Successful decision making in our daily lives requires weighing an option’s costs against its associated benefits. The neuromodulator acetylcholine underlies both the etiology and treatment of a number of illnesses in which decision making is perturbed, including Alzheimer’s disease, attention-deficit/hyperactivity disorder, and schizophrenia. Nicotine acts on the cholinergic system and has been touted as a cognitive enhancer by both smokers and some researchers for its attention-boosting effects; however, it is unclear whether treatments that have a beneficial effect on attention would also have a beneficial effect on decision making. Here we utilize the rodent Cognitive Effort Task (rCET), wherein animals can choose to allocate greater visuospatial attention for a greater reward, to examine cholinergic contributions to both attentional performance and choice based on attentional demand. Following the establishment of baseline behavior, four drug challenges were administered: nicotine, mecamylamine, scopolamine, and oxotremorine . As per previous rCET studies, animals were divided by their baseline preferences, with “worker” rats choosing high-effort/high-reward options more than their “slacker” counterparts. Nicotine caused slackers to choose even fewer high-effort trials than at baseline, but had no effect on workers’ choice. Despite slackers’ decreased willingness to expend effort, nicotine improved their attentional performance on the task. Nicotine also increased measures of motor impulsivity in all animals. In contrast, scopolamine decreased animals’ choice of high-effort trials, especially for workers, while oxotremorine decreased motor impulsivity for all animals. In sum, the cholinergic system appears to contribute to decision making, and in part these contributions can be understood as a function of individual differences. While nicotine has been considered as a cognitive enhancer, these data suggest that its modest benefits to attention may be coupled with impulsiveness and decreased willingness to work hard, especially in individuals who are particularly sensitive to effort costs (i.e. slackers). In our daily lives, we are often confronted with decisions that require weighing each option’s costs against its associated benefits. Disturbances in such cost/benefit decision making have been reported in populations of virtually every severe neuropsychiatric illness per se , wherein animals can choose to allocate greater visuospatial attention for a greater reward, and this task provides measures of both attentional performance and choice based on attentional demand. Previous work with this task indicates that the neurochemical regulation of willingness to work can be dissociated from ability, and that baseline differences in the degree to which animals choose to apply cognitive effort to earn greater rewards is a key determinant of drug response. For example, the psychostimulant amphetamine caused hard-working animals to “slack off”, i.e. choose a greater proportion of trials with lower attentional demands, while “slacker” animals worked harder in response to the drug in the absence of any change in attentional accuracy The goal of this study was therefore to examine how nicotinic and muscarinic acetylcholine receptor agonists and antagonists affected animals’ choice versus their attentional performance on the rCET, paying special consideration to these drugs’ interactions with animals’ existing choice preferences.ad libitum. Animals were pair housed in a climate-controlled colony room on a 12 hr reverse light-dark cycle . All housing and testing was in accordance with the Canadian Council of Animal Care, and all procedures were approved by the University of British Columbia’s Animal Care Committee.Subjects were 24 male Long-Evans rats from Charles Rivers Laboratories , each weighing 275–300 g at experimental commencement. Animals were food restricted to 14–16 g rat chow per day and thus maintained at ∼85% of their free-feeding weight. Water was available All testing took place within 12 standard five-hole operant chambers, each supplemented with two retractable response levers and enclosed in a ventilated, sound-attenuating cabinet . The chambers were controlled by software written in Med-PC by CAW, running on an IBM-compatible computer.All animals were habituated and trained for the rCET as previously described  to commence. After the ITI, one of the five stimulus lights briefly illuminated, with a stimulus duration of 1.0 s for LR trials and 0.2 s for HR trials. Animals were rewarded if they nosepoked the previously illuminated aperture within 5 s (a correct response), and received 1 sugar pellet for a LR trial and 2 sugar pellets for a HR trial. Upon reward delivery, the tray light again illuminated, thus signaling the opportunity to begin the next trial.Trials went unrewarded for a number of reasons: if animals failed to make a lever response within 10 s (a choice omission); if animals nosepoked during the ITI  * 100. When baseline performance on the rCET was deemed statistically stable , the mean choice of the HR option was 68%. Animals were grouped as “workers” if they chose HR for >70% of trials (n = 11) and as “slackers” if they chose HR for ≤70% of trials (n = 13). This subdivision was based on the mean split from the original rCET paper The following variables were also analyzed separately for LR and HR trials: percent accuracy  * 100); percent premature responses  * 100); latency to choose between the LR and HR levers (lever choice latency); latency to correctly nosepoke in the illuminated aperture (correct latency); latency to collect reward (collection latency); percent response omissions  * 100). Failures to choose a lever at the beginning of the trial (choice omissions) and the total number of completed trials were also analyzed.Drug doses were based on previous reports All drugs were prepared fresh daily, and administration adhered to a digram-balanced Latin Square design . All variables expressed as a percentage were arcsine transformed to minimize artificial ceiling effects post-hoc one-way ANOVA or paired-samples t-tests. Any p-values>0.05 but <0.07 were reported as a statistical trend.Pharmacological manipulations were again analyzed using repeated-measures ANOVA. For all drug challenges, dose  and choice were included as within-subjects factors, with group as a between-subjects factor. Any main effects of significance (p<0.05) were further analyzed via 1,22 = 71.338, p<0.001), and workers continued to choose a significantly higher proportion of HR than slackers . The nicotinic acetylcholine receptor (nAChR) agonist nicotine differentially affected choice of HR for workers and slackers , further decreasing choice of HR for slackers but having no effect on workers .Baseline behavior has been discussed at length elsewhere 1,22 = 62.446, p<0.001), indicating that HR trials were indeed more cognitively demanding. As seen in previous cohorts, workers and slackers performed the task equally well , despite workers choosing HR proportionately more, and thus suggesting that differences in choice preference were not a direct result of differences in animals’ visuospatial attentional ability. Despite decreasing choice of HR for slackers, nicotine increased accuracy of HR for slackers, but had no effect on workers’ HR, or LR for all animals .As expected, animals displayed higher accuracy on LR versus HR trials . There were no differences in the level of premature responding between workers and slackers , indicating that choice preference was not guided by motor impulsivity. Nicotine increased premature responding for all animals across both trial types .As seen in previous cohorts, premature responding was higher for HR versus LR trials , suggesting that all animals anticipated a larger reward following the successful completion of HR, but slackers still chose proportionately fewer of these trials than workers. Nicotine dose-dependently increased choice omissions  but decreased response omissions  for all animals across both trial types , and decreased the number of completed trials for all animals by ∼20% .Nicotine had no effect on the latency to choose a lever, nosepoke at the array or collect reward . As consistently seen with the rCET, both workers and slackers collected reward faster following a successful HR versus LR trial . The drug had no effect on premature responding  but did not affect correct or collection latencies . The drug did not affect response omissions  but increased the number of choice (lever) omissions  and decreased the number of completed trials for all animals by ∼10% .For all animals across both trial types, mecamylamine lengthened the latency to choose either the LR or HR lever . When examined separately, scopolamine decreased workers’ HR choice  but had no effect on slackers’ choice . The drug had no effect on animals’ accuracy or premature responding (The muscarinic acetylcholine receptor (mAChR) antagonist scopolamine decreased all animals’ choice of HR . Scopolamine also significantly lengthened all animals’ correct latency  but had no effect on collection latency . The drug significantly increased both response omissions  and choice omissions  and profoundly decreased all animals’ completed trials by over 65% .Scopolamine had an inverted-U-shaped effect on the time taken to choose between LR and HR levers/options, with the lowest dose lengthening choice latency .The mAChR agonist oxotremorine had no effect on animals’ choice or accuracy  but had no effect on correct or collection latencies . The drug increased both response omissions and choice omissions , and also decreased completed trials by ∼40% .Oxotremorine lengthened choice latency for all animals across both trial types  trials for “slacker” rats, despite a modest improvement in these animals’ performance (i.e. accuracy), whereas the drug did not affect workers’ choice. In contrast to its differential choice effects for workers versus slackers, nicotine increased motor impulsivity (i.e. premature responding) for all animals. Interestingly, the mAChR antagonist scopolamine also decreased choice of HR, particularly for workers, without any concomitant effects on performance or motor impulsivity. Finally, the mAChR agonist oxotremorine had no effect on choice but dose-dependently decreased impulsive responding. Taken together, these data support recent findings that nicotinic and muscarinic cholinergic systems subserve cost/benefit decision making Central acetylcholine largely originates from the basal forebrain and pons, and projects to a diffuse set of targets in the central nervous system, including the prefrontal cortex, limbic regions, and the midbrain dopaminergic system Pharmacological studies of cholinergic contributions to decision making have primarily used delay- and risk-discounting tasks, wherein the costs of the HR option were adjusted across blocks within each session In addition to its putative influence on decision making, acetylcholine’s role in attentional processes has also been well described , the precursor to the rCET, which differs from the current task only in its lack of LR/HR options  and, as previously discussed, prefrontal versus striatal acetylcholine likely have dissociable contributions to behavior. Also, amphetamine (which potentiates dopaminergic functioning) has the opposite choice effects to nicotine, instead causing workers to “slack off” and slackers to “work harder” per seIn light of the current and previous data, some tentative, testable models of acetylcholine’s specific contribution to decision making can be made; these models are not mutually exclusive and may in fact complement one another. First, acetylcholine may indirectly influence choice via its interactions with the midbrain dopaminergic system In sum, it appears that both nicotinic and muscarinic cholinergic systems contribute to cost/benefit decision making, and in part their contributions can be understood as a function of individual differences. While nicotine has been considered as a cognitive enhancer by both smokers and researchers File S1Table S1. Nicotine. Table S2. Mecamylamine. Table S3. Scopolamine. Table S4. Oxotremorine.All relevant data in tables. (DOCX)Click here for additional data file.


