Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions  per se does not require or induce a high degree of Gag multimerization, as Gag is present as monomers or dimers in the cytoplasm. In contrast, plasma membrane seems to be the only site at which higher order Gag multimerization occurs. Notably, at least a fraction of the Gag dimers in the cytoplasm are bound to the viral RNA. These results provide deeper insights to our understanding of the molecular details of the initiating events in HIV-1 assembly, which are potential targets for development of new antiviral drugs.Human immunodeficiency virus (HIV) assembles at the plasma membrane of the infected host cell, resulting in the release of infectious virus particles. HIV assembly is directed by the viral structural protein, Gag that performs a number of functions including specific recruitment of viral genomic RNA and multimerization around this RNA to form a virus particle. However, it is currently not clear where in the cell these two key events, Gag-RNA interaction and Gag multimerization, are initiated and whether they are coordinated. In this study we provide strong evidence that recruitment of viral genomic RNA by Gag is initiated in the cytoplasm of the host cell. However, this interaction  During assembly, Gag molecules selectively recruit unspliced viral genomic RNA for packaging into virions from a large pool of cellular RNA molecules. The Gag:RNA interaction is mediated by the nucleocapsid (NC) domain of Gag that binds directly to the packaging sequence (psi), which is composed of four stem loops located within the 5′ UTR and the 5′ end of the Gag multimerization is obviously a key event in HIV-1 assembly and multiple domains of Gag, including MA, CA and NC, have been proposed to play a role in this process. Although biochemical and structural studies The notion that viral RNA plays an important role in HIV-1 assembly raises two important and related questions about early events in the process. The first is where in the cell Gag initially interacts with viral RNA. Two potential sites are the cytoplasm and plasma membrane. Initiation of RNA packaging at the plasma membrane would require the viral RNA and Gag to move separately to this location. Recent live-cell imaging studies indicate that viral genomes that are otherwise highly dynamic in the cytoplasm become anchored at the plasma membrane in the presence of Gag, before particle assembly is detectable The second key question about the initiating steps in particle assembly is whether Gag forms oligomers in the cytoplasm prior to membrane binding. Techniques that rely on epitope masking of Gag upon multimerization Interestingly, in vitro biochemical assays have shown that recombinant Gag exists in monomer-dimer or monomer-trimer equilibrium depending on buffer conditions, particularly the presence of inositol phosphates that might mimic the presence of membrane per se does not induce or require higher order Gag multimerization in the cytoplasm. Instead, membrane interactions appear to be required to induce higher order Gag multimerization in cells.In this study, we aimed to elucidate the initial site of Gag-RNA interaction and whether Gag forms multimers, perhaps on viral RNA, before recruitment to the plasma membrane. To accomplish this, we developed assays in which we combined subcellular fractionation with RNA-immunoprecipitation (RNA-IP) and/or covalent protein crosslinking. Our results indicate that Gag-RNA interaction takes place in the cytoplasm both in transiently transfected and in infected cells. This interaction was not affected by the ability of Gag to localize to the plasma membrane, strongly suggesting that the initial site of Gag-RNA interaction is the cytoplasm. However, absence of CA-CTD led to a decrease in immunoprecipitable Gag-RNA complexes in the cytoplasm and, particularly, at the plasma membrane, suggesting that proper multimerization of Gag might be important for stabilizing Gag-RNA interactions. Crosslinking/subcellular fractionation analyses of Gag molecules in cells showed that Gag forms high-order multimers exclusively at the plasma membrane. In contrast, Gag appeared predominantly as monomers, but did form low-order multimers, in the cytoplasm. We tested whether these low-order multimers form on viral RNA using a sequential RNA-IP assay, which suggested that at least a fraction of Gag molecules multimerize on a given viral genome. Taken together, our results suggest that Gag-RNA interaction initially takes place in the cytoplasm and involves Gag monomers or low-order multimers. Moreover, Gag-RNA interaction The development of live-cell imaging techniques has allowed the visualization of individual virion assembly events that take place at the plasma membrane To address this issue, we developed a quantitative RNA-IP assay where protein-RNA complexes are immunoprecipitated and the amount of RNA that is bound to the protein of interest is analyzed by qRT-PCR. Since coexpression of a packageable HIV-1 genomic RNA carrying binding sites for the bacteriophage MS2 coat protein (V1B-MS2) and the MS2-GFP fusion protein allowed the successful visualization of viral RNA:protein complexes Next, we analyzed the specificity of the RNA-IP assay. First, Gag-GFP was coexpressed with either the V1B-MS2 viral RNA or a mutant derivative, V1B-Δ ψ-MS2, in which stem loops 3 and 4 of the packaging signal were deleted. Association of V1B-Δ ψ-MS2 RNA with Gag was reduced by about 3 to 4-fold as compared to V1B-MS2 RNA . This daWe then tested whether the immunoprecipitated Gag-RNA complexes form in the living cell prior to lysis, or whether they form as an artifact of cell lysis and subsequent experimental steps. Thus, 293T cells separately expressing V1B-MS2 RNA and Gag-GFP were mixed before cell lysis and processed for RNA-IP in parallel with cells coexpressing both viral RNA and Gag-GFP. As indicated in Because HIV-1 assembly requires an intact CA-CTD, and given the possibility that proper Gag multimerization might affect RNA binding, we performed RNA-IP assays using lysates of 293T cells coexpressing V1B-MS2 RNA and a mutated version of Gag-GFP that lacks the CTD of CA (Gag-delCTD-GFP). Notably, about 5-fold less V1B-MS2 was immunoprecipitated by Gag-delCTD, as compared to WT Gag, in terms of the fraction of the input RNA  or the aNext, we determined whether an HIV-1 Gag mutant that should not localize to the plasma membrane could still interact with viral RNA. Mutation of the N-terminal glycine residue of MA to alanine (G2A) blocks the attachment of a myristoyl group to Gag and hence prevents its recruitment to the plasma membrane To analyze more directly whether Gag interacts with viral RNA in the cytoplasm, we coupled the RNA-IP assay to membrane flotation analyses. Lysates of 293T cells transiently expressing Gag-GFP, G2A-Gag-GFP or Gag-delCTD-GFP together with V1B-MS2 were separated using membrane flotation gradients and ten 1 ml fractions of the gradient were collected. In this assay, membranes and associated proteins concentrated mainly in fraction 3, whereas the cytoplasmic content remained in fractions 9 and 10. We isolated total RNA and proteins from each fraction and analyzed each for the presence of the viral and cellular RNA as well as Gag proteins. Additionally, RNA-IP assays were performed on fractions 3 (membranes) and 10 (cytoplasm).In the presence of WT Gag-GFP, viral RNA and Gag-GFP localized to two peaks in the gradient corresponding to the membrane and cytoplasmic fractions . SurprisThe presence of Gag-RNA complexes at the plasma membrane and in the cytoplasm were examined by performing RNA-IP assays using fractions 3 and 10 of the membrane flotation gradients, respectively. As expected, WT Gag was able to efficiently coprecipitate viral RNA from the membrane fraction . SurprisIn parallel with our findings using total cell lysates , Gag-GFPde novo, the YFP-tagged, PR-defective Gag and GagPol proteins. At 48 h after infection, these infected cells were fractionated by membrane flotation and processed as above. As expected, the Gag-MA-YFP protein was present in both the membrane and the cytoplasmic fractions  in the stalk region of MA and an inactive protease (NL4-3 MA-YFP/PR-). This defective genome was introduced into MT2 cells using VSV-G pseudotyped virions that were generated with an excess of complementing WT HIV-1 GagPol proteins. Thus, the virions were infectious for only a single cycle and the infected MT2 cells expressed, ractions . Similarractions . Compareractions , recruitractions . This maractions , suggestNext, viral RNA-Gag complexes were immunoprecipitated from the membrane and cytoplasmic fractions of infected MT2 cells. In agreement with transient transfection based assays, Gag-RNA complexes were successfully isolated from both the cytoplasmic and membrane fractions . The effThe above results strongly suggest that Gag can bind to viral RNA while in the cytoplasm. A number of in vitro The majority of the WT Gag protein was crosslinked and clearly formed high-order multimers at the plasma membrane . A fractNotably, none of the Gag proteins formed high-order multimers in the cytoplasm, although cytoplasmic low-order Gag multimers (i.e. dimers) were apparent in each case , lane 5.Since we observed that a fraction of Gag forms low-order multimers in the cytoplasm, we directly tested whether viral RNA was associated with multimeric, cytoplasmic Gag. Specifically, we performed a sequential RNA-IP (seq-RNA-IP) assay in 293T cells co-expressing