sRNA Molecules Research Articles

The RNA-Binding Chaperone Hfq Is an Important Global Regulator of Gene Expression in Pasteurella multocida and Plays a Crucial Role in Production of a Number of Virulence Factors, Including Hyaluronic Acid Capsule.

The Gram-negative bacterium Pasteurella multocida is the causative agent of a number of economically important animal diseases, including avian fowl cholera. Numerous P. multocida virulence factors have been identified, including capsule, lipopolysaccharide (LPS), and filamentous hemagglutinin, but little is known about how the expression of these virulence factors is regulated. Hfq is an RNA-binding protein that facilitates riboregulation via interaction with small noncoding RNA (sRNA) molecules and their mRNA targets. Here, we show that a P. multocida hfq mutant produces significantly less hyaluronic acid capsule during all growth phases and displays reduced in vivo fitness. Transcriptional and proteomic analyses of the hfq mutant during mid-exponential-phase growth revealed altered transcript levels for 128 genes and altered protein levels for 78 proteins. Further proteomic analyses of the hfq mutant during the early exponential growth phase identified 106 proteins that were produced at altered levels. Both the transcript and protein levels for genes/proteins involved in capsule biosynthesis were reduced in the hfq mutant, as were the levels of the filamentous hemagglutinin protein PfhB2 and its secretion partner LspB2. In contrast, there were increased expression levels of three LPS biosynthesis genes, encoding proteins involved in phosphocholine and phosphoethanolamine addition to LPS, suggesting that these genes are negatively regulated by Hfq-dependent mechanisms. Taken together, these data provide the first evidence that Hfq plays a crucial role in regulating the global expression of P. multocida genes, including the regulation of key P. multocida virulence factors, capsule, LPS, and filamentous hemagglutinin.

A non-canonical RNA silencing pathway promotes mRNA degradation in basal Fungi.

The increasing knowledge on the functional relevance of endogenous small RNAs (esRNAs) as riboregulators has stimulated the identification and characterization of these molecules in numerous eukaryotes. In the basal fungus Mucor circinelloides, an emerging opportunistic human pathogen, esRNAs that regulate the expression of many protein coding genes have been described. These esRNAs share common machinery for their biogenesis consisting of an RNase III endonuclease Dicer, a single Argonaute protein and two RNA-dependent RNA polymerases. We show in this study that, besides participating in this canonical dicer-dependent RNA interference (RNAi) pathway, the rdrp genes are involved in a novel dicer-independent degradation process of endogenous mRNAs. The analysis of esRNAs accumulated in wild type and silencing mutants demonstrates that this new rdrp-dependent dicer-independent regulatory pathway, which does not produce sRNA molecules of discrete sizes, controls the expression of target genes promoting the specific degradation of mRNAs by a previously unknown RNase. This pathway mainly regulates conserved genes involved in metabolism and cellular processes and signaling, such as those required for heme biosynthesis, and controls responses to specific environmental signals. Searching the Mucor genome for candidate RNases to participate in this pathway, and functional analysis of the corresponding knockout mutants, identified a new protein, R3B2. This RNase III-like protein presents unique domain architecture, it is specifically found in basal fungi and, besides its relevant role in the rdrp-dependent dicer-independent pathway, it is also involved in the canonical dicer-dependent RNAi pathway, highlighting its crucial role in the biogenesis and function of regulatory esRNAs. The involvement of RdRPs in RNA degradation could represent the first evolutionary step towards the development of an RNAi mechanism and constitutes a genetic link between mRNA degradation and post-transcriptional gene silencing.

The tracking of intercellular small RNA movement.

RNA silencing is a regulatory mechanism that controls the expression of endogenous genes and exogenous molecular parasites such as viruses, transgenes, and transposable elements. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. In plants, one of the most fascinating aspects of RNA silencing is its mobile nature, in other words its ability to spread from the cell where it has been initiated to neighboring cells, through movement of sRNA molecules. To study this process, a key step is to directly monitor the spread of these nucleic acid species. Here we describe how this can be achieved through biolistic delivery of fluorescently labeled siRNA.

Trans-kingdom cross-talk: small RNAs on the move.