This review summarizes current knowledge on the role in antifungal drug resistance, mode of action and phylogenetic relations of DHA transporters, from the model yeast S. cerevisiae to pathogenic yeasts and filamentous fungi. Through the compilation of the predicted DHA transporters in the medically relevant Candida albicans, C. glabrata, C. parapsilosis, C. lusitaniae, C. tropicalis, C. guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus species, the fact that only 5% of the DHA transporters from these organisms have been characterized so far is evidenced. The role of these transporters in antifungal drug resistance and in pathogen-host interaction is described and their clinical relevance discussed. Given the knowledge gathered for these few DHA transporters, the need to carry out a systematic characterization of the DHA multidrug efflux pumps in fungal pathogens, with emphasis on their clinical relevance, is highlighted.Infections caused by opportunistic fungal pathogens have reached concerning numbers due to the increase of the immunocrompromised human population and to the development of antifungal resistance. This resistance is often attributed to the action of multidrug efflux pumps, belonging to the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). Although many studies have focused on the role of ABC multidrug efflux transporters, little is still known on the part played by the Drug:H The multidrug resistance (MDR) phenomenon, characterized by the simultaneous acquisition of resistance to chemically and structurally different compounds  family of the MFS.There are mainly four mechanisms by which a cell can deal with a toxic compound: (i) drug target alteration, (ii) drug inactivation, (iii) reduced uptake, or (iv) active extrusion  and 2 (DHA2). These families differ mainly in the number of transmembrane spans (TMS), with the first having 12 and the second 14 TMS  the existence and, thus, function of 20 out of 22 of these transporters remained concealed until the genome sequence was disclosed; (2) these transporters were indeed found to confer multidrug resistance, but also to play important roles in S. cerevisiae physiology; and (3) there is significant functional overlap between these transporters, making the discovery of their function a bigger challenge. The functional analysis of the S. cerevisiae DHA transporters provides clues on the function of homolog transporters from pathogenic yeast and filamentous fungi, but also suggests that their role tends to be elusive.The paradigmatic case of the study of DHA transporters in Candida DHA1 and DHA2 transporters considered in this review are those predicted to be encoded in the genomes of C. albicans, C. tropicalis, C. parapsilosis, C. guilliermondii, C. lusitaniae, and C. glabrata  and, based on the BlastP algorithm, using the S. cerevisiae DHA1 and DHA2 proteins to query the Genbank database, respectively. A total of 185 full-size DHA1 proteins and 85 full-size DHA2 proteins were found to be encoded in these eight fungal species, and clustered according to the phylogenetic trees displayed in Figure The A. fumigatus genes (clusters 1–4 and 6–7) or A. fumigatus and C. neoformans genes (cluster 5) . The A. fumigatus genome encodes a total of 33 DHA2 transporters, two thirds of which reside in clusters lacking hemiascomycetous DHA2 members. A. fumigatus genome does not include homologs of Sge1/Azr1/Vba3/Vba5 or Atr1/YMR279C proteins. A total of 7 DHA2 proteins were found in the C. neoformans genome, among which only one S. cerevisiae homolog is found, clustering with Vba1/Vba2 , compared with a maximum of 11 days for the lowest concentration of the wild type (Yamada-Okabe and Yamada-Okabe, C. albicans Qdr1, Qdr2, and Qdr3 transporters was found to lead to defects in biofilm architecture and thickness and to attenuate virulence in a mouse model (Shah et al., C. glabrata DHA transporters CgAqr1 and CgTpo3 were also suggested to contribute to the survival of this pathogen within the host, as they confer resistance to acetic acid (Costa et al., Candida infection prone environments. Indeed, the concentration of lactic or acetic acid can reach up to 125 mM in the vaginal tract, particularly under bacterial vaginosis (Chaudry et al., The clinical impact of the DHA transporters in fungal pathogens is yet to be fully investigated. So far, only a couple of them have been shown to effectively contribute to the development, progression, or persistence of the infection in the host Table . That isC. albicans, the upregulation of the ABC transporters CaCDR1 and CaCDR2 in fluconazole-resistant isolates, which has used to assess their clinical relevance, was assessed mostly by targeted gene expression analysis (Sanglard et al., CaMDR1 have been found among clinical isolates, inducing distinct drug resistance profiles (Gupta et al., Of the high number of MFS transporters that fungal genomes encode, only a reduced amount has been shown to be involved in MDR or to be able to confer resistance to antifungal drugs, and an even lower number has been described as clinically relevant, so far. The lack of information on the clinical impact of fungal DHA transporters may be associated with the types of experiments carried out in this context. For instance, in Given their predicted role in antifungal drug resistance, a few DHA transporters in pathogenic yeasts and fungi have already been characterized. So far, their characterization suggested interesting roles in antifungal drug resistance and, in some cases, in host-pathogen interactions. However, only 5% of the predicted DHA transporters in the 8 fungal pathogens considered in this review have ever been studied. It appears, thus, crucial to study this underappreciated family of transporters, as they may provide decisive insights into the mechanisms underlying antifungal drug resistance that may guide more efficient fungal infection diagnosis, prophylaxis, and therapeutics. Furthermore, unlike what has been observed for ABC drug efflux pumps, which are widespread from bacteria to man, the DHA family appears to be strictly conserved within bacteria and fungi, turning these proteins into interesting candidates as targets for the development of new antifungal drugs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3′ of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5′ untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria.Expression of type four pili (Tfp) is essential for virulence in IMPORTANCE Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT-PCR analysis, we found that the RNA is expressed in stationary phase or following salt stress. Our work also indicates that this RNA does not significantly affect pilE or pilin expression levels but instead appears to modulate pilin variation. Neisseria meningitidis (meningococcus) is a Gram-negative bacterium that is frequently carried asymptomatically in the human upper respiratory tract. Occasionally, the meningococcus causes meningitis and septicemia, which can result in death or devastating sequelae. N. meningitidis has evolved a number of strategies to colonize the upper respiratory tract, including adhesion to epithelial cells and immune evasion. The expression of type four pili (Tfp) by this important human pathogen contributes to both colonization and disease , enabling the bacterium to modulate its pilin sequence  structure is located 5′ to pilE and is essential for gene conversion  RNA within the region 3′ of the pilE gene of N. meningitidis. The aim of this work was to investigate the function of this RNA. AS RNAs have been identified in numerous bacterial species, and their roles are largely to regulate the sense transcript at the level of transcription, mRNA stability, or translation , kanamycin (50 μg/ml), or erythromycin (200 μg/ml) when required. N. meningitidis was grown overnight at 37°C, 5% CO2, either on brain heart infusion  agar supplemented with 5% heat-denatured horse serum and 0.1% starch or in liquid BHI. Erythromycin (2 μg/ml) was added for selection.Strains and plasmids used in this study are described in E. coli or N. meningitidis were grown for approximately 3 h in LB or BHI medium, respectively, to an optical density at 600 nm (OD600) of 0.5 to 0.6 and subjected to acid stress , envelope stress (5% Triton X-100 or 5% ethanol), oxidative stress (0.15% H2O2), salt stress (0.5 M NaCl or 0.5 M KCl), osmotic stress (6% sucrose), or temperature stress (10°C) for 10 min. For analysis of transcript and protein expression during growth, N. meningitidis strains were grown overnight on BHI agar. Bacteria were recovered from the plate, resuspended in phosphate-buffered saline (PBS), and quantified using the A260 to measure DNA in a lysed sample. A volume equivalent to 1 × 109 CFU was inoculated into 25 ml of BHI medium in 125-ml conical flasks (Corning), and cultures were shaken at 180 rpm at 37°C. OD600 readings were taken at periodic intervals to determine growth phase, and samples were taken at various time points for RNA and protein analyses.For stress experiments, liquid cultures of N. meningitidis 8013 using primers pilE_AS-F/pilE_AS-1R and lpxC_frag_F/R . For the construction of strains WT_ery(RecA6) and Mut_ery(RecA6), genomic DNA from strain 8013 containing the recA6 allele (gift from Hank Seifert) was used to transform the strains WT_ery and Mut_ery, respectively. Single colonies of the transformants obtained were confirmed by Western blotting of whole-cell extracts for RecA expression in the presence or absence of isopropyl-β-d-thiogalactopyranoside (IPTG).A 595-bp fragment, including the AS promoter (downstream), and a 752-bp adjacent fragment (upstream) were amplified from frag_F/R , respectminators , into th600 unit. Proteins were separated by SDS-PAGE alongside molecular weight markers  and transferred to nitrocellulose membranes  for Western blotting. PilE was detected using SM1    and IRDyE. coli by using the Fast RNA Blue kit  followed by DNase treatment (Roche) or from N. meningitidis by using the RNeasy midikit and on-column DNase treatment (Qiagen). RNA (20 μg) was separated by electrophoresis in 1.5% agarose formaldehyde gels in HEPES buffer, transferred onto Hybond-N membranes , and cross-linked by UV light. Probes were generated by end-labeling primers , or a PCR product generated using primers 8013_tmRNA_F/R with [γ-32P]ATP (PerkinElmer). Membranes were incubated in hybridization buffer  and hybridized with labeled probes overnight at 64°C. Signals were detected using a fluorescent image analyzer (Fuji FLA-5000) and quantified using AIDA image analyzer software. Experiments were carried out in triplicate using strains from independent transformations.Total RNA was isolated from pilE-specific primers carrying a 5′ tag sequence not found in the N. meningitidis genome  of triplicate experiments performed on WT_ery and Mut_ery strains obtained from three independent transformations. The amount of transcript was expressed as the R value standardized to the transfer-messenger RNA (tmRNA) control. The efficiencies of each primer pair were evaluated by performing a 10-fold dilution series experiment and determining the slope of the standard curves obtained. The primer pairs were found to have efficiencies ranging from 85 to 100%.First-strand cDNA was synthesized from 5 μg total RNA by using AS- or s genome . RNAs weN. meningitidis strains following NaCl stress was mixed with labeled probe and denatured at 80°C for 5 min, and the primer was extended by using avian myeloblastosis virus reverse transcriptase in extension buffer  at 42°C for 1 h. Products were denatured at 80°C for 3 min in formamide sample buffer. A PCR product of the AS