We have solved the three-dimensional crystal structure of the stem-loop II motif (s2m) RNA element of the SARS virus genome to 2.7-Å resolution. SARS and related coronaviruses and astroviruses all possess a motif at the 3′ end of their RNA genomes, called the s2m, whose pathogenic importance is inferred from its rigorous sequence conservation in an otherwise rapidly mutable RNA genome. We find that this extreme conservation is clearly explained by the requirement to form a highly structured RNA whose unique tertiary structure includes a sharp 90° kink of the helix axis and several novel longer-range tertiary interactions. The tertiary base interactions create a tunnel that runs perpendicular to the main helical axis whose interior is negatively charged and binds two magnesium ions. These unusual features likely form interaction surfaces with conserved host cell components or other reactive sites required for virus function. Based on its conservation in viral pathogen genomes and its absence in the human genome, we suggest that these unusual structural features in the s2m RNA element are attractive targets for the design of anti-viral therapeutic agents. Structural genomics has sought to deduce protein function based on three-dimensional homology. Here we have extended this approach to RNA by proposing potential functions for a rigorously conserved set of RNA tertiary structural interactions that occur within the SARS RNA genome itself. Based on tertiary structural comparisons, we propose the s2m RNA binds one or more proteins possessing an oligomer-binding-like fold, and we suggest a possible mechanism for SARS viral RNA hijacking of host protein synthesis, both based upon observed s2m RNA macromolecular mimicry of a relevant ribosomal RNA fold. The SARS RNA genome contains a unique structure that resembles a portion of ribosomal RNA; this may allow the virus to hijack its hosts protein synthesis machinery The virus that causes SARS, like other pathogenic coronaviruses and astroviruses, possesses a linear plus-sense strand RNA genome that has a 5′ methylated cap and 3′ poly-A tail. The viral replicase is translated directly from the genomic sense-strand RNA, and it then creates a full-length complementary (minus-sense strand) copy of the genomic RNA, as well as a nested set of shorter, subgenomic mRNAs having common 3′ UTRs. These 3′ UTRs all share with the genomic SARS RNA a 32-nucleotide element, immediately upstream of the 3′ poly-A tail  2+ ions in the native structure (see cis-[(NH3)2Cl2Pt(IV)]2+ and [Ru(NH3)6]3+ metal complexes that were introduced for heavy atom isomorphous replacement phasing. These highly structured and rigorously conserved features allow us to suggest that SARS pathogenesis might be inhibited by a drug designed to bind to s2m and disrupt one of these structures.G(11), A(12), and A(33), despite their extrusion from the helical base-pair stack, form a well-defined structure that is highly ordered, judging by electron density in the initial map as well as the comparatively low temperature factors these residues have in the refined structure. They conspire with the remaining residues in the asymmetric bubble and the helical region above it to form a rather wide tunnel whose channel runs approximately perpendicular to the main helical axis. The phosphates of G(11) and A(12) are turned inward, creating a negatively charged environment within the tunnel cavity. Consequently, the tunnel forms a binding site for two [Mg modification patterns A of the The intricate three-dimensional structure of the SARS s2m RNA, along with its rigorous sequence conservation, is compelling prima facie evidence for its biological importance in coronaviruses and astroviruses. The structure by itself, however, does not indicate what the function of this motif must be. Hence, comparison of this unique fold with those of known RNA structures is of particular value for formulating testable hypotheses regarding potential biological functions of the s2m RNA. In addition, identification of novel and rigorously conserved tertiary structures that are unique to the viral RNA is of critical importance for future rational design of anti-viral therapeutic agents that specifically target SARS and other coronaviruses and astroviruses.The s2m RNA sequence we crystallized was originally identified from the genomic sense strand within a rigorously conserved region of the 3′ UTR of the RNA. However, because RNA replication and transcription take place via a full-length negative-strand RNA intermediate, it is formally possible that the conserved sequence instead corresponds to a conserved structure at the 5′ end of the anti-sense RNA. We believe this to be improbable because of the energetically unfavorable tertiary structures that would be required to form from the sequence complement. For example, the variant of the energetically stable and rather common GNRA loop structure (GAGUA) would have to be replaced with an energetically unstable and rare CUCAU loop. Similar arguments apply to the other non-Watson–Crick regions of the structure.Crystal packing interactions may potentially distort RNA structures. This effect is sometimes observed for small stem-loop sequences, which often crystallize as duplex dimers rather than as monomeric hairpins. The s2m RNA structure is sufficiently large, and apparently contains enough stabilizing secondary and tertiary interactions, to offset any energetic advantage that might come from crystallizing as a duplex. In addition, the 73% solvent content of the s2m RNA crystals ensures that most of the crystallized RNA is solvent-exposed, rather than involved in extensive packing interactions. At least three inter-molecular contacts are required to form a crystal. The most extensive contact is the base of residue G(11); it stacks upon that of its 2-fold symmetry mate C. It is Crystallographic temperature factors provide direct physical evidence for the relative flexibility or mobility of various regions of a macromolecule. Therefore, based on our chemical probing data, analysis of crystal packing interactions, and consideration of the crystallographic temperature factors, along with the ability to rationalize the sequence conservation pattern and intolerance for nucleotide insertions or deletions based on the structure, we conclude that the crystal structure of s2m is likely to be a close representation of the structure that forms in solution and in the context of the SARS virus RNA genome.Escherichia coli and Thermus thermophilus. However, if we relax the sequence constraints and focus attention upon the conformation of the RNA backbone, we find that the phosphodiester backbone fold accompanying the 90° kink in s2m RNA mimics that found in the 530 stem-loop of 16S ribosomal RNA [The several unique features and unanticipated tertiary contacts we identified in the SARS s2m RNA crystal structure allowed us to reexamine genomic sequences and previously determined RNA tertiary structures for similar motifs with additional constraints imposed by knowledge of the tertiary structure. Our analysis of the human genome, other animal and viral genomes, and the currently available database of RNA three-dimensional structures revealed that the s2m element is found only in astroviruses and coronaviruses; no cellular homologs are immediately apparent. The G(11) to A(33) tertiary contact in the s2m RNA is homologous to the G to A contact in Domain III of the 23S ribosomal RNA, but the context of the interaction in the ribosome is completely different, and the sequence is not conserved between omal RNA  , 100 mM Mg(OAc)2, and 20% MPD. Data from a native crystal diffracting to 2.7-Å resolution, and 3.0-Å cis-(NH3)2(Cl)2Pt(IV)–derivative single-wavelength anomalous dispersion data, were collected at Beamline 9.1 at Stanford Synchrotron Radiation Laboratory on a 3 × 3 CCD detector using 0.98-Å wavelength X rays and crystals that were cryoprotected in the reservoir solution spiked with 12% glycerol and maintained at 100 K. The native and platinum derivative data were processed using CCP4's MOSFLM and reduced and scaled within CCP4 version 5.0 [122 but was unambiguous in P6522, permitting the hand of the space group to be determined. A 47-nucleotide poly-C model was built into the SIRAS map using O, the actual nucleotide-sequence register was then confirmed by inspecting the electron density, and residues 1–47 were built in using O [Crystals of a 48-nucleotide T7 RNA transcript containing the conserved s2m RNA element were obtained via hanging-drop vapor diffusion by equilibrating a solution containing equal volumes of the RNA sample and the reservoir solution against 1-ml of the reservoir solution. The RNA sample solution contained 4.5 mg/ml s2m RNA dissolved in 30 mM Tris (pH 7.6), 100 mM NaCl, and 60 mM MgClsion 5.0 ,33. A sision 5.0 . The ini using O . The pho using O , and the using O . All cry using O ,39 and b using O , and wer using O  and RNAB using O ,43. Tran using O . Primer  using O  using a http://www.rcsb.org/pdb/) under accession number 1XJR and are also available with other supplementary materials at http://www.chemistry.ucsc.edu/%7Ewgscott/sars.Coordinates, native and derivative amplitudes, and experimental phases have been deposited in the RCSB Protein Data Bank (The RCSB Protein Data Bank accession number for the SARS s2m RNA structure reported here is 1XJR. The RCSB Protein Data Bank accession numbers for the other protein and RNA structures discussed in this paper are as follows: the 30S ribosome (1J5E), the 30S ribosome in which prokaryotic IF-1 has been added (1HR0), the eukaryotic analog of prokaryotic IF-1 (1D7Q), and the crystal structure of nsp9 (1QZ8 and 1UW7).


