Dengue virus is a growing global health threat. Dengue and other flaviviruses commandeer the host cell’s RNA degradation machinery to generate the small flaviviral RNA (sfRNA), a noncoding RNA that induces cytopathicity and pathogenesis. Host cell exonuclease Xrn1 likely loads on the 5′ end of viral genomic RNA and degrades processively through ∼10 kB of RNA, halting near the 3′ end of the viral RNA. The surviving RNA is the sfRNA. We interrogated the architecture of the complete Dengue 2 sfRNA, identifying five independently-folded RNA structures, two of which quantitatively confer Xrn1 resistance. We developed an assay for real-time monitoring of Xrn1 resistance that we used with mutagenesis and RNA folding experiments to show that Xrn1-resistant RNAs adopt a specific fold organized around a three-way junction. Disrupting the junction’s fold eliminates the buildup of disease-related sfRNAs in human cells infected with a flavivirus, directly linking RNA structure to sfRNA production.DOI:http://dx.doi.org/10.7554/eLife.01892.001 More than 40% of people around the globe are at risk of being bitten by mosquitoes infected with the virus that causes Dengue fever. Every year, more than 100 million of these individuals are infected. Many develop severe headaches, pain, and fever, but some develop a life-threatening condition where tiny blood vessels in the body begin to leak. If not treated quickly, this more severe manifestation of the illness can lead to death.There are currently no specific therapies or vaccines against Dengue or many other closely related viruses such as West Nile and Japanese Encephalitis. These viruses use instructions encoded in a single strand of RNA to take over an infected cell and to reproduce. The viruses also exploit an enzyme that cells use to destroy RNA to instead produce short stretches of RNA called sfRNAs that, among other things, may help the virus to avoid the immune system of its host. Understanding exactly how Dengue and other viruses thwart this enzyme—which is called Xrn1—may help scientists develop treatments or vaccines for these diseases.Chapman et al. have now shown that Dengue virus RNA contains a number of RNA elements that prevent it being completely degraded by the Xrn1 enzyme. In particular, a junction formed by three RNA helixes is critical for stopping the enzyme in its tracks, leaving the disease-associated sfRNA behind. A single mutation in the Dengue RNA disrupts the structure of the three-helix junction and allows the enzyme to completely destroy the RNA. A similar mutation was also made in the West Nile virus RNA and when human cells were infected with the mutated West Nile virus, the short sfRNAs were not produced. Treatments or vaccines targeting this structure may therefore help reduce illness associated with Dengue and related viruses.DOI:http://dx.doi.org/10.7554/eLife.01892.002 Flaviviruses (FVs) are single-stranded, (+)-sense RNA viruses that include West Nile (WNV), Yellow Fever (YFV), Japanese Encephalitis (JEV) and other human disease-causing viruses . Dengue FVs enter the cell through receptor-mediated endocytosis, culminating in the release of an infectious, ∼10.5 kB genomic RNA (gRNA) into the cytoplasm . The capIn addition to replicated copies of the gRNA, high levels of shorter non-coding viral RNAs are also produced during FV infection . These sExamination of Xrn1 illustrates how remarkable it is that FVs have evolved a way to resist the exonuclease at a specific and defined point within the gRNA. RNA degradation by Xrn1 takes place in the enzyme’s catalytic chamber buried within a conserved active site . ProgresHow do sfRNAs escape degradation by Xrn1? Current evidence suggests that specific RNA sequences and structures located in the 3′UTR of different FVs are the signals that cause Xrn1 to stall. Presumably, these RNAs have unique features that make them impervious to Xrn1. It has been proposed that both stem-loop (SL) and dumbbell (DB) type structures found in the FVs 3′UTRs can halt Xrn1 progression . Of thesHere, we describe our interrogation of the structure and Xrn1-resistant properties of the complete 3′UTR of a serotype 2 Dengue Virus (DENV2). Chemical probing of the global structure of the 3′UTR revealed five independently-folded RNA structures within the longer RNA. We recapitulated Xrn1 resistance in vitro and developed a new fluorescence-based assay capable of quantitative, real-time monitoring of RNA degradation by Xrn1 and resistance to the enzyme. We used this assay to explore the Xrn1-resistant behavior of RNA structures throughout the DENV2 3′UTR, identifying two Xrn1-resistant RNA structures that confer quantitative protection to downstream segments of RNA. Using one of these structures we developed a series of mutants and precisely mapped where Xrn1 halts, revealing that a specifically-structured three-way junction is a functionally critical element of Xrn1 resistance. Similar mutations within the Xrn1-resistant structures of another FV (the Kunjin strain of WNV), also impair the formation of sfRNAs in vitro and in infected human cells. Cumulatively, these results set the stage for detailed mechanistic and structural studies of these unique, disease-related viral RNAs.in silico predictions  and immediately downstream of helix P1  are unreactive to both NMIA and DMS. These data suggest the formation of a stable PK that is predicted by Shapeknots and is therefore included in the refined secondary structure .10.7554/The DENV2 3′UTR is predicted to contain two adjacent ‘dumbbell’ motifs spanning nucleotides 10,450–10,535 (DB1) and 10,541–10,624 (DB2) . SimilarOur chemical probing data are consistent with the formation of a previously-identified stem-loop structure and small upstream hairpin at the extreme 3′ terminus of the viral genome  . In our Bedelovibrio bacteriovorus (BdRppH) to convert the 5′-triphosphate generated during in vitro transcription to a monophosphate (Kluveromyces lactis (KlXrn1)   to degra(KlXrn1) . TruncatUsing this assay we first tested the SL II element of the DENV2 3′UTR for resistance to Xrn1 using a 5′ leader consisting of the 31 nucleotides that naturally precede SL II in the viral genome. Addition of BdRppH and KlXrn1 to a reaction containing this test RNA as well as a synthetically generated 5′-monophosphorylated 24 nucleotide-long internal control RNA (24-mer) resulted in complete degradation of the control RNA and truncation of the test RNA to a discrete product, indicative of Xrn1 resistance . BecauseTo augment the aforementioned assay and enable monitoring of Xrn1 resistance in a quantitative and time-resolved manner, we engineered a fluorescence-based assay based on the malachite green (MG) aptamer . The fluTo assess the products of putative Xrn1 resistance from the fluorescence assay, we analyzed the reactions from the completed experiment using denaturing polyacrylamide gel electrophoresis (dPAGE) . Consistprod) -DVxrRNA1-MG and recovered the truncated product RNA by dPAGE (DVxrRNA1-MGprod) . We thenprod) . This exWe then interrogated the chemical nature of the 5′ end left by Xrn1, which speaks to a possible mechanism for Xrn1 resistance. Substrate recognition by Xrn1 requires a 5′ monophosphate at the end of an RNA and each catalytic cycle regenerates this chemical species. Could Xrn1 resistance be based in part upon malfunction of this process? If so, the products formed by xrRNAs might not bear a 5′ monophosphate and would be precluded from further degradation. We determined the phosphorylation state of the 5′ end of Xrn1-resistant products formed by DVxrRNA1 using the procedure outlined in The results presented above and those in the literature lead to the hypothesis that Xrn1 resistance depends on a specific RNA structure. To link RNA sequence, structure, and function and to identify the elements of the DVxrRNA1 that are important for conferring Xrn1 resistance, we designed eleven mutants targeting conserved xrRNA sequence and secondary structure elements and tested their Xrn1-resistant properties . As befoMutants 1–3 tested the importance of the putative DVxrRNA1 PK. Mutants 1 and 2 cannot form the hypothesized PK and are less resistant to the Xrn1 than WT, but to only a moderate extent . Mutant 3, in which the potential for a PK is restored, has an activity at 90% of WT. The observation that mutants 1 and 2 retain robust Xrn1 resistance is consistent with our chemical probing data that argue against the formation of a stable PK in the resistant RNA . OverallMutant 4 tested the contribution of the L2 loop to Xrn1 resistance. When the 7 nucleotides in this region were mutated to their Watson-Crick (W-C) complement the RNA remained essentially fully resistant to Xrn1. This finding is consistent with the lack of phylogenetic conservation observed in this region.Mutants 5 and 6 tested the importance of conserved nucleotides located within the P1-P2-P3 three-helix junction of DVxrRNA1. Both of these mutations essentially abolish resistance to Xrn1. These findings are consistent with the high conservation of these sequence elements. Furthermore, the dramatic loss of function in these mutants indicates the three-way junction is a critical element in conferring Xrn1 resistance and is likely the central element around which the structure organizes.Mutants 7 and 8 tested the importance of the identities of the base pairs present in the P1 and P2 helices of DVxrRNA1. The motivation for exploring these mutations was based on the conservation of the sequences in these helical regions. In mutant 7, mutation of the natural G-C rich stem of P1 into a series of five A-U base pairs abrogates Xrn1 resistance, consistent with the need for a P1 helix of defined sequence. In contrast Xrn1 resistance is maintained in mutant 8, in which the C-G base at the base of P2 is exchanged for a U-A base pair. This result suggests accurate assignment of secondary structure in this region and reveals tolerance to mutations in this conserved base pair.Mutants 9–11 tested the consequences of truncating the DVxrRNA1 structure from either the 5′ or 3’′ end. Deleting the first 10 nucleotides after the conserved 5′ end of DVxrRNA1  yielded an RNA that wasn’t completely degraded over the course of the experiment but did not yield a truncated product indicative of Xrn1 resistance. This RNA likely has a 5′ end structure that inhibits efficient Xrn1 loading. Mutations 10 and 11 tested truncations of the 3′ end. Despite conservation of several nucleotides in this region, mutants 10 and 11 remain resistant to Xrn1. In conjunction with our experiments which define the 5′ border of xrRNA1 we can therefore localize Xrn1 resistance to a region of the DENV2 genome as small as 59 nucleotides (nucleotides U10299–U10358).2, mutants of DVxrRNA1 display a spectrum of electrophoretic behaviors, indicating a variety of effects on RNA structure. One mutant that stood out was the non-Xrn1-resistant mutant 6 (C10320→G)  gel electrophoresis, which is sensitive to changes in the global structure of a folded RNA . In gels10320→G) . In EDTAferences . These dferences  but indiKUN), we used this virus system to assess the generality and relevance. We first tested two proposed xrRNA structures located within in the WNVKUN 3′UTR  and WNVKUNxrRNA2 , demonstrate resistance to Xrn1 in vitro (KUNxrRNA1 and C10680→G in WNVKUNxrRNA2)  . We obseKUN xrRNA structure in the formation of sfRNAs during infection. We infected human 293T cells with both wild-type and mutant WNVKUN and assessed sfRNA accumulation at 48 hours post infection (h.p.i.). Northern blots of total RNA isolates show that infection by wild-type WNV produces three prominent sfRNA species  is comprised of individual structural elements that, based on our probing data, do not appear to interact with each other to form a higher-order structure. This differs from other viral 3′UTRs that we have shown can fold into a single higher-order structure . The preWe identified two resistant functional RNA structures  located in succession near the 5′ end of the DENV 3′UTR. These elements contain previously-proposed secondary structure stem-loop elements SL II and SL IV. We did not observe resistance from the DB elements in vitro; if they confer Xrn1 resistance during infection this may require interaction with a protein factor. The position of the two confirmed xrRNAs is consistent with a role in protecting downstream sequences and structures, including the DBs and 3′SL from degradation. Many other FV′s also contain two neighboring xrRNA structures at the 5′ end of their 3′UTR′s  . The evoCumulatively, our data suggest that a critical feature of Xrn1 resistance is the formation of a specifically structured three-helix junction. The presence of multiple conserved bases within the three-way junction, including several unpaired nucleotides, suggests that this core organizes the tertiary fold of the RNA. This idea is supported by that fact that this fold can be disrupted by a point mutation made in one of the unpaired nucleotides within the junction. The fold this junction organizes is important for Xrn1 resistance, and while it may be further stabilized by a PK, this tertiary interaction does not appear to be necessary for Xrn1 resistance in vitro (at least in the context of DVxrRNA1).Full understanding of the basis of Xrn1 resistance will require a high-resolution structure of an xrRNA, but our results allow some predictions. First, examination of the three-way junction features and comparisons with other RNA junctions suggest that it forms a ‘type-C’ three-helix junction in which P1 and P2 coaxially stack and P3 assumes an acute angle relative to P1 . Based oin trans  treated milli-Q water and routinely filtered through 0.22 μm sterile filtration systems . DNA primers were purchased from Integrated DNA Technologies  and used without further purification. Nucleic acid concentrations were routinely determined by monitoring a solution’s absorbance at 260 nm using a Nanodrop UV-Vis spectrophotometer.The sequence of a plasmid containing the 3′ UTR of the Jamaica/N.1409 strain of a serotype 2 Dengue virus (GenBank accession number M20558.1) was verified by Big Dye sequencing at UC AMC core facilities. To make dsDNA templates for transcription, appropriate regions of this plasmid were amplified by PCR and transcribed as diagrammed in 4, 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris pH 7.9, 0.1% Triton X and Pfu DNA polymerase. PCR efficiency was verified by 1.5% agarose gel electrophoresis.dsDNA templates were routinely made through two stages of PCR. First stage PCR was routinely carried out on the 50 μl scale. 