FLAG- and HA-tagged Gag proteins together with viral RNA. After dual crosslinking by EGS and formaldehyde, total cell lysates were used in immunoprecipitation assays using anti-HA or anti-FLAG antibodies. The eluates from these first immunoprecipitations were then used in a second round of immunoprecipitation using anti-FLAG or anti-HA antibodies, respectively, and the viral RNA content of first and second round immunoprecipitations was evaluated by qRT-PCR.Analyses of the first immunoprecipitations indicated that WT Gag and G2A-Gag immunoprecipitated viral RNA at similar levels, whereas Gag-delCTD exhibited a decreased level of viral RNA coprecipitation . This reWhen the initial immunoprecipitation with anti-HA antibody was followed by a second immunoprecipitation with an anti-FLAG antibody, viral RNA was easily detected after a second round of immunoprecipitations, regardless of which Gag protein was used . SimilarNotably, even though seq-RNA-IP assays were done on total cell lysates, the G2A-Gag mutant that cannot localize to the plasma membrane , 5 exhibGiven the comparative absence of information about the events that initiate HIV-1 assembly in living cells, we adopted a variety of strategies to analyze this process in cells. First, we developed a quantitative RNA-IP assay that was sensitive, specific and represented Gag-RNA interactions that take place in cells. By coupling this assay to membrane flotation analyses, we were able to show that HIV-1 Gag-RNA complexes form in the cytoplasm of both transiently transfected and infected cells. The fact that formation of these complexes was independent of the ability of Gag to localize to membranes suggests that the cytoplasm is the site where HIV-1 RNA packaging is initiated. Interestingly, the Gag protein of Rous sarcoma virus (RSV) was shown to require nuclear localization for efficient genome packaging The stability of Gag-RNA complexes both in the cytoplasm and plasma membrane seemed to depend on the CTD of CA, as evidenced by the reduced ability of Gag-delCTD to immunoprecipitate viral RNA. It is likely that the Gag-delCTD/RNA complexes disintegrate during immunoprecipitation, rather than fail to form efficiently in the cell, as Gag-delCTD was fully capable of recruiting viral RNA to the plasma membrane. This finding is in agreement with previous imaging experiments, which revealed that some viral RNA molecules recruited by Gag-delCTD dissociate from the plasma membrane Even though a number of FRET and BiFC studies indicated that plasma membrane is the major site of Gag multimerization, there has been inconsistency in the published conclusions about whether and to what extent HIV-1 Gag forms multimers in the cytoplasm Although we cannot exclude the possibility that only a fraction of the Gag dimers detected in the cytoplasm are bound to viral RNA, sequential RNA-IP assays suggest that at least a fraction of Gag molecules form multimers on viral RNA in the cytoplasm. Gag dimerization on viral RNA is likely mediated by RNA sequences around the packaging signal. However, these results do not distinguish between the possibilities that Gag dimers in the cytoplasm form on a single viral RNA molecule or are present on RNA dimers Overall, our findings suggest that HIV-1 genome packaging is initiated in the cytoplasm. This Gag:RNA interaction does not require or induce high-order Gag multimerization in the cytoplasm, but may involve Gag dimers or low-order multimers, that also form in the cytoplasm. This initial Gag:RNA complex is sufficiently stable to survive immunoprecipitation. Subsequently, Gag-viral RNA complex is recruited to the plasma membrane where high-order Gag multimerization occurs and virion assembly is completed.293T (ATCC #: CRL-11268) cells were obtained from ATCC and grown in DMEM supplemented with 10% fetal bovine serum. MT-2 cells, obtained from the NIH AIDS Research and Reference Reagent Program, were cultured in RPMI supplemented with 10% fetal bovine serum.VSV-G pseudotyped NL4-3 (MA-YFP/PR-) viruses were produced by transfection of 293T cells with the proviral plasmid, a complementing codon-optimized Gag-Pol expression plasmid and VSV-G expression vector using polyethyleneimine  as described previously Construction of plasmids expressing codon-optimized HIV-1 Gag-GFP (pCR3.1/HIV-Gag-GFP) Construction of proviral plasmids V1B, V1B-MS2 and V1B-Δ ψ-MS2 was previously described Antibodies used in RNA-immunoprecipitation, sequential-RNA-immunoprecipitation and western blot assays were as follows: mouse monoclonal anti-GFP (Roche), mouse monoclonal anti-FLAG (Sigma), mouse monoclonal anti-HA (HA.11 Covance), rabbit polyclonal anti-HIV-1 MA (NIH), mouse monoclonal anti-HIV-1 p24CA (183-H12-5C).7 cells were lysed in 250 µl of polysome lysis buffer (10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.0, 0.1 M potassium chloride, 5 mM magnesium chloride, 25 mM EDTA, and 0.5% Nonidet P-40, 2 mM DTT) supplemented with SuperaseIN (Ambion) and protease inhibitors (Roche). After preclearing with Protein G-sepharose beads , lysates were diluted in immunoprecipitation buffer . Immunoprecipitations were performed overnight at 4°C in the presence of 5-10 µg of antibody followed by incubation with Protein G-sepharose beads for another 4–5 hours. Parallel immunoprecipitations in the absence of antibody were included as controls. After several washes with NT2 buffer , RNA was eluted by proteinase K (Roche) treatment and purified by phenol:chloroform extraction and ethanol precipitation. RNA was further purified by DNase (Roche) treatment and one more round of phenol:chloroform extraction and ethanol precipitation.RNA immunoprecipitation (RNA-IP) assay was performed as described previously with minor modifications For quantitation of the RNA-IP assay, RNA samples were reverse-transcribed using ImProm-II Reverse Transcription system (Promega). The resulting cDNA was used as template for quantitative real-time PCR (qRT-PCR) using ABI 7500 Fast RT-PCR system. Viral RNA (V1B-MS2 or NL4-3 and its derivatives) was detected by previously described primer-probe pairs  5′ CAAATGGTACATCAGGCCATATCACCT and R: 5′ TCCTTCTGATAATGCTGAAAACATGGGTAT;V1B-MS2ΔΨ: F:5′ AGGTCATCCCTGAGCTGAAC and R: 5′ GCAATGCCAGCCCCAGCGTCGAPDH: F: A standard curve using serial threefold dilutions of input samples (10% to 0.04% of the input) or known copy numbers of NL4-3 plasmid was generated to quantitate the signals from immunoprecipitation samples.7 293T cells were co-transfected with 4 µg of FLAG-tagged Gag, G2A-Gag or Gag-delCTD, 4 µg of HA-tagged Gag, G2A-Gag or delCTD-Gag and 2 µg V1B-MS2 expression plasmids. At 24 hours post-transfection, cells were crosslinked by 1 mM ethylene glycol bis [succinimidylsuccinate]  for 30 min followed by 1% formaldehyde for 10 min at room temperature. Crosslinking was stopped by the addition of 100 mM Tris-Cl and 100 mM glycine. Cells were then washed in 1X PBS three times, resuspended in 500 µl of radioimmunoprecipitation assay buffer , supplemented with protease and RNase inhibitors and sonicated three times for 20 seconds at power setting 2.5 of a model 550 Sonic Dismembrator (Fisher Scientific). Lysates were precleared by centrifugation and protein-G sepharose bead incubation for 1 hour at 4°C. Immunoprecipitations were performed as in the RNA-IP assay described above using anti-HA and anti-FLAG antibodies. After the first immunoprecipitation, protein-RNA complexes were eluted by a 10-minute incubation of Protein G beads in 150 µl elution buffer  at 65°C. Sixty µl of the eluates were then subjected to a second immunoprecipitation. After the second immunoprecipitation, RNA was eluted by Proteinase K treatment. Formaldehyde crosslinking was reversed by incubation at 65°C for two hours and RNA was further purified as in the RNA-IP assay described above.For sequential RNA immunoprecipitation (seq-RNA-IP) assays, 1×107 cells were washed three times with NTE buffer  and resuspended in 500 µl of hypotonic buffer  supplemented with protease inhibitors. Samples were lysed by dounce homogenization and adjusted to 150 mM NaCl and 1 mM MgCl2. Nuclei and intact cells were removed by centrifugation for 10 minutes at 1000× g, 4°C. Thereafter, 350 µl of supernatant was mixed with 1650 µl of 90% sucrose solution  and overlayed with 6.5 ml of 65% and 2.5 ml of 10% sucrose solutions prepared similarly and supplemented with protease inhibitors. When necessary, RNase inhibitors (RNaseOUT and SuperaseIN) were added to the solutions and buffers listed above. Centrifugation was performed in a Beckman SW41 Ti rotor at 35,000 rpm for 18 h. Ten fractions (1 ml each) were collected from the top of the gradient and used for RNA-IP, RNA extraction or protein analyses by western blotting.The membrane flotation assay was based on a previously described protocol rd fraction) or cytoplasmic fractions (10th fraction) were diluted in 2X immunoprecipitation buffer. The remainder of the immunoprecipitation protocol was carried out as described in the above RNA-IP protocol.In cases where membrane flotation assay was coupled to the RNA-IP assay, 400 µl of the membrane  at a final concentration of 10%. Precipitated protein was collected by centrifugation and washed twice with 10% TCA and once with ice-cold acetone. Samples were air-dried briefly and resuspended in SDS-PAGE loading buffer for analysis by western blotting.7 293T cells, transiently expressing Gag and its derivatives were collected in 1X PBS and crosslinked by treatment with 1 mM EGS, a membrane-permeable crosslinker. After 30-minutes of incubation at room temperature, crosslinking was stopped by addition of Tris-Cl at a final concentration of 100 mM. Cells were then subjected to the membrane flotation assay as indicated above. After TCA precipitation of membrane and cytoplasmic fractions (3rd and 10th fractions from the top of the gradient respectively), samples were resuspended in 9M Urea/2% Triton X-100 and delipidated by methanol:chlorofom extraction Approximately 1×10