promoter region was generated using primers pilE_AS-F and pilE_AS-1R, and sequencing was performed using the labeled pilE_AS-1R primer according to standard methods (RNA (10 μg) extracted from  methods . Productin vitro-transcribed AS RNA synthesized with primers T7_iv_temp_F and iv_temp_R using the RiboMAX large-scale RNA production system .Mapping  PCR was performed according to published protocols . Total RrecA6 allele. Each Av assay was carried out using bacteria inoculated from frozen stocks made from single colonies of the respective strains, in order to ensure that each experiment started with a homogenous population of cells containing the same pilE sequence. Strains grown overnight on solid BHI medium without IPTG were used to inoculate BHI broth and grown to mid-log phase. Where relevant, IPTG was then added to a final concentration of 1 mM and the cultures were incubated with shaking at 37°C for 10 min. For NaCl stress, the cultures were incubated with NaCl (0.5 M) together with IPTG for 10 min. The cultures were then serially diluted and plated onto BHI agar containing 1 mM IPTG and grown overnight for 22 h . Ninety-six colonies of each strain under each condition were passaged onto BHI agar and incubated overnight. Lysates were prepared by resuspending single colonies in 100 μl PBS and boiling for 10 min. pilE was amplified from single-colony lysates using primers AS_F and pilE_F, which are specific for amplification of pilE, and the Herculase II fusion DNA polymerase (Agilent). Products were sequenced and compared to pilE progenitor sequence, and changes were mapped to pilS donors. Each variant pilE was considered to have resulted from a single Av event. The assays with 8013 and 8013ΔG4 were performed once. Av assays with WT_ery(RecA6) and Mut_ery(RecA6) were performed three times.Antigenic variation assays in liquid medium were performed with strains 8013 and 8013ΔG4 or with WT_ery and Mut_ery strains containing the t test was used for the quantification of transcript level by strand-specific qRT-PCR. Fisher's exact test was used for the antigenic variation assay with strains 8013 and 8013ΔG4. Results of the antigenic variation assay with Wt_ery(RecA6) and Mut_ery(RecA6) were analyzed by fitting generalized linear models (GLMs) , NaCl induction (yes or no), and assay . R statistical software was used, specifically, the function GLM (The Student s (GLMs) . Binomiation GLM .N. meningitidis strain 8013 (FM999788) revealed the presence of a putative promoter on the AS strand, 5 nucleotides (nt) downstream of the pilE stop codon  (pilS region but not adjacent to the pilE gene (Inspection of the available genome sequence of ain 8013  (accessiop codon . No othelineages . In two  TATAAT) . InteresilE gene .We did not identify any sequences resembling a Shine-Dalgarno ribosome binding site within proximity to an ATG start codon within 350 nucleotides of this promoter. We therefore hypothesized that this promoter drives transcription of a noncoding, AS RNA.E. coli. A 595-bp region from N. meningitidis, including the putative AS promoter and 386 bp of the 3′ end of the pilE coding sequence (but excluding the pilE promoter sequence) was introduced into pEGFP-N2 to make pEGFP-N2(insert1); this plasmid only harbors a cytomegalovirus early-immediate promoter for transcription in eukaryotic but not prokaryotic cells. To examine promoter activity, E. coli strain DH5α was transformed with pEGFP-N2(insert1), grown for 3 h to an OD600 of 0.5 to 0.6 in liquid medium, and then subjected to acid, NaCl, temperature, envelope, or oxidative stress for 10 min. The presence of the AS transcript was detected by Northern blotting by using a 50-nt probe that hybridized 39 nt downstream of the predicted −10 sequence. The AS transcript was significantly more abundant when the E. coli cells were exposed to high NaCl concentrations (0.5 M for 10 min) but not after other stresses  with or without salt stress. A transcript of the expected size was detected in E. coli containing the plasmid with the unmodified promoter sequence (wild type [WT]) or with an alteration in the putative −35 sequence (Mut1), indicating that the promoter is functional and that the −35 sequence is dispensable for expression of the RNA. In contrast, mutation of the −10 sequence (in Mut 2 and Mut 3) was sufficient to abolish transcription.To determine whether the AS promoter is functional, we first examined the ability of this sequence to mediate transcription in stresses . In addistresses . No tranN. meningitidis. Strain 8013 was grown overnight on solid medium and used to inoculate liquid BHI medium. After 3 h growth at 37°C with agitation, bacteria were subject to stress conditions by addition of NaCl or KCl to 0.5 M, sucrose to 6%, or H2O2 to 0.15% for 10 min. Using a probe specific for the AS RNA, a single transcript was detected in bacteria treated with NaCl or KCl, indicating that the AS RNA is also expressed in response to salt stress in wild-type N. meningitidis . There was no difference in growth of WT_ery and Mut_ery (600 of 1.9) (P = 0.0003)  of the AS RNA, we next constructed isogenic strains with either an intact or a mutated AS promoter. The control strain of 8013, designated WT_ery, carries the erythromycin antibiotic resistance marker but an intact AS promoter sequence . We confirmed that AS expression in WT_ery was not affected by the presence of the erythromycin resistance cassette, based on Northern blotting . We also introduced the −10 and −35 AS promoter mutations into  Mut_ery , and Nor of 1.9) . In agrepilE coding sequence and/or 5′ UTR, we mapped the 5′ and 3′ ends of the transcript. The 5′ end of the AS transcript was mapped by primer extension and found to be to 8 nt downstream of the −10 sequence of the predicted promoter. No extension product was obtained from the strain Mut_ery  but not with F+R4 (888 nt) or F+R4.5 (933 nt). Products of 888 nt and 933 nt were amplified from in vitro-synthesized RNA with primers F+R4 and F+R4.5, respectively  of the sense mRNA to alter ribosome binding and thus affect protein expression. To determine whether the AS RNA encompasses the region complementary to the full  Mut_ery . RT-PCR sequence  and C. Ated sRNA . Thus, tpilE coding sequence and 5′ UTR, we next determined whether pilE transcript or pilin protein levels are affected by the presence of the AS transcript. Northern blot analysis was performed using a pilE-specific oligonucleotide probe. Analysis of total RNA isolated from the strains at different growth phases revealed that pilE mRNA was expressed at similar levels throughout growth in both WT_ery and Mut_ery . We found that in the absence of NaCl stress, both Northern blot analysis and strand-specific qRT-PCR indicated a trend toward elevated pilE mRNA levels in Mut_ery compared to WT_ery . Following NaCl stress which induced AS RNA levels up to 280-fold .Given that the AS RNA is complementary to the entire  Mut_ery  and was  Mut_ery . We also280-fold , an apprpilE transcript levels upon deletion or induction of the AS RNA, we investigated whether there were any changes in pilin protein levels, which might result from posttranscriptional regulation of PilE. We performed Western blot analysis of whole-cell lysates using anti-pilin antibody and fluorescence analysis to quantify relative amounts of pilin in whole-cell lysates from WT_ery and Mut_ery either during growth . pilE variation was detected in 8013, while no Av events occurred in 8013ΔG4 . We fitted a binomial GLH which proved to be satisfactory . On their own, neither the presence of NaCl nor the mutation resulted in a statistically significant change in levels of antigenic variation. However, we found that there was a significantly reduced level of antigenic variation when WT_ery was induced with NaCl  but this was not observed for Mut_ery, indicating that the AS RNA transcript or transcription may influence pilin Av.The G4 sequence and position relative to  8013ΔG4 , indicatpilE in N. meningitidis. The sequence and position of the promoter of this RNA are conserved in different meningococcal isolates expressing class I pilE but is absent from the class II pilE locus. Instead, the AS promoter is located in the pilS locus in class II-expressing strains, consistent with this region of the class II pilS locus having homology to the region 3′ of class I pilE . These diverse findings may reflect strain and species specificity; N. gonorrhoeae inhabits a physiologically distinct niche within human hosts compared to meningococcus and thus may employ different strategies for modulating pilE expression and variation. Additionally, changes to pilE CDS and/or 3′ UTR following gene conversion events could result in the introduction of promoter sequences within the pilE region. In strain 8013, we did not identify any additional AS promoters in or adjacent to any of the pilS cassettes, although we cannot exclude the presence of additional, midgene pilE AS promoters in other isolates of N. meningitidis. We note, however, that in an additional 200 genomes of meningococcal isolates from the United Kingdom publically available in the pubMLST database (http://pubmlst.org/neisseria/), the position and sequence of the pilE AS promoter identified in this work are conserved, despite variation in the pilE coding sequence (data not shown).Here we have described the identification of a novel RNA antisense to s I pilE . Analysi in pilE ; other rcis-encoded AS RNAs are abundant in bacteria , the AS RNA levels increase. This increase may result from both increased AS promoter activity and the concomitant reduction of pilE transcription. Changes in the ratio of sense/AS transcripts, either in response to environmental cues or as governed by pilE transcript levels, could serve as a mechanism of regulation of AS functions.While our results do not provide any evidence for the AS RNA affecting N. gonorrhoeae is required for Av of pilE and has been proposed to result in transcription of an RNA that promotes G4 formation (pilE variation in the wild-type strain, which was not observed in the mutant. This suggested that, in contrast to the loss of the G4-associated sRNA which abolishes Av, AS RNA transcript or transcription modulates pilE variation. The mechanism by which this occurs is not yet known. Our mapping experiments revealed that the AS transcript 3′ end overlaps with the promoter of the G4-associated RNA. Overlapping transcription units can influence the expression of the downstream transcript, through promoter occlusion (pilE variation frequency. Further work is under way to examine this possibility.The promoter of the G4-associated sRNA in ormation . In our cclusion . Therefoin vivo, for example competitive conditions of nutrient depletion, high cell density, and oxygen limitation (N. gonorrhoeae, Av rates are not affected by temperature, carbon source, or oxygen availability, but an increase in Av frequency was observed when bacteria were grown under iron-starved conditions (pilE locus in N. meningitidis which influences Av in this important human pathogen. This study provides further insight into the complexity of pilin regulation and variation in pathogenic Neisseria spp.Like many noncoding RNAs, the AS RNA appears to be specifically expressed under certain conditions. As discussed, high concentrations of NaCl were used to induce AS RNA expression in this study. This level of salt is nonphysiological, although certain chronic inflammatory airway diseases, such as cystic fibrosis or rhinitis, have been reported to increase Na and Cl ion concentrations up to almost 300 mM in airway surface liquid . More immitation . Little nditions . Pilin vnditions , 16, 17.


