Borrelia burgdorferi must differentially express genes and proteins in order to survive in and transit between its tick vector and vertebrate reservoir. The putative DEAH-box RNA helicase, HrpA, has been recently identified as an addition to the spirochete's global regulatory machinery; using proteomic methods, we demonstrated that HrpA modulates the expression of at least 180 proteins. Although most bacteria encode an HrpA helicase, RNA helicase activity has never been demonstrated for HrpAs and the literature contains little information on the contribution of this protein to bacterial physiology or pathogenicity. In this work, we report that B. burgdorferi HrpA has RNA-stimulated ATPase activity and RNA helicase activity and that this enzyme is essential for both mammalian infectivity by syringe inoculation and tick transmission. Reduced infectivity of strains carrying mutations in the ATPase and RNA binding motif mutants suggests that full virulence expression requires both ATPase and coupled helicase activity. Microarray profiling revealed changes in RNA levels of two-fold, or less in an hrpA mutant versus wild-type, suggesting that the enzyme functions largely or exclusively at the post-transcriptional level. In this regard, northern blot analysis of selected gene products highly regulated by HrpA  suggests a role for HrpA in the processing and translation of transcripts. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that the post-transcriptional regulatory functions of this enzyme are essential for maintenance of the Lyme disease spirochete's enzootic cycle.The Lyme disease spirochete  Borrelia burgdorferi, must differentially express genes and proteins in order to survive in and transit between its tick vector and animals that it infects. RNA helicases, enzymes that unwind double-stranded RNA, have recently emerged as major players in all types of processes involving RNA in higher organisms. But in spite of the ubiquitous presence of RNA helicases in bacteria, little is known regarding their function. The Lyme disease spirochete, which has a complex lifecycle involving ticks and vertebrate animals, encodes a single putative RNA helicase, HrpA. Here we establish that the purified protein indeed displays both ATPase and RNA helicase activity. In addition to being the first demonstration of RNA helicase activity for a bacterial HrpA, our data indicate that HrpA is involved in RNA processing of some genes. Our findings also show that HrpA is essential for both tick transmission and mouse infection and establish the RNA helicase as an important component in both parts of the spirochete's lifecycle.The bacterium causing Lyme disease,  Borrelia burgdorferi and related species. B. burgdorferi is maintained in nature by a complex enzootic cycle that involves ticks as vectors and vertebrate animals as reservoir hosts. Survival in the very disparate arthropod and animal environments demands changes in the expression of numerous genes in a precise manner 70) in the RNA polymerase holoenzyme to effect transcription in response to environmental signals perceived during tick transmission and the mammalian phase of the enzootic cycle Lyme borreliosis is the most prevalent vector-transmitted disease in the northern hemisphere and has a significant impact on human health see . The disB. burgdorferi. However, a small RNA (DsrA) rpoS/RpoS.In addition to the control of gene expression at the transcriptional level, RNA-mediated regulation has emerged as a burgeoning field E.coli HrpA, based upon sequence similarity with members of the yeast DEAH family (B. burgdorferi) putative DEAH-box RNA helicase. The hrpA gene was initially reported to be required for processing of fimbrial mRNA in E. coliE. coliB. burgdorferihrpA mutant, 187 proteins were differentially regulated: 97 upregulated and 90 downregulated. Disruption of hrpA also resulted in a loss of murine infectivity B. burgdorferi HrpA and demonstrate that it possesses RNA stimulated ATPase activity and RNA helicase activity in vitro. We also report a mutagenic analysis of several domains of HrpA and the effect(s) of these mutations on enzymatic activity in vitro and murine infection. Finally, we demonstrate a role for HrpA on RNA processing of four B. burgdorferi genes and a defect of an hrpA mutant in tick transmission.In the expanding world of RNA regulation, RNA helicases have emerged as major players. RNA helicases, universal enzymes known to play roles in all cellular processes involved in RNA metabolism H family  of RNA hB. burgdorferi hrpA was introduced into the NdeI and BamHI sites of pET-15b  RNA was assayed for all recombinant proteins . As expeThe mutant HrpA proteins were also assayed for their levels of RNA helicase activity . HrpA muB. burgdorferi hrpA was previously shown to result in complete loss of infectivity in wild-type C3H/HeN mice hrpA mutant with a wild-type copy of hrpA provided in trans on a shuttle vector. In an alternative attempt to complement hrpA, the disrupted chromosomal gene was replaced with wild-type hrpA by allelic exchange . Allelic exchange was confirmed by PCR analysis  protein that conceivably may contain functions other than RNA helicase activity. It was, therefore, of interest to test point mutations affecting RNA helicase activity on murine infection to determine whether the inability of an in vitro  and 3 inpPOH62-1 , the con alleles . Using t alleles . This sthrpA-D126A and hrpA-E127A mutations  resulted in a complete loss of B. burgdorferi infectivity in wild-type mice  displayed a less-dramatic decrease in infectivity. Only 50% of blood samples collected 7 days post-infection were positive for spirochetes. For ear punches collected 14, 21 and 28 days post-infection, a delay in dissemination of s mutant .hrpA-S158A  resulted in only a slight delay in the infection, where samples from 3 of 4 mice were positive for spirochetes for weeks one and two. At weeks three, four, and five post-infection, all four mice were positive for B. burgdorferi. This mutant was also less severe in vitro and displayed residual ATPase  may result from the lack of complete inactivation of the helicase activity in the mutant or from some HrpA function that is independent of helicase activity.hrpA is involved in controlling the expression of at least 187 proteins in B. burgdorferihrpA mutant versus the wild-type parent by microarray hybridization. Total RNA extracts of the hrpA mutant and the complemented mutant were prepared in duplicate and each run as a separate sample in the microarray analysis. To control biological variation, total RNA was isolated from three separate 50-ml cultures of each clone grown to a cell density of about 9×107 spirochetes/ml and pooled after the RNA integrity of each sample was verified. RNA samples were then processed for cDNA generation and labeling with fluorescent dyes for hybridization using a NimbleGen 4×72K array of B. burgdorferi B31 genes. A plot depicting the normalized log of hybridization intensities for the hrpA mutant versus the complemented mutant (RhrpA+) is presented in hrpA mutant; out of 1505 genes examined, only 28 showed ≥1.5 - fold changes  of some of the HrpA-regulated proteins, suggesting that HrpA regulation, at least for some genes, was occurring at the post-transcriptional level. The small but significant changes in RNA levels observed in the hrpA mutant versus wild-type may result from small changes in transcription or from post-transcriptional RNA processing.In our previous study, we showed that nges see . Of theshrpA does not result in global transcriptional changes. However, expression of about a dozen proteins were up or downregulated by 4- to 14-fold as determined by mass spectrometry iTRAQ analysis hrpA does not affect major transcriptional changes, then how does it modulate changes in protein expression? To address this question, we used Northern blot hybridization to analyze the processing of RNAs corresponding to some of the more highly regulated proteins or operons: downregulated bb0241 [glpK], bb0242, bb0603, and bba74; and upregulated bb0502, bbb19, bb0443, bbi39, and bba24 [dbpA]. Total RNAs isolated from the hrpA mutant and complement were run on denaturing agarose-formaldehyde gels and then transferred to nitrocellulose. For each transcript, a PCR product targeting the middle of the gene was prepared as a hybridization probe. RNA processing differences between hrpA and hrpA+ spirochetes were detected in five of the 11 transcripts analyzed. As shown in bb0603 (encoding the outer membrane protein P66) was present in both strains, but the band was stronger in the mutant. The wild-type strain displayed a smear from 500–1,500 nt that was not present in the hrpA mutant. Moreover, the mutant strain showed a second difference in processing as demonstrated by the new, intense band running just ahead of the 500 nt marker. For bba74 . The large decrease of visible RNA in the mutant in the Northern blots suggests that the RNA is either in small fragments that have run off the bottom of the gel or in larger fragments of heterogeneous length such that they are not visible on the gel.The data from microarray hybridization indicated that mutation of or bba74  a full ln operon . RNA levdbpA, jag, ospC, rpoA and bbbi39, which are upregulated between 4–12 fold at the protein level in the hrpA mutant Northern blots were also performed on the genes Ixodes scapularis larvae with GCB1164 (hrpA) and GCB549 (complemented mutant) using the immersion method developed by Policastro and Schwan flaB, the mutant and complemented isolates survived at similar levels during the larval blood meal , a time point that enables one to distinguish between a defect in tick-to-mammal transmission and clearance by the host hrpA)-infected nymphs were culture-positive, while 21/30 skin samples were positive for the complement  completely restored infectivity and persistence in mice to a wild–type level. This complementation experiment confirms that hrpA is indeed essential for the infectivity of B. burgdorferi and that the defect observed in the knockout clones is not the result of secondary mutation(s) in the genome.Our previous study showed that disruption of the hrpA gene in the chromosome  may result from the lack of complete inactivation of the helicase activity in the mutant or from some HrpA function that is independent of helicase activity.The approach used for restoring a wild-type