However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences.Trans-splicing, the in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans.Here we report an We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts. Reverse transcriptases (RTs) are enzymes that synthesize complementary DNA (cDNA) from an RNA template, and have evolved in retroviruses to convert single stranded viral RNA into double stranded DNA for integration into host genomes. They are an invaluable tool for molecular biology, being used to copy RNA into DNA for analysis by PCR (RT-PCR), microarrays and high throughput sequencing. It is possible to sequence RNA directly, however, most of the experimentally determined RNA sequences, and all high-throughput data, have been generated by RT-based protocols.RTs lack a proof reading activity , we arbitrarily selected GenBank sequence M14410, a fusion between KRE29 and HXK1, as a substrate for in vitro analysis  polymerase to add a poly(A) tail. These substrates were then used in vitro for reverse transcription as above but primed from oligo(dT). This produced the same pattern of products seen in previous experiments, which now depended on the presence of poly(A) polymerase (SPT7 RNA still occurs in the absence of poly(A) polymerase due to priming of the oligo(dT) on short, encoded oligo(A) stretches in the substrate. These data show that template switching events detected in random hexamer primed RT reactions also occur during oligo(dT) primed cDNA synthesis.Our proposed mechanism for the formation of sense-antisense fusions requires two RT enzymes to be active on the same RNA molecule. This will occur frequently if reverse transcription is primed from random hexamers, but is expected to be less common when oligo(dT) is used to prime synthesis from the poly(A) tail. To test the effect of this change, lymerase . The pp1SPT7 reactions reduced the aberrant products in some experiments, but clearly did not eliminate template switching  and cloned into pGEM-T (Promega). Oligonucleotides used were HXK1 F1/R1 for HXK1, KRE29 F1/R1 for KRE29 and SPT7 F1/R1 for SPT7; sequences of oligonucleotides are given in XhoI and 1 µg transcribed using T7 RNA polymerase (NEB) for 2 h at 37°C. Gels were run in 1x TBE, acrylamide gels contained 8 M urea. Gels were stained with SYBR Safe and imaged using a Fuji FLA5100 scanner. RNA was eluted from acrylamide gel slices by crushing and soaking for 4 h in 0.5 M NaOAc/1 mM EDTA/0.1% SDS, followed by phenol/chloroform extraction and ethanol precipitation with 1 µg glycogen. PCR reactions on reverse transcribed material were performed with Phire (NEB), details of cycle number are given in individual figure legends. Annealing temperature was 50°C for IGS1 and HXK1/KRE29 PCR and 53°C for SPT7 PCR. For poly(A) tailing, 50 ng RNA was incubated with 5U poly(A) polymerase (NEB) and 1 mM ATP, then cleaned on QIAQuick columns (QIAgen).Substrates for 18 was added in place of hexamers, and reactions were heated to 42°C prior to enzyme addition. Superscript III reactions were performed as per manufacturer's instructions at the indicated temperatures. DNA template controls were cDNA from 500 ng HeLa RNA produced as above, with 0.5 ng gel purified XhoI-PvuI fragments of the template plasmids. Where indicated, Actinomycin D  was added to Superscript II RT reactions after the 65°C step at 6 µg ml−1 from a 1 mg ml−1 stock solution.Superscript II RT: 0.5 ng substrate RNA, 500 ng HeLa RNA (Invitrogen), 125 ng random hexamers and 0.5 µl 10 mM dNTPs in 6.5 µl total volume were denatured at 65°C for 5 min before 2 min on ice. 2 µl 5x first strand buffer and 1 µl of 0.1 M DTT were added followed by 0.5 µl (100 U) Superscript II (Invitrogen). Reactions were incubated 10 min at room temperature, 42°C for 50 min and 70°C for 15 min. For oligo(dT) priming, 250 ng oligo(dT)AMV RT: 0.5 ng RNA, 500 ng HeLa RNA (Invitrogen) and 125 ng random hexamers in total volume 8.25 µl were heated 5 min at 70° and 5 min on ice. 1.25 µl 10 mM dNTPs and 2.5 µl 5x buffer were added followed by 0.5 µl (5 U) AMV (Promega). Reactions were incubated for 1 h at 37°C.Table S1Sequencing information. All sequences obtained in this project are shown. Regions of the sequence have been colour coded red, green and blue to indicate that they emanate from different molecules or different regions of the same molecule. Overlapping regions are shown in purple. Sequences were obtained either by direct sequencing of band-purified PCR products, or for complex PCR products by sequencing multiple clones ligated in pGEM-T.(0.04 MB DOC)Click here for additional data file.


Genomic RNA dimerization is an important process in the formation of an infectious lentiviral particle. One of the signals involved is the stem-loop 1 (SL1) element located in the leader region of lentiviral genomic RNAs which also plays a role in encapsidation and reverse transcription. Recent studies revealed that HIV types 1 and 2 leader RNAs adopt different conformations that influence the presentation of RNA signals such as SL1. To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, here we compare the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Prior studies from several groups have employed a variety of constructs and experimental conditions.gag gene.Although some idiosyncratic differences in the dimerization details were observed, we find unifying principles in the regulation strategies of the three viral RNAs through long- and short-range base pairing interactions. Presentation and efficacy of dimerization through SL1 depends strongly upon the formation or dissolution of the lower stem of SL1 called stem B. SL1 usage may also be down-regulated by long-range interactions involving sequences between SL1 and the first codons of the Despite their sequence differences, all three lentiviral RNAs tested in this study showed a local regulation of dimerization through the stabilization of SL1. The 5' untranslated region of the lentiviral genomic RNA is replete with RNA signals involved in different stages of the replication cycle, such as transcription transactivation, polyadenylation, tRNA primer binding, dimerization, encapsidation, splicing, and translation . The RNAin vitro, either through a kissing-loop interaction, or an extended duplex base pairing arrangement . In . In 11])iew, see -4). Roleiew, see , 15]). ). in vit results , the gen results . A ribos results . In this results . Severaln region . Here wen region ,18).In vitro, two dimerization signals exist within the HIV-2 genomic RNA leader region . One. OneIn v region ,20) and  and In v region ,24). Bec. BecIn v region . Alt region -21). We . We In v region . We pro region ,26). Ind. IndIn vbilities . Comparebilities .gag open reading frame that affect both SL1 presentation and overall leader conformation. Most important, all three lentiviral RNAs tested in this study demonstrated a local regulation of dimerization through the stabilization of SL1 by its lower stem structure called stem B.To determine whether common mechanisms of SL1 regulation exist among divergent lentiviral leader RNAs, we compared the dimerization properties of SIVmac239, HIV-1, and HIV-2 leader RNA fragments using homologous constructs and experimental conditions. Overall, it appears that SIV, HIV-1, HIV-2 RNAs have several common features with regard to SL1-mediated dimerization, which points to conserved regulation mechanisms by homologous RNA structures. Truncation analysis revealed that tight dimerization of the three lentiviral leader RNAs is modulated by interactions of nucleotides located between SL1 and the first codons of the BamHI site and the promoter for the T7 RNA polymerase and an antisense primer containing an EcoRI site (Table M15390]), and the first 435, 441, or 550 nucleotides of SIV genomic RNA sequence, mac239 isolate (nt 1 of the genomic RNA sequence corresponds to nt 775 of [GenBank:M33262]). The extended stem B mutation was introduced into a 1–444 HIV-2 ROD sequence using a modified antisense primer . The numbers used to define RNA constructs (for instance 1–561 HIV-2 RNA) are based on genomic RNA numbering. The HIV-2 ROD DNA template (modified plasmid pROD10) was provided by the EU Programme EVA/MRC Centralised Facility for AIDS Reagents, NIBSC, UK (Grant Number QLK2-CT-1999-00609 and GP828102). The SIV mac239 (p239SpSp5' plasmid) and HIV-1 (p83-2 plasmid) DNA templates were obtained from Dr. Ronald Desrosiers through the AIDS Research and Reference Reagent Program ()The formation of HIV-1 RNA dimers, measured by the disappearance of the monomer RNA species, was twice as efficient as HIV-2 or SIV over the whole eight hour experiment, although the interpretation of longer time points was made more difficult by the appearance of two other RNA species that appeared after two hours. One band migrated between the monomer and dimer bands and another one was below the monomer band Figure . Though The relative inefficiency of HIV-2 and SIV RNA tight dimerization suggested that the presentation of SL1 was not optimal in these RNAs. Though more efficient