Laser capture microdissection offers unique possibilities for the isolation of specific cell populations or histological structures. However, isolation of RNA from microdissected tissue is challenging due to degradation and minimal yield of RNA during laser capture microdissection (LCM). Our aim was to optimize the isolation of high-quality RNA from laser capture microdissected fresh frozen prostate tissue on the level of staining and RNA extraction.® Micro (median RIN 7.2), miRNeasy Mini (median RIN 6.6), Picopure® (median RIN 6.0), mirVana™ miRNA (median RIN 6.5) and RNAqueous®-Micro (median RIN 6.3). RNA quality from microdissected samples with either the RNeasy Micro or miRNeasy Mini kit, was comparable to RNA isolated directly from whole tissue slices . Isolated RNA from benign and prostate cancer microdissected tissue demonstrated that RNA quality can vary between regions from the same clinical sample. Additionally, RNA quality (r = 0.89), but not quantity (r = 0.69) could be precisely measured with the Agilent Bioanalyzer.Cresyl violet and haematoxylin were compared as histological stains for LCM. While RNA quality was similar for cresyl violet (median RIN 7.4) and haematoxylin (median RIN 7.6), tissue morphology was more detailed with cresyl violet as compared to haematoxylin. RNA quality from the following kits was compared: RNeasyWe demonstrate that staining with cresyl violet results in the isolation of high quality RNA from laser capture microdissected tissue with high discriminative morphology. The RNeasy Micro and miRNeasy Mini RNA extraction kits generated the highest quality RNA compared to Picopure, mirVana and RNAqueous with minimal loss of RNA quality during LCM.The online version of this article (doi:10.1186/s13104-015-1813-5) contains supplementary material, which is available to authorized users. Biological and histological heterogeneity of tissue samples are challenging issues for the isolation and characterization of individual cell populations that typically comprise tissues such as epithelial, mesenchymal and immune cells. Techniques such as macrodissection produce samples consisting of a mixture of cell types. Laser capture microdissection (LCM) is a method to obtain samples containing one specific cell type from complex tissues based on phenotypical, cytological and histopathological features. Generally, stained tissue slices are observed in an inverted light microscope and under visual guidance, areas of interest are cut loose using a focused laser beam. Excised fragments are catapulted into a specialized tube and protein, RNA or DNA are subsequently extracted for molecular profiling techniques such as gene-expression analysis or proteomics. The extraction of RNA from fresh frozen prostate tissue is particularly challenging due to its rapid degradation by RNases and low quantities of material usually obtained through LCM.In this study, we describe a protocol for the isolation of high quality RNA from fresh frozen prostate tissue including tissue handling, staining and RNA extraction kits of procured RNA Fig. .Fig. 1PrTo obtain high quality RNA from any type of tissue, optimization of steps that precede the LCM process are essential. In order to perform LCM on tissue slides, a histological stain is needed to discern the various cell types and structures present in the tissue. A widely used stain is haematoxylin, a staining method based on the reaction of haematein with DNA . Metal iThe quality of RNA was similar for cresyl violet (median RIN 7.4) and haematoxylin  stained sections. However, the morphology of the tissue was much better visualized in cresyl violet treated slides Fig. b. Studie® Micro (RNeasy), miRNeasy Mini (miRNeasy), Arcturus® Picopure® RNA isolation kit (Picopure), mirVana™ miRNA isolation kit (mirVana) and RNAqueous®-Micro (RNAqueous). Several studies have compared different RNA extraction kits on samples other than microdissected tissue [There are several commercial RNA extraction kits available that allow the isolation of RNA from tissue such as RNeasyd tissue –9. Additd tissue , 10, 11.2). Additionally, RNA was isolated from a whole tissue slice (10 µm) from the same patient in duplicate with each kit, as a control for potential LCM quality loss (n = 30). PCa cell lines PC3, LNCaP and DU145 served as a control for high quality RNA control during each isolation round (n = 20) [RNA was extracted in parallel from microdissected PCa and benign regions from three patients in duplicate using each of the 5 RNA isolation kits . Matching tissue regions (PCa and benign) were microdissected from the prostate tissue with a similar surface area (7 mm(n = 20) . The durFirst, we compared RIN values of RNA isolated with the RNeasy (median RIN 7.2), miRNeasy (median RIN 6.6), Picopure (median RIN 6.0), Mirvana (median RIN 6.5) and RNAqueous (median RIN 6.3) extraction kits, respectively . Overall, these results demonstrates that RNA isolated from LCM samples with our workflow provides high quality RNA, which could be used for further downstream analyses. A complete step by step protocol for the isolation of high quality RNA from prostate tissue is included as a supplementary file , whole tissue sections n = 12) and benign and PCa LCM material n = 24) was compared  and penicillin/streptomycin (P/S) .Radical prostatectomy (RP) specimens were derived from the archive of the department of pathology, Erasmus Medical Centre, Rotterdam, The Netherlands. All RP’s had been performed in our institute between July 2010 and May 2013, prompted by histologically proven hormone-naïve PCa on diagnostic needle-biopsy. After receipt of RP specimens at the department of pathology, a transverse tissue slice was frozen in liquid nitrogen for research purposes and evaluated by a urogenital pathologist (GvL). Patients in Erasmus MC consent with the use of remaining tissue for research purposes based on an opt-out procedure, which was approved by the local Medical Ethical Committee (MEC 2011-195 and 2011-196). The opt-out procedure is a national well-accepted regulatory method during which the patient is informed verbally and can object against the use of the remaining tissue in written form. This procedure was developed by the FEDERA  and described in an official code of conduct . Prostate cancer cells lines PC3, LNCaP and DU145 were used as a control for isolation of high quality RNA. Cell lines were obtained from the American Type Culture Collection  and maintained at 37 °C/5 % CO® Microbeam IV  at room temperature for a maximum of 1 h. Laser focus and energy settings were optimized for every slide to minimize damage from laser cutting of the tissue. Tissue was captured in AdhesiveCap500 opaque tubes , put on dry ice and stored at −80 °C.Fresh frozen prostate tissue was stored in liquid nitrogen or at -80 °C. Polyethylene naphthalate (PEN) membrane slides  were pretreated under a UV light  for 30 min. Tissues from three patients were cut in 10 µm slices in a cryostat  at −26 °C with Tissue-Tek O.C.T. Compound  and mounted on PEN slides. Slides were immediately transferred to dry ice in slide boxes , wrapped in tin foil and stored at −80 °C. One whole tissue slice was cut, transferred to a Eppendorf tube and stored −80 °C as control. For staining and dehydration of tissue, frozen slices were transferred directly to the following pre-cooled solutions on ice: 70 % ethanol for 1 min, distilled water  for 30 s, 1 % cresyl violet in 50 % ethanol  or haematoxylin  for 2 min, 70 % ethanol for 1 min and 100 % ethanol for 1 min. Slides were immediately processed with LCM using a Zeiss PALM® Micro , miRNeasy Mini , Arcturus® Picopure® RNA isolation kit , mirVana™ miRNA isolation kit  and RNAqueous®-Micro . Samples were taken from a −80 °C freezer and lysis buffer from the according RNA extraction kit was immediately added to the sample at room temperature. Buffer and tissue were mixed for 30 s through pipetting and left on the bench for 5 min to assure proper lysis. Extraction was performed according to manufacturer’s protocols with an on-column DNAse digestion step  added after the first wash buffer step for each kit. RNA quality and quantity was measured in duplicate with a 2100 Bioanalyzer and the RNA 6000 Pico Kit according to manufacturer’s protocol . An additional water wash step of the Bionalyzer electrodes was added prior to PicoChip analysis to minimize RNaseZap  associated ghost peaks.The following RNA extraction kits were used to isolate total RNA from LCM acquired material: RNeasyMedian RIN values from cresyl violet and haematoxylin LCM samples were compared with a Wilcoxon matched-pairs signed rank test. The non-parametric Mann–Whitney U test was used to compare median RIN values for RNA quality from LCM, cell lines and tissue samples from the observed differences between groups. Correlation of RIN values and RNA quantity between chips was calculated with Pearson’s r test. All graphs and statistics were performed in GraphPad Prism (version 3.0). p values <0.05 were considered significant.The data sets supporting the results of this article are included within the article and its additional files.