20 μl of the first reaction was then used as a template for a second reaction carried out on a 1 ml scale. Typical PCR conditions: 25 ng plasmid DNA or 0.2–0.4 μM duplex DNA template, 0.5 μM forward and reverse primers, 250 μM dNTPs, 2 mM MgSO2, 30 mM Tris, pH 8.0, 10 mM DTT, 0.1% Spermidine, 0.1% Triton X, and T7 RNA polymerase produced within our lab. 1–3 μl of 0.8 U μl RNasin Plus RNase Inhibitor  was occasionally included. Reactions were typically incubated 8–12 hr at 37°C.1 ml PCR reactions were typically used as a direct input into 5 ml transcriptions. Transcription reactions contained: ∼0.1 μM template DNA, 8 mM NTP’s, 60 mM MgClFollowing transcription, insoluble inorganic pyrophosphate was removed by centrifugation. Reactions were brought to 2 M LiCl, stored at −20°C for 2–16 hr and then precipitated by centrifugation. Pelleted RNA was resuspended in 3 ml of 300 mM EDTA, 100 mM triethylammonium acetate (TEAA), pH 7.0 prior to HPLC purification.RNAs were purified using an Agilent 1260 Infinity HPLC using gradients of 1–30% acetonitrile in 100 mM TEAA, pH 7.0 over a Varian PLRP-S 8 μm 150 × 7.5 mm column. RNA containing fractions were pooled, concentrated and finally buffer exchanged into water using Amicon Ultracel concentrators. RNA was then stored at −20°C until used.Kleuveromyces lactics  and SDS-PAGE SDS-PAGE . We haveCroatalus adamanteus venom  and Phosphodiesterase II from bovine spleen  were obtained as lyophilized solids . To create working stocks of each enzyme approximately ∼5 mg of either white solid was dissolved in 500 μl of 50% glycerol, 200 mM NaCl, 50 mM Tris, pH 7.5. According to the manufacturer’s specifications this results in an ∼8 × 10−4 U/μl solution of CaPdeI and a 0.05 U/μl solution of BtPdeII. Reactions of (+30)-DVxrRNA1 with these enzymes were conducted as described for reactions involving Xrn1 except that they were incubated at 50°C. Reactions using CaPdeI were supplemented with 10 mM Zn(OAc)2, which has been reported to be an essential cofactor of the enzyme.Phosphodiesterase I from During the course of these studies we also tested commercially available versions of Xrn1 and RppH . These enzymes can be used to reproduce our findings.In general, RNA was refolded prior to each experiment described in this manuscript. This was done using a thermocycler program that first ramped to 90°C for 2 min, held at 20°C for 5 min, and finally cooled and held at 4°C until used.2, 50 mM Tris, pH 7.9, 1 mM DTT. For simplicity we will refer to these conditions as 1X EC3 throughout this supplement.The majority of experiments described here were carried out in 100 mM NaCl, 10 mM MgClDenaturing polyacrylamide gel electrophoresis was carried out using gels consisting of 8M urea, 1X TBE .In order to streamline experimentation and accommodate technical aspects of capillary electrophoresis, individual regions of the Dengue 3′UTR were transcribed between adjacent 5′ and 3′ ‘cassettes’. These cassettes were uniformly installed before and after each of the five shorter RNA constructs as diagrammed DMS modification was carried out in 10 μl reactions containing 2 μM RNA and 57 mM DMS in 1X EC3. Reactions were incubated for 5 min at 0°C then quenched by the addition of 1 μl of neat β-mercaptoethanol.NMIA modification was carried out in 10 μl reactions containing 2 μM RNA and 2.5 mM NMIA (Invitrogen) in 1X EC3. Reactions were incubated at 37°C for 1 hr.2, 6 mM DTT, 55 mM Tris, pH 8.3, and 533 μM dNTPs.Following chemical modification, reactions were passed over homemade G-25 Sephadex  spin columns and recovered. 1.25 pmol  of an appropriate 5′ 6-FAM end-labeled primer was then added. Reactions were then re-annealed in order to hybridize these primers to each RNA. Reverse transcription was then carried out using Superscript III reverse transcriptase in a reaction containing 0.1 μM primer and ∼1.5 μM RNA, 80 mM KCl, 3.23 mM MgClFollowing reverse transcription, these reactions were again desalted using G-25 spin columns and brought to 50% formamide . 1 μl of a ROX-labeled DNA ladder  was then added to each ∼40 μl reaction. Reactions were then heated at 90°C for 10 min in order to denature cDNA products from the RNA templates and subsequently transferred to 96-well plates prior to capillary electrophoresis.Capillary electrophoresis was carried out using an ABI 3500 Gene Scanner equipped with a 50 cm capillary unit filled with POP-7 polymer (ABI).In chemical probing experiments 2 μM RNA was folded in 1X EC3 and then reacted with NMIA or DMS. The RNAs mapped in this study were each subjected to a titration of these reagents in order to optimize the extent of modification and the corresponding efficiency of reverse transcription. When the full length 3′UTR was mapped using a primer corresponding to the 3′ end of this RNA we observed significant background pausing in RT just prior to the 3′ component of the DB1 pseudoknot (data not shown). We therefore designed a second, internal primer to monitor chemical modification 5′ of this position in the full length 3′UTR/sfRNA .DMS modiCapillary electrophoresis data were exported as .fsa files and processed using the HITRACE-web online software . This soA standard assay for determining Xrn1-resistance was developed in which 4 μg of RNA  was folded in 36 μl of 1X EC3. In some experiments 300–450 pmol (∼10 μM) of a synthetically prepared 5′-monophosphorylated 24-mer (IDT) was also included as an internal control for Xrn1 activity. Following refolding, 2 μl of >3 U/μl BdRppH was added and the reaction was split between two tubes. 1 μl of >3 U/μl Xrn1 was added to one half of the reaction while the other served as a (−)Xrn1 control. Reactions were incubated 6 hr at 37°C. Reactions were then quenched by the addition of an equal volume of a dPAGE loading dye containing 30 mM EDTA, 8 M Urea and 0.1% (wt/vol) xylene cyanol and bromophenol blue. RNA products were analyzed on 12–20% dPAGE gels and visualized by staining with methylene blue.To assess RNA decay kinetics through monitoring the fluorescence of the MG aptamer, 100 μl reactions of 4 μM RNA  were refolded in 1X EC3. Often several 100 μl reactions using the same RNA were carried out simultaneously and were pooled after being folded separately in an 8-tube PCR strip. While reactions were still pooled, 5 μl of >3 U/ μl RppH per reaction and 7.5–10 equivalents of malachite green  were added. 105 μl aliquots of this mixture were then divided into separate wells of a black, flat bottomed 96-well plate . Fluorescence was monitored using a Glomax Multi+ plate reader using a filter set that allowed excitation at 625 nm and monitoring of emission over 660–720 nm. Prior to the addition of Xrn1, fluorescence was measured for 5 min in order to ensure reactions had reached equilibrium and to allow RppH to start producing 5′-monophosphorylated Xrn1 substrates. At 5 min, 5 μl of >3 U/μl Xrn1 was added and the reaction was then monitored for the next 160 min. Under these conditions RNA decay rates vary linearly with added Xrn1 (data not shown). To produce the gel in In order to characterize the products left by Xrn1 resistance we conducted the experiment described for monitoring Xrn1 decay kinetics using (+31)-DVxrRNA1-MG RNA on an eightfold scale. The products of these reactions, along with an equivalent number of (−)Xrn1 control reactions, were recovered after 160 min, pooled and subjected to 15% dPAGE. RNA products were visualized by staining with methylene blue, carefully excised and eluted from the gel overnight in water. Eluted RNA was concentrated using Amicon molecular weight cutoff filters and frozen at −20°C before additional analyses. Subsequent quantification revealed Xrn1-resistant RNA products were recovered with a 16% overall yield or as 0.52 μmol from a 3.2 μmol prep.prod (product) and (+31)-xrRNA1-MG (control) RNAs were annealed to a 5′ end-labeled primer complementary to nucleotides 10,328 through 10,342 in DVxrRNA1. Reverse transcription was carried out as essentially as described for chemical mapping experiments. Reactions were quenched by the addition of 8 M urea, 10 mM EDTA loading dye and then heated to 90°C for 10 min in order to promote dissociation of cDNAs from the RNA. Reactions were then loaded onto 10% sequencing gels. Electrophoresis was carried out at 50 W for 4 hr. The resulting gels were removed, dried and exposed to Molecular Dynamics phosphor screens before being imaged on a Storm 720 scanner. Gels were visualized using ImageQuant software.Recovered DVxrRNA1-MG32P-ATP and 8 pmol of each RNA , refolded and then held at 4°C. Samples were brought to 1X EC3 by the addition of 2 μl of a 10X buffer and equilibrated on ice for 10 min. An equal volume of 50% (wt/vol) sucrose, 0.1% bromophenol blue, 0.1% xylene cyanol loading dye was added immediately prior to native gel electrophoresis. Electrophoresis was carried out at 4°C using 8%, 39:1 (mono:bis) polyacrylamide 1X TH gels  with 5 mM MgClIn the experiments presented here, 300 pmol of (+31) or (+0) leadered xrRNAs were reacted with 150 pmol of Xrn1 in 1X EC3 for 30 min at the temperature indicated. At 30 min, 450 pmol of a synthetic, 5′-monophosphorylated 24-mer was added and the reaction continued for 30 min. At 1 hr these reactions were quenched by the addition of an 8 M urea, 10 mM EDTA loading buffer and subjected to 10% dPAGE. Gels were visualized by staining with methylene blue.Mutant Kunjin viruses predicted to have defective sfRNA formation during infection were generated by overlap extension PCR. The FLSDX(pro)HDVr Kunjin virus infectious clone  was used to generate Kunjin virus stocks and cells were maintained in MEM plus 10% fetal bovine serum  and 1% streptomycin and penicillin  at 37°C in the presence of 5% CO2. BHK-21 cells were electroporated and virus was amplified by passaging once on BHK-21 cells to generate working stocks. Viral titers were assessed by plaque titrations, infections were performed in human 293T cells (MOI of 10), cells were washed twice after a 2 hr adsorption period, and total cellular RNA was collected at 48 h.p.i using TRIzol . Total RNA was treated with DNase I  to remove residual genomic DNA. Northern blotting was performed as described previously  was separated on a 5% denaturing polyacrylamide gel. RNA was then transferred onto a nylon membrane  for blotting and UV cross-linked before blocking for 30 min at 60°C in hybridization solution . In vitro transcribed, internally radiolabeled RNA probes to the entire 3′ untranslated region of Kunjin virus  were generated using a template containing the Kunjin 3′ UTR inserted into the pGEM-4 vector  in the EcoRI and HindIII restriction sites using these primers: 5′-GAATTCTAAATACTTTGTTAATTGTAAAT; 5’-AAGCTTAGATCCTGTGTTCTCGCACCACCA. Following an overnight incubation at 60°C to hybridize the probe to the membrane, blots were washed twice with wash solution  and twice with stringent wash solution  for 30 min each at 60°C. Hybridized RNAs were visualized by exposing the blot on storage phosphor screens and imaging on the Typhoon Trio Imager . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent . We found that these RNA structures also resist Xrn1 in vitro and their function demonstrates similar sensitivity to mutations made in conserved sequence elements. These data are contained in KUN is a convenient strain for cell-based assays, we used it to correlate the observations we made regarding Xrn1 resistance in vitro with the formation of sfRNAs during infection. Specifically, we introduced mutations in the virus that match those we studied in vitro (in the three-way junction), and observed that these decrease sfRNA formation in infected cells. These data are contained in We chose to address both of these points. First, we interrogated the Xrn1-resistant properties of two putative Xrn1-resistant RNA structures (xrRNAs) present in the 3’UTR of a flavivirus related to Dengue: the Kunjin strain of West Nile Virus (WNV