Small quantities of RNA  available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity.To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility.The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip™ arrays. Here, we present an overview of RNA amplification strategies, identify key limitations to existing techniques, and describe a simple, robust, and cost-effective strategy for single round amplification of RNA in the sense orientation.The increased use of microarray expression profiling to study both the molecular biology of cancer and the cellular physiology of difficult-to-isolate cell types has led to a growing need for methods that allow the use of limiting quantities of RNA. Small surgical biopsies, fine needle aspirates, cyto-lavages, punch biopsies and blood samples often yield only 1–4 μg quantities of RNA as starting material for expression profiling. This limitation has prompted the development of amplification methods that produce the quantities of RNA required for microarray analysis. Changing requirements for the type and quantity of amplified RNA, driven by evolving microarray technologies, have led to the development of novel amplification strategies. While current methods are capable of delivering high-yield RNA amplification, this is often only achieved after complex priming strategies  are coupled with multiple rounds of PCR and/or + sequence on the second-strand cDNA could then be used to facilitate amplification. These methods were confounded by differential amplification of cDNA and by introduction of errors by Taq polymerase. This problem prompted the development of a linear, T7-based in vitro transcription (IVT) method by Van Gelder and Eberwine [Early attempts to amplify RNA employed a strategy based upon the Polymerase Chain Reaction (PCR) -4. TheseEberwine -7.E. coli RNAse H, and priming for second-strand cDNA synthesis occurs by either RNA nicking and priming or cDNA hairpinning6. Second-strand cDNA synthesis is carried out with E. coli DNA polymerase and E. coli DNA ligase followed by blunt-ending with T4 DNA polymerase. Transcription and amplification are then accomplished using the T7 RNA polymerase, which binds to the T7 promoter introduced during first-strand cDNA synthesis, producing antisense RNA (aRNA).In what has now become known as the "Eberwine Method," RNA templates are primed with an oligo(dT) primer that has been 5' modified to contain a promoter for the T7 RNA polymerase and are subsequently reverse transcribed into first-strand cDNA. The RNA-cDNA hybrid is then treated with Technical revisions of the Eberwine method have included changes in first-strand primer concentration to minimize the appearance of non-sequence dependent RNA in the amplified product , supplemOne such alternative  is basedA second alternative to the Eberwine method is the "template-switching" (TS) strategy . This teA third recently introduced alternative is Ginsberg's Terminal Continuation (TC) technique . In this2(ratio) differences within ± 0.5 of the unamplified RNA, they concluded that RNA amplification is useful in expression profiling and is likely to assist in the measurement of low-abundance RNA.The degree to which the pool of amplified RNA generated by these methods reflects the unamplified sample from which it is derived is an obvious concern for microarray applications and other downstream analyses, and the history of attempts to validate RNA amplification methods is summarized by Nygaard et al. . BrieflyDespite the methodological validation provided by these studies, only recently have statistically-based analyses been used to compare RNA amplification methods in a critical, head-to-head fashion. A study by Zhao  examined+ RNA [It is currently accepted that RNA amplification strategies based on the Eberwine method maintain relative mRNA levels between samples when amplifying from either total RNA or poly(A)+ RNA ,25, are + RNA , and are+ RNA . AccordiFirst, most commercial protocols produce RNA only in the antisense orientation. With the emergence of spotted oligonucleotide arrays and mixed cDNA/oligo arrays , appropr+ RNA extracted from both ideal samples (e.g. cell line RNA) and "real-world" samples (e.g. tumors or tissues). This protocol should avoid the need for PCR steps and should require a minimum number of primers (two). Additionally, the protocol should be cost-effective, efficient, and technically simple to conduct. Finally, the method should give results consistent with similar amplification techniques when used with subsequent microarray analysis. We believe these criteria have been met with our protocol, which consequently will be useful in a variety of laboratory applications.Our goal was to develop a strategy based upon the Eberwine method but with the ability to produce sense RNA from small quantities of total or poly-(A)Quantitation of first strand cDNA after hydrolysis of the cDNA/RNA hybrid shows that priming with the T7-Oligo(dT) promoter/primer followed by reverse transcription produces cDNA that totals 5–10% by mass of the original mass of total RNA (data not shown). This is consistent with the average percentage of mRNA in the total RNA sample and therefore suggests that priming proceeds efficiently and that first strand cDNA synthesis successfully copies the population of mRNA in the original total RNA sample. Quantitation of second strand cDNA after priming with the T3N9 promoter/primer indicates a 120% increase in mass versus the mass of the first strand cDNA (data not shown). This suggests that second strand priming proceeds efficiently and that the first strand cDNA is successfully converted to double stranded cDNA by the second strand synthesis reaction. The extra mass (in excess of 100%) is attributable to the use of random nonamer primers which initiates synthesis at multiple positions along each first-strand cDNA template.While inherent constraints of reverse transcription limit the amount of truly "full-length transcript" produced during any RNA amplification scheme, it is nonetheless desirable for an amplification method to favor the production of long transcript. RNA amplified using our method has a median length of 794 nucleotides (range: 70–9000 nt) suggesting that this method is capable of preserving long transcript during amplification Table .) of 26.3 μg of amplified sense mRNA from 4 μg of total RNA (Table Our method produced an average (2(ratio) plots of fluorescence intensity in amplified versus unamplified samples were constructed and analyzed for correlation as described above. Our results show that the amplification bias introduced by this method is small, as reflected in the high average correlation coefficient  between expression profiles from amplified and unamplified RNA samples  plots of fluorescence intensity were constructed and analyzed as described above. Our results demonstrate a high degree of correlation between independent replicates , suggesting that the amplification is highly reproducible and that the amplification bias is introduced consistently when the protocol is repeated.We evaluated the reproducibility of this method and the consistency of the propagation of the amplification bias by comparing the expression profiles of hybridizations of three independently amplified RNA samples. LogWe compared these results to results from the RiboAmp mRNA amplification kit (Arcturus), which produces antisense RNA using a version of the Eberwine method, and from the BDSmart mRNA amplification kit (BD Biosciences), which produces sense RNA using a template-switching strategy. Both protocols were carried out according to the manufacturer's specifications using 4 μg of the pooled cell line RNA and the glioblastoma RNA as the starting material for amplification. The final yield of each method was measured using the NanoDrop spectrophotometer, the median size of the amplified mRNA from each kit was assayed using the Bioanalyzer 2100 (Agilent), and the amplification bias of each method was assessed using microarray analysis (as described above).) and the dispersion of each group  were calculated, demonstrating a high degree of reproducibility for both the RiboAmp  and the BDSmart  protocols. These results suggest that subsequent comparisons of our amplification method to these commercial techniques are not confounded by individual variability among replicates. Additionally, the strong correlation between independent replicates of our method (see above) and of these commercial techniques suggests that performing additional independent replicates would not add significant statistical power to subsequent comparisons.Three independent replicates were conducted for each method, and the reproducibility of the amplification was assessed as described above. The mean correlation coefficient ( = 23.6 μg) was less than that of the Arcturus RiboAmp method  but greater than that of the BDSmart method . The decreased yield versus the Arcturus RiboAmp method is expected, because IVT with the T7 promoter  proceeds more efficiently than IVT with the T3 promoter . The improved yield over the BDSmart method is notable considering that both of these methods involve production of amplified sense RNA (Table The average yield from our method  = 3.6 μg wa = 0.8009) versus amplification with the Arcturus RiboAmp method  and was comparable to the correlation after amplification with the BDSmart method  (Table The median length of the amplified RNA from our method (794 nt) was greater than that of the Arcturus RiboAmp method  and comparable to that of the BDSmart amplification method  Table . The corIn vitro Transcription5–6 became the first standard protocol for RNA amplification designed to fulfill these requirements. The evolution of microarray technology and other RNA techniques have imposed requirements on the type, length, and/or orientation of the starting RNA. Additionally, the growing interest in medical and microbial genomics now requires that data be collected from samples that are becoming progressively smaller and more difficult to acquire. This changing role for RNA amplification has catalyzed the development of multiple institutional and commercial amplification protocols, all of which claim