than HIV-2 and SIV, the yield of HIV-1 tight dimerization during the first hour was low Figure . Since Sgag translation initiation region , while the wild type 1–441 SIV RNA was the slowest . To . To gag Thus, the difference of only a few nucleotides in the SL1 sequence influences tight dimerization rates in SIV and HIV-2 RNAs. However, the incomplete correlation between the SL1 sequence and the dimerization yield in the chimeric RNAs indicates that other features of the leader RNA, like the stem B element, may influence the formation of tight dimers.To test whether formation of stem B directly affects SL1-mediated dimerization, we used the antisense oligonucleotides directed against the region upstream of SL1, including the 5' stem B Figure . We prevThus, binding of antisense oligonucleotides directed against the 5' side of stem B and nucleotides upstream increased the tight dimerization rate for all three lentiviral RNAs tested in this study. However, the rates without oligonucleotide differ and may be related to individual properties of the different lentiviral SL1s, including the apical loop, stem B, and adjacent sequences Figure .To examine the role of stem B stability on tight dimerization, we tested the influence of the incubation temperature on dimerization of RNA constructs ending after stem B  and in constructs lacking the 3' side of stem B . After denaturation and quench-cooling, the RNAs were incubated in dimer buffer for 30 minutes at temperatures ranging from 24° to 60°C (see Methods). The truncation of 3'-stem B induced a shift of the tight dimers temperature profile for all three RNAs , a,15, a4,1in vitro and during viral replication . ,27. in vication . The. Thegag  region . We o Figure  .in vivo by the presence of the loop B structure in SL1. Indeed, several laboratories have shown that loop B  and SIViewed in ,51) genoiewed in . Alternaiewed in . It is piewed in ). They siewed in ). Since iewed in , the majiewed in .Therefore, stem B of SL1 is likely a key regulating element for different conformations of lentiviral leader RNAs, akin to the concept of the riboswitch, proposed in HIV-1 by Berkhout and colleagues . Disruptin vitro, it is likely that these discrete conformational changes represent a conserved regulatory mechanism in vivo.This study demonstrates the ubiquity of structural entrapment as a mechanism of regulation of dimerization among primate lentiviruses and provides a framework for understanding dimerization data in each system through local and long-range RNA arrangements. The 3' leader region exerts an inhibitory effect on lentiviral RNA dimerization by altering the presentation of SL1. Stem B strongly influences SL1 presentation in all three lentiviral RNAs tested in this study. Because a similar set of folding rules can be used to explain the behaviors of three distinct primate lentiviral RNAs gag translation in mutant viruses in cell culture. Conformational changes like the ones described here may help determine the fate of viral RNA with respect to genomic and/or translational duties. Indeed, such viral riboswitches would be expected to be regulated by RNA chaperones like the viral nucleocapsid or Gag proteins and might be influenced by translational events, like ribosomal scanning.In particular, it will be of interest to study the effect of CGI or stem B mutations on packaging and HIV: Human immunodeficiency virus; SIV: Simian immunodeficiency virus; SL1: Stem-loop 1; CGI: C-box – G-box interaction; LDI: Long-distance interaction; BMH: Branched multiple-hairpin structure.The authors declare that they have no competing interests.JSL and JML designed the study. TTB, CLS, and JML carried out the experiments. JSL and JML drafted the manuscript. All authors read and approved the final manuscript.


Emergence of drug-resistant strains of influenza viruses, including avian H5N1 with pandemic potential, 1918 and 2009 A/H1N1 pandemic viruses to currently used antiviral agents, neuraminidase inhibitors and M2 Ion channel blockers, underscores the importance of developing novel antiviral strategies. Activation of innate immune pathogen sensor Retinoic Acid Inducible Gene-I (RIG-I) has recently been shown to induce antiviral state.in vivo in mice.In the present investigation, using real time RT-PCR, immunofluorescence, immunoblot, and plaque assay we show that 5'PPP-containing single stranded RNA (5'PPP-RNA), a ligand for the intracytoplasmic RNA sensor, RIG-I can be used as a prophylactic agent against known drug-resistant avian H5N1 and pandemic influenza viruses. 5'PPP-RNA treatment of human lung epithelial cells inhibited replication of drug-resistant avian H5N1 as well as 1918 and 2009 pandemic influenza viruses in a RIG-I and type 1 interferon dependant manner. Additionally, 5'PPP-RNA treatment also inhibited 2009 H1N1 viral replication Our findings suggest that 5'PPP-RNA mediated activation of RIG-I can suppress replication of influenza viruses irrespective of their genetic make-up, pathogenicity, and drug-sensitivity status. However, other subtypes namely H7, H9, and H5 which primarily cause infections and death among avian species have crossed the species barrier and caused mild to severe or fatal disease in humans [Annual influenza epidemics caused by influenza A and B viruses result in three to five million cases of severe illness with about 250,000 to 500,000 deaths globally every year. In the United States, complications from influenza infections result in approximately 250,000 hospitalizations and 36,000 deaths in an average year, with majority of the fatalities occurring among the elderly population . Influenn humans . Since 2n humans . In factn humans .Vaccination is the primary strategy for reducing the morbidity and mortality associated with human influenza -15. HoweThe immune system has evolved to recognize and eliminate pathogens. A number of pathogen recognition receptor (PRRs) families are involved in pathogen sensing and can be present in the host as soluble molecules in tissue fluids and serum or as molecules on cell membranes, localized in various cellular compartments, or in the cytosol -31. RecoThe 1918 pandemic resulted in 20-50 million deaths worldwide. To test if the activation of RIG-I with 5'PPP-RNA could suppress the replication of the reconstructed 1918 virus, we treated A549 cells with 5'PPP-RNA or CIAP-RNA for 24 hr, and then infected them with 1918 virus at an MOI of 0.01. Supernatants were collected 24 hr post-infection and assayed for viral titer. Figure Next we evaluated the prophylactic antiviral potential of 5'PPP-RNA against avian influenza H5N1 viruses with pandemic potential. A549 cells were transfected with 5'PPP-RNA or CIAP-RNA for 0, 24 or 48 hr, and infected with A/Vietnam/1203, an H5N1virus. Viral titers were determined 24 hr post-infection. As shown in Figure Stockpiling of the antiviral drug, oseltamivir is one strategy for pandemic preparedness, but the emergence of oseltamivir-resistant H5N1 viruses would seriously impede these efforts. Hence, we also tested the ability of 5'PPP-RNA to inhibit the replication of drug-resistant A/Vietnam/1203/2004 viruses that include zanamivir- and oseltamivir-sensitive and adamantanes-resistant and adamantanes- and oseltamivir-resistant virus variants, A/VN/30408/2005 (H274Y) and A/VN/HN/30408/2005 (N294S). As shown in Figure in vivo administration of 5'PPP-RNA will reduce viral titers, we delivered 5'PPP-RNA or PBS formulated in in vivojet-PEI daily for four days and infected mice on day 1 with A/Mexico/4482/09. Daily administration of 5'PPP-RNA alone had no side effects on body weight gain and activity (data not shown). On day 4 post-challenge, lungs from all animals were collected to determine viral titers. Control group had significantly higher viral load in lungs when compared to those that received 5'PPP-RNA  that circumvent host immune responses. Several studies, including our own have shown independently that non-structural protein (NS1) of influenza A viruses inhibits RIG-I function. We, therefore, investigated the expression of IFNβ and RIG-I as well as NS1 by real-time RT-PCR in 5'PPP-RNA or CIAP-RNA transfected A549 cells that were either uninfected or infected with H5N1 virus at an MOI of 0.1 for 24 hr following transfection. As shown in Figure RIG-I activation has been reported to stimulate type 1 interferon expression that induces an antiviral state. To study the direct role of 5'PPP-RNA induced RIG-I and type 1 interferon induction we knockdown RIG-I or IFNαβ-receptor using gene specific siRNA. As shown in Figure We also investigated the therapeutic potential of 5'PPP-RNA in A549 cells infected with seasonal influenza virus A/New York/02/2001 and its oseltamivir-resistant H274Y mutant. As shown in Figure Three influenza pandemics occurred during the 20th century, with varying degrees of severity; outcomes ranged from the high levels of illness and death observed during the 1918 Spanish flu pandemic (estimates of deaths range from 20 to 100 million) to the much lower levels observed during the pandemics of 1957 and 1968 (approximately one million deaths each) ,55. Furtin vitro transcribed 5'PPP-RNA, a ligand for cytoplasmic RNA sensor, RIG-I, in antiviral innate immune response against a panel of influenza viruses including drug-resistant, potential pandemic as well as pandemic strains in human lung epithelial cells. The broad-spectrum prophylactic potential of 5'PPP-RNA against influenza viruses is clearly evident from our studies. 