The RNA world hypothesis, that RNA genomes and catalysts preceded DNA genomes and genetically-encoded protein catalysts, has been central to models for the early evolution of life on Earth. A key part of such models is continuity between the earliest stages in the evolution of life and the RNA repertoires of extant lineages. Some assessments seem consistent with a diverse RNA world, yet direct continuity between modern RNAs and an RNA world has not been demonstrated for the majority of RNA families, and, anecdotally, many RNA functions appear restricted in their distribution. Despite much discussion of the possible antiquity of RNA families, no systematic analyses of RNA family distribution have been performed. To chart the broad evolutionary history of known RNA families, we performed comparative genomic analysis of over 3 million RNA annotations spanning 1446 families from the Rfam 10 database. We report that 99% of known RNA families are restricted to a single domain of life, revealing discrete repertoires for each domain. For the 1% of RNA families/clans present in more than one domain, over half show evidence of horizontal gene transfer (HGT), and the rest show a vertical trace, indicating the presence of a complex protein synthesis machinery in the Last Universal Common Ancestor (LUCA) and consistent with the evolutionary history of the most ancient protein-coding genes. However, with limited interdomain transfer and few RNA families exhibiting demonstrable antiquity as predicted under RNA world continuity, our results indicate that the majority of modern cellular RNA repertoires have primarily evolved in a domain-specific manner. In cells, DNA carries recipes for making proteins, and proteins perform chemical reactions, including replication of DNA. This interdependency raises questions for early evolution, since one molecule seemingly cannot exist without the other. A resolution to this problem is the RNA world, where RNA is postulated to have been both genetic material and primary catalyst. While artificially selected catalytic RNAs strengthen the chemical plausibility of an RNA world, a biological prediction is that some RNAs should date back to this period. In this study, we ask to what degree RNAs in extant organisms trace back to the common ancestor of cellular life. Using the Rfam RNA families database, we systematically screened genomes spanning the three domains of life  for RNA genes, and examined how far back in evolution known RNA families can be traced. We find that 99% of RNA families are restricted to a single domain. Limited conservation within domains implies ongoing emergence of RNA functions during evolution. Of the remaining 1%, half show evidence of horizontal transfer (movement of genes between organisms), and half show an evolutionary history consistent with an RNA world. The oldest RNAs are primarily associated with protein synthesis and export. Following demonstration that RNA can act as genetic material A central tenet of RNA world theory as an account of the early evolution of life on Earth is the Principle of Continuity Assigning relic status to individual RNAs is not without significant complication. First, placing RNAs with non-universal distributions into the common ancestor of archaea, bacteria and eukaryotes requires lineage or domain-specific losses to be invoked These concerns notwithstanding, it remains commonplace for novel RNAs or RNA families to be discussed in regard to their potential relevance to the RNA world. Indeed, there are countless qualitative surveys derived from review of the experimental literature  origins. In addition, we sought to examine whether horizontal transfer between cellular domains (and viruses) is detectable for RNA families. We report an analysis of over 3 million RNAs spanning 1446 families in the Rfam database We first asked whether a systematic analysis of RNA families expands our knowledge of ancient RNAs beyond those identified by traditional experimental work. To examine the degree to which extant RNAs can be traced to earlier evolutionary periods, we performed comparative analyses of annotated RNAs based on data from all three domains of life as well as viruses. To this end, we used the Rfam (RNA families) database To generate a high-quality dataset, we first established the distribution of all individual RNA sequence entries in Rfam by reference to the NCBI taxonomy database, and manually vetted and removed probable false positive annotations. From the resulting dataset, we generated an initial survey of families and clans across bacterial, archaeal, eukaryotic and viral genomes Figure 1We next sought to establish whether the distribution the 12 interdomain Rfam families/clans Figure 1Figure S1), consistent with horizontal transmission of TPP-riboswitch regulated ThiC to the eukaryote lineage from a bacterial donor. Several independent observations are consistent with horizontal transmission: Arabidopsis THIC is nuclear-encoded, but targets to the chloroplast E. coli ThiC mutant Figure S1).A vertical trace is suspected but not demonstrated for the universally distributed TPP riboswitch . Surprisingly, the number of broadly distributed families/clans within each domain is small , though among eukaryotes there are a high number of clans, which may encompass multiple RNA families with a shared evolutionary history. Two patterns emerge from this analysis . However, closer inspection reveals most are singleton false positives or artefactual family groupings. Our dataset therefore does not allow the placement of any individual miRNA families in LECA.A different picture emerges for miRNAs however. The broad distribution of miRNAs is consistent with the suggestion that RNAi pathways trace to the LECA  & , Dataset S4), possibly attributable to the smaller number of experimental screens for novel RNAs across members of this domain.A broad qualitative difference between bacteria compared to archaea and eukaryotes is the preponderance of conserved regulatory elements, primarily riboswitches Figure 3Figure S2).In comparing the RNA repertoires of the three domains, a key question is whether the underlying Rfam data cover a reasonable spread of species within each domain, or whether data from a few species or phyla dominate. This is important in that the low number of broadly distributed families/clans we observe within each domain could be the result of an underlying sampling bias. A priori we may expect a significant bias, given current genomic coverage of microbial biodiversity. For instance, a recent survey of snoRNAs indicates there is broad, though nevertheless patchy coverage across major eukaryotic and archaeal groups For protein-coding genes, discovery of novel proteins has been significantly enhanced by sequencing of genomes chosen for maximal phylogenetic diversity How should we interpret these data? The limited distribution of domain-specific RNAs is likely to be biased by sampling, a problem that affects all genomic data, and is even more acute for detailed experimental data. On available data, we find that only a minority of domain-specific RNAs exhibit a broad distribution. A broad distribution could result from vertical inheritance, but it could also be the result of horizontal gene transfer. Taxonomic biases might underestimate the number of RNAs vertically traceable to the ancestor of a domain, whereas horizontal gene transfer might be expected to expand the distribution of some RNAs. Assuming that current sampling has gaps, but is not completely uninformative We have examined the evolution and diversity of RNAs across the entire tree of life, an important complement to previous comparative studies on RNA metabolism It is commonplace for novel RNAs or RNA families to be discussed in regard to their potential relevance to the RNA world, yet RNAs with limited distribution are difficult to reconcile with a very ancient evolutionary origin unless massive losses are invoked. Excepting the possibility of losses (which cannot be readily tested since the evidence for antiquity has been erased), our study shows that direct evidence for the RNA continuity hypothesis remains scant; there is undoubtedly an RNA ‘palimpsest’  & biological distinctness of the three domains Conversely, we find clear evidence of distinct domain-level repertoires, but limited evidence of inter-domain transfers, consistent with a recent analysis indicating a detectable vertical trace amidst ongoing HGT http://rfam.sanger.ac.uk/). The distribution of Rfam families (Dataset S1) was established in two steps. First, for a given family, all annotations across the EMBL database http://www.ebi.ac.uk/embl/) were binned into domains using the taxonomic information attached to each sequence. We then inspected annotations from families whose distribution spanned more than one domain to identify possible false annotations. For all Rfam families with annotations spanning two or more domains (including viruses) we first confirmed the taxonomic affiliation of each sequence through reciprocal blasts against the GenBank database and removed any cases where sequences were clearly misannotated . Next, we inspected the quality of each annotation with reference to Rfam seed alignments. Any sequences with a bitscore within +10 bits of the individual bitscore cutoffs for curated seed alignments, and where sequence similarity was deemed insufficient to reliably establish homology, were discarded.Annotated noncoding RNA data used in this study was derived from data curated in Release 10.0 of the Rfam database , Dataset S1), we used the top-level classifications within each domain in the NCBI Taxonomy Database. At the time the analyses were performed, the proposed archaeal phylum Thaumarchaeota In assigning Rfam entries to specific taxonomic groups of bacteria and archaea (http://molevol.cmima.csic.es/castresana/Gblocks_server.html), with the settings ‘Allow smaller final blocks’ and ‘Allow gap positions within the final blocks’ selected. ProtTest All sequences annotated as THIC in Genbank were retrieved (8 Feb 2011). The resulting list of 4508 sequences were examined for sequence similarity by generating a blast network using the blastall program from the BLAST package (version 2.2.18), with an E-value cutoff of 0.1. The network of blast results was visualized with CLANS Dataset S1Distribution of Rfam families across domains and major phylogenetic groups.(XLS)Click here for additional data file.Dataset S2Distribution of archaeal, eukaryote and bacterial Rfams.(XLS)Click here for additional data file.Dataset S3Distribution of eukaryotic miRNAs in Rfam.(XLS)Click here for additional data file.Dataset S4Numbers and taxonomic sources of annotations associated with RNA functional groups.(XLS)Click here for additional data file.Figure S1Unrooted PhyML phylogeny of TPP-regulated gene product THIC. (A) Tree in landscape format so labels are legible. The phylogeny shows good support for a close affinity between Plant and green algal (green) and a clan of proteobacterial homologs (red), to the exclusion of archaeal sequences (dark blue), consistent with possible HGT from bacteria to eukaryotes. Monophyletic groups are not recovered for either archaea or bacteria, suggestive of horizontal transmission events. All tips are labeled with the following information: MCL_cluster|Domain|gi_number|species_name. Bootstrap values are out of 108 (t of 108 . (B) Sam(TIF)Click here for additional data file.Figure S2Analysis of taxonomic distribution of Rfam entries within the EMBL nucleotide database. Data for each of the three domains (A) Eukarya (B) Archaea (C) Bacteria are binned by indicated major taxonomic groupings (see ings see . The x-a(TIF)Click here for additional data file.Figure S3Discovery curves for Rfam. These curves plot the oldest reliable electronic date (EMBL entry or publication) associated with a particular Rfam family. Domain distribution  is based on current distributions. To generate discovery curves for all RNA families in Rfam 10.0 (which includes families built before January 2010), we extracted the oldest dates from the literature references contained in the corresponding Stockholm file and from the EMBL accessions – the oldest date of the two is plotted.(TIF)Click here for additional data file.Text S1PDF with supporting text and references, and supplementary tables S1–S4.(DOC)Click here for additional data file.


Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons (DN) in the substantia nigra (SN). Several lines of evidence suggest that apoptotic cell death of DN is driven in part by non-cell autonomous mechanisms orchestrated by microglial cell-mediated inflammatory processes. Although the mechanisms and molecular network underlying this deleterious cross-talk between DN and microglial cells remain largely unknown, previous work indicates that, upon DN injury, activation of the β2 integrin subunit CD11b is required for microglia-mediated DN cell death. Interestingly, during brain development, the CD11b integrin is also involved in microglial induction of neuronal apoptosis and has been shown to act in concert with the DAP12 immunoreceptor. Whether such a developmental CD11b/DAP12 pathway could be reactivated in a pathological context such as PD and play a role in microglia-induced DN cell death is a tantalizing hypothesis that we wished to test in this study.in vitro and in vivo toxin-based experimental models of PD recapitulating microglial-mediated non-cell autonomous mechanisms of DN cell death. In vitro, enriched mesencephalic neuronal/microglial co-cultures were exposed to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) whereas in vivo, mice were administrated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) according to acute or subchronic mode. Mice deficient for DAP12 or CD11b were used to determine the pathological function of the CD11b/DAP12 pathway in our disease models.To test the possibility that DAP12 could be involved in microglia-associated DN injury, we used both in vitro. Yet, in vivo, mice deficient for either of these factors develop similar neuropathological alterations as their wild-type counterparts in two different MPTP mouse models of PD.Our results show that DAP12 and CD11b partially contribute to microglia-induced DN cell death in vitro but not in vivo in the MPTP mouse model of PD. Therefore, the CD11b/DAP12 pathway may not be considered as a promising therapeutic target for PD.Overall, our data suggest that DAP12 and CD11b contribute to microglial-induced DN cell death  Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons (DN) in the substantia nigra (SN). Although the mechanism by which these neurons degenerate is still uncertain, several lines of evidence suggest that apoptotic cell death of DN is driven in part by non-cell autonomous mechanisms implicating microglial cells and inflammatory processes . CurrentThe molecular signals from the dying/suffering DN neurons that trigger microglial cell activation and the oxidative burst are still largely unknown. Yet, recent evidence indicate that microglial CD11b, the alpha M subunit of the MAC1 β2 integrin, is likely to be involved in NADPH-oxidase activation and superoxide production ,5. This in vitro and in vivo models of PD recapitulating microglia-mediated non-cell autonomous mechanisms of DN neurodegeneration. We found that whereas DAP12 and CD11b partially contribute to microglia-induced DN cell death in vitro, mice deficient for either of these factors develop similar neuropathological alterations to their wild-type (WT) counterparts in two different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models of PD.To test this hypothesis, we used both KI) on C57BL/6 genetic background [tm1Myd/J on C57BL/6 background obtained from the Jackson Laboratory) were used. For in vitro experiments, primary mesencephalic neurons were prepared from gestational age 13 days C57BL/6J mice embryos whereas primary microglial cells were obtained from newborn C57BL/6J, DAP12KI or CD11b−/− mice. For in vivo experiments, ten- to twelve week-old male C57BL/6J, DAP12KI and CD11b−/− mice, weighing 25 to 30 g were used. Mice were kept in a temperature-controlled room (23°C ± 1°C) under a 12-hour light/dark cycle and had ad libitum access to food and water. All animals were further genotyped after their sacrifice. Animal handling was carried out according to ethical regulations and guidelines  and the European Communities Council Directive 86/609/EEC. Experiments using vertebrates were approved by the Services Vétérinaires de Paris.C57BL/6J mice , DAP12-mutated mice (DAP12+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCl) were purchased from Sigma-Aldrich . Dihydrorhodamine-123 (DHR-123), the cell permeant probes used for the detection of ROS was purchased from Molecular Probes .Lipopolysaccharide (LPS),1-methyl-4-phenylpyridinium . The cultures were then maintained in N5 medium supplemented with 5 mM glucose, 5% horse serum, and 0.5% fetal calf serum, except for the first 3 days in vitro (DIV) during which the concentration of fetal calf serum was set at 2.5% to favor initial maturation of the cultures [+) neurons represent approximately 1 to 2% of the total number of neuronal cells present in these cultures.Cultures were prepared from the ventral mesencephalon of gestational age 13 days C57BL/6J mice embryos. Dissociated cells in suspension obtained by mechanical trituration of midbrain tissue pieces were seeded at a density of 1.2 to 1.5 × 10KI or CD11b−/− mice, according to procedures described previously [6 cells per 35 mm dish).Microglial cells were derived from the cerebral cortex of newborn C57BL/6J, DAP12eviously . Pure am5 cells/well) were added to 4-DIV primary neuronal cultures grown in 24-well plates. Control neuronal cultures were supplemented with an equal volume of cell-free medium. LPS  or MPP+, at 0.1 μM, were applied directly to the mixed cultures at 5-DIV, that is, one day after the seeding of microglial cells on primary neurons.Freshly isolated microglial cells , and then washed twice with PBS before an incubation step at 4°C for 24 to 72 h with the following antibodies. A monoclonal anti-TH antibody diluted 1/5000  was used to assess the survival of DN. Microglial cells were characterized using a mouse anti-CD11b antibody . All antibodies were diluted in PBS containing 0.2% Triton X-100 except the mouse anti-CD11b antibody, which was diluted in PBS only. Detection of the primary antibodies was performed with a cyanin-3 conjugate of an anti-mouse IgG antibody  or with an Alexa Fluor 488 conjugate of an anti-rabbit antibody . Cell counting was performed at 200× magnification using a 20× objective matched with a 10× ocular. The number of TH2.-) levels were measured using dihydroethidium (Invitrogen) as fluorescent probes at 6-DIV, that is, when oxidative stress is at its peak. Mixed microglia-neuron cultures were exposed for 30 minutes to 1 μM dihydroethidium, washed three times, and then maintained in serum-free supplemented medium. The fluorescent signal, visualized by epifluorescence microscopy , was quantified using the Simple-PCI software from C-Imaging Systems  and a Nikon TE-300 inverted microscope equipped with an ORCA-ER digital camera . Fluorescent images of randomly chosen fields (six to eight in each culture condition) were acquired with a 20× fluorescent objective. The average pixel intensity over the surface of each cell body was determined under the different test conditions. Background fluorescence was subtracted from raw data, and the results were expressed as a percentage of the mean fluorescence intensity per cell in control cultures. A minimum of 60 cells was analyzed under each test condition.Superoxyde ion  injection of 30 mg/kg (free base) MPTP-HCl per day for 5 consecutive days and were then euthanized at 1, 2, 4, 7 or 21 days after the last MPTP injection. For acute MPTP intoxication, mice were i.p.-injected with 4 doses of 20 mg/kg (free base) MPTP-HCl at 2-h intervals and were then euthanized 2 or 7 days after the last MPTP injection. Control mice received an equivalent volume of 0.9% NaCl solution. For immunohistochemistry, mice were injected with a lethal dose of pentobarbital (100 mg/kg) and then transcardially perfused with 50 mL of heparin solution (5 U/mL) followed by 100 mL of ice-cold 4% paraformaldehyde (PFA) solution. After extraction from the skull, brains were further post-fixed overnight in fresh 4% PFA/PB solution, and cryoprotected with 30% sucrose in PB. Coronal free-floating striatal and mesencephalic sections (30-μm thick) were prepared using a freezing microtome (Leica) and collected in 10 regularly spaced series. For quantitative PCR, brains were rapidly removed from the skull and striata, ventral mesencephalon and cortex were dissected on humidified filters at 4°C. Tissues were then frozen in liquid nitrogen and kept at −80°C until use.Dat forward 5-CGC TGG AGG CAG TCG AA-3, and reverse 5-CGG AGC ATT TGC TTT TAC TCA TG-3; mouse Cd11b forward 5-GAT GCT TAC CTG GGT TAT GCT TCT-3, and reverse 5-CCG AGG TGC TCC TAA AAC CA-3; mouse Dap12 forward 5-TGG TGT TGA CTC TGC TGA TTG C-3, and reverse 5-CCT TCC GCT GTC CCT TGA C-3. Primer sequences of housekeeping gene were as follow: mouse Hprt forward 5-CTT CCT CCT CAG ACC GCT TTT-3, and reverse 5-AAC CTG GTT CAT CAT CGC TAA TC-3; mouse Gapdh forward 5-TGT GTC CGT CGT GGA TCT GA-3, and reverse 5-CCT GCT TCA CCA CCT TCT TGA-3.Real-time quantitative (q)PCR was performed as described . The priMice were euthanized 90 minutes after one i.p. injection of 30 mg/kg MPTP-HCl, and their striata were recovered and treated with 500 μL 0.1 N HClO4 before being processed for HPLC using UV detection (295-nm wavelength).Seven days after the last MPTP injection, mice were euthanized and their striata were dissected out and treated with 0.1 N perchloric acid containing 0.05% disodium ethylenediaminetetraacetic acid (EDTA) and 0.05% sodium metabisulfite. Striatal tissue content in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was assessed by high performance liquid chromatography (HPLC) using electrochemical detection with a potential set at +0.65 V.Immunohistochemical staining on mouse brain sections was performed as previously described . The folFor double-staining experiments, brain sections were simultaneously incubated with two primary antibodies: anti-CD11b , anti-DAP12 . Sections were then incubated in specific CY3- or Alexa488-conjugated secondary antibodies  at 1:1000 dilution for 120 minutes at room temperature.