to be high-yield, low-bias techniques capable of amplifying RNA with the specific characteristics required for downstream applications.The emergence of methods to study the transcriptome initially necessitated the production of relatively large quantities of RNA from experimental systems, and Eberwine and Van Gelder's T7-based system of Template switching, terminal continuation, and other novel techniques for RNA amplification that have been developed over the past five years have become increasingly technically complex, and we feel that this is a significant disadvantage. In particular, they stray from the Eberwine approach, which has been extensively validated, and often rely on the use of proprietary (i.e. unspecified) enzymes, primers, and reaction components, which limit the utility of the methods (as well as protocol modification and fine-tuning). While proponents of these methods argue that the limitations are offset by improved yield, production of long transcript, reproducibility, and reduced amplification bias, we have found in practice that no single protocol is able to perform consistently in all four areas.The technique that we have described affords the advantages of all of the aforementioned protocols while eliminating the major limitations and controlling the technical complexity. Several aspects of this protocol make it a robust tool that we believe will be useful in conjunction with a variety of experimental systems and downstream applications. First, the method we present is based upon the Eberwine technique. The methods are both straightforward and validated and do not require the use of custom enzymes, multiple (more than 2) proprietary primers, or PCR steps to complete the amplification. Second, this new method produces amplified mRNA in the sense orientation and can be used for production of aRNA with minor modification. This corrects for the downstream limitations imposed by antisense amplification in both the original Eberwine method and the Arcturus RiboAmp kit and allows amplified mRNA produced by our method to be used in conjunction with both cDNA (sense and antisense orientation) and long oligonucleotide (sense orientation) arrays. Third, our method requires only one round of amplification to produce RNA in the sense orientation. We believe that this represents a significant advantage over previously described, T7-based amplification strategies (most notably the method of Xiang ), where Another advantage of our protocol is that it is readily adaptable to a variety of experimental systems. The approach that we have described synthesizes amplified RNA from eukaryotic total RNA in the sense orientation. However, because this method adds unique promoters to both the first and second cDNA strand , either orientation of RNA can be produced from the same population of cDNA simply by selecting the appropriate RNA polymerase for IVT. Moreover, changing the two primers allows the protocol to be adapted to produce sense or antisense RNA from either prokaryotic or eukaryotic total RNA without any additional adjustments. The protocol can also be modified for use with either T3 or T7 IVT systems by changing the promoter sequence of the promoter-modified primer, it can be used to salvage partially-degraded RNA (as described by Xiang ), and itIn clinical and research settings using laser capture microdissection or other strategies where very small amounts of starting RNA (10–100 ng) are available, multiple rounds of amplification may still be necessary. A final advantage of our protocol, as discussed previously, is that a second round of amplification can be easily incorporated and can be used to produce additional RNA in either the sense or antisense orientation. Future testing of this protocol will focus on the quantity and fidelity of the RNA population after multiple rounds of amplification and will include appropriate comparisons to commercial, multi-round techniques. However, the purpose of the present technique is to minimize bias while maintaining yield after a single round of amplification. In this setting, our results show that our method outperforms two commercial, single-step protocols when using identical quantities of initial RNA. We believe the performance and versatility of this method make it particularly beneficial to investigators attempting to conduct microarray analysis from 1–4 μg of initial RNA.Neither this nor any other current strategy for RNA amplification is optimally suited to every experimental setting, and each technique has its own advantages and limitations. The challenge of selecting an amplification strategy is choosing a method that provides sufficient amplification and appropriate RNA orientation for downstream requirements yet minimizes the labor, cost, and bias introduced by the process. The method that we have described has been designed and validated for use in experiments where microarray analysis is to be performed with approximately 1 μg of starting total RNA. In this setting, our method produces a population of amplified RNA having minimal and consistent amplification bias, orientation compatible with both spotted cDNA and oligonucleotide arrays, and quantity sufficient for several downstream assays from a single amplification. Additionally, the protocol we present here can be easily adapted for use in a variety of experimental systems. This includes production of either sense or antisense RNA in prokaryotic or eukaryotic systems (simply changing one or both primers). None of these variations require multiple rounds of amplification to produce RNA of a specific orientation, but the protocol can easily be modified to enable multiple rounds when necessary.Finally, we would like to note that there are some limitations to the method we have outlined here. Readers may note that several amplification strategies have been reported to produce greater than 1,000-fold increases in RNA yield, one aspect of our method that may be viewed as a limitation is its ~130-fold amplification. However, most higher-yield amplification strategies use multiple rounds of amplification and are subject to additional amplification bias. While we recognize that researchers using submicrogram quantities of starting material may consider strategies incorporating multiple rounds of amplification, in our experience even small tissue biopsies yield more than 1 μg of total RNA. The ability to amplify this quantity of RNA efficiently and effectively was one of our major design considerations. In this setting, 130-fold amplification produces more than enough RNA for multiple downstream microarray analyses and confers the benefit of reduced bias inherent in a single-round amplification protocol.We have developed a robust method for amplification of mRNA in the sense orientation. This protocol is based upon the Eberwine method and incorporates the advantages of more recent amplification techniques while eliminating many of the limitations of these strategies. Our method allows the production of sense mRNA with one round of IVT, yields 130-fold amplification, preserves long transcript, and produces mRNA that is well suited for downstream microarray applications. Microarray assays performed with RNA amplified using our protocol demonstrate that the method results in low amplification bias and is highly reproducible. Additionally, our method is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded samples of prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of downstream microarray platforms . We feel that these advantages make our method a robust tool with the potential for application in a variety of research settings.24 primer followed by heat denaturing and then cooling to facilitate primer annealing. Reverse transcription produces cDNA-mRNA hybrids which are then subjected to alkali hydrolysis to remove template mRNA. First-strand cDNA (fs-cDNA) is separated from residual enzymes, nucleotides, and mRNA fragments using a spin column technique.The protocol that we have designed is based on the Eberwine method but uses novel priming strategies to produce amplified RNA in the sense orientation. First-strand cDNA synthesis from total RNA is primed with an Oligo(dT)E. coli DNA polymerase and ligase followed by blunt-ending with T4 DNA Polymerase. The double-stranded cDNA is purified using a spin column technique, and in vitro transcription (IVT) from the T3 promoter sequences incorporated into the ss-cDNA produces amplified RNA in the sense orientation . The sample is heated to denature the fs-cDNA and to eliminate secondary structure. The temperature is rapidly dropped to the upper limit of the annealing range and then ramped more slowly to a final temperature of 4°C. We believe that this strategy minimizes "self-priming" of the fs-cDNA and, in the absence of competitive priming from RNA fragments, facilitates optimal annealing of the second-strand primer. Second-strand cDNA (ss-cDNA) synthesis is carried out using n Figure .2 and maintained accordingly. When culture plates achieved ~90% confluence, RNA was extracted and purified following TIGR standard operating procedures based on the Trizol method (Invitrogen) [260/280 and OD230/260 in TE (pH 8.0) using a NanoDrop spectrophotometer, and RNA integrity was assessed by measurement of the 28S/18S ratio with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. The final values for the RNA pool were: OD260/280 = 2.04, OD230/260 = 2.20, 28S/18S = 2.13, indicating high purity, non-degraded RNA. Superas-In RNAse inhibitor (Ambion) was added to a final concentration of 1U/μL according to manufacturer's recommendations. RNA was stored at -80°C until it was used in the RNA amplification process.Total RNA used in this study was derived from three cell lines: PA-1 ovarian teratocarcinoma, CaOV-3 ovarian adenocarcinoma, and U118MG glioblastoma. All cell lines were grown in a monolayer under Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell lines were held at 37°C in an atmosphere of 4% COitrogen) . RNA extAll temperature controlled steps in this protocol were conducted using a Peltier thermal cycler  in thin-walled, nuclease-free PCR tubes (BioRad). All enzymes, buffers, and other reaction components