5'PPP-RNA significantly inhibited in vitro growth of not only wild-type drug-sensitive and resistant H5N1 strains with comparable efficacy but also highly pathogenic 1918 pandemic strain. We also included 2009 pandemic H1N1 viruses in our studies to see the effect of 5'PPP-RNA both in vitro and in vivo. Data shown Figure in vitro suppression level of A/California/08/09 was similar to that of other viruses used in this study. Furthermore, 5'PPP-RNA also inhibited A/California/08/09 viral replication in vivo in Balb/c mice. These data suggest that irrespective of type or subtypes, drug-susceptibility status, or in vivo virulence, the replication of influenza viruses was inhibited by 5'PPP-RNA. The dose and frequency of administration of 5'PPP-RNA may influence the extent of inhibition of viral replication.In the present study, we explored the role of Treatment with 5'PPP-RNA not only induced the expression of IFN-I but also upregulated RIG-I expression. 5'PPP-RNA induced expression of IFN-I and RIG-I was not affected by subsequent infection with H5N1 Figure  or 1918 Viruses have acquired mechanism(s) to escape host immune surveillance by inhibiting IFN induction pathways. The NS1 protein of Influenza A virus inhibits host antiviral defenses by direct interaction with RIG-I -44. 5'PPTo evaluate the therapeutic potential of 5'PPP-RNA, we treated pre-infected (with wild-type and drug-resistant A/New York/02/2001) A549 cells with 5'PPP-RNA at different time-points. We observed relatively weak antiviral effect of 5'PPP-RNA, when A549 cells were transfected with 5'PPP-RNA post 8 hr infection. Interestingly, at this time point we also observed significantly low RIG-I and IFNβ expression  treatment was performed to remove tri-phosphate groups from in vitro synthesized RNA. Briefly, 100 μg of in vitro transcribed RNA was treated with 150U of CIAP for 3 hr at 37°C in a buffer containing 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA and 50U of RNase inhibitor. RNA was purified as described above. Chemically synthesized RNA with same sequence and length that contains an OH-group at its 5'-end was purchased from Dharmacon .5'PPP-RNAs were synthesized using T7 RNA polymerase. Using annealed complimentary oligo nucleotides, a DNA template was constructed that contains a T7 RNA polymerase promoter followed by the sequence of interest (AGCUUAACCUGUCCUUCAA) to be transcribed ,61. Twenin vitro transcribed 5'-PPP-RNA has been shown to contain dsRNA as well that activates RIG-I [in vitro transcribe 5'PPP-RNA is single stranded RNA (ssRNA) and/or double stranded RNA,(dsRNA) we used RNase A treatment under the conditions that degrades ssRNA, but not dsRNA. This confirmed that in vitro transcribed RNA used in our study was RNA only  supplemented with 10% fetal bovine serum (FBS), 100? U/ml penicillin and 100 μg/ml streptomycin. Normal human bronchial epithelial (NHBE) cells  were maintained under the conditions and media specified by the supplier.Seasonal viruses used in this study include the laboratory H1N1 strain A/Puerto Rico/8/34, wild-type H1N1 , H3N2 , and B viruses (wild-type B/Memphis/20/96 and its R152K mutant resistant to NA inhibitors zanamivir and oseltamivir), HPAI H5N1 viruses , and A/Vietnam/HN30408/2005 (N294S),, 2009 pandemic H1N1 viruses, A/California/08/09 and A/Mexico/4482/09, and the reconstructed 1918 Spanish influenza pandemic virus (H1N1) generated by plasmid-based reverse genetics . All humin vitro transcribed 5'PPP-RNA, chemically synthesized RNA or CIAP-treated RNA using Lipofectamine 2000. This dose was determined by dose-kinetics studies using 1, 2, 4 and 6 μg of RNA. After 24 hr, cells were infected with the different viruses used in this study in a 6-well plate. Unless specified, infection of cells was performed at a multiplicity of infection (MOI) of 0.1 with or without trypsin supplement. Each treatment was carried out in duplicate cultures. After 24 hr, cells were harvested for RNA and protein analysis and cell-culture supernatants were collected and stored at -80°C for determination of viral titer by plaque assay as described previously using MDCK cells[Cells were transfected with 2 μg of DCK cells. This tith of each reaction) were analyzed in using syber green Q-PCR reagents . PCR condition was kept as 94°C for 15 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s for a total of 45 cycles. The threshold cycle number for cDNA was normalized to that of β-actin mRNA, and the resulting value was converted to a linear scale. Data from three independent experiments were taken account for analysis. All data points fell into a normal distribution and there were no outliers.Total RNA was isolated from cells using the RNAeasy kit  and real time RT-PCR was conducted using a Stratagene Q3005 PCR machine for mRNA expression of RIG-I, IFNβ, NS1, and β-actin. For each sample, 2 μg of RNA was reverse transcribed using Superscript II Reverse Transcriptase  according to the manufacturer's directions. Parallel reactions without reverse transcriptase were included as negative controls. Reverse transcription reactions  for 20 minutes at 4°C. The protein content of different samples was determined using a protein assay reagent . Equal quantities of solubilized protein were resolved by 10% SDS-PAGE, blotted to nitrocellulose membrane and probed with the indicated primary antibodies. Anti-RIG-I was purchased from Axxora, LLC . Anti-β-actin antibody was purchased from Sigma, St. Louis, MO, USA. Antibody signals were detected by chemiluminescence using secondary antibodies conjugated to horseradish peroxidase and an ECL detection kit .A549 cells grown on cover-slips were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr. Cell monolayers were then washed with cold PBS and fixed with 2% paraformaldehyde for 15 min at room temperature. Cells were permeablized with 0.05% saponin in PBS for 30 min at room temperature, and then blocked with blocking buffer  at room temperature. Cells were incubated with primary antibodies (1 μg/ml) at 4°C overnight, and were washed 3 times with wash buffer (0.05% saponin in PBS). Cell monolayers were further incubated with secondary antibodies (Alexa 594 or 488) in blocking buffer for 1 hr at room temperature followed by three washes with buffer. To stain nuclei, cells were incubated with Hoechst dye (5 μM in PBS containing 0.05% saponin). Cells were further washed (3 times) with PBS, and were mounted on slides using Prolong antifade mounting media  and were observed under a Zeiss LSM 510 fluorescence microscope.in vivo-jetPEI according to manufacturer's protocol  in a volume of 200 μl for 4 days. On day 1, mice were challenged intranasaly with 1000 MID50 of 2009 pandemic virus (A/Mexico/4482/09). A separate group of animals that received only 5'PPP-RNA complexed with in vivo-jetPEI without viral challenge were observed to determine changes in body weight and activity. Lungs from control and treated challenged animals were collected on day 4 to determine viral titers using MDCK cells as described earlier.Female Balb/c mice 6-12 weeks old purchased from Jackson Laboratories were intravenously injected 100 μg of 5'PPP-RNA, capped 5'-RNA or PBS complexed with P < 0.05 was considered significant. All data points were included in the analysis and there were no outliers.To determine the statistical significance among the 5'PPP-RNA or CIAP-RNA treated and untreated groups, we used Analysis of variance and a value of The authors declare that they have no competing interests.PR participated in the planning and execution of the experiments, analyses of the results, and manuscript writing. LJ, VD, and MAH performed studies with human and avian viruses. VJ, WGD, JBB, MEW and MBP assisted in making 5'PPP-RNA and tested mRNA samples for the expression of various host innate immune response genes with cytokine arrays. SG provided assistance in assessing the functionality of cytokines and chemokines. HZ and TMT performed studies with the 1918 pandemic virus. LG, JMK, AG, and TF provided advice in the planning and execution of the studies. SS participated in the planning, coordination, supervision, and execution of the experiments that led to the present manuscript. All the authors saw and approved the final version of the manuscript.Figure S1. 5'PPP-RNA inhibits replication of drug-resistant human viruses. A459 cells (1 × 106 cells/well) in a 6-well tissue culture plate were mock-transfected or transfected with 2 μg of 5'PPP-RNA or CIAP-RNA for 24 hr and then infected with (a&b) wild-type and drug-resistant human H1N1 viruses; (b&c) drug-resistant H3N2 viruses and (d&e) wild-type and drug-resistant B viruses. Supernatants collected were assayed for viral titers as indicated in material and methods. Results shown are mean ± SD from three independent experiments and are expressed as viral titer (pfu/ml).Click here for fileFigure S2. Testing viral colonies if they are interferon escape mutant. A/NewYork/02/2001 colonies from 5'PPP-RNA treated A549 cells grown on MDCK cell were isolated and viral stocks were made by growing them in MDCK cells. Subsequently, these viruses were tested for 5'PPP-RNA sensitivity in A549 cells transfected 24hr earlier with either CIAP-RNA or 5'PPP-RNA. Culture supernatants were collected 24hr post-infection to determine viral titers in MDCK cells as described in materials and methods.Click here for fileFigure S3. Analysis of size, integrity and single or double-strandness of RNA. In vitro transcribed RNA (1 μg) generated by T7 polymerase was digested with 0.1 μg/ml RNase A (Ambion) or DNase I (10U/ml) (Ambion) at 37C for 1 hr, separated on agarose gel, and visualized by ethidium bromide staining.Click here for file


Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle.In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication.We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may contribute to the stimulation of viral RNA replication via clearance of ribosomes from viral RNA. The regulation of mRNA translation by RNA-binding proteins is an essential mechanism for the control of gene expression. RNA-binding proteins form functional ribonucleoprotein complexes (RNPs) that determine the fate of the cognate mRNAs. They have been shown to play crucial roles in RNA metabolism, such as pre-mRNA splicing, capping and polyadenylation, tRNA maturation, mRNA localization and translation, which in turn affect the vast majority of cellular processes.Xenopus homologue of PTB regulates Vg1 mRNA localisation Polypyrimidine tract binding (PTB) protein, one of the best studied RNA-binding proteins, is a 57 kDa protein with four RNA recognition motifs (RRMs) and an affinity for pyrimidine-rich RNA sequences Caliciviridae family of positive-strand RNA viruses is divided into four currently defined genera: Norovirus, Sapovirus, Lagovirus and Vesivirus. Human caliciviruses (HuCV), from the Norovirus and Sapovirus genera are a major cause of acute gastroenteritis and are responsible for more than 85% of non-bacterial gastroenteritis outbreaks in Europe in vitroThe Calicivirus protein synthesis is thought to occur via a unique mechanism not found in any other animal RNA viruses. This mechanism relies on ribosomal recruitment by the VPg (NS5) protein, which is covalently linked to the 5′ end of the viral RNA, through the interaction of VPg with various translation initiation factors FCV, strain Urbana, was generated by transfection of RNA transcripts derived from the full-length infectious clone pQ14 E. coli cells were transformed with a pQE9 plasmid expressing his-PTB1 provided by Stephen Curry . The protein purification was performed using Ni-NTA (Qiagen) according to the manufacturer's instructions. His-PTB was dialyzed against 50 mM HEPES pH 7.6, 1 mM DTT, 1 mM MgCl2 and 20% glycerol. GST-PTB was purified from DH5α E. coli cells transformed with the pGEX6P-1 PTB plasmid, provided by Richard Jackson . Cells were grown in LB at to an OD600 of 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added  and the protein expression was allowed to occur for 2 hours. The cells were then lysed using a French press in PBS containing 0.5 mM DTT, 10 mM EDTA, 3 µg/ml leupeptine and pepstatin, 1% Triton X-100, 1 mM PMSF. GST-PTB was then purified from the clarified lysate using glutathione-sepharose, washing with PBS containing 0.5 mM DTT, 10 mM EDTA and 1% Triton X-100. A second wash with 50 mM Tris pH 8.0, 150 mM NaCl and 0.5 mM DTT followed and the protein then eluted in 10 mM reduced glutathione, 50 mM Tris pH 8.0, 150 mM NaCl and 0.5 mM DTT. Eluted protein was dialyzed against 50 mM HEPES pH 7.6, 1 mM DTT, 1 mM MgCl2 and 20% glycerol and stored at −80°C until further use.For the purification of his-PTB SG13009 in vitro transcribed RNA derived from the full-length cDNA clones using Lipofectamine 2000 (Invitrogen). High titre virus stocks were prepared by an additional passage in CRFK cells and viral titers were determined by TCID50. RT-PCR was used to confirm the virus stocks retained the introduced mutations.Mutagenic oligonucleotides were synthesized and used for site directed mutagenesis of the full-length infectious clone pQ14 5cells/well. Single step growth curves were performed using an initial multiplicity of infection (m.o.i.) of 3 TCID50 per cell, based on the titre in CRFK cells. Samples were taken in triplicate for TCID-50 titration at 0, 4, 6, 8, 10 and 12 hours post infection (hpi). Multi-step growth curve analysis was performed using a m.o.i. of 0.01 and harvesting at 0, 4, 8, 12, 24 and 28 hpi.Growth curves of wild type and mutant virus (mBS2+3) were performed in AK-D , CRFK  and FEA  cells to assess viral growth kinetics. Cells were plated in 24 well plates at a density of 2×10in vitro transcription from the dicistronic plasmid pGEM-CAT/FMDV/LUC E.coli Poly(A) polymerase (Ambion). The FCV 5′G1–245 RNA was produced by PCR using primers which introduced a T7 RNA polymerase promoter at the 5′ end of the PCR product (details available upon request) followed by in vitro transcription. The in vitro translation assays were performed in Flexi rabbit reticulocyte lysates (RRLs) (Promega). Each 12.5 µl translation reaction included 6.25 µl of RRL, 20 µM amino acid mixture minus methionine, 100 mM KCl, 2 mM DTT, 0.5 mM MgOAc, 5 µCi 35S methionine (1000 Ci/mmol)  and 312.5 ng of an RNA preparation containing FCV VPg-linked RNA or in vitro transcribed dicistronic RNA. Translation reactions occasionally contained 1 µl of his-PTB at various concentrations, and 5′G1-245 RNA transcripts at various concentrations. The reactions were incubated at 30°C for 1.5 h.FCV VPg-linked RNA was purified from FCV replication complexes isolated from FCV infected CRFK cells as described previously In vitro transcribed, gel purified and refolded RNA (1 µg) was mixed with yeast RNA (1 µg) and structure buffer provided with the RNases (Ambion). Enzymatic cleavage reactions were performed at 30°C for 10 minutes with enzyme concentrations empirically determined to generate single cleavage products. The reactions were stopped using inactivation/precipitation buffer, the RNA precipitated, subsequently washed with 70% ethanol and resuspended in 10 µl of nuclease free water. An aliquot of the digested RNA (2 µl) was then used for primer extension with end labeled primers and AMV reverse transcriptase as described by the manufacturer (Promega). A sequencing ladder generated using the same primer was run along with the primer extension products on a 7M urea 6% acrylamide sequencing PAGE. The gel was dried and exposed to a phosphorimager screen.Biochemical RNA structure probing was performed using the conformational specific RNases V1, A, T1 (Ambion) and T2 (MoBiTec GMBH). in vitro transcription of PCR products containing the region of interest under the control of a T7 RNA polymerase promoter (primer details available upon request). Radioactively labeled RNA was subsequently gel purified by denaturing urea PAGE, the probe excised, eluted and precipitated prior to quantification by spectrophotometry. RNA was refolded by heat denaturation followed by slow annealing (∼1°C/min) prior to use. Typically a 7.5 µl EMSA reaction contained 85 nM radioactive RNA probe, GST-PTB at various concentrations (see figures for more details), 0.67 µg/µl yeast tRNA (Ambion), 50 mM HEPES pH 7.6, 25 mM KCl, 2.5 mM MgCl2, 1 mM DTT and 4% glycerol. All reactions were incubated for 10 min at 30°C and the reactions analyzed on a 4% acrylamide gel (acrylamide:bis-acrylamide 19:1) containing 5% glycerol. The EMSA gels were dried and visualized on a phosphorimager screen. Band quantification was performed using the ImageQuant 5.0 software package (Molecular Dynamics) and the amount of protein required to shift 50% of the probe was determined using SigmaPlot. Note that relative, rather than absolute, affinity was determined due to the difficulty in determining the active fraction of the purified GST-PTB.Radioactive probes were generated by The specific digestion of the FCV genomic RNA in the viral RNA preparation from infected cells was carried out using RNase H and an oligonucleotide that is complementary to NS6-7 coding region (ntds positions 3215-3244). RNase H reactions contained 1.26 µg of VPg-linked RNA preparation, 25 pmol of primer, 10 U RNAsin (Promega), 37.5 mM KCl, 25 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 5 mM DTT and 50 U of RNase H (Ambion). The reaction was incubated at 37°C for 20 min. M13/Puc R was used as non-specific control oligonucleotide to control for any effects of RNase treatment and subsequent extraction on the replication/translation of the viral RNA.6) grown on glass chamber slides were transfected with the pEGFP-PTB  or a combination of pEGFP-PTB and pDsRed2-Nuc (Clontech). 