We report the draft genome sequence of Laverivirus UC1, assembled from San Francisco wastewater. This dicistronic RNA virus bears some similarity to dicistroviruses; however, it appears to have a unique genome organization relative to all other known RNA viruses. Solenopsis invictavirus-1 (30% amino acid identity), the ATPase/helicase of the dicistrovirus Formica exsecta virus 1 (26% amino acid identity), and the capsid protein of the dicistrovirus aphid lethal paralysis virus (23% amino acid identity) (Dicistroviridae members that have the two ORFs in the opposite configuration (The picorna-like superfamily is a taxonomic unit of positive-stranded RNA viruses with conserved RNA-dependent RNA polymerase (RdRp), capsid, and helicase proteins that infect a broad range of hosts, including animals, plants, and insects –3. While–dentity) , 5. The Dendroctonus ponderosae (–We have given the name Laverivirus UC1 to this virus due to its discovery in wastewater and the Latin root “laver” for a water basin, as well as the lack of a known host. The host organisms of dicistroviruses are arthropods, though given the unique genome organization of Laverivirus this may not be the case. Of note, the plurality of nonchordate eukaryotic reads in the non-DNAsed sample aligned to the mountain pine beetle nderosae , 8. In tnderosae –14.This viral genome was recovered from a wastewater sample that was taken on 25 January 2010, one week after a large rainstorm. This same sample also contained novel ciliate and marine RNA viruses, along with a new picalivirus –18. Samp–KF510029.The GenBank accession number for Laverivirus UC1 is


Coronaviruses are the diverse group of RNA virus. From 1960, six strains of human coronaviruses have emerged that includes SARS-CoV and the recent infection by deadly MERS-CoV which is now going to cause another outbreak. Prevention of these viruses is urgent and a universal vaccine for all strain could be a promising solution in this circumstance. In this study we aimed to design an epitope based vaccine against all strain of human coronavirus.in silico tools to predict consensus immunogenic and conserved peptide. We found that conserved region is present only in the RNA directed RNA polymerase protein. In this protein we identified one epitope WDYPKCDRA is highly immunogenic and 100% conserved among all available human coronavirus strains.Multiple sequence alignment (MSA) approach was employed among spike (S), membrane (M), enveloped (E) and nucleocapsid (N) protein and replicase polyprotein 1ab to identify which one is highly conserve in all coronaviruses strains. Next, we use various in vivo study of our identified novel peptide antigen in RNA directed RNA polymerase protein for universal vaccine – which may be the way to prevent all human coronavirus disease.Here we suggest  Coronaviruses are the diverse group of virus which infects domestic animals, birds as well as human . CoronavThe first coronavirus 229E was identified in 1960. Since then different types of coronaviruses have emerged. HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV are the coronaviruses which infect human . The firMost infections caused by human coronaviruses were relatively mild. Among all the human coronaviruses, SARS-CoV and MERS-CoV are much more deadly. The severe acute respiratory syndrome (SARS) outbreak caused by SARS-CoV in 2002 to 2003. The SARS-CoV created outbreak resulted in a total of 916 deaths among more than 8000 confirmed cases in over 30 countries ,11. The in vivo[In 2003 after the discovery of SARS-CoV, there were a significant increase in research on coronavirus, but no definitive antiviral or therapeutic treatment for coronavirus infections came from these researches . From thin vivo.The identification of therapeutics is a high priority and though there is currently no specific therapy or vaccine for human coronaviruses, this disease has been severe with a high case-fatality rate . As thesWith the disclosure of huge sequence information, epitope based vaccine design now has become a most promising approach for viral vaccine preparation . In ordeIn this study, we design an epitope based universal vaccine which can be use to prevent all kind of human coronaviruses. For this, bioinformatics analyses of viral proteins were done for finding the conserved peptide region and for mapping the evolutionary conserved epitope. The 3D structure of RNA directed RNA polymerase was determined by threading modeling technique and a highly immunogenic, accessible and conserved epitope was identified. This epitope can be used as a universal vaccine against all human coronaviruses.To find a conserved region, MSA by clustalW  and protFor vaccine design the peptide has to be immunogenic and antigenic . The conTo become a vaccine, an epitope should be accessible to the antibody. If the antibody can bind to the epitope that will be able to induce an immune response . The surThe conservancies of all epitopes were determined by IEDB conservancy analysis tools . From thHydrophilicity is desired feature of B cell epitope which indicates the accessibility of the epitope. The WDYPKCDRA epitope was found to be hydrophilic Figure  in naturAs the experimental tertiary structure of the RNA directed RNA polymerase is not available, we modeled a 3D structure by I-TASSER server  by multiCoronaviruses are one of the most diverse groups of virus which are becoming a deadly virus day by day. Though the first two strains were not so much deadly but the other members were pathogenic. After SARS outbreak, a new coronavirus strain called MERS-CoV is now going to cause another outbreak . The celThe concept of prevention of viruses by designing a universal vaccine has also been reported previously, for example against Influenza virus. In case of influenza virus, universal vaccine against matrix 2 protein which was found to be conserved among all Influenza subtypes was reported . An atteVaccine development has been one of the most important contributions of immunology to public health to date. Traditionally vaccines were based on the intact pathogen, either inactivated or live attenuated. These types of vaccines had some crucial drawbacks like safety consideration and the loss of efficacy due to the genetic variation of the pathogen. But now a day’s these vaccine concepts are greatly replaced by novel vaccine approaches like naked DNA vaccine, epitope based vaccine. The main benefit of immunization with an epitope-based vaccine is the ability to immunize with a minimal structure and it will stimulate an effective specific immune response, while avoiding potential undesirable effects .in silico analyses, the actual immunogenicity, stability, efficacy and their delivery strategy inside the recipients body can’t be determined by this in silico analysis. To address these questions in vitro and in vivo experiments are essential.In this study, we aimed to design an epitope-based universal vaccine for all human coronavirus strain. For this purpose we did multiple sequence alignment of the spike (S), envelope (E), membrane (M), nucleocapsid (N) protein and replicase polyprotein 1ab of all six human coronaviruses. Replicase polyprotein 1ab was taken to check whether there is any conservancy among the non structural protein as this replicase polyprotein cleaved into 15 non-structural proteins. In case of S, E, M and N protein, no putative conserved region was found. But conserved region was found in case of replicae polyprotein 1ab where the conserved region was in the RNA directed RNA polymerase. This indicates that this protein is less mutating than the S, E, M and N protein. This RNA directed RNA polymerase protein was targeted to determine antigenic sites based on immunogenicity and surface accessibility using different bioinformatics analyses. The consensus antigenic sites were the desired one and their conservancy was also determined. From the conservancy analysis it was found that Bepipred  and IEDBin vitro and in vivo studies can be performed.This study shows that though the human coronaviruses are not structurally related but it is possible to design an epitope-based universal vaccine for all human coronavirus strains. Our results are based on sequence analysis and computational predictions show predicted epitope would be a candidate target for the universal vaccine; and to determine the actual effectiveness of the peptide for mounting an immune response both A total available 46 replicase polyprotein 1ab, 17 spike (S) protein, 18 envelope (E) protein, 18 membrane (M) and 18 nucleocapsid (N) protein sequence data were retrieved from NCBI GenBank sequence database    and B ceTo predict the surface accessible epitope of the conserved peptide, Emini surface accessibility prediction tool  of the BThe epitope conservancy analysis tool from the IEDB analysis resource was employed for epitope conservancy prediction  of all pThe conserved epitope was then also analyzed to determine the hydrophilicity of the predicted epitopes. Parker hydrophilicity prediction tool  of ImmunAs the experimental structure of RNA directed RNA polymerase protein of any human coronavirus isolate was not found in protein data bank (PDB), a 3D structure was predicted using I-TASSER server . I-TASSEHCoV: Human coronavirus; SARS: Severe acute respiratory syndrome; SARS-CoV: SARS coronavirus; MERS-CoV: Middle east respiratory syndrome coronavirus; HCoV-229E: Human coronavirus 229E; HCoV-OC43: Human coronavirus OC43; HCoV-NL63: Human coronavirus NL63; HCoV-HKU1: Human coronavirus HKU1; ACE2: AngiotensinI-converting enzyme 2; DPP4: Dipeptyl peptidae 4.The authors declare that they have no competing interests.RS & AI performed the analysis. AI conceived the project idea. RS and AI wrote the manuscript. Both authors read and approved the final manuscript.Multiple sequence alignment of Spike (S) protein: Multiple sequence alignment of total 17 numbers of sequences of human coronaviruse isolates indicate that there is no conservation in their spike protein. This alignment was visualized by Jalview 2.8  and coloClick here for fileMultiple sequence alignment of envelope (E) protein: Figure legend as in supplementary Figure S1.Click here for fileMultiple sequence alignment of membrane (M) protein: Figure legend as in supplementary Figure S1.Click here for fileMultiple sequence alignment of nucleocapsid (N) protein: Figure legend as in supplementary Figure S1.Click here for fileConserved peptide found in RNA directed RNA polymerase by multiple sequence alignment of replicase polyprotein 1ab: All human coronaviruses are found to be conserved in their replicase polyprotein 1ab.Click here for fileRamachandran plot for RNA directed RNA polymerase protein: Red colored region is the most favored region, brown and yellow colored regions are additionally allowed region and generously allowed regions respectively.Click here for fileSequence sources and other sequence related information.Click here for file