were purchased from Invitrogen unless otherwise noted. Four (4) μg of pooled total cellular RNA (described above) was as starting material for the amplifications. Priming for first strand cDNA synthesis is carried out by combining the total RNA with 1 μL of T7-Oligo(dT) primers  and 1 μL of Superas-In RNAse inhibitor . The mixture is diluted to 17.8 μL in DEPC treated water, heated at 70°C for 10 minutes, and cooled to 4°C for five minutes. First strand cDNA synthesis is accomplished by adding 6 μL of 5× First Strand Buffer, 3 μL 0.1M DTT, 1.2 μL of dNTP mix (50 mM), and 2 μL of SuperScript II reverse transcriptase followed by incubation at 42°C for 2.5 hours. After incubation, the reaction is cooled to 4°C for 2 minutes. Template RNA is hydrolyzed by adding 10 μL of 1N NaOH (Sigma) and 10 μL of 0.5M EDTA (Sigma) followed by incubation at 65°C for 15 minutes. The pH is subsequently neutralized by adding 10 μL of 1N HCl (Sigma). Hydrolyzed RNA and residual dNTPs are removed using the Minelute reaction cleanup kit (Qiagen) according to the manufacturer's protocol. Two rounds of elution from the column, each in 10 μL of Buffer EB (Qiagen) are performed to improve recovery.11 (Operon) followed by incubation at 95°C for 3 minutes. Immediately following this incubation, the samples temperature is decreased as quickly as possible to 50°C followed by a temperature ramp from 50°C to 4°C at the rate of 0.4°C/s (~120s ramp time) and then a hold at 4°C for 2 minutes. Second strand cDNA synthesis is accomplished by adding 7μL of 5× Second Strand Buffer, 2 μL of dNTP mix (50 mM), 4 μL of E. coli DNA polymerase I (10U/μL), and 1 μL of E. coli DNA ligase (10U/μL) followed by incubation at 16°C for 2 hours. After incubation, blunt-ending is carried out by adding 2 μL of T4 DNA polymerase (5U/μL) and incubating at 16°C for 5 minutes. The reaction is terminated by adding 3.5 μL of 0.5 M EDTA (Sigma). Double stranded cDNA is purified using the Minelute reaction cleanup kit (Qiagen) following the manufacturer's protocol. Two rounds of elution with Buffer EB (Qiagen) are carried out to improve yield. The first elution is in 10 μL of Buffer EB, and the second is in 7 μL of Buffer EB. This provides the eluted cDNA in a final volume of 16 μL (corrected for 1 μL of retention on the column), which is the appropriate volume for use in the subsequent IVT reaction.Priming for second strand cDNA synthesis is accomplished by combining the eluted first-strand cDNA with 2 μL of random nonamer primers modified by the addition of a T3 promoter sequence at the 5' end . A double reaction is used to improve yield, so 16 μL of NTP mix, 4 μL of 10× Reaction Buffer, and 4 μL of T3 MegaScript Enzyme Mix are added to the sample. The reaction is incubated at 37°C for 14 hours (overnight). The amplified RNA is purified using either the RNEasy Mini or the RNEasy Minelute kit (Qiagen) according to the manufacturer's protocol. Superas-In RNAse inhibitor (Ambion) is added to the amplified RNA to a final concentration of 1U/μL according to the manufacturer's recommendations.260/280 and OD230/260 in the corresponding elution buffers (pH 8.0) using a NanoDrop spectrophotometer. RNA length distribution and integrity were assessed with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. Agilent's Bioanalyzer Expert 2100 software was used to quantify transcript length, and the software's automatic integration tool was used to determine the area under the curve. The median transcript length for each method was taken as the point at which half of the integrated area was achieved.cDNA and RNA quantity and purity were assessed by measurement of OD28. cDNA target synthesized from the cell line RNA was indirectly labeled with Cy-5 while target derived from the glioblastoma was labeled with Cy-3. The labeled cDNA from the cell line pool and from the glioblastoma were cohybridized to human 32,000 element spotted cDNA arrays. The array production, cDNA synthesis, target labeling, and hybridization were conducted following TIGR standard operating procedures [Microarray analysis was conducted to determine the magnitude of amplification bias. Pooled human cell line RNA (as described above) as well as RNA extracted from one human glioblastoma were amplified as described. The amplified sense RNA (2 μg) was used as the starting material for synthesis of cDNA target, and a spiking control consisting of RNA transcripts of 10 genes from the Arabidopsis thaliana genome was added to each sample prior to cDNA synthesis in order to provide a consistent positive reference in subsequent hybridizationsocedures . IndepenIn order to compare our method to commercially-available RNA amplification kits, microarray analysis was conducted using identical pooled cell line and glioblastoma RNA that was amplified with two commercial products . Amplification of 4 μg of total RNA was carried out according to the manufacturer's protocol for both kits, and 2μg of amplified RNA from each sample was used for microarray analysis as described above. Independent replicates were conducted for all amplifications and hybridizations.Unamplified total RNA from both the pooled cell line and the glioblastoma was used as the starting material for the control arrays. Ten (10) μg of total RNA from each sample was used for cDNA synthesis, and the remainder of the protocol was carried out as described for the amplified samples.2 transformed, and  was calculated for each array element. Assessments of bias were conducted by comparing the log2(ratio) values for each element in the amplified sample arrays to their counterparts in the unamplified control arrays. Measurements of reproducibility were conducted by comparing the log2(ratio) values of corresponding elements in independent replicates.Microarray data was analyzed using the TIGR TM4 software package for microarray analysis . TIGR Sp against the  and calculating the correlation coefficient (r) for each independent replicate. The mean correlation coefficient  was calculated for each method, and differences between  for each amplification method were tested for statistical significance. Welch's T-Test [The degree of bias introduced by an amplification method was assessed by plotting s T-Test  was used2(ratio) values for paired hybridizations derived from independent amplifications of the same starting RNA, calculating the correlation coefficient (r) for a series of independent replicates, and computing the mean correlation coefficient . Reproducibility was further investigated by calculating the variance (σ2) and the coefficient of variation  for  (CV), which describe the dispersion of the values contributing to .The degree of reproducibility of our amplification method was assessed by plotting the logThis method is based on an idea by Norman Lee. John Quackenbush and Nicholas Marko further developed the approach. Marko reduced this to practice in the laboratory, greatly expanding on the method and creating a robust protocol. Bryan Frank contributed significantly to the development, implementation, and optimization of the protocol. Marko conducted the data analysis and was largely responsible for the present manuscript.


To understand mycobacterial pathogenesis analysis of gene expression by quantification of RNA levels becomes increasingly important. However, current preparation methods yield mycobacterial RNA that is contaminated with chromosomal DNA.Mycobacterium smegmatis genomic DNA is efficiently removed by DNaseI in contrast to untreated samples.After sonication of RNA samples from This procedure eliminates one of the most prevalent error sources in quantification of RNA levels in mycobacteria. Mycobacterium tuberculosis is the leading cause of death from a single infectious disease with approximately 8.8 million new cases and two million deaths per year. To understand the pathogenesis of M. tuberculosis, analysis of gene expression by relative or absolute quantification of RNA levels using microarrays and RT-PCR  becomes increasingly important [Mycobacterium smegmatis is especially refractory to lysis and therefore was chosen as a model organism.mportant . Widely mportant . Howevermportant . Since tmportant . This camportant ,6. VirtuM. smegmatis SMR5 [600 of 0.8 and mixed with 5 ml killing buffer  [-1 into several aliquots. One aliquot containing 10 μg RNA was left untreated. The second aliquot was directly treated with 10U of RNase-free DNaseI (Roche) for 1 h at 37°C, while the third aliquot was sonicated two times for 20 sec with 0.9 sec intervals at 20 % power  prior to DNaseI treatment. Between the two sonication steps the cell suspension was chilled on ice for 5 min. DNaseI was removed by precipitation with polyethylene glycol (PEG) 6000. As a control for the RNA quality, cDNA was synthesized by Omniscript reverse transcriptase and sensiscript reverse transcriptase  from total RNA (100 ng) for 35 min at 50°C followed by an inactivation step of 15 min at 95°C. The 16Sr RNA was then amplified with the primers 16S-FP (5'-TGCTACAATGGCCGGTACAAA-3') and 16S-RP (5'-GCGATTACTAGCGACGCCGACTT-3') using up to 30 cycles of 1 min at 94°C, 30 sec at 53°C, and 1 min at 72°C before a final extension step of 7 min at 72°C. As a control for DNA contamination, standard PCRs were performed, to which RNA was added after the RT inactivation step. PCR products were analysed at cycles 23, 25, 27 or 30 to check the purity of the RNA. All samples apparently contained 16S rRNA (Fig. tis SMR5  was growM. smegmatis and will be of great value for RNA preparations from other microorganisms, including M. tuberculosis.This result shows that sonication improved DNA degradation by DNaseI most likely by rendering the chromosomal DNA more accessible to enzymatic action. This work describes a simple and efficient procedure to improve the quality of RNA preparations from JS carried out the RNA preparations, the RT-PCR experiments and wrote a draft of the manuscript. JGB performed RNA preparations and RT-PCR experiments. FT participated in coordinating and supervising the study. MN conceived of the study, participated in coordinating and supervising the study, and wrote the final manuscript. All authors read and approved the final manuscript.


Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS.To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications. Cytokines are a family of low-molecular weight proteins secreted by various cell types, with pleiotropic functions and constitute a tightly regulated network that plays a central role in the immune system. Cytokines, classified into different groups such as interleukins (IL), interferons (IFN), colony-stimulating factors (CSF), tumour necrosis factors (TNF), tumour growth factors (TGF) and chemokines are implicated in the differentiation, proliferation, migration and effector functions of immune cells. Interacting one with the others, they have polarizing effects on the target cells and are pivotal in tuning immune responses . TherefoCytokines are often produced locally so that the concentration of circulating cytokines in the plasma is usually low. Their half-life and turnover may vary complicating the delineation of informative cytokine profiles. Although transcription of messenger RNA is not strictly correlated to protein secretion and activity, detection of cytokine RNA by real time PCR is now considered a reference technique for analysis of small-size samples with high sensitivity . It can The already available techniques, which offer a so-called "absolute quantification" of the target cytokine mRNA, achieve quantification by reference to an external standard curve based on serial dilutions of a known amount of the corresponding cDNA . MoreoveIn the present study, we first show that the efficiency of the RT step depends on the target mRNA and on the experiments and that these variations have critical impact on the reliability of mRNA quantification. To overcome this, we describe here CyProQuant-PCR, a new technique for absolute measurement of cytokine mRNA based on an external RNA standard curve. Primer pairs have been designed for allowing amplification of a set of cytokine mRNA using the same conditions both in terms of thermocycling parameters and master mix components, a prerequisite for multiple cytokine mRNA measurements with high throughput.in vitro stimulated PBMC and its intra and inter-experimental variability were assessed. Finally, CyProQuant-PCR was used to quantify TGF-β1 transcripts in small blood samples from children with acute Plasmodium falciparum malaria.In the present paper, we describe i) the construction of the synthetic RNA standard, ii) the primer pairs specific for the following human cytokines IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, and for the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), the β2-microglobulin (β2-MG) and the ubiquitin-C (UBC) to be used as internal standards and iii) the conditions for efficient real time amplification of multiple cytokine specific mRNA. The technique was validated using Primer pairs were designed from published genomic sequences using Primer Express software (Applied Biosystems), except for the UBC and the YWHAZ genes for which the primers had already been described . When poT) and the maximum signal-to-noise fluorescence ratio (ΔRn) while minimising non-specific amplification (data not shown). Among the final concentrations of 50, 300 and 900 nM tested for each primer, the optimal final concentration was set at 900 nM for every primer. The primer pairs are shown in Table Primer concentrations were optimised to determine the minimum primer concentrations giving the lowest threshold cycle . The molecular mass of each RNA standard was calculated on the basis of its sequence and solutions ranging from 101 to 1012 copies of standard RNA were made.To generate the standard RNA corresponding to each target sequence, gene specific primers were fused in their 5' end to the RNA polymerase T7 promoter sequence. The PCR performed with these modified primers pairs lead to a larger PCR product with the T7 promoter sequences upstream and downstream from the specific amplicon. The T) against the logarithmic value of the starting RNA copy number for each dilution. Figure These serial diluted solutions were reverse transcribed and the cDNA amplified in duplicate to generate a standard curve by plotting the threshold cycle . The slope of the standard curves generated by these three ranges as well as the corresponding efficiency are shown in Table CyProQuant-PCR was compared to the classical approach based on a DNA standard curve for the quantification of TNF-α, IL-1β and IFN-γ transcripts. DNA templates for RNA transcription of TNF-α, IL-1β and IFN-γ were used to generate a range of concentration from 10 to 10In order to show that these intergenic differences were not due to interassay variation, we compared amplification efficiencies of TNF-α, β2-MG, MIF and UBC from i) a single cellular RNA sample, ii) a pooled RNA sample containing standard RNA for each of the targets and iii) a pooled DNA sample corresponding to the cDNA obtained from the pooled RNA sample containing standard RNA for each of the targets. Results obtained are summarised in Table Altogether, these data demonstrate that RT efficiency displays intergenic variations that are not due to interassay differences.RT efficiency has critical impact on transcript quantification as shown in Table Taken together, these results support the use of RNA as external standard for reliable and reproducible quantification of transcripts.Since RT-PCR efficiency varies, absolute mRNA quantification can only be reliably obtained if the external RNA standard and the cellular RNA are retro-transcribed and amplified with the same efficiency. This was secured by comparing the standard curves obtained after amplification of a range of 10X serial dilution of cellular RNA and β2-MG external RNA standard. The slopes obtained were of -3,307 and -3,305 for the cellular RNA and for the external RNA standard, respectively. This corresponds to efficiencies of 100,6% and 100,7% respectively (data not shown).in vitro conditions. Moreover, based on the approach recently described by Pachot et al.  were N is the number of bases composing the standard DNA. Stock solution of 10in vitro transcribed . The standard RNA generated was purified  and loaded on a 4% agarose gel for electrophoresis. The concentration of the standard RNA was determined spectrophotometrically at 260 nm. The molecular weight of the transcript was calculated by N*500-[(N-1)*175] were N is the number of bases composing the standard RNA. Stock solution of 1012 copies of standard RNA /3,85 μL were made in RNA storage solution , split in single-use aliquots and stored at -80°C in safe-lock tubes. External RNA standard range was made extemporaneously by 1:10 serial dilutions in water.Gene specific primers were fused on their 5' end to the sequence of the RNA polymerase T7 promoter to generate modified primers. These primers were used to amplify a gene specific PCR product flanked by transcription initiation sites. Five hundred nanograms of this construct were For CyProQuant-PCR assays, 100 ng of total cellular RNA from PBMC and serial dilution of external RNA standard were reverse transcribed simultaneously in a parallel procedure using Reverse Transcription TaqMan reagents  on a MasterCycler Gradient . The final volumes were set at 100 μL for the cellular RNA samples and 50 μL for the external RNA standard range. The thermocycling parameters were as follows: 25°C, 10 min.; 48°C, 60 min. and 95°C, 5 min. cDNA were immediately used for PCR amplification.T) against the logarithmic values of the starting RNA standard copy number.Reverse-transcribed standard RNA and cellular RNA were amplified simultaneously on the same PCR plate on an ABI Prism 7700 . An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix . Thermocycling conditions were 50°C for 2 min., 95°C for 10 min. and 40 cycles of . The sample target RNA copy numbers were calculated using SDS 1.9 Software . The baseline fluorescence was set manually to correct for differences in initial cDNA concentration and the threshold was positioned at a fluorescence level that was 10 times higher than the background signal. Target mRNA copy numbers in cellular samples were calculated based on a standard curve generated by SDS 1.9 Software  by plotting cycles at threshold . Results are expressed as pg/mL for one million living cells.Tests for significance were done using Stata software  by Kruskal-Wallis rank test.Results disclosed in this manuscript have been protected in French patent application FR0408645.in vitro stimulation experiments. DJ gave technical assistance. SL helped in RNA extraction of malaria samples. JCB introduced PB to molecular biology techniques and provided critical advices. BDA designed and conducted the study that yielded the malaria samples. OMP revised the manuscript and supported the work. CB conceived the strategy and coordinated the study. PB and CB drafted the manuscript. All authors read and approved the final manuscript.PB developed the entire technique. IV did the