+ and Nissl+ cell bodies were quantified stereologically on regularly spaced sections covering the whole substantia nigra pars compacta (SNpc) using the VisioScan stereology tool. The investigator performing the quantification was blinded to the treatment and genotype groups during the analysis. Fluorescent sections were analyzed on a Zeiss Axioplan 2 using ExploraNova FluoUp 1.0 software. Striatal TH optic densitometry was measured by image analysis software .DAB-immunostained sections were analyzed by bright-field microscopy, using a Leitz microscope equipped with image analysis software . THt-test, or when data were not normally distributed, with the nonparametric Mann–Whitney U-test. Differences in means among multiple datasets were analyzed using one- or two-way analysis of variance (ANOVA) with time, treatment, or genotype as the independent factors. When ANOVA showed significant differences, pairwise comparisons between means were tested by the Tukey post hoc analysis. When data were not normally distributed, ANOVA on ranks was used . In all analyses, P-values of less than 0.05 were considered significant .All values are expressed as the mean ± standard error of the mean (SEM). Differences in means between two groups were analyzed using the two-tailed Student’s in vitro using mixed cultures of mesencephalic neurons and microglial cells . These results show that like CD11b, DAP12 contributes also to the LPS-induced neurotoxicity of microglia.It has been shown that CD11b is involved in LPS-induced neurotoxicity of microglia . In addi+[+ treatment of mesencephalic neuronal cultures induces, on average, a 20% DN cell loss  . These results show that microglial DAP12 and CD11b participate in non-cell autonomous mechanisms of DN cell death induced by MPP+.We next investigated the role of microglial DAP12 and CD11b in a more relevant model of PD-associated dopaminergic cell death. We thus cultured mesencephalic neurons alone or with WT, DAP12- or CD11b-deficient microglia and monitored the selective death of DN induced by the neurotoxin MPP+. As expe+-stimulated microglia is mediated, at least in part, by the DAP12- and CD11b-dependent production of ROS. To test this hypothesis, we first established an assay to measure microglial cell-derived ROS production. We took advantage of the property of O2.- to specifically oxidize dihydroethidium (DHE) to ethidium, which then binds to the nucleic acids in the cells in which it has been produced [+. As expected, exposure of neuron/microglia co-cultures with MPP+ induced a significant increase in the intensity of ethidium production by microglial cells  . This result shows that in contradiction with a previous in vitro study [+-induced DN injury is not dependent on DAP12 or CD11b.In some systems, the mechanism of neuronal death induced by activated microglial cells involves the CD11b-dependent production of ROS ,9. This produced . We thusin vitro data show that DAP12 and CD11b contribute to the toxicity of microglia stimulated by MPP+-induced dopaminergic injury. These results suggest that microglial DAP12 and CD11b could be involved in dopaminergic neurodegeneration in the MPTP mouse model of PD. We thus investigated the involvement of DAP12 and CD11b in this pathological process.Our Dap12 and Cd11b mRNA expression in the mesesencepalon of mice acutely intoxicated with MPTP. In agreement with the time course of DA neuron injury previously described, we found that dopamine transporter (Dat) mRNA level was consistently decreasing from day 1 onward , but also provide further evidence that microglial CD11b and DAP12 may act in concert to induce neurotoxicity as in the developing hippocampus [in vitro. Such a difference might be explained by the fact that we directly measured the intracellular microglial superoxide ions whereas in the other studies total extracellular ROS was measured [We have now shown that microglial DAP12- or CD11b-deficiency is associated with increased DN survival in a mouse pocampus ,9. Yet, pocampus ,5, deficmeasured ,5.+-exposed mesencephalic neurons cultured with microglia. In addition, DAP12 and CD11b were upregulated in nigral microglial cells following MPTP-induced nigrostriatal pathway injury. This prompted us to test the possibility that a microglial CD11b/DAP12 pathway could be involved in non-cell autonomous mechanisms of DN cell death in the MPTP mouse model of PD. Surprisingly, neither DAP12- nor CD11b-deficiency in the mouse were associated with neuroprotection in this experimental model of PD. However, studying the same CD11b−/− transgenic line with the same genetic background, gender and age, it has been found that CD11b-deficient mice were strongly protected against MPTP toxicity [in vivo, an original finding of our study is that the DAP12 is not crucially involved in the demise of DN in the MPTP mouse model of PD. Therefore, while the developmental CD11b/DAP12 pathway is likely to be reactivated in a PD-like neuropathological context, it is unlikely to play a primary role in neurodegeneration in vivo.DAP12- and CD11b-deficiency lead to partial neuroprotection of MPPtoxicity . Such diin vitro but not in vivo. Yet, one may expect that cellular interactions and signaling are much more complex in an integrated biological system than in culture. Hence, inhibition of the CD11b/DAP12 pathway in vivo could be compensated by alternative mechanisms, such as activation of Toll-like receptor-4 (TLR4), which is exclusively expressed by microglial cells in the CNS [in vivo. Since activation of the TREM2/DAP12 pathway has been shown to inhibit TLR responses in macrophages [in vitro data do not support this latter scenario, this will need to be tested in vivo in WT and DAP12-deficient mice challenged by LPS infusion into the SN.It is currently unknown why microglial CD11b or DAP12 deficiency is protective  the CNS . Previou the CNS . Interes the CNS . Since H the CNS , one mayrophages ,29, DAP1in vitro and that DAP12 is reactivated during PD-like nigrostriatal pathway injury induced by MPTP intoxication in adult mice. Yet, genetic ablation of either DAP12 or CD11b in mice does not improve MPTP-associated neuropathological alterations suggesting that this developmental microglia-induced neuronal death mechanism is unlikely to play a significant role in the pathophysiology of PD and should not, therefore, be considered further as a promising therapeutic target for this neurodegenerative disorder.In summary, we have shown that microglial DAP12 and CD11b contribute partially to microglia-associated DN cell death in vitro; DMEM: Dulbecco’s modified Eagle’s serum; DN: Dopaminergic neuron; DOPAC: 3,4-dihydroxyphenylacetic acid; EDTA: Ethylenediaminetetraacetic acid; HPLC: High performance liquid chromatography; HVA: Homovanillic acid; ITAM: Immunoreceptor tyrosine-based activation motif; LPS: Lipopolysaccharide; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NAPDH: Nicotinamide adenine dinucleotide phosphate-oxidase; PBS: Phosphate-buffered saline; PD: Parkinson’s disease; PFA: Paraformaldehyde; qPCR: Quantitative polymerase chain reaction; ROS: Reactive oxygen species; RNS: Reactive nitrogen species; SEM: Standard error of the mean; SN: Substantia nigra; TH: Tyrosine hydroxylase; TLR4: Toll-like receptor-4; WT: Wild-type.ANOVA: Analysis of variance; CNS: Central nervous system; DAB: 3,3-diaminobenzidine; DHR: Dihydrorhodamine-123; DIV: Days The authors declare that they have no competing interests.in vivo studies including mouse perfusion, brain processing, tissue immunohistochemistry, cell counting, double immunofluorescent staining on mouse brain tissue sections and data analysis, helped to set up in vitro models, performed the statistical analysis and draft and wrote the manuscript. YM set up the in vitro models, carried out cell cultures and treatments, cell immunostaining and counting, and performed data analysis. CB performed immunofluorescent staining on mouse brain tissue sections, image acquisition, and wrote the manuscript. DAF performed HPLC analysis. ECH gave a critical reading of the manuscript. AB participated in data analysis, draft and wrote the manuscript. SH conceived the study, participated in its design and coordination, performed MPTP intoxications, drafted and wrote the manuscript. All authors read and approved the final manuscript.KK carried out the KK present address: UPMC University Paris 06, CNRS, UMR7102, Team Development and Aging of the Nervous System, F-75005 Paris, France. ECH, AB and SH are investigators at the Centre National de la Recherche Scientifique (CNRS).