romosome , coupledromosome , facilithrpA mutant hrpA mutant may have escaped identification by the limited proteomics approach and proteins that are expressed in the mouse and/or the tick would not have been identified.The precise mechanism by which the RNA helicase is involved in mouse infectivity remains speculative and is likely through its role in regulating important genes, such as the integrin-binding protein and porin P66, which is required for mouse infectivity B. burgdorferihrpA itself does not appear to be controlled by the RpoN-RpoS pathway. The minimal changes observed by microarray analyses comparing the hrpA mutant and complemented isolates contrasted with the significantly larger changes observed by iTRAQ, which for some proteins were as high as 14-fold downregulation and 12-fold upregulation. Thus, post-transcriptional RNA processing by HrpA represents an additional mechanism by which spirochetes could modulate their proteome throughout the enzootic cycle. Of note, there appears to only minimal overlap between the RpoS and HrpA regulons. Of the 187 proteins differentially-regulated by HrpA, only 11 are regulated by RpoS mcp1, ospC, dbpA and bb0689) are transcribed by RpoS but were present at higher protein levels in the hrpA mutant. Conversely, transcripts for seven genes  that are repressed in an RpoS-dependent manner appear to be stabilized by HrpA .Numerous studies to date have examined the contribution of environmental signals to differential gene expression in E. coli, resulting in the generation of a stable mRNA and the upregulation of daaE, which encodes a fimbrial adhesin B. burgdorferi, we looked for changes in RNA processing in an hrpA mutant versus wild type, in the most highly HrpA-regulated genes. Changes in RNA processing were observed in five downregulated genes: bb0603 [p66], bba74, bb0241 [glpK], bb0242, and bb0243 [glpA]  appeared to show a generalized RNA decay in the absence of HrpA, suggesting that HrpA either directly or indirectly protects these transcripts from cellular nucleases. The absence of full-length transcript in these cases correlates well with the decrease in protein expression.RNA helicases are known to play a role in all aspects of RNA metabolism and in ribosome biogenesis 3 [glpA] . In contin vitro. It is of noteworthy that four E. coli DEAD-box proteins have recently reported to have ATP-independent RNA annealing, strand displacement and RNA chaperone activity in vitroOur work here shows that the DEAH-box protein HrpA displays RNA helicase activity hrpA mutant organisms replicated normally within feeding nymphs, suggesting that the transmission defect is likely due to an inability to disseminate within the vector during the blood meal. Two of the proteins most affected by loss of HrpA are GlpK and GlpA, both of which are involved in glycerol utilization during the tick phase of the enzootic cycle hrpA mutant, glpA and glp operon mutants do not persist through the larval molt and display modest to severe growth defects, respectively, within feeding nymphs glp transcripts does not appear to be responsible for the transmission defect we observed with the hrpA mutant. A survey of the HrpA regulon did not identify any obvious candidate proteins involved in spirochete migration per se. Therefore, further studies will be needed to define more precisely the point(s) during transmission that are controlled by HrpA-regulated gene products.Spirochetes lacking HrpA are avirulent in mice infected via needle . Here, wGuide to the Care and Use of Experimental Animals by the Canadian Council on Animal Care. The animal protocol (AC12-0070) was approved by The Animal Care Committee of the University of Calgary. All animal experimentation for tick transmission studies was conducted following the Guide for the Care and Use of Laboratory Animals (Eighth Edition) and in accordance with protocols reviewed and approved by the University of Connecticut Health Center Institutional Animal Care and Use Committee.All animal experimentation for murine infection was carried out in accordance with the principles outlined in the most recent policies and E. coli strains used are presented in B. burgdorferi strains used are listed in All plasmids and hrpA in clone pASD1 (hrpA in pET-15b (Novogen). Site-directed mutagenesis and purification of PCR products were performed as described previously E. coli Rosetta DE3 cells carrying either wild-type or mutant hrpA genes were overexpressed and lysed as described previously 2PO4, 0.5 M NaCl, 5 mM imidazole, 10% glycerol (w/v), pH 8.0]. Samples were diluted in sample preparation buffer . Samples were then loaded on columns and washed with 20 ml of wash buffer  and 1 ml fractions were collected. Samples were subjected to 7% SDS-PAGE and visualized by Coomassie blue staining. Fractions containing purified His-tagged HrpA were pooled. Salt concentration and pH values of the samples were adjusted to 0.5 M salt concentration through addition of HAP buffer  and were loaded on a 3 ml Hydroxyapatite  column prepared in HAP buffer containing 0.5 M NaCl. Loaded samples were washed with 10 ml of HAP buffer containing 0.5 M NaCl, followed by 50 mM potassium phosphate, 0.5 M NaCl. Samples were eluted through a gradient of HAP buffers containing 0.5 M NaCl and 0.6 M potassium phosphate buffer. Once the fractions containing purified HrpA were pooled , and specified concentrations of HrpA were incubated for 1 h at 37°C. The reactions were ended by addition of 20 mM EDTA. Reaction products were analyzed by spotting onto DE 81 paper (Whatman) strips. Descending chromatography was performed using 0.35 M ammonium formate. After the run, the strips were dried and exposed to a Cyclone phosphor screen for 30 min and ATP hydrolysis quantified using a Cyclone phosphorImager. The plotted results are the average of two separate experiments for each sample.Reaction mixtures of 20 µl containing 40 mM Tris-HCl (pH 8.0), 2 mM DTT, 2 mM MgClin vitro yielding an 84 nt RNA strand, AACGGUUUCCCUCUAGAAAUAAUUUUGU UUAACUUUAAGAAGGAGAUAUACAUAUGG-3′5′-UAAUACGACUCACUAUAGGGAGACCAC. In vitro transcription and removal of template DNA were according to manufacturer instructions. The transcript was then extracted with acidified phenol∶chloroform∶isoamyI alcohol (125∶24∶1), pH 4.5 (Ambion) and precipitated with 100% ethanol. Precipitated RNA was washed once with 70% ethanol, dried in a Speedvac and dissolved in DEPC-H2O (diethylpyrocarbonate treated water). The 37-mer short strand, ACAAAAUUAUUUCUAGAGGGAAACCGUU-3′5′-AUCAGACUC, was synthesized  complementary to the middle region of the long strand RNA (bold characters indicate the double stranded portion of the substrate). The short strand RNA was 5′ end labelled with [γ-32P]ATP using T4 polynucleotide kinase (New England Biolabs). Labelled short and long RNA strands were hybridized at a ratio of 1.3∶1 (Long strand: Short strand) in 20 mM Hepes-KOH buffer (pH 7.6) containing 0.5 M NaCl, 1 mM EDTA and 0.1% SDS in a 50 µl reaction volume. The reaction mixture was heated to 95°C for 5 min followed by a slow cooling to room temperature. Partially double-stranded RNA was cleaned from unbound excess radiolabel by Sephadex G-25 Superfine  spin columns. The radiolabelling quality and substrate integrity was confirmed by running the purified substrate on a 12% native polyacrylamide  gel in TBE buffer at room temperature and visualizing with a Cyclone phosphorImager.Radiolabelled partially double-stranded RNA helicase substrate  was prep2, 1 mM ATP, 0.1 mg/ml BSA, 2 units RNasin, 0.5 pmoles helicase substrate and specified concentrations of purified enzyme. Reaction mixtures were incubated 1 h at 37°C, then stopped by the addition 5 µl of 5× RNA loading buffer containing 100 mM Tris-HCl, pH 7.5, 50% glycerol, 5 mM EDTA, 0.5% SDS, 0.1% NP-40, 0.1% (w/v) bromophenol blue and xylene cyanol FF. Samples were analyzed by 12% native polyacrylamide (29∶1 acrylamide∶bis-acrylamide) gel in TBE buffer with running conditions of 8 volts/cm. Double stranded and unwound RNA strands were visualized and quantified using a Cyclone phosphorImager and ImageQuant software. The background from the no enzyme control was subtracted from the data points to obtain helicase activity levels.Helicase activity of the samples was assayed in 20 µl reaction mixtures containing 20 mM Hepes-KOH (pH 7.6), 2 mM DTT, 50 mM KCl, 2 mM MgClB. burgdorferi strains were cultured at 35°C in BSK-II prepared in house 2 incubator. Cell counts were taken using a Petroff-Hausser chamber in a dark-field microscope.Except when specified, all hrpA was performed as previously described for the complementation of B. burgdorferi uvrBhrpA gene, a PflgB-driven kanamycin-resistance cassette and 500 bp of sequence downstream from hrpA. The overlap PCR product was cloned into pJET1.2/blunt cloning vector (Fermentas), sequenced and then was used to transform B. burgdorferi strain GCB1164 (hrpA KO) hrpA that was deleted in the knockout strain and to confirm that the size of complemented hrpA was similar to wild-type hrpA  as described B. burgdorferi GCB1164 (hrpA KO) and possible clones were grown in the presence of kanamycin (200 µg/ml). A PCR strategy B. burgdorferi  and 41 clones for I285A (from 4 transformations) were recovered. For the D126A mutation, four out of the six clones tested by PCR contained the mutation. From the 12 clones tested for the D127A mutation, nine appeared to contain the mutation by PCR screen. For T160A mutation, 30 clones were recovered over four transformations and all contained only the wild-type hrpA sequence. For I285A, 41 clones were recovered over four transformations and only two contained the expected mutation.To insert point mutations in on sites . For thegdorferi . Brieflyeri hrpA . Restoraentation  and by DB. burgdorferi clone was determined by multiplex PCR as previously described B. burgdorferi genomic DNA, 1 unit of Phusion DNA polymerase (New England Biolabs), 1× HF buffer, 250 µM dNTPs (Fermentas) and 1× of circular or linear primers mix. PCR products were then analysed by electrophoresis in a 3% Metaphor Agarose gel , DNA was stained with GelRed Nucleic Acid Gel Stain  and visualized under UV light. All clones used retained all plasmids that were present in the parental GCB1164 strain, which lacks lp28-2 and cp9.The plasmid content of each confirmed 4 spirochetes by both intraperitoneal and subcutaneous injection. At 7 days post-infection, 50 µl of blood was