RNA interference (RNAi) is an evolutionarily conserved mechanism that is involved in the post-transcriptional silencing of genes. This process elicits the degradation or translational inhibition of mRNAs based on the complementarity with short interfering RNAs (siRNAs) or microRNAs (miRNAs). Recently, differential expression of specific miRNAs and disruption of the miRNA synthetic pathway have been implicated in cancer; however, their role in autoimmune disease remains largely unknown. Here, we report that anti-Su autoantibodies from human patients with rheumatic diseases and in a mouse model of autoimmunity recognize the human Argonaute (Ago) protein, hAgo2, the catalytic core enzyme in the RNAi pathway. More specifically, 91% (20/22) of the human anti-Su sera were shown to immunoprecipitate the full-length recombinant hAgo2 protein. Indirect immunofluorescence studies in HEp-2 cells demonstrated that anti-Su autoantibodies target cytoplasmic foci identified as GW bodies (GWBs) or mammalian P bodies, structures recently linked to RNAi function. Furthermore, anti-Su sera were also capable of immunoprecipitating additional key components of the RNAi pathway, including hAgo1, -3, -4, and Dicer. Together, these results demonstrate an autoimmune response to components of the RNAi pathway which could potentially implicate the involvement of an innate anti-viral response in the pathogenesis of autoantibody production. The exact mechanisms and causes of autoimmune diseases remain unknown. They are thought to develop when self-reactive lymphocytes escape from tolerance and are activated or when incomplete thymic and/or bone marrow clonal selection or disruption of the anergy of autoreactive lymphocytes perturb the delicate balance of non-self-antigen and self-antigen recognition . The disAlthough our understanding of these specific disease processes is incomplete, human autoantibodies have proven very useful for the discovery, identification, and elucidation of newly described cellular components and macromolecules . For exaPatients with systemic rheumatic diseases commonly produce antibodies against specific classes of highly conserved RNA-protein complexes. These include several known RNA-binding autoantigens, such as SS-A/Ro, SS-B/La, Sm, and U1 RNP . RNA-binRecently, human autoantibodies were used to identify and characterize a new protein named GW182 . GW182 iRNAi is an evolutionarily conserved mechanism involved in the post-transcriptional regulation of gene expression in many eukaryotes . It was RNAi is triggered by double-stranded RNA (dsRNA) precursors that are rapidly processed into small RNA duplexes of approximately 21 nucleotides in length by a dsRNA-specific endonuclease termed Dicer . These sSome of the proteins most consistently found in RISC are the highly conserved Argonaute (Ago) proteins . There aTo date, the most commonly identified diagnoses of patients with autoantibodies to GW182 and GWBs are Sjögren's syndrome, mixed motor/sensory neuropathy, and systemic lupus erythematosus (SLE) -22, in pHuman sera used in this study were obtained from serum banks at the Advanced Diagnostics Laboratory, University of Calgary , the University of Florida Center for Autoimmune Diseases , and the University of North Carolina Hospitals . Murine sera were obtained from BALB/c mice prior to or after pristane injection . Murine in vitro transcription and translation (TnT) reaction. Briefly, the hAgo1 plasmid was digested with EcoRI and SmaI, purified, filled in with Klenow polymerase, and religated. Similarly, the hAgo4 plasmid was digested with NotI and NcoI, purified, and ligated with a linker containing an EcoRV cut site: 5'-GGCCGATATCGTGC-3' and 5'-CATGGCACGATATC-3'. The hAgo3 cDNA  in the pCMV-SPORT6 vector was purchased from Invitrogen . The Dicer insert  in pBluescript was obtained from the Kazusa DNA Research Institute . The Dicer coding region was recloned by polymerase chain reaction (PCR) amplification using the following two PCR primers: 5'-CGATACAGTCGACGCCACCATGGAAAGCCCTGCTTTGCAACC-3' and 5'-CCAATACGGCACGACAGTC-3'. All constructs were confirmed by DNA sequencing performed by the University of Florida Interdisciplinary Center for Biotechnology Research core laboratory.The hAgo2 cDNA in pCMV-SPORT vector was obtained from Dr. Tom Hobman  . The hAgIndirect immunofluorescence (IIF) analysis was previously described . Brieflyin vitro TnT and IP was previously described [35S]-methionine. The IPs of hAgo1-4 and Dicer TnT products were performed using 0.5 M NaCl NET/NP40 buffer , 0.3% Nonidet P-40) and NET2+F buffer  containing Complete protease cocktail inhibitors , respectively. Cell labeling and IP of proteins from [35S]-methionine-labeled HeLa or K562 cell extracts using human and/or murine sera were described previously [The protocol for escribed . TnT reascribed 3S-methioneviously .During our exploration of the biology of GWBs and RNAi, hAgo2, an approximately 100-kDa protein, was demonstrated to coimmunoprecipitate with GW182 and localize to GWBs . Based oAutoantibodies to the Su antigen were initially identified and characterized in 1984 and reported to be specifically associated with SLE in both humans and mice ,27. HoweSeveral monospecific anti-Su human and mouse sera have been demonstrated to exhibit a negative fluorescent anti-nuclear antibody staining, although in a number of cases the sera have been shown to stain the cytoplasm of cells . Knowingin vitro translated hAgo2. SDS-PAGE analysis clearly demonstrated that the Su antigen immunoprecipitated by the human anti-Su serum comigrated with the in vitro translated hAgo2  sera similarly demonstrated that each serum was capable of recognizing the other three Ago family members. These results were not surprising and are consistent with the concept of inter-molecular epitope spreading [Human and mouse anti-Su autoimmune sera recognize other members of the Ago protein family. Because the Su antigen has been characterized as a macromolecular complex that consists of a doublet of approximately 100- and 102-kDa proteins , it is ppreading , becausepreading ,17. It ipreading . Regardlin vitro translated Dicer. SDS-PAGE analysis demonstrated that the 200-kDa Su antigen immunoprecipitated by the human anti-Su serum comigrated with the in vitro translated Dicer  or normal human (n = 10) and mouse (n = 6) sera  of the seven pristane-induced autoimmune mouse sera also stained GWBs, and of the tested sera, 100% (5/5) were hAgo2-positive; however, none (0/2) were Dicer-positive. None (0/16) of the normal human and pre-immune mouse sera stained cytoplasmic structures characteristic of GWBs, and none (0/10) of the Su-negative autoimmune sera stained GWB structures. Of the tested sera, none were reactive with in vitro translated hAgo2 (0/24) or Dicer (0/9). The significance of these results is that anti-Su autoimmune sera appear to be uniquely defined by their ability to stain GWBs within the cytoplasm of cells and recognize the hAgo2 protein, thus making them readily identifiable. During the course of our serological studies, we also determined that one of the two prototype human anti-GW182 sera was also positive for anti-Su antibodies, further demonstrating the close association and potential overlap between these two sets of autoimmune sera.To ascertain a broader perspective of the serological properties of anti-Su sera, 22 human and seven pristane-induced mouse anti-Su sera were further characterized and compared with other human Su-negative autoimmune n = 0 or normIn summary, these data identify the Su autoantigen as a macromolecular complex closely associated with the GWB structure and the RNAi pathway. Furthermore, these data demonstrate that anti-Su autoantibodies target key components of the RNA silencing machinery, in particular hAgo2. However, further studies will be required to better ascertain the extent of anti-Su autoantibody reactivity with other members of the Ago family and definitively determine whether Dicer is unambiguously the 200-kDa protein commonly associated with the Su antigen. Regardless, the association between RNAi and systemic autoimmune diseases is potentially significant due to the growing connection between RNAi and viruses. RNAi is an innate anti-viral response found in certain plant and invertebrate species and remains an evolutionarily conserved mechanism in many eukaryotes. Its role as a natural anti-viral response in mammals has been postulated and is supported by the evidence that mammalian viruses encode suppressors of RNAi ,30. EvenOur work links key components of the RNAi machinery with autoimmune disease. Moreover, our study identifies a murine model of autoimmunity that has the potential to advance our understanding of autoimmune responses to components of the RNAi pathway. Interestingly, pristane treatment of mice has been demonstrated to activate endogenous retroviruses . TherefoAgo = Argonaute; dsRNA = double-stranded RNA; EDTA = ethylenediaminetetraacetic acid; G = glycine; GWB = GW body; IIF = indirect immunofluorescence; IP = immunoprecipitation; miRNA = microRNA; PCR = polymerase chain reaction; RISC = RNA-induced silencing complex; RNAi = RNA interference; siRNA = short interfering RNA; SLE = systemic lupus deerythematosus; TnT = transcription and translation; W = tryptophan.The authors declare that they have no competing interests.AJ carried out the initial IIF and TnT-IP studies, participated in the serological testing and in the design of the study, and drafted the manuscript. KI carried out TnT-IP studies, completed the serological testing, and participated in the design of the study. MS carried out the IP studies and participated in the design of the study. MJF, WHR, and MS collected and classified the sera and also helped edit the manuscript. EKLC conceived of the study, participated in its design and coordination, and edited the manuscript. All authors read and approved the final manuscript.