This review focuses on the mobility of small RNA (sRNA) molecules from the perspective of trans-kingdom gene silencing. Mobility of sRNA molecules within organisms is a well-known phenomenon, facilitating gene silencing between cells and tissues. sRNA signals are also transmitted between organisms of the same species and of different species. Remarkably, in recent years many examples of RNA-signal exchange have been described to occur between organisms of different kingdoms. These examples are predominantly found in interactions between hosts and their pathogens, parasites, and symbionts. However, they may only represent the tip of the iceberg, since the emerging picture suggests that organisms in biological niches commonly exchange RNA-silencing signals. In this case, we need to take this into account fully to understand how a given biological equilibrium is obtained. Despite many observations of trans-kingdom RNA signal transfer, several mechanistic aspects of these signals remain unknown. Such RNA signal transfer is already being exploited for practical purposes, though. Pathogen genes can be silenced by plant-produced sRNAs designed to affect these genes. This is also known as Host-Induced Genes Silencing (HIGS), and it has the potential to become an important disease-control method in the future.

Physiological roles of small RNA molecules

Unlike proteins, RNA molecules have emerged lately as key players in regulation in bacteria. Most reviews hitherto focused on the experimental and/or in silico methods used to identify genes encoding small RNAs (sRNAs) or on the diverse mechanisms of these RNA regulators to modulate expression of their targets. However, less is known about their biological functions and their implications in various physiological responses. This review aims to compile what is known presently about the diverse roles of sRNA transcripts in the regulation of metabolic processes, in different growth conditions, in adaptation to stress and in microbial pathogenesis. Several recent studies revealed that sRNA molecules are implicated in carbon metabolism and transport, amino acid metabolism or metal sensing. Moreover, regulatory RNAs participate in cellular adaptation to environmental changes, e.g. through quorum sensing systems or development of biofilms, and analyses of several sRNAs under various physiological stresses and culture conditions have already been performed. In addition, recent experiments performed with Gram-positive and Gram-negative pathogens showed that regulatory RNAs play important roles in microbial virulence and during infection. The combined results show the diversity of regulation mechanisms and physiological processes in which sRNA molecules are key actors.

Identification of Novel Regulatory Small RNAs in Acinetobacter baumannii

Small RNA (sRNA) molecules are non-coding RNAs that have been implicated in regulation of various cellular processes in living systems, allowing them to adapt to changing environmental conditions. Till date, sRNAs have not been reported in Acinetobacter baumannii (A. baumannii), which has emerged as a significant multiple drug resistant nosocomial pathogen. In the present study, a combination of bioinformatic and experimental approach was used for identification of novel sRNAs. A total of 31 putative sRNAs were predicted by a combination of two algorithms, sRNAPredict and QRNA. Initially 10 sRNAs were chosen on the basis of lower E- value and three sRNAs (designated as AbsR11, 25 and 28) showed positive signal on Northern blot. These sRNAs are novel in nature as they do not have homologous sequences in other bacterial species. Expression of the three sRNAs was examined in various phases of bacterial growth. Further, the effect of various stress conditions on sRNA gene expression was determined. A detailed investigation revealed differential expression profile of AbsR25 in presence of varying amounts of ethidium bromide (EtBr), suggesting that its expression is influenced by environmental or internal signals such as stress response. A decrease in expression of AbsR25 and concomitant increase in the expression of bioinformatically predicted targets in presence of high EtBr was reverberated by the decrease in target gene expression when AbsR25 was overexpressed. This hints at the negative regulation of target genes by AbsR25. Interestingly, the putative targets include transporter genes and the degree of variation in expression of one of them (A1S_1331) suggests that AbsR25 is involved in regulation of a transporter. This study provides a perspective for future studies of sRNAs and their possible involvement in regulation of antibiotic resistance in bacteria specifically in cryptic A. baumannii.

The Diversity, Biogenesis, and Activities of Endogenous Silencing Small RNAs in Arabidopsis

In eukaryotic RNA silencing, RNase-III classes of enzymes in the Dicer family process double-stranded RNA of cellular or exogenous origin into small-RNA (sRNA) molecules. sRNAs are then loaded into effector proteins known as ARGONAUTEs (AGOs), which, as part of RNA-induced silencing complexes, target complementary RNA or DNA for silencing. Plants have evolved a large variety of pathways over the Dicer-AGO consortium, which most likely underpins part of their phenotypic plasticity. Dicer-like proteins produce all known classes of plant silencing sRNAs, which are invariably stabilized via 2'-O-methylation mediated by HUA ENHANCER 1 (HEN1), potentially amplified by the action of several RNA-dependent RNA polymerases, and function through a variety of AGO proteins. Here, we review the known characteristics and biochemical properties of the core silencing factors found in the model plant Arabidopsis thaliana. We also describe how interactions between these core factors and more specialized proteins allow the production of a plethora of silencing sRNAs involved in a large array of biological functions. We emphasize in particular the biogenesis and activities of silencing sRNAs of endogenous origin.