RNA hairpins are a common type of secondary structures that play a role in every aspect of RNA biochemistry including RNA editing, mRNA stability, localization and translation of transcripts, and in the activation of the RNA interference (RNAi) and microRNA (miRNA) pathways. Participation in these functions often requires restructuring the RNA molecules by the association of single-strand (ss) RNA-binding proteins or by the action of helicases. The Drosophila MLE helicase has long been identified as a member of the MSL complex responsible for dosage compensation. The complex includes one of two long non-coding RNAs and MLE was shown to remodel the roX RNA hairpin structures in order to initiate assembly of the complex. Here we report that this function of MLE may apply to the hairpins present in the primary RNA transcripts that generate the small molecules responsible for RNA interference. Using stocks from the Transgenic RNAi Project and the Vienna Drosophila Research Center, we show that MLE specifically targets hairpin RNAs at their site of transcription. The association of MLE at these sites is independent of sequence and chromosome location. We use two functional assays to test the biological relevance of this association and determine that MLE participates in the RNAi pathway. In virtually all RNA molecules, single stranded regions undergo complementary base-pairing with neighboring regions to form double-stranded structures called stem-loops or hairpins. During the fundamental processes of transcription and translation, these RNA structures are reshaped by helicases—enzymes that separate paired regions of nucleic acids. In addition, small non-coding RNA molecules involved in regulating gene expression rely on sequences that allow them to form hairpin structures. In this paper we present evidence that the Drosophila helicase MLE, well known for its role in dosage compensation, participates in the processing of hairpin RNAs in the pathway that leads to induced RNA interference. Hairpins can display a high level of heterogeneity in stem length, loop size, the number of bulges or internal loops present in the stem, and their thermodynamic properties . Their frocesses . Even whrocesses ,10. The rocesses .in vitro [in vivo functions of MLE have been reported. In the first, a mutant allele of mle, mlenapts, results in a paralytic phenotype due to a splicing abnormality of the para mRNA in a portion of the transcript subject to RNA editing [Helicases are ubiquitous enzymes that participate in all of the steps related to nucleic acid metabolism. Maleless (MLE), a Drosophila helicase, exhibits single-stranded RNA or DNA binding activities and is an RNA:DNA helicase/adenosine triphosphatase (ATPase) in vitro . Ortholo editing . A sugge editing , where i editing ,16. Rece editing .in vivo. Our results suggest that MLE may play a broad and significant role in the structure-function relation of regulatory RNAs during development and differentiation.The ability of the MLE helicase to interact with RNA polymerase II (RNAPII) , and itsIn addition, our results have direct relevance to mammalian RNAi. The mammalian ortholog of MLE, RHA/DHX9, was shown to play a role in RNA interference ,26. ThisHrb87F RNAi stock containing an integrated pValium1 plasmid expressing a dsRNA under the control of an inducible UAS-promoter [Hrb87F RNAi plasmid  only when its transcription was activated by an Actin5C-Gal4 driver carried on the second chromosome . These studies used an promoter . MLE staromosome . MLE wasromosome . Thereforomosome . Curiousromosome . Hrb87F elomeres ; therefoion site . SurprisHrb87F gene and one targeting mof, the gene that encodes the histone acetyl transferase present in the MSL complex in males, but that is also found in both sexes [Hrb87F RNAi line and the mof RNAi line, indicating that its localization is independent of the sequence of the dsRNA being transcribed. Moreover, the successful MOF knock down  with stems ranging from 331 to 570 bp . We nextIt is possible that MLE is recruited to the integration site of the plasmid by a DNA alternative structure formed during the inverted repeat’s transcription and not by the architecture of the transcript. Therefore, in order to establish the RNA-specific nature of the binding, we incubated polytene chromosomes with RNase A. We chose to perform this experiment on male larvae in order to use the RNA dependent localization of MLE on the X chromosome as a positive control for the RNase treatment. As previously reported , cell peA parallel treatment with RNase III, which specifically cleaves dsRNA, did not result in a measurable effect on MLE localization . Howeverwhite intron and it was then amplified with two different primers sets targeting the stem region  experiment followed by a qRT-PCR reaction. Extracts from larvae expressing Hrb87F lhRNA were used for immunoprecipitation with either anti-MLE or generic IgG antibodies. The immunoprecipitated RNA was isolated and quantified by gene-specific qRT-PCR with IgG as a negative control. In this reaction, the cDNA was obtained using a primer targeting the loop structure formed by the m region . Hrb87F Hrb87F hairpin RNA did not show any binding of Rm62 helicase at the integration site of the plasmid, although the protein is present at other sites such as developmental puffs  in nearly 100% of the 260 wings examined, with the remaining wings showing an even more severe phenotype (class 6) . Critically, in an mle1/mle1 mutant background we observed a substantially less severe Notch RNAi phenotype: all 115 wings analyzed retain most of their margins, with 70% of them exhibiting extensive notching (class 4) and approximately 30% showing mild or no notching (class 2 and 3). In this case, as well, the difference is highly significant . To test whether the effect of MLE on the wing phenotype induced by Notch RNAi was due to the specific mle allele used, females with an mle1/mleY203 heteroallelic combination were obtained. Here again the absence of MLE leads to an improvement in wing development . These results suggest that MLE normally enhances the efficiency of the RNA machinery.The ability of MLE to target hairpin RNAs at their sites of transcription and our published observations that MLE interacts with the components of the RNAi machinery, Argonaute 2 and Dicer-2 , led us ng blade ,23. In aclass 6) . This reckground , suggestelopment  that is Egfr  dsRNA synthesis in the wing with the ms1096-GAL4 driver. Egfr is involved in wing vein development [ms1096-GAL4 leads to profound adult vein defects. By classifying the observed vein phenotypes according to the number of veins affected we defined the 7 classes listed in ms1096-Egfr RNAi wings examined (n = 92) were concentrated in the 3 classes with the most severe phenotypes  and no wings in class 2 (the least severe phenotype) were detected. In contrast, in an mle1/mle1 mutant background, more than 80% of the ms1096-Egfr RNAi wings (n = 86) fall in the 3 mildest phenotypic classes ; no wings belonging to class 7 were found. The observed effect of the absence of MLE on the Egfr RNAi phenotype is significant .In order to demonstrate that this effect is reproducible with other dsRNAs, we performed a similar experiment by driving elopment , and itsIn vitro, it was shown to unwind short double-stranded RNA or RNA/DNA substrates [in vivo RNA remodeling properties was offered by the phenotype of the mlenapts allele, which suggested that MLE may play a role in resolving loop structures in the para gene transcript [MLE is a Drosophila helicase required for dosage compensation in males. bstrates . An earlanscript . Such a anscript ,16. As Manscript , we askeovo gene null allele have no or very few germ cells. Introduction of the mle1 mutation largely restores the germ line in these females [Argonaute 1 (Ago 1), Dicer-1 (Dcr-1), pasha and Drosha—interrupt germ-line cell division and oocyte formation [Of some interest is a set of published observations that may suggest a link between an effect of MLE in females and some observed phenotypes of mutants in the RNAi pathway. Adult females homozygous for an  females . Mutatioormation . Connectormation . In maleormation . Recentlormation . AlthougMLE’s targeting the transcripts of RNAi transgenes indicates that it may play an active role in the RNA interference pathway. In support of such a potential role, we have demonstrated that MLE is required for optimal RNAi efficiency. RNA helicase A (RHA/DHX9), the mammalian ortholog of MLE, plays a role in RNA interference. RHA associates with Ago 2 and Dicer in human cells , it is aAnother possibility is that MLE interferes with the editing activity of the ADAR enzyme. ADAR and the RNAi pathway are in an antagonistic relationship, likely due to the fact that they compete for the same substrates ,46. ADARAll the discussed scenarios are not mutually exclusive. Therefore, it is not unreasonable to consider that MLE could play a role in the Drosophila immune response against inverted repeat viruses, as well as in retrotransposon silencing and in the regulatory pathways controlled by the recently identified endogenous hairpin RNAs ,44. MoreHrb87F RNAi), 31401 (mof RNAi), 32872 (Dp1 RNAi), 32944 (Pep RNAi), 34665 (Chd1 RNAi), 28981 (Notch RNAi), 25781 (Egfr RNAi), 31603 (Luciferase RNAi), 35789, and 35788 (Luciferase overexpression) were obtained from the Bloomington Drosophila Stock Center (BDSC). TRiP RNAi stocks 26772 (CG14962 RNAi), 25993 (CG4617 RNAi) and 31922 (CG3838 RNAi) were a gift from B. Yedvobnick and are available at BDSC. Stocks v107001 (Jil1 RNAi) and 31994 (Hp1 RNAi) were obtained from the Vienna Drosophila Resource Center (VDRC). The C96-GAL4, (stock 43343) and the Actin5C-Gal4 (stock 3954) drivers were obtained from the BDSC and the ms1096-GAL4,UAS-GFP driver was a gift from K. H. Moberg.TRiP collection stocks 31244 and 31472 . Wings from adult females were removed and mounted in Euparal mounting medium (BioQuip Products). The Chi-square test was used for statistical analysis.To test the effect of MLE on the wing phenotype induced by RNA was isolated from 10 larvae per sample using the Qiagen RNeasy mini-kit following manufacturer’s instructions. iScript One-step RT-PCR kit with SYBR Green (BIO-RAD) was used for the real-time, reverse transcription-PCR. Transcription measurements were normalized to the Pka-C1 transcript and the ddCt method was used to calculate the fold difference. The results of three independent biological replicates were averaged. The primers used to detect Luciferase are: forward 5’-AGGTTCCATCTGCCAGGTATCAG-3’ and reverse 5’-ACACACAGTTCGCCTCTTTGATTAAC-3’. The primers used to detect Pka-C1 are: forward 5'-TTCTCGGAGCCGCACTCGCGCTTCTAC-3' and reverse 5'-CAATCAGCAGATTCTCCGGCT-3'Larvae were frozen at -80°C, thawed and homogenized in 100 μl of Promega Passive lysis buffer. The lysates were then frozen at -80°C and thawed at 37°C for three times then were spun at 13000 rpm for 5’. 20 μl were used for the luciferase assay according to manufacturers protocol. Relative light units (RLU) were normalized for protein content. The results are the average of three independent biological replicates50 of larvae per sample were homogenized in 1ml of lysis buffer containing 25 mM Tris-HCL pH 7.5, 300 mM NaCl, 2mM MgCl2, 5mM DTT, 0.5% NP40, protease inhibitor , and RNasin  and incubated on ice for 10 min. Lysates were then sonicated at 0-3W and 2V for 27 seconds x 4 on ice before spinning at 14000 rpm for 10’ at 4°C. The supernatant was pre-cleared with Dynabeads Proteinase G  for 1h at 4°C. 10% of the pre-cleared lysates was saved for protein quantitation for final normalization and the remaining 90% then incubated with 50 μl of Dynabeads Protein G slurry and equal volumes of either pAb-anti-MLE, or generic anti-mouse IgG (Jackson IR) antibody as IP control, for 1-2h at 4°C. After being washed five times with lysis buffer, the IPed beads were digested with RNase-free DNase RQ1  and RNasin in digestion buffer at 37°C for total 30min to remove genomic DNA resulting from non-specific binding. After washing with lysis buffer, the DNase-digested beads were treated with Trizol  and chloroform to extract the IPed RNA that was precipitated with glycogen, 3M NaAc, and isopropanol O/N at -80°C followed by spinning down at maximum speed, at 4°C, for 20 min. After washing with 75% ethanol, the IPed RNA pellets were resuspended in nuclease-free water for reverse transcription using SuperScript III reverse transcriptase  to generate the first strand cDNAs using the primer 5’-TGAGTTTCAAATTGGTAATTGGACCCT -3’. The first strand cDNAs were applied as templates for real-time PCR using SYBR green and the following set of primers were used: Hrb87F-P1 forward 5’-TGCTTGGCAATAGCCTTCTTCA-3’, Hrb87F-P1 reverse 5’-TCGATGACTACGATCCCGTTGACA-3’, Hrb87F-P3 forward 5’-GCATTGTCGATCATGTACGACT-3’, Hrb87F-P3 reverse 5’-CTTCGGTTTCATCACGTACT-3’. Three independent biological replicates are presented for P1 and P3 primer pairs.The MLE-IP efficiency and the lhRNA enrichment in the MLE-IPed RNAs were calculated based on two levels of normalizations. First, concentrations of the crude protein in the starting lysates were normalized to make sure equal amount of protein were used for both the MLE-IP and the IgG-IP. Second, the average Ct numbers from the IgG-IPs were used for normalization to compare the lhRNA enrichment by the MLE-IP relative to the IgG-IP, expressed in terms of fold difference 2^-dCt.Salivary glands from third instar larvae were dissected in PBS buffer and fixed for 10’ with 2% formaldehyde in 45% acetic acid. After squashing, the slides were frozen in liquid nitrogen, washed twice in TBS-Triton 0.1% and incubated in TBS-Triton containing 5% milk for 30’. Polytenes