24 hrs post transfection, cells were then infected with either FCV-LC-DsRed CRFK cells were plated on round glass cover slips and infected with FCV (m.o.i. of 0.5) for 16 h at 32°C . The glass cover slips were washed with chilled PBS and fixed in 4% paraformaldehyde solution containing 250 mM HEPES pH 7.4 for 10 min on ice. An 8% paraformaldehyde solution containing 250 mM HEPES pH 7.4 was applied for 20 min at room temperature and the cells were washed 3 times in PBS. 50 mM ammonium chloride was applied for 10 min at room temperature and the cells were washed 3 times in PBS. Permeabilization of the cells was achieved using 0.2% Triton-X 100 in PBS for 10 min at room temperature followed by three 5-minute washes in block buffer . NS6-7 staining was performed using a rabbit polyclonal antibody and PTB staining was performed using a mouse monoclonal antibody for PTB (DH17). Image-iT FX signal enhancer was applied according to the manufacturer's instructions (Invitrogen). α-rabbit Alexa Fluor 488 (Invitrogen) and α-mouse Alexa Fluor 546 (Invitrogen) were used as secondary antibodies. The cover slips were mounted on a glass slide with 40 µl of Mowiol 4–88  containing 1 µg/ml of DAPI. The cells were viewed under a Zeiss LSM 510 Meta confocal microscope, and the images were processed with Zeiss Meta 510 software. For the quantification of staining intensity in specific areas of the confocal images Image Quant 5.0 software (Molecular Dynamics) was used as described previously 7 CRFK cells were infected with FCV for 6 h using an m.o.i. of 0.1. Ribonucleoprotein complexes were extracted from FCV infected CRFK cells and subjected to immunoprecipitation following the previously described procedure 10Caliciviridae family. RNase cleavage sites were determined using primer extension with two different primers  1-245 and 1-284 of the 5′ ends of the FCV gRNA and sgRNA, respectively  primers  and then primers . Clearly primers ; for exa primers . The posTo further characterize the interaction between PTB and the 5′ end of the FCV gRNA, truncations of the region were used as radiolabeled probes for EMSA analysis . The preAs expected, PTB was unable to bind to the transcript 1–111, containing none of the predicted PTB binding sites . AdditioTo clarify the contribution of the other potential binding sites, mutations were introduced in order to disrupt the PTB consensus binding sequence in BS1, BS2 and BS3 in all possible combinations . As all To begin to determine the role that PTB plays in the FCV life cycle we first examined how virus infection altered PTB localization. Infected CRFK cells were stained for PTB and the viral polymerase, NS6-7, as a marker for the viral replication complexes. The localization of PTB in uninfected cells is predominantly nuclear . We obsein vitro data demonstrated efficient PTB binding to the FCV genome, it was important to demonstrate that this interaction also occurred during viral infection. To address this question, RNA immunoprecipitation was used to determine if PTB was physically associated with the viral RNA during replication. Two monoclonal antibodies (DH7 and DH17) to PTB that efficiently immunoprecipitated PTB from cell extracts (data not shown) were able to co-precipitate the FCV RNA from infected CRFK cells as determined by RT-PCR  was titrated back into reactions containing inhibitory levels of recombinant PTB ) was used to assess the effects of PTB on the general cap-dependent and IRES-dependent translation  co-purified with PTB from E.coli, rather than PTB itself. Alternatively the FCV G1–245 sequence may be unable to compete for PTB binding to the dicistronic RNA or high levels of PTB may have resulted in competition for other factors required for general translation initiation in the RRL.In order to define the stage of the virus life cycle at which PTB might be involved, the uncoupling of the two main processes, translation and replication, was required. These two processes are inherently linked for all positive-strand RNA viruses since the viral RNA functions both as a template for replication by the RNA polymerase and as a template for translation. In addition, the unique mechanism of calicivirus VPg-dependent calicivirus translation initiation which relies on the interaction of host cell translation initiation factors with the VPg protein covalently linked to the 5′ end of viral RNA on assay . Additioproteins , indicatnant PTB . FCV translation . Upon adnslation . Under tnslation . At the nslation . In contin vitro transcribed RNA for the study of authentic calicivirus translation initiation. During virus replication a sgRNA is produced from the 3′ half of the viral genomic RNA, which, similarly to the genomic RNA, is translated following the recruitment of translation initiation factors by the VPg protein. However, the sgRNA does not produce the viral polymerase and encodes only the major capsid precursor protein (LC-VP1) and the minor capsid (VP2) protein. Thus, transfection of cells with VPg-linked sgRNA alone allows the analysis of virus translation in the absence of replication as there is no source of the NS7 RNA polymerase. We have previously demonstrated that PTB also binds the 5′ end of the sgRNA in vitro translation of the digestion reaction in RRLs . Hence the observed defect in replication is likely the result of an inability to coordinate translation and replication. To further elucidate the nature of the cell type specific effect and examine if there was any correlation between the observed effects of the binding site mutations and the levels of cellular PTB, we examined the steady-state levels of PTB in the various cell lines using both monoclonal and polyclonal antibodies  in three different cell lines; CRFK (feline kidney cell line), FEA (feline embryonic airway) and AK-D cells . The effect of mutations in the 5′ end of the genomic RNA were examined as we speculated that any alteration of the efficiency of translation and/or replication of the viral genomic RNA, which encodes the replicase components, would have a more apparent effect on virus titre than similar mutations in the viral sgRNA, which encodes the structural components only. Single step growth curve analysis indicated that the mBS2+3 virus had a cell type specific defect in virus replication, displaying reduced replication kinetics in the fetal lung cell line AK-D . A slightibodies . We obsetibodies . Howevertibodies . There wtibodies . This mavice versa. Thus, the reduction in FCV virus replication and subsequent yield of infectious virus that we previously observed upon PTB knockdown could not be attributed to one specific function of PTB within the viral life cycle.Our previously published data revealed that PTB was required for the efficient replication of FCV, as siRNA mediated knockdown of the protein significantly reduced the virus yield in CRFK cells Drosophila, highlighting that the action of any RNA-binding protein largely depends on the RNP that it is a component of Previous studies have highlighted that the primary role for PTB in the life cycle of several positive-strand RNA viruses is at the level of viral translation, primarily functioning as an enhancer of translation initiation in vitro RRL translation system Drosophila Oskar protein. During these studies we found that although at low levels of exogenous PTB, a limited increase of the FMDV IRES activity in vitro was observed, higher levels led to a significant reduction, presumably through the titration of additional factors. The original investigations into the involvement of PTB in FMDV translation, revealed that dependence on PTB was observed only in RRLs depleted of PTB in vitro translation is significantly lower than the temperature for the normal virus infection. The inhibitory effect of PTB was more apparent at temperatures lower or higher than the optimal 30°C (data not shown) providing additional evidence towards the RNA chaperone activity of PTB under suboptimal conditions.This study was designed to try to address the fundamental role of PTB in the calicivirus life cycle using feline calicivirus as a model system. Prior to this study, the only available system to allow the examination of FCV translation, in the absence of genome replication was the Vaccinia virus can result in the processing of the FCV LC-VP1 into the mature VP1 protein Vaccinia virus encoded protease or the induction of a cellular stress leading to the expression of a protease responsible for the observed processing. It is therefore possible that the multiple transfections required to obtain efficient PTB knockdown, followed by subsequent transfection with viral sgRNA also resulted in the induction of a similar cellular stress related protease which processed LC-VP1 to produce a protein of approximately the same mass as the mature capsid protein. Indeed previous studies have highlighted that caspase 2 induced during FCV infection can cleave the mature capsid protein (60kDa) during FCV infection to produce a ∼40kDa product Due to the unique mechanism of calicivirus translation, an attempt to verify the role of PTB in FCV translation in cell culture was made by selective digestion of the genomic RNA in the mixture of viral RNAs using RNase H. The inactivation of the genomic RNA allowed only translation from the sgRNA that, in the absence of polymerase, cannot be replicated. It is known that both genomic and subgenomic RNA are translated by the same VPg-dependent mechanism At first glance the increased levels of viral sgRNA translation in PTB depleted CRFK cells is contrary to our previously observed reduced viral replication  As PTB is predominantly nuclear, our observed cytoplasmic relocalization during FCV infection would add to our hypothesis on the involvement of PTB in the maturation of the viral replication complexes. It is noteworthy that only partial colocalization of PTB with NS6-7 was observed during our study . Whilst The cell type specific effect of the PTB binding site mutations is somewhat intriguing and may be due to the apparent heterogeneity of PTB expression in AK-D cells . PreviouIn light of these data, we propose a speculative model for the function of PTB in the FCV life cycle detailed in Figure S1Time-lapse analysis of the effect of FCV infection on PTB localization. GFP-PTB expressing CRFK cells were infected with recombinant FCV expressing a LC-dsRED fusion protein (m.o.i. 2) and examined during the course of the infection. XVID file.(1.01 MB AVI)Click here for additional data file.Figure S2Time-lapse analysis of the effect of FCV infection on nuclear localization. Cells transfected with cDNA constructs expressing PTB-GFP and dsRED fused to 3 copies of the nuclear localization sequence of the SV40 large T-antigen (DsRED2-Nuc) were infected with FCV at a m.o.i. of 2. Cells were then examined over the course of the infection. XVID File.(1.38 MB AVI)Click here for additional data file.