Neural Development describes the use of a synapse-specific label in Caenorhabditis elegans to implicate local UNC-6/netrin signaling in this developmental process. Thus, as well as their well known roles in cell migration and axon guidance, UNC-6/netrin signals distinguish an appropriate synaptic partner from other potential targets.Forming synaptic connections of the appropriate strength between specific neurons is crucial for constructing neural circuits to control behavior. A recent paper in http://www.neuraldevelopment.com/content/6/1/28See Research article:  Constructing neural circuits to perform biological functions is an intricate multi-step process that includes generating neurons, moving them to the right location, ensuring that they send out axons and dendrites to certain destinations, delivering pre- and postsynaptic components to the right parts of these neuronal outgrowths, and finally making the appropriate synaptic connections of the correct strength.Specificity is crucial throughout this developmental process. In the final step of circuit formation, when axons or dendrites reach their target region, they are likely to be confronted with many potential synaptic partners within a relatively small space, from which they need choose the correct one. As circuit construction occurs in the context of the complex developing nervous system, multiple steps in the formation of multiple circuits might overlap in time and space, posing an additional requirement that distinct circuits do not cross-interfere. That neurons robustly form synapses with the correct partner, while avoiding unsuitable targets, is therefore especially important to ensure correct information transmission and eliminate inappropriate crosstalk between circuits.Neural Development , which efficiently identifies specific synaptic contacts in living animals [What are the signals that govern this level of synapse specificity? A paper from Miri VanHoven and colleagues in k et al. ) adroitl animals .et al. report the first application of the GRASP technique for identifying molecules that influence synaptic partner choice  and its postsynaptic interneuron (AVA)  Figure , bottom.Using GRASP-labeled PHB-AVA synapses to facilitate a screen of candidate mutants, the authors identified UNC-6/netrin and its receptor UNC-40/DCC as central players in the formation of this synaptic connection. They also find that a mutant in UNC-40/DCC shows the predicted increase in posterior movement consistent with reduced transmission between PHB and AVA, thus providing a behavioral correlate to structural synaptic changes in living animals to demonstrate biological relevance .et al. indicate that the graded strength of synaptic connections can be controlled by UNC-6/netrin levels. Bidirectional control of graded transmission across two neurons also occurs during neuronal plasticity, raising the question as to whether proteins such as UNC-6/netrin are also involved in synaptic plasticity after the circuit is constructed.The number of PHB-AVA synaptic contacts is reduced in UNC-6/netrin or UNC-40/DCC mutants, whereas overexpression of UNC-6/netrin increases their number . TogetheDrosophila neuromuscular junction [C. elegans . In theet al. [UNC-6/netrin first gained prominence as an axon guidance molecule ,6; additet al.  now showet al. reveals that several downstream effectors of UNC-40/DCC are not essential for synaptic partner recognition even though they play important roles in axon guidance [First, how does the same signal lead to different biological outcomes? The work of Park guidance . Perhapsguidance ), and doDrosophila neuromuscular junction [Second, how is neural development compartmentalized such that early signals do not influence late steps, and that the development of one circuit does not interfere with another nearby? Signaling specificity is likely to require a combination of several developmental and molecular mechanisms. UNC-6/netrin is deployed in different cells for guiding PHB axon outgrowth versus synapse formation ,8. Thus,junction , might aFinally, would the repeated use of the same molecule require developmental programs to minimize cross-interference and thus constrain the types of structures and anatomy that could develop? Or would it be an economical way of coordinating complex structures so that related structures are organized around a single cue? An integrated analysis of the multiple processes involved in neural circuit formation will be an important next step in addressing these big picture questions in development, and tools like GRASP  are very


Over the past decade, the classifications and volume of non-coding RNA (ncRNA) transcripts have grown profoundly, spurred by transcriptomic sequencing efforts Bartel, . This haa priori information and annotation have the advantage of flexibility. This flexibility, however, comes at some expense in terms of accessibility, analysis, and quantification.One of the challenges with the emerging field of ncRNAs is the frequent updates to the current database of annotated transcripts and occasional changes in nomenclature, making it essential to keep current with available sequence information. For example, the miRBase server, which contains all annotated miRNAs, has already been updated through 19 different versions to date. Importantly, these updates can include addition or deletion of transcripts due to various reasons. Technology suppliers usually lag behind databases with their ncRNA platforms, although some have the capacity to include custom content if required for a particular study. The fluctuation in transcript annotation is also important to consider in the context of lncRNAs, as fixed platforms take longer to accommodate changes in this rapidly emerging genomic dimension. For this reason, RNA sequencing (RNA-Seq) approaches that don't rely on Despite considerable advances in ncRNA analysis, there remains technical challenges and key differences between platforms and approaches that may add further variation to an area already subject to heterogeneity. For example, postmortem miRNA profiling in schizophrenia has revealed significant changes associated with the disorder (Perkins et al., RNA-Seq is contributing to significant discoveries in neuroscience research. One of the key advantages of this approach is that poorly characterized RNA variants, particularly of the non-coding variety that lack comprehensive annotation, are now captured and can be analyzed. Previously, these molecules were only seen at great expense on genome tiling arrays and a few custom non-coding arrays. This technology has the potential to make a tremendous impact in the ncRNA world as they emerge for the first time with an equal billing as the coding transcripts. This technology, however, is also subject to some limitations that need to be considered for experimental design, in addition to the issue of ligation, amplification, and normalization bias mentioned already. Firstly, there are no one-size-fits all strategy for ncRNAs. Small RNAs, such as miRNA, need to be fractionated and treated separately to their longer counterparts. Standard RNA-Seq, also poly-A selection during library preparation and as such, will not enrich for RNA transcribed by pol III promoters and other intergenic promoters. Total RNA-Seq requires a ribosomal RNA depletion step, and to fully appreciate the influence of NATs it may be necessary to use strand-specific RNA-seq. Even more selection may be necessary to find rare non-coding transcripts. For example, ncRNAs were recently found in “gene deserts” after targeted enrichment using tiling arrays (Mercer et al.,


Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 “melting types” (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype  Enterococcus faecium emerged as a hospital-associated pathogen in the 1990s in the USA, Europe and Australia. The acquisition of resistance to ampicillin and vancomycin and spread of successful vancomycin-resistant clones has resulted in an increasing burden of vancomycin-resistant E. faecium (VREfm) as a difficult to treat nosocomial infection E. faecium means that using PFGE data to track strains over long time periods and to infer the large scale population structure can be problematic Pulsed-field gel electrophoresis (PFGE) has been considered the reference method for tracking outbreaks due to its high discriminatory power E. faecium genotyping method that provides results concordant with MLST would be useful for E. faecium monitoring and the initial investigation of suspected VREfm outbreaks. In this study a typing method was developed based upon the identification and high-resolution melting (HRM) interrogation of DNA fragments containing resolution-optimized sets of SNPs derived from the E. faecium MLST database. The use of such SNP sets in allele specific real-time PCR (AS kinetic PCR) based bacterial genotyping has been previously described E. faecium bloodstream isolates.However, in the meantime, a rapid, simple and low cost E. faecium blood culture isolates previously described and typed by MLST were included in this study E. faecium over a 12 year period at a single Australian institution. The isolates were collected as part of an infection control quality improvement project and formal approval by the local ethics committee was not required.A collection of 85 DNA was prepared using the DNeasy Tissue Kit  and the modified protocol for isolation of genomic DNA from Gram-positive bacteria as described in the manufacturer's instructions.SmaI-digested genomic DNA agarose plugs was performed as previously described PFGE of E. faecium MLST database at http://efaecium.mlst.net/ were derived using the program ‘Minimum SNPs’ Resolution optimized SNPs from the concatenated m) for these three isolates was determined, and the mean Tm difference to the closest 0.1°C was used to calibrate the other samples. Use of the three known control isolates in each run also facilitated the accurate assignment of HRM alleles (see below).Reactions were performed on a RotorGene 6000 device . Reactions volumes comprised 5 µL 2× Platinum® SYBR® Green qPCR SuperMix-UDG , 1 µM of each primer and 2 µL of DNA (1∶10 dilution) in a final volume of 10 µL. The temperature cycling parameters were: 95°C for 2 min; 30 cycles of 95°C for 5 s, 55°C for 20 s; followed by 72°C for 2 min and 50°C for 30 secs. Amplification was followed by HRM (72–88°C) increasing by 0.1°C at each step. Runs were performed in laboratories in two separate institutions by multiple operators and results were completely concordant and reproducible. Normalization temperatures for analysis of each fragment were standardized Table 1.m of each amplified fragment is in large part dependent on the absolute G+C content. HRMType® converts each MLST database-specified variant of each HRM fragment into a predicted HRM allele on the basis of G+C content. For example, for the atpA95 fragment, in the current E. faecium MLST database, the numbers of G+Cs are 79, 80, 82, 83, 84, 85. HRMType® then converts each known ST into a predicted multilocus HRM type that is termed a “Melting Type” (MelT), and displays each ST with a corresponding MelT. Our convention is that the HRM alleles are numbered according to the absolute number of G+C residues and the MelTs are numbered sequentially commencing with ST1 corresponding to MelT 1. If during experimental work a fragment  does not produce a curve as predicted according to G+C content, the unpredicted curve can be assigned a new HRM allele number. For this fragment , this new HRM allele number is assigned for all other STs that have the identical sequence in the fragment . HRMType® also calculates the cumulative Simpson's Index of Diversity (D) We used a previously described Stata/IC 10.1  “do_file” named HRMType® to assemble a predicted key for translating between MLST and multilocus HRM data in silico analysis of the MLST sequence.We adapted HRMType® to allow the input of the specific eight SNPs used by Rathnayake et al. http://goeburst.phyloviz.net/. This allows for the overlaying of the MelTs onto a snapshot of the MLST derived population structure of E. faecium.The Adjusted Rand Indices (ARI) were calculated using Comparing Partitions D. The SNPs were (locus name followed by nucleotide position within that locus): atpA 95, atpA 239, atpA 485, ddl 204, ddl 456, purK 460, pstS 95 and pstS 452. Amplified fragments incorporating these SNPs ranged in size from 79 to 180 bp  currently in the MLST database into 230 Melting Types (MelTs) with a D of 0.991, where the MLST database is regarded as providing D = 1. This indicates a probability of 0.991 that any two randomly selected STs will be discriminated according to their MelT.A set of eight SNPs within the MLST loci was identified on the basis of maximized o 180 bp Table 1.D for the seven loci are (in order of discriminatory power): atpA , ddl , purK , pstS , gdh , adk , gyd .Notably, the resolution optimized SNPs were identified in only four of the seven MLST loci. Examination of the MLST loci determined that these are the four most polymorphic loci with the largest number of alleles and individually they also provide the most discriminatory power compared to MLST. Specifically, the number of alleles (n) and E. faecium isolates pstS95, the raw data from the curves could have been exported without calibration and the curves correctly assigned. For fragment pstS95 , we feel that there continues to be an important role for rapid, low cost methodologies that make use of existing instrumentation present in many less resourced laboratories.Depending on the monetary, time and capillary sequencing resources available, MLST can be resource intensive for many laboratories. HRM typing utilizes eight PCR reactions with little downstream processing and is thus significantly cheaper than MLST, which requires an initial seven PCR reactions followed by capillary sequencing and analysis. Although less discriminatory than MLST, HRM typing can be used as an initial genotyping scheme to rapidly screen isolates at low cost while providing results that are concordant with MLST. For example, it can be used to identify globally recognized and endemic strains of Results S1Melting type and multilocus sequence type conversion key.(XLS)Click here for additional data file.Results S2Results for HRM typing, MLST, PFGE and SNP genotyping for 85 isolates.(XLS)Click here for additional data file.


