small RNAs that regulate gene expression transgenerationally. In this study, we show that a neuronal process can impact the next generations. Neurons-specific synthesis of RDE-4-dependent small RNAs regulates germline amplified endogenous small interfering RNAs (siRNAs) and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among the targets of these small RNAs, we identified the conserved gene saeg-2, which is transgenerationally downregulated in the germline. Silencing of saeg-2 following neuronal small RNA biogenesis is required for chemotaxis under stress. Thus, we propose a small-RNA-based mechanism for communication of neuronal processes transgenerationally.

Adolescent Alcohol Exposure Epigenetically Suppresses Amygdala Arc Enhancer RNA Expression to Confer Adult Anxiety Susceptibility.

BACKGROUND: Adolescent intermittent ethanol (AIE) exposure is an emerging risk factor for adult psychopathology, such as anxiety disorders. Enhancer RNAs (eRNAs) are short noncoding RNAs transcribed from enhancer regions that regulate synaptic plasticity-associated gene expression, including Arc, but their role in AIE-induced susceptibility to anxiety in adulthood is unknown. METHODS: Rats were exposed to AIE (ethanol exposure 2 days on/off) or intermittent normal saline during postnatal days 28 to 41 and allowed to grow to adulthood for analysis of behavior and biochemical measures. Some AIE rats and rats with intermittent normal saline exposure were exposed to an acute challenge with ethanol in adulthood. Cohorts of alcohol-naïve adult rats were cannulated in the central nucleus of amygdala and infused with either Kdm6b small interfering RNA or an antisense locked nucleic acid oligonucleotide specific to Arc eRNA before behavioral and biochemical analysis. RESULTS: AIE adult rats displayed heightened anxiety and decreased Arc eRNA expression, which is regulated epigenetically through decreased Kdm6b expression. This triggered condensed chromatin at the synaptic activity response element site and promoter of the Arc gene, facilitating increased negative elongation factor binding to the Arc promoter and decreasing Arc expression in the amygdala. Knockdown of Kdm6b or Arc eRNA expression in the central nucleus of amygdala provoked anxiety in alcohol-naïve adult rats and recapitulated the molecular and epigenetic phenotypes of AIE. CONCLUSIONS: These data suggest that eRNA regulation via epigenetic reprogramming in the amygdala, particularly at the Arc synaptic activity response element site, contributes to adult anxiety after adolescent alcohol exposure.

Small non-coding RNA expression in mouse nephrogenic mesenchymal progenitors.

MicroRNAs (miRNAs) are small non-coding RNAs that are essential for the regulation of gene expression and play critical roles in human health and disease. Here we present comprehensive miRNA profiling data for mouse nephrogenic mesenchymal progenitors, a population of cells enriched for nephron progenitors that give rise to most cell-types of the nephron, the functional unit of the kidney. We describe a miRNA expression in nephrogenic mesenchymal progenitors, with 162 miRNAs differentially expressed in progenitors when compared to whole kidney. We also annotated 49 novel miRNAs in the developing kidney and experimentally validated 4 of them. Our data are available as a public resource, so that it can be integrated into future studies and analyzed in the context of other functional and epigenomic data in kidney development. Specifically, it will be useful in the effort to shed light on molecular mechanisms underlying processes essential for normal kidney development, like nephron progenitor specification, self-renewal and differentiation.

Small noncoding RNA profiles along alternative developmental trajectories in an annual killifish.

Embryonic development of Austrofundulus limnaeus can occur along two phenotypic trajectories that are physiologically and biochemically distinct. Phenotype appears to be influenced by maternal provisioning based on the observation that young females produce predominately non-diapausing embryos and older females produce mostly diapausing embryos. Embryonic incubation temperature can override this pattern and alter trajectory. We hypothesized that temperature-induced phenotypic plasticity may be regulated by post-transcriptional modification via noncoding RNAs. As a first step to exploring this possibility, RNA-seq was used to generate transcriptomic profiles of small noncoding RNAs in embryos developing along the two alternative trajectories. We find distinct profiles of mature sequences belonging to the miR-10 family expressed in increasing abundance during development and mature sequences of miR-430 that follow the opposite pattern. Furthermore, miR-430 sequences are enriched in escape trajectory embryos. MiR-430 family members are known to target maternally provisioned mRNAs in zebrafish and may operate similarly in A. limnaeus in the context of normal development, and also by targeting trajectory-specific mRNAs. This expression pattern and function for miR-430 presents a potentially novel model for maternal-embryonic conflict in gene regulation that provides the embryo the ability to override maternal programming in the face of altered environmental conditions.

Next-generation sequencing reveals differentially expressed small noncoding RNAs in uterine leiomyoma.