preparations were then incubated overnight with primary antibody in humid chambers at 4°C, washed twice in TBS-Triton 0.1% and incubated with a secondary antibody at room temperature for 1hr. After washing twice in TBS-Triton 0.1% the slides were mounted using VECTASHIELD mounting medium with DAPI. For the RNase A and RNase III treatments, salivary glands were dissected in PBS, transferred to PBS-Triton 0.1% for 5’ and incubated in PBS with or without 0.5 mg/ml RNase A (Qiagen) for 10’. Primary antibodies were used at the following concentrations: MSL1 (1:300), MSL3 (1:50), MLE (1:300), MOF (1:50), GAL-4 by Babco/BioLegend (1:50), Rm62, a gift from E. Lei, (1: 100). For the co-staining experiments an MLE antibody raised in Guinea pig was used at 1:50. The secondary antibodies are: Rodamin Red-X anti-Rabbit (Jackson IR) (1:500) and FITC anti-Guinea pig (Jackson IR) (1:500).The quantitative fluorescence analysis of polytenes treated with RNase A was performed using imageJ software. For each polytene chromosome analyzed, three areas adjacent to the fluorescent band were selected to measure background levels, the mean background fluorescence was multiplied by the area of the fluorescent band and subtracted from the integrated density of the band in order to obtain the corrected total band fluorescence (CTBF). The T-Test was applied for statistical validation.S1 FigHrb87F (Hrb87F RNAi). Co-staining with Guinea-pig anti-MLE and respectively rabbit anti-MSL1, MSL3 or MOF. The white arrows indicate the integration site of the plasmid.Polytene chromosomes from male larvae expressing a dsRNA targeting (PDF)Click here for additional data file.S2 FigHrb87F (Hrb87F RNAi). MLE staining results in a clear signal at the integration site of the plasmid, indicated by a white arrow, and at the 3R telomere, indicated by an asterisk.Polytene chromosomes from female larvae expressing a dsRNA targeting (PDF)Click here for additional data file.S3 FigHrb87F (Hrb87F RNAi) has been induced by an Act5C-GAL4 driver on the third chromosome. MLE staining results in a clear signal at the integration site of the plasmid expressing the dsRNA. The white arrows indicate the plasmid integration site.Polytene chromosomes from female and male larvae in which the expression of a dsRNA targeting (PDF)Click here for additional data file.S4 FigPolytene chromosomes from male RNAi larvae co-stained with anti-MLE and anti-MOF. MLE is present at the integration site of the plasmid (white arrow) and at the X-chromosome in both Hrb87F RNAi and MOF RNAi larvae. MOF is present on the X-chromosome of Hrb87F RNAi larvae while it is completely absent in MOF RNAi larvae, indicating that it was successfully knocked down.(PDF)Click here for additional data file.S5 FigMLE staining of polytene chromosomes from female larvae expressing three different dsRNA inserted in a pValium10 plasmid.(PDF)Click here for additional data file.S6 Fig(A) MLE and MSL1 co-staining of polytenes chromosome from male larvae expressing a dsRNA targeting Hrb87F induced by an Act5C-GAL4 driver. Incubation with RNaseA reduces MLE’s enrichment at the site of hairpin transcription. The white arrows indicate the integration site of the plasmid. (B) Magnification of the area indicated by the arrow in portion (A) of the figure. (C) Quantitative analysis of fluorescence levels. MLE signal at the integration site of the plasmid, expressed in terms of corrected total band fluorescence (CTBF), is significantly reduced after RNase A treatment . The analysis was performed on 6 polytene chromosomes treated with RNase A and 8 control chromosomes.(PDF)Click here for additional data file.S7 FigHrb87F induced by an Act5C-GAL4 driver. Incubation with RNase III does not appear to affect MLE’s enrichment at site of hairpin transcription. The white arrows indicate the integration site of the plasmid.MLE staining of polytenes chromosome from male larvae expressing a dsRNA targeting (PDF)Click here for additional data file.S8 FigHrb87F induced by an Act5C-GAL4 driver. (A) MLE is highly enriched at the integration site of the plasmid (indicated by the white arrow) while Rm62 does not appear to be enriched at the same site. (B) MLE and Rm62 are both enriched at developmental puffs.MLE and Rm62 co-staining of polytene chromosomes from female larvae expressing a dsRNA targeting (PDF)Click here for additional data file.S9 FigC96-GAL4. No difference is observed between the homozygous mutant mle1/mle1 and the heteroallelic combination mle1/mleγ203. The slight difference in phenotype distribution observed in the mle1/mle1 flies versus the one reported in Wing phenotypes from female flies, raised at room temperature, in which Notch dsRNA expression is induced by (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex.Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the  The singular configuration of the bases in the U-turn loop structure is thought to facilitate the initial base-pairing and the progression of the intermolecular helix, causing this motif to act as a general enhancer of RNA/RNA binding rates  half-life , and it determines strong incompatibility against pMV158  antisense RNA. These findings suggested that a “kissing” step was not strictly required for the RNA pairing mechanism in pMV158  was employed as the host for all the in vivo assays. Pneumococcal cells were grown and transformed as described  sequencing gels. All oligonucleotides employed in this work are listed in Supplementary Table Oligonucleotides (oligos) used for amplification of DNA, S. pneumoniae  from cells harboring plasmids were used to determine plasmid copy number as described . DNA concentration was quantified using a NanoDrop™ spectrophotometer.Enzymes for the modification of the DNA  as well as DNA polymerases were purchased from New England Biolabs and Fynnzymes, respectively, and used as specified by the suppliers.Total RNAs were prepared from exponentially growing pneumococcal cultures harboring the different pCGA9 plasmid variants , essentially as described  program Zuker, 1.rnaII gene of pCGA9  employed for the binding experiments. DNA templates encoding the different antisense RNA species were generated by extension of the common primer P-RNAII annealed to the following 83-mer oligos . Samples were cleaned up by filtration through Sephadex G-25 columns  and successive extraction with phenol/chloroform and chloroform, followed by ethanol precipitation. RNAs were quantified from their absorbance at 260 nm in a NanoDrop™ spectrophotometer.Double-stranded DNAs containing an improved promoter for T7 RNA polymerase (T7 RNAP)-mediated transcription  following the manufacturer's indications. Then, the full-length RNA molecules were isolated from 12% PAA sequencing gels, extracted with phenol/chloroform and chloroform, and ethanol-precipitated, previous to the labeling of their 5′-end with T4 polynucleotide kinase (New England Biolabs) and [γ−32P] ATP. Labeled RNAs were re-isolated from 12% PAA sequencing gels as above.The three 17-mer oligos  in TMN buffer 2. Binding was allowed for 30 min at 37°C, and terminated by adding an equal volume of loading buffer  and placing the mixture on ice. The samples were applied to non-denaturing 10% PAA gels. Gels were run at 300 V for 2 h.Previous to the binding reaction, the antisense and target RNAs were incubated separately at 65°C for 10 min and then at 37°C for 10 min, in order to obtain their native conformation. The binding mixtures (10 μl) contained 0.2 nM of any of the 5′-end-labeled antisense RNA variants and various concentrations (0–400 nM) of the unlabeled target RNA (mRNAdK) was obtained with the Sigmaplot 9.0 software from data point fitting to the equation:Radioactive bands were visualized and quantified from fixed and dried gels using the storage phosphor technology, with the aid of a FLA-3000 (FUJIFILM) imaging system and the Quantity One software (Bio-Rad). The mRNA concentration required for half-maximal binding under the described experimental conditions  was calculated as follows  on ethanol-dry ice. Sample electrophoresis as well as visualization and quantification of the bands were performed as above. Least-squares analysis of the data from a log-linear plot of the fraction of free antisense RNA as a function of the incubation time yielded a straight line, at least until ~10% of the antisense RNA remained unbound. The slope of the curve was used to obtain the pseudo-first-order rate constant (b). The second-order rate constant  was employed to provide the host machinery needed for protein synthesis. Previous to the addition of the antisense and messenger RNAs, translation mixtures were prepared as directed by the manufacturers, maintained at 0°C, and treated with rifampicin (25 μg/ml) to prevent subsequent transcription from DNA traces that might be present in the mRNA preparation. Then, mRNA (2.5 μg) synthesized from pALTER-T7-copG-repB-cat (see above) and one of the antisense RNA variants (at a 20-Fold molar excess over the mRNA) were added, and the translation mixtures were incubated at 37°C for 90 min. De novo-synthesized proteins, labeled with [35S]-methionine, were separated on 15% SDS-PAA gels. Radioactivity in the bands was detected and quantified as described above. Values of the radioactivity in RepB were normalized with respect to those in CopG, whose synthesis is not regulated by the antisense RNA. Similar results were obtained when the values were normalized with respect to the levels of T-Cat.To test whether wt and mutant antisense RNAs exert incompatibility toward the pMV158 replicon, type I quantitative incompatibility tests  cells was analyzed as above after each 10 generations.Whenever the presence in vitro synthesized antisense RNA variant were hybridized with the appropriate 5′-end-labeled oligo, and the samples were processed and analyzed in the same way as those containing the cellular RNA.To determine the relative intracellular amount of the different antisense RNA variants, “oligo-RNA band-shifts” experiments were performed basically as described  harboring 8-nt sequence substitutions in different elements of the RNAII secondary structure have been synthesized both s Figure  correspos Figure . All theCis-encoded antisense RNAII is complementary to the untranslated intergenic region of the bicistronic copG-repB mRNA  by T7 RNAP-mediated run-off transcription of the intergenic region of the copG-repB operon  or 80 nt (mRNA80) on the copG-repB mRNA. However, run-off target products were longer-than-expected and contained 3′ extensions that arose most likely from the RNA-dependent polymerase activity of T7 RNAP and were involved in terminal hairpins , we studied the efficiency of formation of RNA-RNA complexes that persist throughout electrophoresis on non-denaturing PAA gels. Two parameters were determined to analyze the efficiency of RNA-RNA binding: the concentration of free target RNA for half maximal binding (dK) and the association rate constant (ak).To know the contribution of the intermolecular pairing through different RNAII regions to the binding with the target RNA (mRNARNA-RNA binding efficiency was most impaired by substitution of the RNAII region-2 (antisense RNA m2), which resulted in a 45-Fold increase in the concentration of free mRNA for half-maximal binding, and a 200-Fold decrease in the association rate constant  only slightly impaired the inhibitory ability of the antisense RNA. In contrast, significant levels of RepB were synthesized in the presence of the m1 or m2 mutant RNAs, indicating that substitution of region-1 or, particularly, of region-2 resulted in a severe decrease of the inhibitory activity of the antisense RNA , and the DNA of this plasmid could not be detected in any of the clones  of cells containing the resident plasmid after ~37 generations, and again no detectable pLS1 DNA in the bacterial extracts  of the cells containing pCGA9-m3, and its average copy number was estimated to be ~3 , was a goal. Therefore, investigation on the participation of the RNAII 3′ U-tail is missing in the present work, although no indications of a main role of the equivalent region have been reported for other antisense RNA systems  or even into the lower portion (as in pIP501) of the stems  might account for the ~2-3-Fold higher efficiency of binding of the antisense RNA to mRNA80 relative to mRNA60  or to full-length copG-repB