The RNA polymerase (3Dpol) domain of 3CDpro modulates these activities of the protein. We have recently shown that the level of 3CDpro in HeLa cell-free in vitro translation-RNA replication reactions is suboptimal for efficient virus production. However, the addition of either 3CDpro mRNA or of purified 3CDpro protein to in vitro reactions, programmed with viral RNA, results in a 100-fold increase in virus yield. Mutational analyses of 3CDpro indicated that RNA binding by the 3Cpro domain and the integrity of interface I in the 3Dpol domain of the protein are both required for function. The aim of these studies was to determine the exact step or steps at which 3CDpro enhances virus yield and to determine the mechanism by which this occurs. Our results suggest that the addition of extra 3CDpro to in vitro translation RNA-replication reactions results in a mild enhancement of both minus and plus strand RNA synthesis. By examining the viral particles formed in the in vitro reactions on sucrose gradients we determined that 3CDpro has only a slight stimulating effect on the synthesis of capsid precursors but it strikingly enhances the maturation of virus particles. Both the stimulation of RNA synthesis and the maturation of the virus particles are dependent on the presence of an intact RNA binding site within the 3Cpro domain of 3CDpro. In addition, the integrity of interface I in the 3Dpol domain of 3CDpro is required for efficient production of mature virus. Surprisingly, plus strand RNA synthesis and virus production in in vitro reactions, programmed with full-length transcript RNA, are not enhanced by the addition of extra 3CDpro. Our results indicate that the stimulation of RNA synthesis and virus maturation by 3CDpro in vitro is dependent on the presence of a VPg-linked RNA template.Poliovirus protein 3CD This has been attributed to insufficient concentrations of viral proteins for RNA synthesis or encapsidation, to differences in membranous structures or the instability of viral particles in vitro CTP for 1 hour. RNA synthesis was measured by the incorporation of label into polymer using a filter-binding assay. As shown in Fig. pro(3CproH40A) and those to which no additional protein has been added.We have previously shown that translation of 3CDt 3 fold . The addyprotein ,9. We hapro is the precursor of both proteinase 3Cpro and polymerase 3Dpol. The 3Cpro domain contains both the proteinase and the RNA binding site CTP. After 1 hr of incubation the RNA products were applied to a nondenaturing agarose gel together with a [α-32P]-labeled full-length poliovirus RNA transcript as a size marker  or by mutant 3CDpro protein  is required for the stimulation of plus strand RNA synthesis but mutation R455A/R456A in the 3Dpol domain of the protein is not important for this process.To examine the effect of 3CDomplexes  were isoker Fig. , lane 1.ein Fig. , lane 3.pro(3CproH40A) on both minus and plus strand RNA synthesis we used preinintiation replication complexes [32P]CTP. The RNA products were resolved on a nondenaturing agarose gel. Minus strand RNA synthesis was estimated from the amount of replicative form (RF), in which the minus strand is hybridized to the plus strand template RNA. As shown in Fig. pro(3CproH40A). Poliovirus RF and ssRNA obtained from a reaction in which HeLa extracts were replaced by crude replication complexes (CRCs), isolated from PV-infected HeLa cells UMP incorporated into polymer. As shown in Fig. pro(3CproH40A) stimulates RNA synthesis up to 3-fold while 3Cpro(C147G) alone exhibits no significant enhancement of the RNA yield. When the two proteins are added together there is essentially no increase in the total amount of RNA produced over a period of 16 hours. Control reactions, lacking a viral RNA template exhibited very little, if any, incorporation of label into a polymeric product  inhibits plus or minus strand RNA synthesis we labeled with [α-32P]CMP the RNA products formed in preinintiation replication complexes during a 1 hr incubation period, as described above. The samples were analyzed on a nondenaturing agarose gel and as a size marker we used [α-32P]CMP-labeled RNA products made in CRCs  alone has very little, if any, effect on the yield of either of the 2 kinds of RNA products  and 3CDpro(3CproH40A), however, the synthesis of both products is completely inhibited  whereas the production of infectious virus was stimulated about 100 fold. The fact that there is such a large discrepancy between the extent of stimulation of RNA synthesis and virus production by 3CDpro(3CproH40A) suggested to us the possibility that this protein has an additional role at a subsequent step in the viral life cycle, the encapsidation of the progeny viral RNAs. To examine at which step of assembly this might occur, we labeled the viral proteins with [35S]-methionine in the in vitro reactions and analyzed the viral particles produced after 15 hr incubation either in the absence or presence of 3CDpro(3CproH40A). The samples were first loaded on a 5–20% sucrose gradient and sedimented for 15 hr, which resulted in the separation of the 5S protomers and 14S pentamers from the large capsid precursors and mature virions [35S]-labeled PV-infected HeLa cell lysate was applied  in the reactions  or at interface I in 3Dpol(R455A/R456A), exhibited very little increase in the total amount of 5S and 14S particles, when compared to reactions lacking 3CDpro(3CproH40A) , we used Western blot analyses with anti VP2 polyclonal antibody  on the formation of 80S (empty capsids) and 155S particles (provirion and mature virus). As we discussed before, the role of the 80S particle in viral assembly is unclear. The experimental evidence available at this time favors the hypothesis that empty capsids are dead-end products rather than true intermediates of particle assembly -methionine in vitro, were separated by sedimentation in a 5–20% sucrose gradient for 80 min. Under our experimental conditions the provirions (125S) and mature virus (155S) comigrate -methionine into capsid precursors, empty capsids or particles sedimenting at 155S. These results are difficult to reconcile with the 100-fold increase in infectious virus observed in translation RNA-replication reactions that are supplemented with extra 3CDpro(3CproH40A) -methionine into particles sedimenting at 80S and 155S was determined in reactions treated with SDS. It has been previously demonstrated that only mature virions but not provirions are stable in SDS -label in the pellet, derived from reactions with no extra 3CDpro, was 10-fold higher than in pellets of reactions lacking the extra protein (data not shown). A Western blot analysis of the particles in the pellets indicated the presence of VP0 but no VP2 (data not shown).To obtain further proof that the extra 3CDe in SDS . As show0A) Fig.  suggestipro(3CproH40A) produced 3–7-fold higher amounts of 155S particles than those that lacked the extra protein  or 3CDpro disappeared upon SDS treatment  in translation-RNA replication reactions enhances the yield of mature virus about 40-fold. Western blot analyses with anti VP2 antibodies of gradient samples 8–9 from the 155S peak confirmed the presence of VP0, indicating provirions in reactions lacking extra 3CDpro(3CproH40A)   yielded a 155S peak containing predominantly VP2, as judged by the Western analysis  enhances the specific infectivity of the viral particles produced.As we discussed above, reactions containing extra 3CDin Figs. , 6. Thesin Figs.  suggestient Fig. . These r0A) Fig.  or conta6A) Fig. . Faster sis Fig. . Therefocis-active hammerhead ribozyme resulted in templates that have regained most of their ability to support efficient plus strand RNA synthesis in the translation-RNA replication system CTP for 1 hr and the products were applied to a nondenaturing gel. As shown in Fig. pro(3CproH40A) in such reactions has no stimulatory effect on plus strand RNA synthesis (compare lane 2 with lane 3). As a size marker for plus strand RNA we have included the [α-32P]-labeled full-length PV ssRNA product made in CRCs  to translation reactions of transcript PV RNA had no effect either on the efficiency of translation or the processing of the polyprotein (data not shown).Previous studies have demonstrated that in the in