We have developed a process for transcriptome analysis of bacterial communities that accommodates both intact and fragmented starting RNA and combines efficient rRNA removal with strand-specific RNA-seq. We applied this approach to an RNA mixture derived from three diverse cultured bacterial species and to RNA isolated from clinical stool samples. The resulting expression profiles were highly reproducible, enriched up to 40-fold for non-rRNA transcripts, and correlated well with profiles representing undepleted total RNA. Microbial communities are known to play significant roles in human health, development, and disease -4, and DMicrobial transcriptome sequencing poses significant challenges. Messenger RNA typically constitutes a very small fraction of the total RNA in bacterial cells, as vast amounts of ribosomes and, hence, rRNA are required to meet the cells' demand for protein synthesis. Moreover, the majority of bacterial mRNA is not polyadenylated, as it is in eukaryotes, and can, therefore, not be isolated using oligo-dT selection. Thus, specialized approaches are needed to enrich the desired transcripts for sequence-based characterization.Numerous rRNA-depletion methods have been developed. These include commercially available kits such as MICROBExpress (Ambion), which uses capture oligonucleotides targeting specific regions of the 16S and 23S rRNAs, and mRNA-ONLY (Epicentre), which utilizes a 5´-monophosphate-dependent exonuclease to degrade processed 5'-phosphorylated RNA molecules such as rRNAs. These kits are widely used, albeit with limited success for meta-transcriptomic purposes. For example, a recent study comparing MICROBExpress and mRNA-ONLY, either alone or in combination, achieved only a modest (1.9- to 5.7-fold) enrichment of bacterial mRNA with less than 25% of aligned sequencing reads representing transcripts other than rRNA . Similaret al. [Escherichia coli transcriptome [Recently, several alternative methods for removal of rRNA have become available: Ribo-Zero, a new hybrid-subtraction kit from Epicentre, promises to remove all species of rRNAs, including the 5S rRNA, from intact and partially degraded total RNA preparations from both Gram-negative and Gram-positive bacteria; the Ovation Prokaryotic RNA-seq System from NuGEN uses a proprietary set of 'not so random' primers to avoid rRNA as template during first and second strand cDNA synthesis similar to the strategy of Armour et al. ; and deget al.  has beencriptome .Our goal was to establish a robust and scalable RNA-seq process applicable to cultured bacteria as well as to complex community transcriptomes. An effective process should a) reduce rRNA sequences to very low levels; b) accurately maintain relative representation of transcript sequences; c) be equally successful for any species; d) work well with RNA of varying quality; and e) be highly reproducible. To this end, we evaluated rRNA depletion methods and chose a protocol that eliminates rRNA reads efficiently and robustly, largely irrespective of the quality of the RNA input sample. We paired this protocol with a strand-specific cDNA synthesis and RNA-seq approach  that helProchlorococcus marinus, Escherichia coli, and Rhodobacter sphaeroides) that cover a wide range of base compositions . We prepared total RNA from each organism and used these samples separately or as a 'PER' pool (mixed 1:1:1 by mass) of input material.To provide a benchmark for method evaluation, we prepared RNA from three well characterized organisms , a commercial kit for 'not-so-random' primed cDNA synthesis of non-rRNA templates (Ovation Prokaryotic RNA-Seq System), and a protocol for removing highly abundant cDNAs by low-csing DSN .We constructed RNA-seq libraries from total (undepleted) RNA and rRNA-depleted samples using each of the methods, sequenced them using the Illumina platform, mapped the reads to the three reference genome sequences and separately counted reads that aligned to rRNA and to the coding DNA sequence (CDS) of annotated genes . Read counts for protein-coding genes were normalized for CDS lengths and for the total number of sequencing reads by calculating RPKM (reads per kb per million mappable reads) values  for each2 R= 0.88, 0.95, and 0.88 for P. marinus, E. coli, and R. sphaeroides, respectively), indicating little, if any, systematic skewing caused by the rRNA-subtraction procedure.Without depletion, for all three organisms almost all mapped reads (>98%) aligned to rRNA ; however, it did not perform well for the GC-rich R. sphaeroides transcriptome . A detailed analysis of the CDSs for R. sphaeroides pointed to a high GC content as a major adverse factor  had a moderate mean GC content of 57%. The majority (83%) of transcripts were poorly enriched (<2-fold) or even depleted (68% GC on average within this group). It is possible that the high-GC fraction anneals faster or forms hairpins during the re-association reaction and, thus, becomes a substrate for degradation by DSN, thereby making it more difficult to enrich GC-rich CDSs relative to R. sphaeroides rRNA, which is only about 56% GC.Light normalization (DSN) reduced the proportion of rRNA reads appreciably for 2 R= 0.96, 0.97, 0.90 for P. marinus, E. coli, and R. sphaeroides, respectively); however, it was not effective in removing P. marinus and R. sphaeroides rRNAs  and was only slightly better for E. coli (70% rRNA reads remaining despite rRNA depletion). Neither mRNA-ONLY nor Ovation reduced the fraction of rRNA reads below 70%. Moreover, neither protocol generated CDS-expression profiles that correlated well with the undepleted total RNA control .MICROBExpress maintained a strong correlation between the total and rRNA-depleted PER pool . Cases of CDSs being present in only one or the other of two experiments were confined to rare transcripts with RPKM values <10 .To evaluate the effectiveness of the Ribo-Zero method on low quality RNA samples, we created a simulated low quality sample by fragmenting total PER RNA artificially such that the mode of the fragment size distribution was approximately 300 bases , 99.8% (E. coli) and 99.9% (R. sphaeroides). Results for other quartiles are presented in Figure S5 in Additional file To measure the strand specificity of our method, we calculated separate sense and antisense read densities for all members of a curated list of protein-coding genes. The median strand specificity for genes that were expressed in the top 10% expressed members was 99.8%  whereas RNA from stool B was partially degraded (RIN = 7).Methanobrevibacter smithii ATCC 35061). Subsequent alignment of the RNA-seq reads showed that the majority of the reads mapped to the 19 most abundant genomes in the stool samples  without dropouts over four orders of magnitude  and a concomitant increase of mapped reads to We developed a robust RNA-seq process that is applicable both for bacteria grown in culture and for complex bacterial communities from clinical samples, and in so doing have overcome the major challenge to microbial meta-transcriptomics: a universal rRNA depletion method that does not require high quality RNA. First, we compared five different methods for rRNA depletion and identified one protocol, Ribo-Zero, that works well for both intact and fragmented total RNA representing a wide range of base compositions. Second, we combined this method with a strand-specific RNA-seq approach  to distiPrior to our evaluation, MICROBExpress was considered the standard for microbial meta-transcriptomics applications despite limitations that included results being strongly affected by both community composition and RNA integrity, and that the method did not produce RNA-seq data sets containing less than 75% rRNA reads . By contTo increase the power of the method, we incorporated dUTP marking and degradation of second strand cDNA to generate RNA-seq libraries that preserve the orientation of the expressed transcripts . This prDespite our technical advances, the RNA-seq process is still limited by RNA input requirements. For cultured bacterial cells or microbial communities from stools, sufficient RNA starting material is usually obtainable, but input RNA can be limiting with other human or environmental samples. Based on our results, 5 µg of total RNA give excellent results. We have shown that libraries can be made effectively with as little as 5 ng input RNA; however, the Ribo-Zero kit does not perform effectively below microgram amounts of RNA and should be avoided. Several meta-transcriptomics studies resorted to polyadenylation and RNA amplification  or multiCurrently, large-scale metagenomics projects are rapidly cataloging microbial genes from habitats of the human body, including the gut (The European MetaHIT consortium ), nasal We note that nearly all steps in our process are amenable to automation, including nucleic acid extraction, cDNA synthesis and construction of Illumina sequencing libraries, and can thus be easily scaled to process large numbers of samples. With the exception of the rRNA depletion step with Ribo-Zero, all RNA fragmentation and enzymatic steps are cleaned up using automation-friendly paramagnetic beads; therefore, cDNA synthesis can be automated, as has been demonstrated with a similar protocol . Our IllWe have devised a robust and scalable process for bacterial RNA-seq that combines an efficient rRNA-removal protocol, automation-friendly cDNA-library construction, and maintenance of strand information. This process represents a significant improvement over previous methods and can be applied to profile gene expression in both simple and complex, naturally occurring bacterial communities.E. coli MG1655 and R. sphaeroides 2.4.1  were grown in 250 ml LB broth with shaking at 37°C and 30°C, respectively, to an OD600 of about 0.5. Each culture was divided into 50 ml aliquots. Cells were harvested by centrifugation at 4,000 × g for 10 minutes at room temperature. Pellets were resuspended in 25 ml of RNAlater . The tubes were agitated on a rotator at 4°C overnight, centrifuged at 4,000 × g for 10 minutes, placed in an ethanol/dry ice bath to flash freeze the pellet and stored at -80°C.P. marinus (MED4) cultures were a kind gift from Sallie Chisholm . Cells were grown in 8 L of PRO99 Sargasso sea water medium [Fresh r medium  in a 24°r medium -37. CellBacterial cell pellets stored at -80°C in RNAlater (25 ml) were thawed on ice, resuspended and re-pelleted in 1 ml aliquots for 10 minutes at 4,000 × g in a microcentrifuge. The supernatant was removed and 200 μl bacterial lysis buffer  and 15 μl proteinase K  were added to each tube. Samples were incubated at room temperature for 10 minutes, and vortexed for 10 s before and every 2 minutes during the incubation. QIAGEN RLT Plus buffer (750 μl) supplemented with 1 % v/v beta-mercaptoethanol (Sigma) was added to each tube and vortexed briefly to mix.Two stool samples were collected 7 months apart from a single human donor . Approximately 200 mg of stool were placed in approximately 2 ml RNAlater buffer, briefly mixed to disperse matter, and stored at room temperature during transport to the lab. The first sample (stool A) was extracted within 5 h of collection. The second sample (stool B) was stored at 4°C upon arrival for 24 h and then frozen and stored at -20°C for approximately 5 days until extracted. Prior to extraction, samples were vortexed briefly and centrifuged for 10 minutes at approximately 16,000 × g in a microcentrifuge at room temperature. Bacterial lysis buffer (100 μl) plus 10 μl proteinase K (20mg/ml) was added to half the stool sample (approximately 100 mg). Samples were incubated at room temperature for 10 minutes and vortexed for 10 s before and every 2 minutes during the incubation. QIAGEN RLT Plus buffer (1.2 ml) containing 1 % v/v beta-mercaptoethanol was added to each tube and vortexed briefly to mix. Samples were transferred into 2 ml sterile bead beating tubes  filled with 1 ml of 0. 