collected from the saphenous vein and cultured in 1.7 ml of culture medium. At weeks two, three and four, two ear punches per mouse were collected and incubated in 1.5 ml of culture medium. At day 35 post-infection, one ear, the bladder, the heart and one knee joint were recovered. For all tissue sample cultures, 1× Borrelia antibiotic cocktail  was added to the culture medium. Cultures were considered positive for B. burgdorferi when spirochetes could be observed using a dark-field microscope.For mouse infection studies, three to four week old C3H/HeNCrl male mice  were infected with 2×10B. burgdorferi strains GCB549 and 1164 were inoculated into 20 ml of BSK-II prepared in-house supplemented with 6% rabbit serum and incubated at 35°C until they reach a density of around 5×106 spirochetes/ml. The cultures were then diluted to a concentration 1×106 spirochetes/ml and incubated at 23°C in the dark. At this stage, each culture was prepared in duplicate. When the cultures reached around 2×106 spirochetes/ml at 23°C, one replicate was prepared for 35°C shift and the other was kept at 23°C until it reached a density of about 8×107 spirochetes/ml and then harvested. The cultures to be shifted to 35°C were diluted to a density of 1×106 spirochetes/ml. Following the dilutions, cultures were supplemented with final concentrations of 1% antibiotic cocktail and 5% human blood from which the buffy coat was removed and containing 10% 0.1 M sodium citrate. Blood containing cultures were periodically mixed to keep blood cells in suspension and incubated at 35°C for 48 h where they reached a density of about 8×107 spirochetes/ml. To harvest the Borrelia cells, cultures were centrifuged at 600 rpm for 10 min at 4°C and the culture media containing Borrelia cells were pipetted out gently into a new centrifuge tube without disrupting the pelleted blood cells. Borrelia were harvested by centrifugation at 6000× g for 20 min at 4°C after two successive PBS wash steps. An aliquot from each culture was saved to check for expected OspC induction by Western blotting. To control biological variation for the microarray analysis, samples prepared by pooling 4 independent cultures. To prepare whole cell lysates, B. burgdorferi cells were lysed using SDS-PAGE loading buffer 7 spirochetes was loaded per each well of 12% SDS PAGE gels for separation. Samples were then blotted onto nitrocellulose membranes (Amersham Hydrobond-ECL). OspC was detected using rat anti-B31 OspC-His in a dilution of 1/1000 as a primary antibody and peroxidase-conjugated AffiniPure donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories) in a dilution of 1/5000 as the secondary antibody. 3,3′,5,5′-Tetramethylbenzidine (TMB) liquid substrate (Sigma) was used as a colorimetric detection reagent according to manufacturer's instructions. Once the OspC induction was confirmed, total RNA was extracted from hrpA mutant and the complemented mutant clones harvested at their late exponential phase with centrifugation at 6000× g for 20 min at 4°C. RNA samples were extracted using Qiagen RNeasy Total RNA extraction kit according to the manufacturer's instructions, Possible remaining contaminant DNA was removed from the samples by incubation with DNase I (BioRad) at 37°C for 2 h. DNase I was inactivated by incubation at 70°C for 20 min. RNA samples were further concentrated with ethanol precipitation and resolubilized in DEPC-H2O at a concentration of 2 µg/µl. The integrity of the RNA samples were checked on 1% Agarose gels. Total RNA extracts of the hrpA mutant and the complemented mutant were prepared in duplicate and each run as a separate sample in the microarray analysis. To control biological variation, total RNA was isolated from three separate 50 ml cultures of each clone grown till their late exponential phase and pooled after RNA integrity was proved. Samples were then sent to the Vancouver Prostate Centre, Laboratory for Advanced Genome Analysis, Vancouver, BC, Canada for microarray (NimbleGen 4×72K arrays) and statistical analysis. Gene level t-tests and absolute fold changes with a 1.4-fold change cut off, are given for RNA samples  in Supplementary Sample preparation for microarray analysis was performed as previously described with slight modifications B. burgdorferi strains GCB549 and 1164 were inoculated in 20 ml of BSK-II prepared in-house supplemented with 6% rabbit serum, incubated at 35°C until they reached a density of around 5×106 spirochetes/ml. The cultures were then diluted to a concentration 1×106 spirochetes/ml and incubated at 35°C until they reached the density of approximately 8×107 spirochetes/ml. To harvest the Borrelia cells, cultures were centrifuged at 6000× g for 20 min at 4°C and washed once with PBS. Resulting pellets were then extracted using an RNeasy Mini Kit (Qiagen) following the manufacturer's instructions. The RNA quality was checked on a 1% agarose gel run in RML buffer.Frozen stock PCR conditions for gene specific Northern probes for selected genes were as follows: 1× Phusion High Fidelity (HF) buffer (New England Biolabs), 0.5 mM dNTPs, 0.02 units/µl Phusion DNA polymerase (New England Biolabs), 0.5 mM of each forward and reverse primer  in 20× SSC buffer  by using capillary transfer overnight at room temperature. RNA was UV cross-linked to the membrane by using a UV Stratalinker 1800 (Stratagene). Membrane strips were incubated for 2 h in prehybridization buffer , 1 mM EDTA) at 85°C, followed by addition of labelled gene specific probes and overnight hybridization at 68°C. Membranes were washed twice with 1× SSC, 0.1% SDS for 10 min at 68°C. Dried strips were visualized by using a Cyclone Phosporimager. Autoradiography images were produced at different exposure times for each gene. The sizes of the transcripts were estimated by comparison with RNA Millenium molecular weight standards (Ambion).For loading of Northern blots the amount of RNA loaded per lane was equivalent as determined by spectrophotometry and verified by agarose gel elctrophoresis and RNA staining with ethidium bromide. For Northern blots ten micrograms of RNA denatured in 50% formamide containing 1× MOPS [3-(N-morpholino)propanesulfonic acid] buffer  by incubation at 85°C for 10 min. Samples were immediately chilled on ice for 10 min followed by a short centrifuge spin to deposit all of the liquid at the bottom of the tubes. For each sample 2 µl of 10× formaldehyde gel loading buffer  bromophenol blue, and xylene cyanol FF) was added. RNA samples were separated in 2.2 M formaldehyde agarose gel using 1× MOPS running buffer for 4 h at 40 V (4 V/cm). Running buffer was refreshed at the half time of the run. The lane containing RNA Millenium molecular weight standards (Ambion) was cut from the visualized gel and the gel was incubated with DEPC-HIxodes scapularis larvae  were infected with GCB1164 (hrpA mutant) and GCB549 (complemented mutant) B. burgdorferi using the immersion method as previously described 8 spirochetes/ml) for 1–2 h at room temperature. Following immersion, larvae were washed twice with 1 ml of sterile PBS and allowed to recover overnight. Larvae infected by immersion were fed to repletion on a naïve C3H/HeJ mouse housed over water and collected daily. Replete larvae (3 pools of 15 larvae per isolate) were surface-sterilized by successive washes with 1 mL of water, 0.5% bleach, 3% hydrogen peroxide, 70% ethanol and water prior to being processed for qPCR. Total genomic DNA was isolated from replete larvae at drop-off using the Gentra Puregene Yeast/Bact. Kit (Qiagen) according to the manufacturer's instructions. DNAs were diluted 1∶10 in water prior to being analyzed by qPCR using a TaqMan assay for flaB as previously described Borrelia antibody , and semi-solid phase plating. Tick-to-mammal transmission was monitored in two separate experiments by (1) culturing the skin surrounding the bite site immediately following drop-off (∼96 hours post-placement and/or within 24 hours of repletion) (5 mice/isolate) Pathogen-free Figure S1SDS PAGE analysis of purified recombinant HrpA proteins. Proteins were run on a 7% SDS PAGE gel and stained with Coomassie blue. Purification steps are described in Materials and Methods Panel A shows a molecular mass marker (M); Panel B contains steps for HrpA purification: lane 1, soluble lysate; lane 2, Ni column flow-through; lane 3, Ni column elution; lane 4, hydroxylapatite column pooled elution fractions; Panel C contains hydroxylapatite column pooled elution fractions of D126A, E127A, S158A, T160A and I285A HrpA mutants in lanes 5–9, respectively. Ten microliters of each pooled elution fraction was loaded on the gel.(TIF)Click here for additional data file.Figure S2Strategy used to screen for point mutations in B. burgdorferi. The strategy is adapted from  A) For each mutant, a primer with a sequence that is conserved between the mutants and the wild-type hrpA (B1220) was used in conjunction with a second primer containing the mutated base as the last nucleotide (B2176 is shown as an example). B) Example of a PCR screen for B. burgdorferi hrpA D126A point mutant (clones 1 and 2). The construct containing the mutated hrpA (pPOH87) and genomic DNA from wild-type B. burgdorferi GCB920 strain were used as templates for positive and negative controls, respectively. A PCR reaction lacking template was also included. The presence of point mutations was confirmed by DNA sequencing.(TIF)Click here for additional data file.Figure S3Spirochete burdens in pools of B. burgdorferi-infected nymphs (8–10 ticks per pool) fed to repletion on individual mice (5 mice per isolate). Genome copy numbers were assessed by qPCR using a TaqMan-based assay for flaB. Values represent the average flaB copy number per nymph ± standard error of the mean in each pool. Values below each column indicate the number of culture-positive bite site skin samples  obtained by culturing the area surrounding the feeding site . Skin samples were collected and cultured within 24 hours of tick drop-off (∼96 hours post-placement).(TIF)Click here for additional data file.Table S1Plasmids used in this study.(PDF)Click here for additional data file.Table S2Primers used in this study.(PDF)Click here for additional data file.Table S3B. burgdorferi strains used in this study.(PDF)Click here for additional data file.Table S4Partial list of B. burgdorferi genes regulated based upon microarray analysis.(PDF)Click here for additional data file.