mRNA (in vitro translation experiments). These findings contrast with those reported for the antisense control system of ColE1, where inhibitory binding of the antisense RNA occurs when nascent pre-primer RNA is 100-150 nt long. Once the pre-primer has grown longer, binding of the antisense RNA is still possible, but it does not prevent folding of the pre-primer RNA into the set of secondary and tertiary structures that are crucial for primer maturation  antisense RNAII (48-nt long) is the smallest characterized so far; (ii) contrarily to other systems that also involve pairing of complementary sequences located on cognate secondary structure elements of the inhibitor and target RNAs, formation and progression of a “kissing complex” is not a critical step in this plasmid; (iii) efficient binding to the mRNA requires intermolecular pairing through the RNAII region-2 5′-adjacent to the hairpin; (iv) in contrast to what has been reported for other systems (Wagner and Simons, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae. While not capable of depositing histones onto DNA on its own, Hif1p can act in conjunction with a yeast cytosolic extract to assemble nucleosomes onto a relaxed circular plasmid.Hif1p is an H3/H4-specific histone chaperone that associates with the nuclear form of the Hat1p/Hat2p complex (NuB4 complex) in the yeast To identify the factor(s) that function with Hif1p to carry out chromatin assembly, multiple steps of column chromatography were carried out to fractionate the yeast cytosolic extract. Analysis of partially purified fractions indicated that Hif1p-dependent chromatin assembly activity resided in RNA rather than protein. Fractionation of isolated RNA indicated that the chromatin assembly activity did not simply purify with bulk RNA. In addition, the RNA-mediated chromatin assembly activity was blocked by mutations in the human homolog of Hif1p, sNASP, that prevent the association of this histone chaperone with histone H3 and H4 without altering its electrostatic properties.These results suggest that specific RNA species may function in concert with histone chaperones to assemble chromatin. Our understanding of chromatin assembly has grown rapidly in the past several years. It is now apparent that in addition to the assembly of chromatin structure that occurs during DNA replication, which is known as replication-coupled chromatin assembly, a significant amount of chromatin assembly also occurs outside of S-phase and is known as replication-independent chromatin assembly (or histone exchange)While many proteins have been found to participate in chromatin assembly, it is likely that additional factors have yet to be identified. This is particularly apparent in yeast, where many of the proteins that are thought to play central roles in the histone deposition process are not essential for viability. For example, the CAF-1 and HIR complexes are histone H3/H4 chaperones that are integral components of the replication-coupled and replication-independent chromatin assembly pathways, respectively. Genes encoding the subunits of these complexes can be deleted both individually, and in combination, without a complete loss of viabilityIn vivo, Hif1p has been shown to be involved in the reassembly of chromatin structure that occurs following the recombinational repair of a DNA double strand break and some of its involvement in this process is independent of Hat1pin vitro experiments demonstrated that Hif1p is not capable of depositing histones onto DNA alone but can facilitate the deposition of histones on a closed circular plasmid in the presence of a yeast cytosolic extractHif1p, which shows sequence similarity to the N1/N2 family of histone chaperones, is a histone chaperone with specificity for histones H3 and H4Recombinant Hif1p can specifically interact with histone H3/H4 complexes but is not capable of depositing the H3/H4 complexes onto DNA . However, the addition of a cytosolic extract allows rHif1p to facilitate histone deposition. This suggests that the cytosolic extract contains a factor (or factors) that function in conjunction with rHif1p in the deposition of histones.hif1Δ yeast spheroplasts that are osmotically disrupted in buffer with a low concentration of salt (50 mM NaCl). As seen in The cytosolic extract used in these experiments is generated from The synergistic activity seen between Hif1p and the cytosolic extract was, in some ways, similar to the increased levels of chromatin assembly that are seen when Asf1p is assayed with either CAF-1 or the HIR complexWe used conventional protein purification techniques to isolate the cytosolic extract factor(s) biochemically. The cytosolic extract was loaded onto a DEAE Sepharose anion exchange column equilibrated in buffer containing 50 mM NaCl. Proteins that flowed through the column (FT) were collected and then the column was washed and successively step eluted with buffers containing 100 mM NaCl, 250 mM NaCl and 500mM NaCl. Hif1p-enhanced the chromatin assembly activity that was found in the FT fraction but not in any of the step elution fractions . AdditioAs the chromatin assembly activity flowed through a DEAE Sepharose column at low salt concentration, we applied this fraction to a carboxymethyl (CM) Sepharose cation exchange column. The FT fraction (50 mM NaCl) was collected along with 100 mM NaCl, 250 mM NaCl, 500 mM NaCl and 1.0 M NaCl step elution fractions. Again, all of the chromatin assembly activity was found in the FT fraction . The matThe observation that the chromatin assembly activity in the yeast cytosolic extracts did not bind to either DEAE Sepharose, CM Sepharose or Heparin Sepharose columns in low salt buffer was somewhat unusual behavior for a protein. To confirm that the chromatin assembly activity was protein-based, the material that had passed through the DEAE, CM and heparin columns was boiled and the denatured proteins removed by centrifugation. Surprisingly, this had little effect on the chromatin assembly activity . To detein vitro. A crucial question to address was whether this was an effect of bulk RNA or whether specific species of RNA were responsible for the chromatin assembly activity. To address this issue, we determined whether the RNA-mediated chromatin assembly activity fractionated as a discreet entity or whether it co-eluted with bulk RNA. The material that flowed-through the DEAE, CM and heparin columns was briefly incubated at 95 degrees and denatured proteins were removed by centrifugation. The soluble fraction was then resolved on a Superose 6 column. Fractions from the column were then phenol/chloroform extracted and nucleic acids were isolated by precipitation. The nucleic acids were then resuspended and assayed for chromatin assembly activity in both the absence and presence of rHif1p. As seen in The results shown in To further test whether the chromatin assembly activity was due to the presence of specific RNA species or whether it was attributable to a bulk RNA effect, the isolated RNA that flowed through the DEAE, CM and heparin columns was resolved by polyacrylamide:urea electrophoresis. The gel lane was then cut into a series of 1.0 cm sections and the RNA was eluted from each. Aliquots from each pool of RNA were then either re-run on a gel or assayed for chromatin assembly activity. As seen in As is the case for most histone chaperones, Hif1p is an acidic protein that contains a large domain that is highly enriched in glutamic acid and aspartic acid residues. Hence, the increased level of chromatin assembly seen when Hif1p is added to the cytosolic extract (or isolated RNA) may simply be a function of Hif1p providing additional negative charge. To test this electrostatic model, we used the human member of the N1/N2 family of histone chaperones, Nuclear Antigenic Sperm Protein (NASP)NASP was originally identified as a histone H1 chaperone but was subsequently shown to also interact specifically with H3/H4 complexesIn 1981, Brutlag and colleagues reported that RNA can mediate chromatin assemblyBased on our results, we would like to argue that this prevailing view should be challenged and that the possibility that specific RNA molecules can function in the cell to mediate chromatin assembly should be considered. A number of observations indicate that the RNA-dependent chromatin assembly activity that we have detected is not due to a non-specific effect of bulk RNA. First, unlike the RNA-dependent activity from Drosophila embryos, the activity that we have isolated does not bind to a DEAE column at low salt, as would be expected for the majority of RNA in a cytosolic extract. Second, a highly purified sample of this activity resolves on a gel filtration column as a distinct entity. The fact that the peak of this activity did not coincide with the elution pattern of bulk RNA strongly argues that a specific RNA (or group of RNAs) is capable of functioning as a chromatin assembly factor. Third, the RNA-dependent activity that we have isolated can function in conjunction with protein-based histone chaperones. While the RNA-based activity could generate nucleosomes on its own, the rate at which nucleosomes are formed is significantly enhanced by the presence of the histone chaperones. Importantly, the ability of histone chaperones to function with this RNA-based activity is sensitive to mutations in the histone chaperones that do not alter their electrostatic properties. Finally, our understanding of the breadth of functions that are performed in the cell by RNA has exploded in the decades since RNA was first shown to mediate chromatin assembly in vitro. For example, it is now abundantly clear that specific RNA molecules play a critical role in the targeting and function of chromatin modifying factors at specific sites in the genome600∼0.5) and harvested by centrifugation (∼4000×g for 5 minutes)2). Lysed spheroplasts were centrifuged at 1500×g for 15 minutes to pellet nuclei and cell debris and the supernatant used as the cytosolic extractYeast strain XAY4 (Hif1Δ) was grown to mid-log phase (ODRecombinant Hif1p was produced as describedSupercoiled pUC18 plasmid DNA was isolated from E. coli and relaxed with topoisomerase I using manufacturers instructions (Sigma). In vitro chromatin assembly assays were performed in a total volume of 25 µL. The assay buffer contained 10 mM Tris (pH8.0), 1 mM EDTA, 100 mM NaCl and 100 µg BSA. Reactions also contained 0.2 µg relaxed pUC18 DNA, 0.5 to 1.0 µg purified chicken erythrocyte histones and rHif1p and yeast cytosolic extract or column fraction as appropriate. Reactions were incubated at 37 degrees for 1 hour and then stopped by the addition of 6.25 mL of 5× stop buffer  and an additional 30 minute incubation at 37 degrees. DNA from each reaction was then isolated by phenol/chloroform extraction followed by ethanol precipitation. DNA was resolved on a 1.5% agarose gel run overnight at 12V. DNA was then visualized by staining with Sybr Gold.Cytosolic extract was diluted with DN(0) buffer to match the conductivity of DN(950) buffer and then clarified by centrifugation at 16,000×g for 10 minutes . Clarified extract was applied to 25 mL DEAE Sepharose column (using an AKTA Purifier) and the column was extensively washed with DN(50). The column was then successively step eluted with DN(100), DN(250) and DN(500). Material that flowed through the DEAE column (FT) was dialyzed against 50 mM HEPES (pH 6.0), 50 mM NaCl buffer and applied to a 5 mL carboxymethyl Sepharose column (CM). After washing, the CM column was successively eluted with buffer containing 100 mM, 250 mM, 500 mM and 1 M NaCl. The FT from the CM column was directly applied to a 1 mL Heparin Sepharose column. Isolated RNA was resuspended in TE and 250 µL was applied to a 25 mL Superose 6 column, which was run at 0.3 mL per minute and 1 mL fractions were collected.RNA was isolated from extracts or column fractions by phenol/chloroform extraction followed by ethanol precipitation. RNA was isolated from gels following electrophoresis of RNA samples on a 6% polyacrylamide/8M urea gel. Gels were visualized with Sybr Gold. Gel slices were excised with a razor blade and crushed in a microfuge tube. Gel pieces were incubated with 1.0 mL of 0.3 M NaCl overnight at 4 degrees on a rotator. Supernatant was separated from the gel pieces and RNA precipitated with isopropyl alcohol.