1 mm glass beads (BioSpec Products), and placed in a bead beater  for 3 minutes on 'homogenize' setting.The lysed bacterial and stool samples were homogenized using QIAshredder spin columns (QIAGEN) and added to the AllPrep DNA spin columns for RNA and DNA isolation following the manufacturer's protocol. RNA integrity values   were detAn early access version of Meta-Bacteria Ribo-Zero rRNA removal kit  was used according to the manufacturer's instructions. RNA input amounts determined the amount of Ribo-Zero rRNA removal solution to add . Samples in Ribo-Zero rRNA removal solution were incubated at 68°C for 10 minutes followed by a 15 minute incubation at room temperature. To remove the hybridized rRNA molecules from the mRNA, the RNA/rRNA solution reactions were incubated with the prepared microsphere beads, mixed well and placed at room temperature for 10 minutes (mixing every few minutes), then at 50°C for 10 minutes. The mRNAs were separated from the microspheres bound with rRNAs by a filter column provided in the kit. The final purification of eluted mRNA was performed using Agencourt RNAClean XP beads .MICROBExpress  was used according to the manufacturer's specifications. Briefly, total RNA (5 to 10 μg) was combined with binding buffer (200 μl) and capture oligonucleotide mix (4 μl). The RNA mix was heated to 70°C for 10 minutes then incubated at 37°C for 15 minutes to hybridize the capture oligos. The RNA/capture oligo mix was equilibrated with oligomag beads  and incubated at 37°C for 15 minutes. Tubes were placed on a magnet to separate the supernatant containing the enriched mRNA from the oligomag beads. The enriched mRNA was purified and concentrated by ethanol precipitation according to the manual with precipitation at -80°C for 1 h.Enzymatic reactions using the mRNA-ONLY Prokaryotic mRNA Isolation Kit (Epicentre) were performed according to the manufacturer's specifications. Briefly, total RNA (5 to 10 μg) was combined with 2 μl mRNA-ONLY 10× reaction buffer, 0.5 μl ScriptGuard RNase Inhibitor, 1 μl Terminator Exonuclease (1 U) and nuclease free water in a final volume of 20 μl and incubated at 30°C for 60 minutes. Reactions were terminated with the addition of 1 μl of mRNA-ONLY stop solution (100 mM EDTA). Agencourt RNAClean XP beads (0.8× of the reaction volume) were used to purify the reaction according to the manual.E. coli DNA) and negative (nuclease free water) amplification control. The plates were sealed, centrifuged briefly, and placed in the thermal cycler  with the following cycling conditions: 95°C for 2 minutes, 30 cycles of 95°C for 20 s, 50°C for 30 s, 72°C for 5 minutes. If an amplification product of approximately 600 bp was observed, the RNA was treated again with DNase.The TURBO DNA-free kit (Ambion) was used for the DNase treatment. Total RNA or rRNA depleted RNA  was treated using a rigorous protocol that includes a second addition of DNase (2 to 4 units) and incubation at 37°C for 30 minutes according to the manufacturer's specifications. Reactions were terminated with the addition of the DNase inactivation reagent (0.2× the reaction volume) and purified using Agencourt RNAClean XP beads (0.8× of the reaction volume) according to the kit instructions. The presence of DNA contamination was assessed by PCR with 16S-specific primers. Each reaction included 1× AccuPrime PCR buffer II (10×), 0.75 U of AccuPrime Taq High Fidelity polymerase  and 200 nM of each primer (357F: 5'- CCTACGGGAGGCAGCAG -3' and 926R: 5'- CCGTCAATTCMTTTRAGT -3'). The DNase treated RNA (2 μl) was added to each reaction in a final reaction volume of 20 μl. Each reaction was run in parallel with a positive  in a final concentration of 1× per reaction. A maximum of 5 μg of RNA was added to each 10 μl fragmentation reaction, incubated at 80°C for 4 minutes on a thermal cycler, and placed on ice. Agencourt RNAClean XP beads (2.0× of the reaction volume) were used to purify the reactions according to the manual. An RNA fragment size distribution with a mode of approximately 300 bases was achieved with these conditions.E. coli DNA polymerase I , 10 U of E. coli DNA ligase , 5 U of RNase H  to the first strand reaction . After 2 h at 16°C, the reaction was stopped by adding 10 μl of 0.5 M EDTA and purified using MinElute PCR Clean up columns (QIAGEN) according to the manufacturer's instructions.cDNA synthesis was performed as previously described . Total oet al. [E. coli DNA polymerase I , 10 U of E. coli DNA ligase  and 5 U of RNase H  in a final volume of 150 μl. The second strand reaction was incubated at 16°C for 2 h. The reaction was stopped by adding 10 μl of 0.5 M EDTA and purified using MinElute PCR clean up columns (QIAGEN) according to the manufacturer's instructions or Agencourt AMPure XP beads (2.0× of the reaction volume).Strand-specific cDNAs were made by dUTP marking and degradation of second strand cDNA  using a et al. .. Total The Ovation Prokaryotic RNA-Seq System  was used as follows. Intact RNA was DNase treated as described above and synthesized into cDNA according to the manufacturer's protocol. Briefly, the first strand primer was mixed with the intact RNA (500 ng/reaction), incubated at 65°C for 5 minutes, and placed on ice. First strand buffer and enzyme mix were added to each tube, mixed well, and incubated at 40°C for 30 minutes, 85°C for 5 minutes and a 4°C hold. Reaction Enhancement Enzyme mix was added to each tube and incubated at 37°C for 15 minutes with a 4°C hold. Second strand primer mix was added to the first strand reaction and incubated at 65°C for 5 minutes with a 4°C hold. The second strand master mix was added to each tube, mixed well and incubated at 25°C for 60 minutes with a 4°C hold. The cDNA was purified using a MinElute column (QIAGEN) and eluted in 1× low TE . The cDNA was sheared using the Covaris S2 adaptive focused acoustics instrument  with the following conditions: duty cycle 5%, intensity 10, cycles/burst 200, time 6 minutes. The sheared products were purified and concentrated with Agencourt AMPure XP beads (2× the reaction volume).Libraries for Illumina sequencing  were madThe enrichment protocol following adaptor ligation was modified using 0.5 μM of each truncated paired end adaptor primer (SBS3_8 nt_F: 5'- TACACGACGCTCTTCCGATCT-3' and SBS8_7nt_R: 5' - CTGAACCGCTCTTCCGATCT-3'), 1× of AccuPrime PCR buffer I (10×), 0.5 U of AccuPrime Taq High Fidelity polymerase  in a final volume of 25 μl. Reactions were run on an ABI 9700 thermal cycler (Applied Biosystems) with the following cycling conditions: 98°C for 3 minutes, 20 cycles of 98°C for 30 s, 55°C for 30 s, 65°C for 1 minute with a final extension of 65°C for 10 minutes. Enriched reactions were purified using Agencourt AMPure XP beads (0.8× the reaction volume).The hybridization reaction was prepared on ice in a 96-well plate with 100 ng enriched cDNA plus 1× hybridization buffer  in a final volume of 18 μl. The plate was incubated in a thermal cycler (ABI 9700) at 98°C for 10 minutes and 68°C for 4 h. A 68°C pre-heated mix of 2× DSN buffer (20 μl) and 2 μl DSN enzyme  was added to each reaction  and incubated for another 25 minutes at 68°C. The reaction was stopped by adding 40 μl of the 2× DSN stop solution (10 mM EDTA) and purified with Agencourt AMPure XP beads (1.6× of the reaction volume). Samples were enriched with full-length Illumina PE1.0 and PE2.0 primers (1 μM each), 1× of AccuPrime PCR buffer I (10×), 0.5 U of AccuPrime Taq High Fidelity polymerase  in a final volume of 25 μl. Enriched reactions were purified using Agencourt AMPure XP beads (0.8× of the reaction volume) and sequenced.(Prochlorococcus marinus subsp. pastoris str. CCMP1986 (MED4), Escherichia coli, K12 substr. MG1655, Rhodobacter sphaeroides 2.4.1) were aligned to the respective genome sequences. BWA-aligned reads were then analyzed and assigned to individual genes according to the genome annotations provided by GenBank . The normalized read counts for each gene, RPKM, was calculated by 1,000 × (The sum of reads/Gene length) × .Libraries were sequenced on either Illumina GAII or Hi-Seq instruments. Sequencing mode (single or paired end) and read lengths for each experiment are available in Additional file DNA-seq reads were aligned to a reference set of 649 selected bacterial genomes  as described . BrieflyThe sequencing data have been submitted to the Sequence Read Archive, and accession numbers are listed in Additional File Prochlorococcus marinus: Escherichia coli: and Rhodobacter sphaeroides; RIN: RNA integrity value; RPKM: reads per kilobases per million mappable reads; RPKMO: reads per kilobases per million mappable reads aligning to annotated open reading frames.CDS: coding DNA sequence; DSN: duplex-specific nuclease; ORF: open reading frame; PER pool: a pool (1:1:1 by mass) of total RNA extracted from The authors declare that they have no competing interests.DC and GG carried out research in the lab. KH, BJH, JL, DG and GG analyzed data. JZL advised on lab methods and data analyses. JI provided the human stool samples. AME and DW coordinated the transfer of stool samples to the Broad and participated in project discussions. BWB, CN and AG directed the project and coordinated the research. GG and AG wrote the paper. All authors have read and provided helpful comments for the manuscript, and they have approved the manuscript for publication.Figures S1 through S8 and legends. Figure S1: linear correlation of gene expression profiles before and after different rRNA depletion methods. Figure S2: detrimental effect of high GC content on enrichment by low-c0t normalization using duplex specific nuclease (DSN). Figure S3: fragmentation profile of total RNA prior to Ribo-Zero treatment. Figure S4: PER CDS detection sensitivity before and after Ribo-Zero treatment of intact and fragmented RNA. Figure S5: antisense read densities for two technical replicates of strand-specific RNA seq of Ribo-zero treated PER RNA. Figure S6: representation of bacterial species in sequencing data sets from two human stool samples. Figure S7: RNA-seq data for Prevotella copri, Bacteroides vulgatus, and Eubacterium rectale in stool A. Figure S8: RNA-seq data for Prevotella copri and Bacteroides vulgatus in stool B.Click here for fileList of 649 reference genomes with GenBank accession numbers.Click here for fileTotal mapped reads to top 19 species in stool samples.Click here for fileList of unannotated rRNAs in stool samples.Click here for fileSample recovery through the RNA-seq process assessed by the Agilent Bioanalyzer RNA 6000 Pico and DNA High Sensitivity Kits.Click here for fileRNA-seq metrics and Sequence Read Archive accession numbers.Click here for file