ZC3HAV1L and CHMP1A from RNA-seq data of normal human mammary epithelial cells, and experimental confirmation of the chimeric transcript in multiple human cells and tissues. Our experimental characterization also detected three variants of the ZC3HAV1L-CHMP1A chimeric RNA, suggesting that these genes are involved in complex splicing. The fusion sequence at the novel exon-exon boundary, and the absence of corresponding DNA rearrangement suggest that this chimeric RNA is likely produced by trans-splicing in human cells.Interchromosomal chimeric RNA molecules are often transcription products from genomic rearrangement in cancerous cells. Here we report the computational detection of an interchromosomal RNA fusion between This article was reviewed by Rory Johnson (nominated by Fyodor Kondrashov); Gal Avital and Itai Yanai


Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways. Small RNAs play a variety of regulatory roles, including highly conserved developmental functions.  Caenorhabditis elegans. First, microRNAs (miRNAs) were identified as transcribed RNAs corresponding to mutant loci that disrupted stage-specific developmental processes [Drosophila, Arabidopsis, and mammalian cultures, have identified additional non-coding RNA pathways, including piwi-interacting RNA (piRNAs) and long non-coding RNA (lncRNAs), both of which have also been found in C. elegans [Non-coding RNAs regulate a myriad of biological processes ,2. Two brocesses ,4. Theserocesses ,4,5. Subrocesses . Second,rocesses ,9,10,11. elegans ,13,14. T elegans , protect elegans , multi-g elegans , and dev elegans .C. elegans with enhanced exo-RNAi responses (Eri mutants) [Genetic screens have identified mutants in mutants) ,21,22,23mutants) . One promutants) ,25 resulmutants) ,21. Addimutants) . C. elegans has only one dicer homolog—dcr-1—this competition model has been hypothesized to include other small RNA pathways, particularly the microRNA pathway [dcr-1-dependent non-coding RNAs compete for limited common enzymatic resources, including RNA-dependent RNA polymerases (RdRPs) that produce abundant secondary siRNAs and secondary siRNA binding Argonouates (SAGOs) [lin-4 and let-7, are developmentally regulated [dicer causes sickly phenotypes in animals [Although competition has only been inferred between endo- and exo-RNAi pathways, because  pathway ,26. This (SAGOs)   ,26. Howeegulated ,4, which animals ,27 may seri-1 mutant partially rescues the let-7 mutant’s physiological defects, thus apparently enhancing residual let-7 activity, Eri mutants also show increased expression of let-7 targets lin-14, lin-41, and daf-12, thus appearing to simultaneously decrease let-7 activity. Similarly, we show that engaging the exo-RNAi pathway, by exposing animals to gfp dsRNA, also appears to reduce the efficacy of both endoRNAi and miRNAi pathways. Finally, we observed that the relative expression of stage-specific developmental genes differs amongst small RNA pathway mutants, suggesting that in addition to competition for common limiting RNAi resources, perturbing small RNA flux also impacts developmental regulation. This may have large and indirect effects on the activity of endogenous small RNA pathways.Here, we examine the relationship between endo-RNAi and miRNAi pathways and show that, although the eri pathway competes with the miRNA pathway (let-7(n2853) single and let-7(n2853);eri-1(mg366) double mutant animals. The n2853 allele is a partial loss-function mutation that remains viable at 15 °C [eri-1;let-7 double mutant exhibited a significant reduction in the burst vulva phenotype (let-7(n2853) single mutant  in an eri-1 mutant background is indirect, then no effect on let-7 target mRNA levels is expected. An advantage of assaying target mRNA levels is that the competition model does not make any predictions about which stage of small RNA processing or activity the competition occurs. This means the production, stability, or even efficacy of small RNAs could be rate-limiting. A complication is that let-7 regulates temporally expressed genes, thus it is critical to measure RNA levels from precisely staged animals. Therefore, RNA was extracted from mid-fourth larval stage (L4) worms hatch-synchronized to within 1 hour and then raised at 15 °C for an additional 69 hours. As an additional level of control, we normalized gene expression to bli-1, an L4-specific collagen gene that is not known to be regulated by endogenous small RNAs [bli-1 mRNA expression. Surprisingly, we observed that the let-7-regulated genes lin-14, lin-41, and daf-12 [eri-1 mutant background , we measured the abundance of  mutants . If eri-all RNAs . Thus, if let-7283 in an eckground A. To enslization . Again, ckground A, suggeslet-7 target mRNA levels is specific to eri-1(mg366), or represents a more general effect of reduced function in the endo-siRNA pathway, we determined the expression levels of lin-14 and lin-41 in rrf-3(pk1426), dcr-1/eri-4(mg375), eri-6/7(tm1917), and ergo-1/eri-8(gg100) mutant backgrounds. Consistent with our eri-1 observations, these mutants also showed increased expression of the two let-7 targets , siRNases (ERI-1), RNA binding Tudor domain proteins (ERI-5), helicases , and Agos (ERI-8). To determine whether the observed increase in  targets B, suggeslin-14 and lin-41 expression increased between 20 to 60 fold in let-7(n2853) mutants compared to N2 wild type. Similarly, the viable dcr-1(mg375) allele showed a very strong upward effect on the let-7 target mRNA levels, as would be expected for a mutation that directly affects miRNA biogenesis [lin-14 and lin-41 expression levels in a sid-1(qt9) background. Because C. elegans miRNAs seem to act cell autonomously [lin-14 and lin-41 expression levels were indistinguishable between sid-1(qt9) and N2 animals , but dropped to undetectable levels at later L4 stages (hours 69 to 73) [eri-1(-) background, lin-41 transcript levels remained at the higher level for at least an additional four hours. In wild type, the decrease in lin-41 levels was detectable at 65 hours, whereas in eri-1 mutants, the decrease was first detectable at 69 hours. This seems to suggest that there was a temporal delay in the decrease of lin-41 expression  because the eri-1(-) worms haven’t reached the equivalent worm time (mid-L4) for a maximum let-7 efficacy. Consistent with previous observations, in a wild type background, pression C. Althoulet-7 transcripts itself because of its relative rarity and the relatively subtle differences in expression between mutants, which prevented us from drawing additional support for this delayed development hypothesis.However, other trivial explanations may also fit the data, including a very likely possibility that data points at hours 65 and 69 were noise fluctuations in two genes’ expression between the two strains C. Unfortbli-1 is transcribed specifically during the L4 stage [bli-1 transcript levels relative to gpd-3 transcript levels—which are transcribed stably throughout development [let-7 target genes, the systemic RNAi mutant sid-1(qt9) had no detectable effect. Furthermore, both let-7(n2853) and dcr-1/eri-4(mg375) mutants, which are known to have significant sickness [bli-1 versus gpd-3 differences than the other endo-RNAi mutants (ergo-1/eri-8(gg100) allele, which causes the least sterility and brood size reduction [bli-1 transcript levels  and pmp-3 (a measure of peroxisome activity) [gpd-3, to normalize bli-1 transcript levels  mutants. This analysis confirms that at 69 hours post hatching, eri-1(mg375) worms compared to N2 and sid-1(qt9) worms likely exhibit slowed development. Therefore, these results further suggest that the endo-RNAi mutants reduce the accumulation of L4 specific bli-1 transcript due to a delay in development. These findings suggests that either endo-RNAi mutants slow development leading to lower ctivity) , in addit levels B. We foulin-41 transcripts  or gfp-dsRNA expressing bacteria (exo-RNAi(+)) ) A. RNA waNAi food B. Similad (gpd-3 B.rrf-3-siRNA targets F14F7.5 and Y43F8B.9 [rrf-3(pk1426) mutant than in wild type mid-L4 worms, similar to previously published findings [gfp(RNAi)-treated animals exhibited higher expression levels of the Y43F8B.9 gene (gfp(RNAi)-treated animals did not exhibit significant differences in expression levels of F14F7.5 compared to vector L4440-treated animals , (GR1373) eri-1(mg366), (HC195) nrIs20 (sur-5::NLS-GFP), (HC196) sid-1(qt9), (HC793) eri-1(mg366);let-7(n2853), (MT7626) let-7(n2853), (NL2099) rrf-3(pk1426), (YY168) ergo-1/eri-8(gg100), and (YY470) dcr-1/eri-4(mg375). All strains and assays were maintained and performed at 15° C as previously described [The following strains were used: (FX1917) escribed .gfp(RNAi) assays were performed as previously described [gfp were prepared as previously described [escribed . Bacteriescribed .-synchronized to within 1 hour, worms from NG large plates, gfp(RNAi) plates, or L4440 vector plates grown at 15 °C for needed number of hours were pooled, washed extensively (M9) and then allowed to swim for 20 minutes to clear gut content. RNA was isolated with proteinase K (Omega) followed by phenol:chloroform extraction (Amresco). The RNA pellets were subjected to DNase I (Roche) treatment, and cleaned by RNeasy (Qiagen) per manufacturer’s instructions. All initial RNA input concentrations were normalized to ~30−200 ng/µL.HatchReverse transcription was performed using 20 µL reactions of ~150–600 ng of input RNA by Thermoscript RT (Invitrogen), using gene specific RT primers (available upon request). cDNA quantification was performed using 2 µL of the RT reaction in a 50 µL QuantiTect SYBR Green (Qiagen) reaction with nested PCR primers. The PCR reaction cycles were: 15 minutes 95 degrees, 15 seconds 94 degrees, 30 seconds 52 degrees, 1 minute 72 degrees, read, cycle to step 2 for 45 cycles, using an Eppendorf Mastercycler Realplex4 and Noiseband quantification. Subsequent analysis was performed using the ΔCT approach for expression normalized to another gene, or the ΔΔCT approach for expression normalized to another gene and then relative to wild type’s expression . Three to five biological replicates of worms were combined for an RNA prep. This RNA was then quantified in triplicates (error bar generation), and repeated two to four times (representative shown), to determine the precision of relative RNA abundance, similar to prior small RNA qPCR experiments .C. elegans small RNA competition model [C. elegans, which suggest that developmental timing is intricately tied to small RNA expression, provides a cautionary caveat to these approaches. That is, while the functions of these ceRNAs are robustly demonstrated in vitro, when and how they are expressed in vivo for their putative regulation of miRNAs is not fully known. It is reasonable to assume that these ceRNAs are spatially and temporally regulated. Defects in tumors growth can impact small RNA expression, thus attributing causal relationships when developmental factors may indirectly affect both miRNAs and ceRNAs should be done with caution.Recent publications report on competitive regulation schemes akin to the on model  for RNAson model ,45,46,47on model . The useC. elegans limiting RNAi resources are largely involved in the biogenesis or execution of the secondary response, analogies to the C. elegans competition model were deemed inapplicable. However, our observation that engaging the exoRNAi pathway via experimental dsRNA can cause developmental delays suggests that the indirect aspect of the C. elegans small RNA competition can still serve as a model. Off-target effects have long been assumed to result from promiscuous siRNA binding, but our results suggest that perturbed small-RNA flux may also indirectly misregulate biological, processes, including development. Therapeutic delivery of small RNAs has been a commercial goal of RNAi for many years . Because