vitro translation-RNA replication system the amount of plus strand RNA product obtained from PV ribozyme-treated transcript RNA or viral RNA is about 100-fold higher than what is produced in reactions with ribozyme-deficient transcript RNAs . To examRCs Fig. , lane 1.in vivo complementation rarely occurs, and if it does, it is very inefficient . Oligomerization of 3CDpro might also be aided by intermolecular contacts between the 3Cpro domains of two molecules . The expossible . In addicontacts . Whetherolecules . Howeverd system .pro(3CproH40A). According to the first model 3CDpro(3CproH40A) interacts with the progeny plus strand RNA, possibly at the cloverleaf, and causes an important conformational change. This step requires the RNA binding activity of the 3Cpro domain of the protein but binding might also be enhanced by the oligomerization of the polypeptide along interface I in the 3Dpol domain. Subsequently the RNA interacts either with the pentamers or the empty capsid and it is encapsidated, yielding a provirion while 3CDpro(3CproH40A) leaves the complex. The correct conformation of the RNA inside the provirions affects the shape of the capsid such that now the cleavage of the VP0s is favored to complete maturation. The second model is similar to the first one except that now 3CDpro(3CproH40A) itself is encapsidated, bound to the progeny RNA. This keeps the RNA in the correct conformation inside the capsid so that the maturation cleavage of VP0 can occur. The second model is supported by previous studies by Newman and Brown who observed that 3CDpro, 3Dpol and 2CATPase proteins were contained within isolated poliovirus and foot-and-mouth disease virus particles [pro in our 155S peak derived from reactions with extra 3CDpro(3CproH40A) using Western blot analysis with either anti 3Cpro or anti 3Dpol antibodies [data not shown]. In any case, the suboptimal concentration of functional 3CDpro in translation RNA-replication reactions might lead to progeny RNA molecules lacking the proper conformation for encapsidation and efficient virus maturation.On the basis of these observations we propose 2 possible models for efficient virus maturation in the in vitro translation-RNA replication reactions supplemented with extra 3CDarticles . In thisarticles . Howeverpro(3CproH40A) the synthesis of plus strands, and consequently the production of mature virus could be enhanced. To our surprise, this does not happen. The simplest explanation of these observations is that the level of endogenous 3CDpro is sufficient for the synthesis of the low level of plus strand RNA that is produced in the system. Therefore supplying the reactions with extra 3CDpro(3CproH40A) would have no stimulatory effect. However, this explanation does not account for the fact that virus synthesis is not stimulated by 3CDpro(3CproH40A) in reactions containing ribozyme-treated transcript RNAs. The yield of virus in such reactions is 50-fold higher than in samples in which ribozyme-deficient transcripts were used as template for translation and RNA replication. The only known difference between viral RNA and ribozyme-treated transcript RNA is the lack of VPg in the latter structure. Therefore our results indicate that the presence of VPg at the 5' end of the input viral RNA CTP  but lacking unlabeled CTP. At the indicated times samples were taken and the reactions were stopped by the addition of SDS to a final concentration of 0.5%. The samples were extracted with phenol-chloroform and the RNA was precipitated with ethanol. The pellets were resuspended in 10 mM Tris pH 7.5, 1 mM EDTA and were loaded on a DEAE-81 filter papers (Whatman). The filters were dried and subsequently washed three times with 5% Na2HPO4, once with water and once with 70% ethanol, as described before [35S]CTP was added and incubation was continued for 1 hr. The samples were treated and analyzed as described in Method I.Method I. Translation-RNA replication reactions (125 μl) were incubated at 34°C in the presence of 62.5 μC of [α-d before . Method Preinitiation RNA replication complexes were prepared as described previously  except f32P]CTP. The reactions were incubated at 34°C for 1 hr, the labeled RNAs were separated by native agarose gel electrophoresis, and the products were visualized by autoradiography. The reaction products were quantitated with a Phosphorimager (Molecular Dynamics Storm 800) by measuring the amount the amount of [α-32P]CMP incorporated into RNA.Plus and minus strand RNA synthesis were determined as described previously . Transla32P]CTP. The samples were incubated for 1 hr at 34°C and the RNA products were separated on a native agarose gel. The products were visualized by autoradiography.Alternatively, plus strand RNA synthesis was measured in translation-RNA replication reactions that were incubated for 4 hr at 34°C, in the absence of guanidine HCl, and the initiation complexes were isolated by centrifugation. They were resuspended in translation-RNA replication reactions lacking viral RNA but supplemented with [α-EcoRI prior to transcription by T7 RNA polymerase. The transcript RNAs were purified by phenol/chloroform extraction and ethanol precipitation. Translation reactions (25 μl) containing 8.8 μC of Trans [35S]Label  were incubated for 4 hours at 34°C [All plasmids were linearized with actions 2 μl contaCrude replication complexes (CRCs) were prepared by a method similar to what has been described before . HeLa ce32P]CTP.RNA synthesis by CRCs was measured as described before . In vitr35S]TransLabel  for 12 hr at 34°C. The excess unincorporated label was removed by dialysis. The samples were introduced into a Slide-a-lyzer (Pierce Endogen) dialysis cassette with a M.Wt cut-off of 10 kD and were dialyzed several times against phosphate buffer at 4°C until essentially all the excess label was eliminated. After dialysis the samples were centrifuged at 14,000 × g to remove any precipitated material. The samples were diluted to 500 μl and were centrifuged in a 5–20% sucrose density gradient in phosphate buffered saline containing 0.01% bovine serum albumin in a SW41 rotor at 40,000 rpm at 4°C. To separate 80S empty capsids and 155S virus particles (provirions and virions) the gradients were centrifuged for 80 min [35S]-labeled PV-infected HeLa cell extracts.HeLa S10 translation-RNA replication reactions (25 μl) were incubated in the presence of 8.8 μC of [r 80 min . To idenFor the identification of the capsid proteins present in sucrose gradient fractions Western blot analysis was used . Samples2 area.Standard electron microscopy processing techniques were used for negative staining. Briefly, formvar coated, 200 mesh nickel grids were prepared. Grids, sample side down were floated on droplets of suspended poliovirus, followed by fixation in a solution of 1% glutaraldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4. Samples were washed in PBS, then in water followed by phosphotungstic acid. The samples were viewed with a F. E. I. Tecnai 12 BioTwin electron microscope and digital images were captured with an ATM camera system. In each sample the viral particles were counted within a 20 mmE. coli and purified by nickel column chromatography (Qiagen): 3CDpro(3CproH40A), a proteinase active site mutant [pro(3CproC147G), a proteinase active site mutant [pro was described previously [proH40G) and a mutation (3DpolR455A/R456A) at interface I in the 3Dpol domain of the protein.The following PV proteins with a C-terminal his tag were expressed in e mutant ; 3Cpro(3e mutant . The pureviously . This prproR84S/I86A) was described before [RI prior to transcription with T7 RNA polymerase.Poliovirus sequences were derived from plasmid pT7PVM, which contains the full-length (nt 1–7525) plus strand poliovirus cDNA sequence . All cond before . Both plDF carried out all the experiments and made substantial contributions to the design of the experiments. HP contributed purified mutant enzymes for the study. CEC has contributed to the interpretation of the data and revised the manuscript critically. BR initiated the studies on this subject. EW contributed to the design of the experiments and revised the manuscript critically. AVP planned the experiments and wrote the manuscript. All authors read and approved the final manuscript.