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

Small noncoding RNA (sRNA) molecules are integral components of the regulatory machinery for many bacterial species and are known to posttranscriptionally regulate metabolic and stress-response pathways, quorum sensing, virulence factors, and more. The Yop-Ysc type III secretion system (T3SS) is a critical virulence component for the pathogenic Yersinia species, and the regulation of this system is tightly controlled at each step from transcription to translocation of effectors into host cells. The contribution of sRNAs to the regulation of the T3SS in Yersinia has been largely unstudied, however. Previously, our lab identified a role for the sRNA chaperone protein Hfq in the regulation of components of the T3SS in the gastrointestinal pathogen Yersinia pseudotuberculosis. Here we present data demonstrating a similar requirement for Hfq in the closely related species Yersinia pestis. Through deep sequencing analysis of the Y. pestis sRNA-ome, we found 63 previously unidentified putative sRNAs in this species. We identified a Yersinia-specific sRNA, Ysr141, carried by the T3SS plasmid pCD1 that is required for the production of multiple T3SS proteins. In addition, we show that Ysr141 targets an untranslated region upstream of yopJ to posttranscriptionally activate the synthesis of the YopJ protein. Furthermore, Ysr141 may be an unstable and/or processed sRNA, which could contribute to its function in the regulation of the T3SS. The discovery of an sRNA that influences the synthesis of the T3SS adds an additional layer of regulation to this tightly controlled virulence determinant of Y. pestis.

Plant Mobile Small RNAs

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.

Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm

Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.

C/D box sRNA, CRISPR RNA and tRNA processing in an archaeon with a minimal fragmented genome

The analysis of deep sequencing data allows for a genome-wide overview of all the small RNA molecules (the 'sRNome') that are present in a single organism. In the present paper, we review the processing of CRISPR (clustered regularly interspaced short palindromic repeats) RNA, C/D box sRNA (small non-coding RNA) and tRNA in Nanoarchaeum equitans. The minimal and fragmented genome of this tiny archaeon permits a sequencing depth that enables the identification of processing intermediates in the study of RNA processing pathways. These intermediates include circular C/D box sRNA molecules and tRNA half precursors.

Characterization of polyplexes involving small RNA

The purpose of the present study is to provide a tool for an efficient design and synthesis of non-viral vectors for small RNA delivery. The effects of properties of the polycation, such as molecular weight, charge density and backbone structure, to polyplex structure and physicochemical behavior were systematically evaluated. The condensing agents, polyethylenimine (PEI), chitosan (CS) and poly(allylamine) (PAA) were added to sRNA molecules at different N/P ratio. The efficiency of encapsulation and protection of sRNA, as well as polyplex size, zeta potential and morphology were followed and compared. The results show that PEI/sRNA polyplexes display a small size and positive zeta potential. However, for low molecular weights, this polycation is unable to protect sRNA in the presence of a decompacting agent. With chitosan, sRNA is efficiently compacted at high N/P ratios. The CS/sRNA complexes display small sizes, ca. 200nm, positive surface charge and also good stability. Finally, the PAA/sRNA polyplexes were found to be the smallest at low N/P ratios, displaying a good encapsulation efficiency and high stability. A rationale for the experimental observations is provided using Monte Carlo simulation for systems with polycations of different length and charge density. The simulations showed that there is an interplay between the size of polycation chains and its charge density that define the degree of condensation for sRNA.

Differential Impact of the HEN1 Homolog HENN-1 on 21U and 26G RNAs in the Germline of Caenorhabditis elegans