Summary: Here we introduce catRAPID omics, a server for large-scale calculations of protein–RNA interactions. Our web server allows (i) predictions at proteomic and transcriptomic level; (ii) use of protein and RNA sequences without size restriction; (iii) analysis of nucleic acid binding regions in proteins; and (iv) detection of RNA motifs involved in protein recognition.Results: We developed a web server to allow fast calculation of ribonucleoprotein associations in Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Xenopus tropicalis . The catRAPID omics was benchmarked on the recently published RNA interactomes of Serine/arginine-rich splicing factor 1 (SRSF1), Histone-lysine N-methyltransferase EZH2 (EZH2), TAR DNA-binding protein 43 (TDP43) and RNA-binding protein FUS (FUS) as well as on the protein interactomes of U1/U2 small nucleolar RNAs, X inactive specific transcript (Xist) repeat A region (RepA) and Crumbs homolog 3 (CRB3) 3′-untranslated region RNAs. Our predictions are highly significant (P < 0.05) and will help the experimentalist to identify candidates for further validation.Availability:catRAPID omics can be freely accessed on the Web at http://s.tartaglialab.com/catrapid/omics. Documentation, tutorial and FAQs are available at http://s.tartaglialab.com/page/catrapid_group.Contact:gian.tartaglia@crg.eu Several options are available to refine the type of analysis in eight model organisms or custom libraries (see online documentation):catRAPID omics takes as input the protein sequence (FASTA format): full-length or, alternatively, nucleic acid binding regions. In the case of a protein query, For a transcript query (FASTA format), the server uses the full-length sequence if below 1200 nt, or, alternatively, uses fragments with predicted stable secondary structure  and ranks RNA interactions (downloadable by the user).Using the interaction propensities distribution, The http://service.tartaglialab.com/static_files/shared/faqs.html). As for the reference sets, ENSEMBL (version 68) is used for retrieval and classification of coding and non-coding RNAs, whereas protein sequences are gathered from the UniProtKB database (release 2012_11). Finally, catRAPID omics uses hmmscan, a Hidden Markov Model-based algorithm from the HMMER3 package . Good performances are found for low-throughput experiments on murine non-coding X inactive specific transcript (Xist) repeat A region (RepA)   as well as human Crumbs homolog 3 (CRB3) 3′-untranslated region . To evaluate catRAPID’s performances on high-throughput data, we collected positive interactions  as well as negative controls (same numbers as positives and generated in four random extractions). Comparing the interaction scores of positives and negatives, we found enrichment  in 72% (TDP43), 88% (FUS), 74% (SRSF1) and 56% (EZH2) of cases. On the same datasets, SVM RPIseq showed enrichment in 58% , 83% , 47%  and 41%  of cases. The n (RepA)   B. To ill domains C, we used region  (P-valuecatRAPID omics server offers unique features such as organism-specific proteomic and transcriptomic libraries, possibility to generate custom datasets, analysis of long sequences and calculation of interaction specificities. Moreover, we implemented an algorithm for the detection of RNA motifs as well as protein RNA binding domains, which will help to retrieve recognition motifs embedded in sequences. Our server enables fast calculations of ribonucleoprotein associations and predicts RNA binding activity of proteins with high accuracy, thus resulting in a powerful tool for designing new experiments.Despite recent technical developments, detection of protein–RNA associations remains a challenging task. For this reason, we developed an algorithm that can be used to complement experimental efforts (


HSPA8 pre-mRNAs into human cells induced suppression of the target mRNA expression in a time- and dose-dependent manner. The artificial box C/D RNA directed to the branch point adenosine of the second intron, as well as the analogue directed to the last nucleotide of the second exon of the HSPA8 pre-mRNA caused the most prominent influence on the level of HSPA8 mRNAs. Neither box D nor the ability to direct 2′-O-methylation of nucleotides in target RNA was essential for the knockdown activity of artificial snoRNAs. Inasmuch as artificial box C/D RNAs decreased viability of transfected human cells, we propose that natural snoRNAs as well as their artificial analogues can influence the maturation of complementary pre-mRNA and can be effective regulators of vital cellular processes.Box C/D small nucleolar RNAs (snoRNAs) are known to guide the 2′-O-ribose methylation of nucleotides in eukaryotic ribosomal RNAs and small nuclear RNAs. Recently snoRNAs are predicted to regulate posttranscriptional modifications of pre-mRNA. To expand understanding of the role of snoRNAs in control of gene expression, in this study we tested the ability of artificial box C/D RNAs to affect the maturation of target pre-mRNA. We found that transfection of artificial box C/D snoRNA analogues directed to  Small nucleolar RNAs (snoRNA) are commonly known to be involved in the processing of precursor ribosomal RNA (pre-rRNA) and small nuclear RNAs (snRNAs). Box C/D snoRNAs direct 2′-O-methylation of rRNA nucleotides, and box H/ACA snoRNAs guide the conversion of uridine to pseudouridine –4. The sBox C/D RNAs contain the following functional elements: boxes C and D, which are essential for snoRNA interaction with specific proteins, and a guide sequence that determines the nucleotide to be modified . Some borRNAs and snRNAs are the main natural targets of 2′-O-methylation that is mediated by box C/D RNAs. However, a number of snoRNA-like transcripts do not have guide sequences that are complementary to rRNA or snRNA, so they have unknown functions and are called “orphan snoRNAs” , 9. BioiIn earlier studies, the development of snoRNA expression vectors was one of the main approaches to study the influence of box C/D snoRNAs on target genes in mammalian cells. The developed DNA vectors encoded mature snoRNAs or artificial pre-mRNA fragments that were processed to snoRNA , 13. UsiTherefore, recent results indicate that box C/D RNAs that include a sequence element complementary to a pre-mRNA could control the posttranscriptional maturation of the pre-mRNA, with antisense elements located in different parts of the snoRNA molecules. In our study, we proposed and employed a novel approach to investigating the snoRNA ability to control gene expression. The approach involves direct application of artificial RNA constructions containing structural features of snoRNAs instead of DNA vectors expressing snoRNAs. Such snoRNA analogues can be addressed to different pre-mRNA or mRNA targets and delivered to the cells using transfection methods.HSPA8 gene encoding the human heat shock cognate 71 kDa protein (HSC70) as a model target of snoRNA-directed action. Stress-induced proteins related to the heat shock protein 70 (HSP70) family perform numerous functions in the cell, including folding of nascent cellular proteins, regulation of intracellular transport, and directed degradation of proteins. Moreover, heat shock proteins take part in controlling repair systems, the cell cycle, cell proliferation, and differentiation -labeled with T4 polynucleotide kinase . RNA forms were separated on a 10% denaturing polyacrylamide gel, and individual RNA forms were eluted from gel bands into 0.3 M NaOAc. Transfection of radiolabeled RNAs into human cells was performed as described above. The transfected cells were cultured for time intervals ranging from 3 h to 72 h. Total RNA was isolated with Trizol and analyzed in a 10% denaturing polyacrylamide gel followed by autoradiography of radiolabeled RNA fragments.Synthetic RNAs were 5′-[6) in 6-well plates were transfected with 160 nM FAM-labeled (FAM—carboxyfluorescein) artificial RNA. Control cells were incubated with Lipofectamine only. After incubation for 24  hours, the cells were detached with 0.025% trypsin and washed twice with IMDM containing 10% FBS. The transfected cells were scanned using a FACSCanto II flow cytometer  and the data were analyzed by FACSDiva Software (BD Biosciences).MCF-7 cells . The data were analyzed using iQ5 system software (Bio-Rad). Mean values (±SD) from 3 independent experiments were represented. The PCR products were analyzed in a 4% denaturing polyacrylamide gel or 1.5% agarose gel.The reverse transcription and cDNA amplification (RT-PCR) were performed in the one-tube reaction mixture “Real Best Master Mix RT”  with the addition of Sybr Green  in the case of real-time RT-PCR. The following gene-specific primers were used: 3 cells were plated onto 96-well plates and incubated at 37°C with 5% CO2. After 24 h, the cells were transfected with 40–160 nM artificial RNAs. After incubation for 72 h, MTT was added to the cells at a final concentration of 0.5 mg/mL and the plates were incubated at 37°C for 3 h. The medium was removed, followed by the addition of 0.15 mL of DMSO to each well. The plates were read at 570 and 620 nm using an Apollo LB912 plate reader . Cell viability was determined as the absorbance at 570 nm with reference to 620 nm and expressed as a percentage of control (control cells were incubated with Lipofectamine only) ±SD for triplicate independent experiments.To estimate the influence of the artificial box C/D RNA on MCF-7 human adenocarcinoma cell viability and proliferation we used MTT assay -2,5-diphenyl-2Htetrazolium bromide), Sigma-Aldrich). Aliquots (150 mL) containing 2.5 × 10HSPA8 pre-mRNA. Human U24 is a 76 nt long