OBJECTIVE: To determine the expression profile of small noncoding RNAs (sncRNAs) in leiomyoma, which has not been investigated to date. DESIGN: Laboratory-based investigation. SETTING: Academic center. PATIENT(S): Women undergoing hysterectomy for benign indications. INTERVENTION(S): Next-generation sequencing and screening of an sncRNA database with confirmatory analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). MAIN OUTCOME MEASURE(S): Expression profile of sncRNAs in leiomyoma and matched myometrium. RESULT(S): Screening our previously determined RNA sequencing data with the sncRNA database resulted in identification of 15 small nuclear (sn) RNAs, 284 small nucleolar (sno) RNAs, 98 Piwi-interacting (pi) RNAs, 152 transfer (t) RNAs, and 45 ribosomal (r) RNAs, of which 15 snoRNAs, 24 piRNAs, 7 tRNAs, and 6 rRNAs were differentially expressed at a 1.5-fold change cutoff in leiomyoma compared with myometrium. We selected 5 snoRNAs, 4 piRNAs, 1 tRNA, and 1 rRNA that were differentially expressed and confirmed their expression in paired tissues (n = 20) from both phases of the menstrual cycle with the use of qRT-PCR. The results indicated up-regulation of the snoRNAs (SNORD30, SNORD27, SNORA16A, SNORD46, and SNORD56) and down-regulation of the piRNAs (piR-1311, piR-16677, piR-20365, piR-4153), tRNA (TRG-GCC5-1), and rRNA (RNA5SP202) expression in leiomyoma compared with myometrium (P<.05). The pattern of expression of these sncRNAs was similar to RNA sequencing analysis, with no menstrual cycle-dependent differences detected except for SNORD30. Because Argonaute 2 (AGO2) is required for sncRNA-mediated gene silencing, we determined its expression and found greater abundance in leiomyoma. CONCLUSION(S): Our results provide the first evidence for the differential expression of additional classes of sncRNAs and AGO2 in leiomyoma, implicating their roles as a gene regulatory mechanism.

Small non-coding RNAs are altered by short-term sprint interval training in men.

Small non-coding RNAs (ncRNAs) are emerging as important molecules for normal biological processes and are deregulated in disease. Exercise training is a powerful therapeutic strategy that prevents cardiometabolic disease and improves cardiorespiratory fitness and performance. Despite the known systemic health benefits of exercise training, the underlying molecular mechanisms are incompletely understood. Recent evidence suggests a role for epigenetic mechanisms, such as microRNAs, but whether other small ncRNAs are modulated by chronic exercise training is unknown. Here, we used small RNA sequencing to explore whether sprint interval training (SIT) controls the abundance of circulating small ncRNAs in human whole blood samples. Ten healthy men performed SIT three times a week for 6 weeks. After training, subjects showed marked improvements in maximal oxygen consumption and cycling performance with concurrent changes to the abundance of diverse species of circulating small ncRNAs (n = 1266 small ncRNAs, n = 13 microRNAs, q < 0.05). Twelve microRNAs altered by 6 weeks of SIT were ubiquitously expressed microRNAs and two regulated important signaling pathways, including p53, thyroid hormone and cell cycle signaling. MicroRNAs altered by 6 weeks of SIT were unchanged after a single session of SIT (n = 24, all P > 0.05). Relative to older individuals, younger subjects exhibited an increased acute SIT-induced fold change in miR-1301-3p (P = 0.02) - a microRNA predicted to target mRNAs involved in alternative splicing, phosphoprotein and chromosomal rearrangement processes (all P < 0.001). Our findings indicate many species of circulating small ncRNAs are modulated by exercise training and that they could control signaling pathways responsible for health benefits achieved from exercise.

[Biogenesis of small non-coding RNAs in animals]. / La biogenèse des ARN courts non codants chez les animaux.

In recent years, the discovery of small non-coding RNAs has opened an all new field in molecular biology. Indeed, these non-coding sequences give rise to powerful regulators of gene expression. Nowadays, different types of small non-coding RNAs have been described. Of these, the best-characterized types are microRNAs, piRNAs (Piwi-interacting RNAs) and siRNAs (small interfering RNAs). Because of their fine-tuning important function in the regulation of gene and genome expression, an aberrant expression level of those small non-coding RNAs are associated to several pathologies. While this new research field is attracting attention, many aspects to be discovered. This review focuses on the biogenesis pathways of microRNAs, piRNAs and siRNAs in animals.

Transfer RNA-derived small RNAs in plants.

Rather than random degradation products, the 18 to 40 nucleotides (nt) transfer RNA-derived small RNAs (tsRNAs) are RNA species generated specifically from pre-RNAs or mature tRNAs in archaea, bacteria and eukaryotes. Recent studies from animal systems have shown that tsRNAs are important non-coding RNAs that regulate gene expression at the transcriptional and/or post-transcriptional levels. They are involved in various biological processes, such as cell proliferation, tumor genesis, stress response and intergenerational epigenetic inheritance. In this review, we will summarize the discovery, biogenesis, and function of tsRNAs in higher plants. In addition, analysis on tsRNAs from lower plants is shown.

Global transcriptional landscape and promoter mapping of the gut commensal Bifidobacterium breve UCC2003.

BACKGROUND: Bifidobacterium breve represents a common member of the infant gut microbiota and its presence in the gut has been associated with host well being. For this reason it is relevant to investigate and understand the molecular mechanisms underlying the establishment, persistence and activities of this gut commensal in the host environment. RESULTS: The assessment of vegetative promoters in the bifidobacterial prototype Bifidobacterium breve UCC2003 was performed employing a combination of RNA tiling array analysis and cDNA sequencing. Canonical -10 (TATAAT) and -35 (TTGACA) sequences were identified upstream of transcribed genes or operons, where deviations from this consensus correspond to transcription level variations. A Random Forest analysis assigned the -10 region of B. breve promoters as the element most impacting on the level of transcription, followed by the spacer length and the 5'-UTR length of transcripts. Furthermore, our transcriptome study also identified rho-independent termination as the most common and effective termination signal of highly and moderately transcribed operons in B. breve. CONCLUSION: The present study allowed us to identify genes and operons that are actively transcribed in this organism during logarithmic growth, and link promoter elements with levels of transcription of essential genes in this organism. As homologs of many of our identified genes are present across the whole genus Bifidobacterium, our dataset constitutes a transcriptomic reference to be used for future investigations of gene expression in members of this genus.