RNA interference (RNAi)–related pathways affect gene activity by sequence-specific recruitment of Ago proteins to mRNA target molecules. The sequence specificity of this process stems from small RNA (sRNA) co-factors bound by the Ago protein. Stability of sRNA molecules in some pathways is in part regulated by Hen1-mediated methylation of their 3′ ends. Here we describe the effects of the Caenorhabditis elegans HEN1 RNA–methyl-transferase homolog, HENN-1, on the different RNAi pathways in this nematode. We reveal differential effects of HENN-1 on the two pathways that are known to employ methylated sRNA molecules: the 26G and 21U pathways. Surprisingly, in the germline, stability of 21U RNAs, the C. elegans piRNAs, is only mildly affected by loss of methylation; and introduction of artificial 21U target RNA does not further destabilize non-methylated 21U RNAs. In contrast, most 26G RNAs display reduced stability and respond to loss of HENN-1 by displaying increased 3′-uridylation frequencies. Within the 26G RNA class, we find that specifically ERGO-1–bound 26G RNAs are modified by HENN-1, while ALG-3/ALG-4–bound 26G RNAs are not. Global gene expression analysis of henn-1 mutants reveals mild effects, including down-regulation of many germline-expressed genes. Our data suggest that, apart from direct effects of reduced 26G RNA levels of henn-1 on gene expression, most effects on global gene expression are indirect. These studies further refine our understanding of endogenous RNAi in C. elegans and the roles for Hen1 like enzymes in these pathways.

Novel iron‐regulated and Fur‐regulated small regulatory RNAs in Aggregatibacter actinomycetemcomitans

Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01-JA04) identified by bioinformatics was significantly upregulated in iron-limited medium compared with iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate Escherichia coli ferric uptake regulator (Fur) from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. The A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNAs have the ability to modulate biofilm formation, but their functional target genes remain to be confirmed.

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6. Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5. Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence <em>in situ</em> Hybridization (LNA flow-FISH): A Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

The origin and effect of small RNA signaling in plants

Given their sessile condition, land plants need to integrate environmental cues rapidly and send signal throughout the organism to modify their metabolism accordingly. Small RNA (sRNA) molecules are among the messengers that plant cells use to carry such signals. These molecules originate from fold-back stem-loops transcribed from endogenous loci or from perfect double-stranded RNA produced through the action of RNA-dependent RNA polymerases. Once produced, sRNAs associate with Argonaute (AGO) and other proteins to form the RNA-induced silencing complex (RISC) that executes silencing of complementary RNA molecules. Depending on the nature of the RNA target and the AGO protein involved, RISC triggers either DNA methylation or chromatin modification (leading to transcriptional gene silencing, TGS) or RNA cleavage or translational inhibition (leading to post-transcriptional gene silencing, PTGS). In some cases, sRNAs move to neighboring cells and/or to the vascular tissues for long-distance trafficking. Many genes are involved in the biogenesis of sRNAs and recent studies have shown that both their origin and their protein partners have great influence on their activity and range. Here we summarize the work done to uncover the mode of action of the different classes of sRNA with special emphasis on their movement and how plants can take advantage of their mobility. We also review the various genetic requirements needed for production, movement and perception of the silencing signal.

Sequence-based analysis uncovers an abundance of non-coding RNA in the total transcriptome of Mycobacterium tuberculosis.

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5’ and 3’ untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.

Orientation and expression of methicillin-resistant Staphylococcus aureus small RNAs by direct multiplexed measurements using the nCounter of NanoString technology

Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. Several genomes of this major human pathogen have been publicly available for almost 10 years, but comprehensive links between virulence or epidemicity and genome content of the bacterium are still missing. This project aims at characterizing a set of small transcribed molecules currently ignored by standard automated annotation algorithms. We assessed the NanoString's nCounter Analysis System for its ability to determine the orientation and quantity of the expressed small RNA (sRNA) molecules that we recently detected with RNA-Sequencing (RNA-Seq). The expression of approximately seventy small RNAs, including sRNA localized in pathogenic islands, was assessed at 5 time points during growth of the bacterium in a rich medium. In addition, two extraction strategies were tested: RNA was either purified on columns or simply prepared from crude lysates in the presence of a chaotropic buffer. The nCounter System allowed us to perform these 64 measurements in a single experiment, without any enzymatic reaction, thus avoiding well-known technical biases. We evaluated the reproducibility and reliability of the nCounter compared to quantitative RT-PCR (RT-qPCR). By using two different designs for the two coding strands, we were able to identify the coding strand of 61 small RNA molecules (95%). Overall, the nCounter System provided an identification of the coding strand in perfect concordance with RNA-Seq data. In addition, expression results were also comparable to those obtained with RT-qPCR. The sensitivity and minimal requirements of the nCounter system open new possibilities in the field of gene expression analysis, for assessing bacterial transcript profiles from complex media (i.e. during host–pathogen interactions) or when starting from poorly purified RNA or even directly from lysed infected tissues.

The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci.

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 5' adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 5' nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGO-associated proteins in influencing epigenetic modifications.