conserved RNA which contains two pairs of C/D boxes . It is predicted to guide the 2′O-ribose methylation of both 28S rRNA C2338 and C2352 [To investigate the influence of box C/D RNA on pre-mRNA maturation, we constructed and synthesized U24 snoRNA analogues containing guide sequences complementary to HSPA8 pre-mRNA nucleotides that were critical to splicing the second intron of the pre-mRNA-target , the last nucleotide of the second exon (RNA8), the first and the last nucleotides of the second intron , and the first nucleotide of the third exon of the target pre-mRNA  and two guide sequences. The first guide sequence (from the 5′-terminus) was directed to G1702 in 18S rRNA, which was one of the key nucleotides of the ribosome-decoding center . EarlierA RNA10, . ArtificA intron . AnaloguA intron , were taHSPA8 pre-mRNA.We have also obtained box C/D RNA analogue directed to two rRNA nucleotides, namely, G1702 in 18S rRNA and U4502 in 28S rRNA RNA4502, . In otheTo investigate the efficiency of artificial box C/D RNAs transfection into human cells, we synthesized FAM-labeled RNA analogues and studied the internalization and compartmentalization of the RNAs in MCF-7 cells via fluorescence microscopy. It was found that the fluorescent RNAs were detected in the cytoplasm as well as in nuclei. These results were confirmed with RT-PCR of nuclear and cytoplasmic total RNA from MCF-7 cells transfected with the artificial box C/D RNA .Here, to estimate the efficiency of transfection of box C/D RNA into human cells we treated MCF-7 cells with artificial FAM-labeled RNA in complex with Lipofectamine and analyzed cell populations using flow cytometry. The analysis of cells transfected with FAM-RNA8 revealed that 50% of the treated cells reliably contained the fluorescent snoRNA analogue . The simAnalysis of radiolabeled box C/D RNA analogue accumulation and integrity in human cells showed that full-length RNA (79 nt long) molecules were detected in the transfected cells at least 72 h after RNA transfection  and to the last nucleotide of the second exon of the HSPA8 pre-mRNA (RNA8) caused the decrease in target mRNAs level by about 50% and 40%, respectively. In the same conditions, box C/D RNA analogues directed to other nucleotides in the HSPA8 pre-mRNA , and the first nucleotide of the third exon of the target pre-mRNA  into MCF-7 human mammary adenocarcinoma cells, isolated total RNA 21 h after transfection, and performed RT-PCR with primers specific to A RNA10, ) had smaHSPA8 pre-mRNA  in MCF-7 cells  (We next examined the time- and concentration-dependence of -7 cells . Increasecrease) .HSPA8-specific RT-PCR was accompanied by increased yield of minor products corresponding to aberrant (shorter) products of HSPA8 pre-mRNA splicing. The primary structures of RT-PCR products were determined with Sanger sequencing and are schematically represented in HSPA8 mRNA splicing variants that resulted from skipping the second or simultaneously skipping both the third and fourth exons of the major mRNA isoform. The skipping of exons resulted in a shift in the mRNA reading frame and the appearance of premature stop-codons. It can be supposed that the aberrant splice variants undergo the rapid degradation through the nonsense-mediated mRNA decay pathway [The decreased yield of the major product of  pathway , 27.The directional influence on the pre-mRNA splicing is one of the promising approaches for gene expression modulation. Such an approach has some advantages; for instance, it provides control of the gene expression in stages of mRNA maturation prior to mRNA appearance in the cytoplasm –30. ThusHSPA8 pre-mRNA decreased MCF-7 viability  caused the most prominent influence on MCF-7 viability and reduced MTT-index by more than 28% at a final concentration of artificial RNAs 80 nM (HSPA8 mRNAs (We also estimated the influence of the artificial box C/D RNA on MCF-7 cell viability and proliferation using the MTT assay. We found that box C/D RNA analogues directed on nucleotides in iability . Some inAs 80 nM . As showA8 mRNAs .HSPA8 mRNA suppression effect and MCF-7 viability reduction was not observed (HSPA8 mRNA expression but nevertheless reduced MCF-7 viability (by 22%, 35%, and 37% at 40 nM, 80 nM, and 160 nM, resp., HSPA8 mRNA level there was some accessory process affecting the viability of transfected human cells. It is most likely that accompanying process induced in response to artificial box C/D RNA was activation of the innate immune system in transfected cells. The combination of the gene-specific knockdown and the activation of the interferon response system were reported earlier for a small interfering RNA-dependent regulation of gene expression [When RNA concentration was increased up to 160 nM correlation between observed . Moreovepression –33.The questions of whether box C/D snoRNAs direct 2′-O-methylation during their interaction with pre-mRNAs and whether modification of the target nucleotide is essential for the ability of the snoRNA to regulate gene expression remain open. Classical methods to detect 2′-O-methylated RNA nucleotides do not reveal modified nucleotides in minor RNA forms such as pre-mRNA. At the same time, box D is known to be an essential element for snoRNA-guided 2′-O-methylation.HSPA8 pre-mRNA, respectively.To study the participation of box D of artificial RNAs in pre-mRNA knockdown activity, we constructed U24 RNA analogues RNA7D* and RNA8D*, where both the box D and D′ CUGA sequences were replaced with AAAA . RNA7D* HSPA8 mRNA level to 53 ± 9.3% and 68 ± 5.6%, respectively (We found that transfection of RNA7D* or RNA8D* into human cells decreased the ectively . The decThere are numerous RNA transcripts that are complementary to different pre-mRNAs in eukaryotic cells, but their functions are not well understood. Antisense transcripts could take part in a variety of vital processes including chromatin remodeling, regulation of transcription, posttranscriptional modifications, and intracellular transport –37.Our results suggest that posttranscriptional pre-mRNA maturation could be affected by artificial analogues of snoRNAs with antisense sequences that are complementary to pre-mRNAs. Furthermore, our results suggest that the observed reduction of mRNA level occurs independently of the D/D′ boxes of the analogues and thus independently of their potential ability to guide 2′-O-methylation of the target pre-mRNA.The study provides a novel tool to offer insights into the question of whether snoRNAs that are complementary to pre-mRNAs could affect the processes of target pre-mRNA maturation. Our results showed that transfection of human cells with artificial snoRNAs containing guide sequences directed to pre-mRNA could impair the splicing and affect overall mRNA isoform levels. Transfection with artificial snoRNAs decreased human cell viability, suggesting that snoRNA analogues are able to activate regulatory processes affecting vital cellular functions.


Plant RNA viruses have effective strategies to infect host plants through either direct or indirect interactions with various host proteins, thus suppressing the host immune system. When plant RNA viruses enter host cells exposed RNAs of viruses are recognized by the host immune system through processes such as siRNA-dependent silencing. Interestingly, some host RNA binding proteins have been involved in the inhibition of RNA virus replication, movement, and translation through RNA-specific binding. Host plants intensively use RNA binding proteins for defense against viral infections in nature. In this mini review, we will summarize the function of some host RNA binding proteins which act in a sequence-specific binding manner to the infecting virus RNA. It is important to understand how plants effectively suppress RNA virus infections via RNA binding proteins, and this defense system can be potentially developed as a synthetic virus defense strategy for use in crop engineering. RNA binding proteins (RBPs) play critical roles in post-transcriptional gene regulation by controlling splicing, polyadenylation, mRNA stability, RNA trafficking and translation Moore, . FurtherInfection of host cells by plant RNA viruses has long been puzzling because they usually encode only a few proteins. However, plant RNA viruses suppress host innate immunity or utilize host proteins for their successful replication and movement  positively contributes to hemibiotrophic pathogen defense, and possibly regulates genes involved in the salicylic acid (SA) signaling pathway in the cytosolic region, via direct binding of target RNAs , necrotrophic bacterium Pectobacterium carotovorum SCC1 as well as the biotrophic virus Tobacco mosaic virus , was determined to be directly involved in plant defense against viral RNA infection, and showed enhanced ribonucleolytic activity to viral RNAs upon infection  family has been identified as ribonuclease-like PR proteins  directly bound to the “UGUA”–containing nucleotides in the 3′ untranslated region (UTR) and internal regions of Cucumber mosaic virus (CMV) and suppressed replication of CMV. Furthermore, APUM5 had a function of suppressing translation  core sequence (5′-UGUACAUA-3′) within the 3′ UTR of hunchback mRNA  also contains putative PHD-binding core motifs in its genome. When TuMV was inoculated in APUM5-overexpressing transgenic plants, the transgenic plants exhibited reduced TuMV RNA levels and slightly increased resistance compared to wild-type plant at the early stage  RNA 1 (BTR1), negatively affects ToMV multiplication, and suppresses the spread of the virus via interaction with the 5′ terminal region, which contains the initiation codon for the ToMV replication proteins -binding proteins (PCBPs) were known to contain three hnRNP KH RNA binding domains, and these domains are essential for the multiplication of polioviruses. PCBPs interact with the cytidine-rich RNA region of poliovirus RNA 5′ UTR , as the dicer-like 4 (DCL4) interacting partner, was involved in antiviral defense against Turnip yellow mosaic virus (TYMV) infection (Jakubiec et al., Some RBPs are differentially regulated at the translational or post-transcriptional level depending on the cell type, developmental stage, or biotic/abiotic interactions (Woloshen et al., Plant RNA viruses belong to the large group of all known viruses and are responsible for a vast variety of plant diseases. Plant genomes encode hundreds of RBPs, which are believed to bind to specific target mRNAs and affect plant physiology. However, only a few RBPs have been characterized so far. We briefly reviewed some RNA binding proteins which positively regulate plant innate immunity via direct binding to the viral RNAs Table . NormallThe 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.


Closteroviridae. Members in this family are restricted to the phloem and rely on whitefly vectors of the genera Bemisia and/or Trialeurodes for plant-to-plant transmission. All criniviruses have bipartite, positive-sense single-stranded RNA genomes, although there is an unconfirmed report of one having a tripartite genome. Lettuce infectious yellows virus (LIYV) is the type species of the genus, the best studied so far of the criniviruses and the first for which a reverse genetics system was developed. LIYV RNA 1 encodes for proteins predicted to be involved in replication, and alone is competent for replication in protoplasts. Replication results in accumulation of cytoplasmic vesiculated membranous structures which are characteristic of most studied members of the Closteroviridae. These membranous structures, often referred to as Beet yellows virus (BYV)-type vesicles, are likely sites of RNA replication. LIYV RNA 2 is replicated in trans when co-infecting cells with RNA 1, but is temporally delayed relative to RNA 1. Efficient RNA 2 replication also is dependent on the RNA 1-encoded RNA-binding protein, P34. No LIYV RNA 2-encoded proteins have been shown to affect RNA replication, but at least four, CP (major coat protein), CPm (minor coat protein), Hsp70h, and P59 are virion structural components and CPm is a determinant of whitefly transmissibility. Roles of other LIYV RNA 2-encoded proteins are largely as yet unknown, but P26 is a non-virion protein that accumulates in cells as characteristic plasmalemma deposits which in plants are localized within phloem parenchyma and companion cells over plasmodesmata connections to sieve elements. The two remaining crinivirus-conserved RNA 2-encoded proteins are P5 and P9. P5 is 39 amino acid protein and is encoded at the 5′ end of RNA 2 as ORF 1 and is part of the hallmark closterovirus gene array. The orthologous gene in BYV has been shown to play a role in cell-to-cell movement and indicated to be localized to the endoplasmic reticulum as a Type III integral membrane protein. The other small protein, P9, is encoded by ORF 4 overlaps with ORF 3 that encodes the structural protein, P59. P9 seems to be unique to viruses in the genus Crinivirus, as no similar protein has been detected in viruses of the other two genera of the Closteroviridae.Criniviruses comprise one of the genera within the family  Closteroviridae possess the largest and most complex ssRNA genomes which vary in size from ca. 15–20 kb . Criniviruses are exclusively transmitted by whiteflies of two genera: Bemisia and Trialeurodes. The genus Ampelovirus has members with monopartite genomes, and the viruses are transmitted by mealybugs. The newly proposed genus, Velarivirus, contains members formerly within the genus Ampelovirus, but which represent a different phylogenetic clade (Most plant viruses have positive-sense single-stranded RNA (ssRNA) genomes that vary in size among viruses in different taxa. Members in the family 15–20 kb . Closter15–20 kb . The genic clade . HoweverLettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus. Studies on LIYV date back to the late 1970s when several crops in California and Arizona , were severely affected by this newly discovered virus, resulting in losses exceeding $20 million in a single growing season . Primary work focused on characterizing LIYV whitefly transmission properties, host range, examination of virion morphology, and its effect on host cells (Bemisia tabaci biotype A (New World) by a more competitive, and more aggressive non-LIYV vector whitefly, Bemisia tabaci biotype B  population in southern California and Arizona in the late 1970s. Although whiteflies, and particularly, Bemisia tabaci had been recognized as a plant virus vector for many years, LIYV was recognized as a novel type of virus at that time. Bemisia tabaci-mediated LIYV transmission was semi-persistent. Transmission electron microscopic studies on purified virions and LIYV-infected plants showed that LIYV virus like particles (virions) were similar to those of the closteroviruses known at that time. The virions were long, flexuous rods  and Citrus tristeza virus (CTV), subsequent studies revealed that LIYV has shorter particle lengths of approximately 800 nm ] all of which had a single large, single-stranded genomic RNA . Later sequencing of other crinivirus genomes showed that they also have bipartite genomes with conservation of most of the gene content and order  with the possible exception of Potato yellow vein virus (PYVV), which is suggested to have a tripartite genome  and RNA helicase (HEL) motifs .) motifs . Althoug) motifs . Agranov) motifs . Further) motifs . ORF 1B ) motifs . Genome ) motifs . However) motifs . Studiesoviruses . Other coviruses  that belSweet potato chlorotic stunt virus (SPCSV) has been shown to encode a protein, P22, from the 3′ terminal ORF on RNA 1. P22 exhibits RNase III endonuclease activity and in vitro has been shown to cleave double-stranded small interfering RNAs (Sweet potato feathery mottle virus (Tomato chlorosis virus (ToCV). One of these, P22, is encoded by the ToCV RNA 1 3′ ORF, similarly to that seen for SPCSV .ed cells . Subsequed cells . FurtherLettuce infectious yellows virus RNA 2 is 7,193 nt, 5′ capped, not polyadenylated and does not encode proteins necessary for RNA replication. RNA 2 contains a 5′untranslated sequence of 326 nt. This sequence only shows limited homology with LIYV RNA 1 including the first five nucleotides (5′-GGUAA-3′) and a stretch of 23 nucleotides (5′-UCUUGGAGAAUUUCGAUGGCACU-3′). These 23 nucleotides in RNA 1 are from positions 83 to 105 and surround the first AUG which begins at nucleotide 99. However, in LIYV RNA 2 this 23 nt stretch is found upstream (at positions 121–143) of the first AUG start codon that is located at position 327. The significance of the common 23 nucleotide sequence is not known. It is important and worthwhile to note here, that LIYV RNA 1 and RNA 2 show no nucleotide sequence homology in their 3’-termini, unlike that found for most multipartite plant viruses and most of the other criniviruses sequenced so far. ORF 1 of LIYV RNA 2 encodes for a small protein of ~5 kDa. This is the first ORF of the conserved closterovirus quintuple gene block, and the putative LIYV protein, as well as those for other closteroviruses is predicted to be highly hydrophobic and to form a transmembrane helix. ORF 2 encodes the Hsp70h. This is conserved among closteroviruses, and before many closterovirus genomes were sequenced, conservation among the specific motifs shared among heat shock 70 proteins was used to design degenerate oligonucleotide primers that proved to be very useful for generic closterovirus detection , but no function has yet been determined for this protein. Amino acid alignment of crinivirus P9 homologs does not show significant amino acid similarity, but the predicted secondary structures of these proteins are very similar  for various members. LIYV RNA 2 ORF 7 encodes a 26 kDa protein, P26. Similarly positioned ORFs encoding similarly sized proteins are found among all criniviruses, and while, like for P9, the amino acid sequences do not show significant similarity, their predicted secondary structures are similar .etection . Interesetection . LIYV RNetection  and BYV etection . ORF 4 o similar ,b. ORF 5 similar . It is iis virus . Within  similar . LIYV RN similar . The genFigure 4). The CPm is localized to a short terminal region while the CP makes up the majority of the capsid. However, a surprising result was finding that the Hsp70h and P59 also are LIYV virion components suggesting even further complexity to LIYV and other closterovirus virions. This was first demonstrated when stringently purified LIYV virions were analyzed by SDS-PAGE and immunoblotting using antisera specific to four LIYV-encoded proteins: CP, CPm, P59, and to Hsp70h  and immunogold labeling analyses confirmed that the LIYV virions, like those of BYV and CTV are morphologically polar . This suggested that perhaps one or more of the four virion proteins might be a determinant of Bemisia tabaci transmissibility. in vitro acquisition and subsequent transmission of LIYV by Bemisia tabaci biotype A to plants. Results from several experiments indicated that LIYV transmission efficiency was not affected by antisera to P59, CP, or Hsp70h, however, the CPm antiserum specifically and completely eliminated LIYV transmission by Bemisia tabaci. Only, when the CPm antiserum was diluted to 1% final concentrations was there incomplete neutralization of LIYV transmission by Bemisia tabaci biotype A . This has been supported by subsequent studies of Bemisia tabaci biotype A . These data suggest that LIYV virions lacking intact CPm can systemically invade N. benthamiana plants, but when any of the other three virion proteins are deleted, the ability to establish systemic infections was abolished.Purified LIYV virions proved to be transmissible to plants by the whitefly, N. tabacum suspension cell culture . The RNA 1 3′ ORF complement of other criniviruses is extremely variable  did not show obvious temporal regulation/accumulation differences among LCV RNAs 1 and 2 , and LIYV infection induces re-organization of the ER (N. benthamiana protoplasts (unpublished). Cerulenin inhibits de novo fatty acid and steroid biosynthesis. Cerulenin binds in equimolar ratios to β-keto-acyl-ACP synthase, thus blocking its interaction with malonyl-CoA thereby affecting fatty acid synthesis. In contrast, brefeldin A inhibits the transport of proteins from the ER to Golgi, and also induces retrograde protein transport from the Golgi to the ER . In contrast, brefeldin A, although resulting in earlier cell death, showed no effects on LIYV, but showed a slight increase in TMV replication in protoplasts as measured by GFP fluorescence. The observations give additional support that LIYV replication depends on both ER-derived membrane recruitment and de novo biosynthesis of lipids.Although it has been realized for many years that viruses utilize host membranes as scaffolds for replication , how clof the ER . Togetheo the ER . We usedFigures B). Plasmalemma deposits were first described by Figure 1F) and speculated that the plasmalemma deposits might have roles in transporting LIYV virus particles from companion to sieve tube cells. Recent studies have shown that the plasmalemma deposits also are formed in LIYV-infected protoplasts, and they also contain aggregates of LIYV virions arranged perpendicular to the plasmalemma. “Sacks” of LIYV virions can also be found external to the plasmalemma of LIYV-infected protoplasts immediately adjacent to the plasmalemma deposits . Moreover, further studies showed that P26 still associates to plasmalemma deposits when expressed from the heterologous TMV vector indicating that no other crinivirus protein is needed for this association. Hence, P26 association to the plasmalemma deposits is unique (Agrobacterium tumefaciens to deliver specific LIYV mutants to N. benthamiana plants (N. benthamiana plants (unpublished), further supporting a role for LIYV P26 in systemic plant infection.While much recent research has focused on studying the genomic RNA sequences, genome organization, and phylogenetic relationships for many newly discovered criniviruses, there are still many crinivirus-encoded proteins whose roles in infections have not been elucidated. For example, LIYV RNA 2 encodes for a protein at its 3′ terminus, P26. Similarly positioned genes encoding similarly sized proteins are found among all criniviruses sequenced to date. LIYV P26 is not a virion component  and micrs unique . Interess unique . It is ps unique . Whole ps unique . More rea plants  have shoFigure 2).It is worthwhile to note that LIYV RNA2 encodes two other proteins. ORF 1 encodes a small hydrophobic protein, P5, with a transmembrane helix . The ortCrinivirus sequenced so far . However, this protein shows high sequence variability among these viruses. Yeast two hybrid studies of P5 and P9 with each other and with the other LIYV RNA 2-encoded proteins showed that P9 is self-interacting  and hinders even opportunities to rapidly screen germplasm for virus resistance. Effective delivery to natural hosts of specific criniviruses by agroinoculation, or some other means not relying on the whitefly vector would yield great practical as well as fundamental benefits.Despite their economic importance and widespread incidence in various host plants almost worldwide, reverse genetics systems are now available for only two criniviruses: LIYV and LCV . Both caClosteroviridae, such with CTV (Although many criniviruses presently cause important diseases in many crop plants, successful strategies for their control are limited. In areas where vector populations are high, insecticides are often used but generally are ineffective in preventing crinivirus inoculation to susceptible plant hosts. It is interesting that no successful genetically engineered approaches capable of inducing RNA interference-based immunity are known for criniviruses. This is despite efforts at least with SPCSV in sweet potatoes . Howeverwith CTV . GeneticThe 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.


Exosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.From the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs  collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA , long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5This study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases. Many cells produce exosomes -3, smallThe existence of exosomes has been known for many years; however, it is only recently that these lipid-rich vesicles have been reported to contain an abundance of nucleic acids, in particular small non-coding RNAs . StudiesExosomal RNAs have been implicated in many exosome-mediated biological functions . For exaWhile exosomes have been shown to play functional roles in recipient cells, the RNA content of the exosomes may provide unique molecular signatures for disease diagnosis and prognosis ,36-39. IMost of the current studies have used microarray or real time quantitative PCR (qPCR) assays to examine exosomal RNAs, with a focus on miRNAs. Because of the inherent limitations of these technologies, unknown miRNAs or other RNA species are often undetectable. Importantly, no systemic analysis of exosomal RNAs in peripheral blood has been reported until now. Blood is an important medium that allows exosomes to circulate and deliver cell signaling molecules to any part of the body. In this study, we performed a sequencing-based RNA profiling analysis using the blood from three blood donors. We evaluated three small RNA library preparation protocols and systemically characterized the extracellular RNA species. This study will provide a general guideline for blood-based exosomal RNA sequencing analysis and contribute to an understanding of exosome-mediated biological functions and mechanisms.8 and the RNA yields from each of the samples were similar, ranging from 10–15 ng . The RNAs sizes ranged from 18–28 nucleotides (nt)  produced more target fragments that were separated from adaptor dimers . From the 14 libraries, we obtained a total of 101,804,712 raw single-end reads, an average of about 7.27 million reads per library. Among the raw reads, we found 73,112,422 (71.82%) sequences with insert lengths ≥16nt (query sequences) and of these, 55.54%  could be mapped to known RNAs and to the human genome  Figure A. The hiFor sample-sample  variations, we compared pooled samples  prepared using the Bioo Scientific and NEB kits. Overall, we found that there was significant correlation between samples B and C (r = 0.986) Figure B, followTo better demonstrate the technical, biological, and methodological variations, we performed an unsupervised hierarchical clustering analysis using the log2-transformed sequence counts of the 100 most abundant miRNA transcripts. As expected, the heat map showed that there was a clear separation between groups composed of replicates, samples and library preparation kits Figure D. NearlyTo examine if the miRNAs were unique to or common to the different preparation protocols, we first removed the low abundant miRNAs  and then compared the remaining miRNAs among the three kits. Sample A was used for the comparison because this was the only sample that was tested in all three kits. The Illumina, Bioo Scientific and NEB kits detected 317, 364 and 370 known miRNAs, respectively. Of these, 287 were detected by all three kits  predicted a total of 1205 target genes for the top five exosomal miRNAs. We found significant enrichment of these genes in gene ontology (GO) terms, including protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. For example, we found a 1.33 fold enrichment of phosphoproteins (Bonferroni p = 1.35E-17), a 1.23 fold enrichment of splice variants (Bonferroni p = 3.43E-7), and a 2.46 fold enrichment of genes involving chromosomal rearrangement (Bonferroni p = 5.81E-5) . Therefore, the mRNAs and lncRNAs that were identified in this study should all be treated as fragmented RNAs. The procedures that were used for RNA extraction and library preparation may have caused partial RNA degradation, enabling the detection of fragments of the long RNAs in the small RNA libraries. Another possible explanation for the presence of long RNA fragments is that the exosomes also function as a “reservoir” to remove degraded mRNA and lncRNA derived from the cytosol. The exact mechanism underlying the presence of fragmented long RNAs in exosomes remains to be unraveled.The current study demonstrated the reproducibility for each library preparation kit. Both Pearson correlation and hierarchical cluster analysis showed highly correlated RNA profiles between technical replicates, suggesting the consistency of these commercial kits. However, the study also showed significant biases between the library preparation methods. Each kit preferentially captured specific RNA sequences. For high abundant RNAs, this bias does not seem to be problematic because all three kits detected these RNAs. For low abundant RNAs, however, the bias could be an issue because these RNAs may be detected by one kit but not by another. Protocol-based bias may also create problems in data interpretation if different commercial kits are used. We suggest that separate validation using qPCR should be performed for all sequencing-based detections.The ever growing number of novel sequences in the miRNA database implies that human miRNA annotation is far from complete . To idenThe plasma exosomes are believed to be derived from a variety of cell populations. Their heterogeneous origin may limit the detection of disease-specific exosomes in peripheral blood samples. Vast numbers of exosomes shed from other cell types may dilute the exosome population derived from tumor cells, significantly reducing the proportion of tumor-derived miRNAs in the sequencing libraries. Because the less common tumor-derived miRNA may be a direct reflection of the disease status and critical for tumor development, the increased read depth of RNA sequencing is required. It is worth mentioning, that although the detection of rare RNA transcripts will increase as sequencing depth increases, the rare sequences still account for a tiny fraction of the exosomal RNAs. Whether or not the rare exosomal miRNAs are functional remains to be determined.We developed a comprehensive data-generation and data-analysis pipeline that includes exosome isolation, RNA extraction, library preparation, RNA sequencing, and RNA annotation. Our data show that plasma-derived exosomes contain diverse RNA species, in particular, miRNA. The abundance of the exosomal RNAs varies dramatically. Some highly abundant miRNAs may play critical roles after being transferred to target cells. The three commercial small RNA preparation kits that we tested generated sufficient DNA fragments for sequencing but had significant biases towards capturing specific RNAs. The use of large-scale RNA sequencing will ensure the discovery and characterization of the whole transcriptome (known and unknown RNAs) of the blood-derived exosomes, which has not been completely examined so far. A fully characterized transcriptome will help gain a better understanding of exosome-mediated molecular mechanisms and will contribute to biomarker discovery. It is expected that the blood-based sequencing assay described here will find clinical applications as a biomarker discovery tool for disease diagnosis and prognosis.The objective of this study was to provide general guidelines for profiling analysis of exosomal RNA in peripheral blood. To accomplish this goal, we selected plasma samples  from three anonymous blood donors and split each sample into two for technical replication. We tested the six samples  using two small RNA library preparation kits: NEBNext multiplex small RNA library preparation kit  and NEXTflex small RNA sequencing kit . We also tested samples A1 and A2 using the TruSeq small RNA sample preparation kit . Altogether, we tested the six plasma samples by sequencing the 14 indexed libraries prepared using the three kits as described above. This study allowed the direct comparison of three currently available small RNA library preparation protocols and identified the most suitable strategy for future exosomal RNA sequencing analysis. A flowchart of the study design is shown in Figure Human plasma samples were obtained from the Mayo Clinic and stored at -80°C before use. Exosomes were isolated from 250 μL of plasma using the ExoQuick exosome precipitation solution  according to the manufacturer’s instructions with minor modifications. Briefly, the plasma was incubated with thromboplastin D  for 15 min at 37°C. After centrifugation at 10,000 rpm for 5 min, the supernatant was mixed with 75 μL of ExoQuick solution and RNase A  to a final concentration of 10 μg/mL. The mixture was kept at 4°C overnight and then further mixed with 150 units/mL of murine RNase inhibitor (NEB) before centrifugation at 1500 g for 30 min. The exosome pellet was dissolved in 25 μL 1 × PBS; 2 μL of the solution was reserved for evaluation of exosome size and concentration using the NanoSight LM10 instrument , and RNA was extracted immediately from the remaining solution.The concentration and size distribution of the isolated exosomes were measured using NanoSight. Prior to sampling, the sample solutions were homogenized by vortexing, followed by serial dilution to a final dilution of 1:100,000 in 0.2 μm-filtered 1x PBS. The National Institute of Standards and Technology (NIST) traceable 97 nm ± 3 nm polystyrene latex standards were added and analyzed along with the diluted exosome solution to validate the operation of the instrumentation. A blank 0.2 μm-filtered 1x PBS was also run as a negative control. Each sample analysis was conducted for 90 seconds. The Nanosight automatic analysis settings  were used to process the data. All samples were evaluated in triplicate.6 HEK293 cells were mixed with 700 μL QIAzol lysis buffer, and the mixture was processed according to the manufacturer’s standard protocol. The extracted RNA was eluted with 14 μL of RNase-free water. The quantity and quality of the RNA were determined by Agilent Bioanalyzer 2100 with a Small RNA Chip for exosomal RNA, and a RNA 6000 Pico Kit for cellular RNA .Exosomal or HEK293 cellular RNA was prepared using a miRNeasy Micro Kit . Twenty-three μL of exosome suspension or 1 × 10RNA isolated from the plasma exosomes was first incubated at room temperature, either with 30 units/μL of DNase I (QIAGEN) for 10 min or with 10 μg/mL RNase A for 30 min. The RNase A digestion was terminated by adding 150 units/mL of murine RNase inhibitor. The resultant RNA samples were processed with the Agilent Bioanalyzer. In another enzyme protection assay, before the addition of murine RNase inhibitor, plasma samples were incubated with 10 μg/mL RNase A under various conditions, namely, at 37°C for 15 min, at room temperature for 30 min, or at 4°C overnight, followed by exosome isolation and RNA extraction. The same procedure was carried out using commercially available small RNA, which acted as a control for this assay. The RNA eluents along with the naked small RNase A-treated RNA were then evaluated with the Agilent Bioanalyzer.For each library, 2 ng of small RNA was used in all the experimental procedures. Each library was prepared with a unique indexed primer so that the libraries could all be pooled into one sequencing lane. The 14 RNA libraries were prepared and amplified following the instruction of each manufacturer. The amplified libraries were resolved on a native 5% acrylamide gel. DNA fragments from 140–160 bp (the length of miRNA inserts plus the 3′ and 5′ adaptors) were recovered in 12 μL elution buffer (QIAGEN). The indexed libraries were quantified on the Bio-Rad 1000 qPCR instrument using the KAPA Library Quantification Kit in triplicates, according to the manufacture’s protocol . Ten μL of the pooled library at a final concentration of 2 nM were then sent to the Core Facility at Medical College of Wisconsin for sequencing using Illumina HiSeq2000 DNA sequence analyzer.Perl scripts (available upon request) were developed to process the data from the RNA sequencing. Raw reads were first extracted from FASTQ files, and trimmed using a sequencing quality control of Q >13 . Then th6. multiplied by 1 × 10The RNA sequencing data are available from the NCBI Gene Expression Omnibus database [GEO: GSE45722].To validate the RNA sequencing data, we performed a qPCR analysis of miR-92a-3p, miR-191-3p, miR26b-5p, and β-actin. The miRNA-specific miScript Primer Assays and the primer set specific for β-actin were purchased from QIAGEN . First, 5 ng exosomal RNA or 20 ng cellular RNA was reverse transcribed by the miScript II RT kit (QIAGEN) at 37°C for 60 min, and then the enzyme was inactivated at 95°C for 5 min. After the activation of the polymerase enzyme at 95°C for 15 min, 40 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 30 s were performed on the SteponePlus instrument (ABI). Melting curve analysis was used to confirm the specificity of the amplification reactions.To find novel miRNAs, we applied miRDeep2 and processed the raw sequencing data independently . Predicthttp://mirdb.org/miRDB/), an online database for miRNA target prediction and functional annotations. All the targets were predicted using MirTarget2 [http://david.abcc.ncifcrf.gov/) provides a comprehensive set of functional annotation tools to understand biological meaning behind large list of genes [We downloaded all miRNA target genes from miRDB  generated more sequences with 21–23 nt inserts than did the other two kits that were tested. Overall represents the averages of the three different kits that were tested.Click here for fileRead counts of the miRNAs detected in the 14 libraries .Click here for fileTop 20 RNAs in other RNA species .Click here for filePutative miRNAs predicted by miRDeep2.Click here for file