In recent years, the number of available RNA structures has rapidly grown reflecting the increased interest on RNA biology. Similarly to the studies carried out two decades ago for proteins, which gave the fundamental grounds for developing comparative protein structure prediction methods, we are now able to quantify the relationship between sequence and structure conservation in RNA.Here we introduce an all-against-all sequence- and three-dimensional (3D) structure-based comparison of a representative set of RNA structures, which have allowed us to quantitatively confirm that: (i) there is a measurable relationship between sequence and structure conservation that weakens for alignments resulting in below 60% sequence identity, (ii) evolution tends to conserve more RNA structure than sequence, and (iii) there is a twilight zone for RNA homology detection.The computational analysis here presented quantitatively describes the relationship between sequence and structure for RNA molecules and defines a twilight zone region for detecting RNA homology. Our work could represent the theoretical basis and limitations for future developments in comparative RNA 3D structure prediction. The view of RNA as a simple information transfer molecule has been challenged since the discovery of ribozymes, a class of RNA with enzyme-like functions -3. RNA mFor RNA, the axiom of "function is more conserved than structure and structure is more conserved than sequence" has been adopted since the end of the sixties  and evenWe begin this article by describing the results of an all-against-all comparison of a non-identical RNA structure set (Results). Next, we discuss the impact that our findings could have in the development of comparative approaches for RNA structure prediction (Discussion). Finally, we end by detailing the methodology used for aligning and assessing RNA alignments .i.e., percentage of C3' atoms within 4.0 Å RMSD) higher than 60% and only 3 alignments resulted in less than 75% PSI. The average PSI value for the 589 pair-wise alignments was 90.4%, which corresponded to a core RNA size ranging from ~50 to ~2,800 nucleotides. The superimposition of the Staphylococcus phage group I ribozyme  and a fragment of the synthetic construct group I Intron  resulted in 60.9% aligned C3' atoms, which corresponded to a structural core of 120 nucleotides  and the tRNA(Met) of Acuifex aeolicus  resulted in 65 core nucleotides within 1.9 Å RMSD  resulted in 134 core nucleotides within 1.8 Å RMSD  and 23S ribosomal RNA of Thermus thermophilus  resulted in 2,347 core nucleotides within 1.7 Å RMSD  higher than 34.2%. Structure identity ranged from 60.9 to 100.0%, with 95% of the alignments with PSI higher than 81.2%. The same trend is observed by comparing sequence conservation (PID) with structure diversity (structure core RMSD)  for 30-60% and 0.63 (p = 3.8·10-15) for 90-100%, which indicates that as sequence identity decreases, the relationship between sequence and structure conservation weakens. Furthermore, to assess the impact of the sequence high identity alignments (95-100%) in the fitting of our exponential curves, we removed all pair-wise alignments with sequence identity higher than 95%. The correlation coefficient between RMSD and PID decreased to 0.66 but remained statistically significant (p = 1.5·10-57). A similar clustering of the sequence space was previously observed for proteins [All the alignments in the HA-RNA09 set superposed a medium to large structurally conserved RNA core. The structure diversity between the selected pairs of RNA structure cores had a measurable relationship to the sequence diversity, which best fitted an exponential decay curve with a 0.67 correlation coefficient Figure . Similar) Figure . The relproteins ,50, wher1hr2 and 1x8w PDB identifiers, chains A) resulted in 75.6% sequence identity and only 54.7% secondary structure identity.As previously observed, this analysis quantitatively confirmed that RNA secondary structure largely determines tertiary structure. The relationship between secondary structure and tertiary structure conservation in RNA best fitted an exponential decay curve with a correlation coefficient of 0.73 Figure . The medln(PPID), -ln(PPSS), and -ln(PPSI) cut-offs, which were calculated using the background distribution of similarity scores from a set of 3D structural alignments between RNA molecules with sequence identity lower than 25% (Methods). The resulting groups included: (i) true positive alignments between related structures with all three cut-offs higher than 4.5, (ii) true negative alignments between dissimilar structures with all three cut-offs lower than or equal to 4.5, and (iii) medium accuracy alignments with one or two cut-offs higher than 4.5. The sequences of all pair-wise alignments within each group were aligned using the sequence-based alignment program Infernal [i.e., Infernal e-value) and the length of the alignment  as well as to assess the difficulty detecting homology based solely on sequence information. Similar to proteins [-10 e-value for RNA sequences of ~50 nucleotides and leveled at ~5·10-4 e-value for RNA larger than 100 nucleotides  and only 152 false positives . There were a total of 262 RNA structure pairs with all three p-values higher than or equal to 4.5 that were very difficult to detect based on the Infernal alignment score. Those pairs of sequences, which corresponded mainly to tRNA molecules, aligned between 67 and 78 nucleotides with mean sequence identities of 41.7% and constitute a 23.2% of false negative rate . The analysis of the 262 false negative pair-wise alignments showed that their average PID was much lower (41.7%) than for the pairs of related structure above the "twilight zone" (71.2% average PID). However, the secondary structure of those pairs resulted in an 83.1% average PSS indicating that even though their sequences diverged, their secondary and tertiary structures were conserved. This set thus becomes a very difficult set of related RNA structures to detect by sequence alignment methods.The 101,475 pair-wise alignments in the RNA09 dataset were divided into three different groups depending on the -Infernal . This diproteins , we obses Figure . Pair-wii.e., structural divergence >20%) for pairs of RNA structures that superimpose with sequence identity below 40%. Moreover, the exact relationship between sequence and structure conservation for pairs of distant RNA molecules  is less evident, which results in a correlation coefficient of 0.34. Homologous pairs of RNA molecules will diverge into different structures when there is a significant decrease in the identity of their sequences. Therefore, it is more difficult to assess structure conservation based on sequence diversity in the low regime of sequence identity . Highly similar structures conserve their base pairing. The degree of conservation between tertiary and secondary structure in RNA results in a correlation of 0.73. However, the relationship between sequence and secondary structure conservation is weakly correlated, which agrees with the difficulty of predicting secondary structure from RNA sequence alone. Similar conclusions were obtained with the ARTS program, which used a >90% secondary structure identity threshold for structural classification of RNA [Our analysis indicate that two related RNA molecules conserve a structure core of at least ~50 nucleotides, which can be superimposed within 4.0 Å RMSD. Such conserved structure core starts diverging as sequence identity decreases below 50% and becomes noteworthy  as well as removing alignments between a structure and its sub-structures. Moreover, such problem will become less relevant with time given the increased pace of deposition of new RNA structures in the PDB. Second, RNA motif comparison has classically been centered on small RNA fragments of about 10 to 30 nucleotides. However, given the intrinsic difficulty of discerning significant alignments from random pairs of short RNA structures or motifs, our work has focused on identifying structural cores of at least ~50 nucleotides.Despite the increasing interest on RNA function and the accelerated deposition of RNA structures, there was a gap between the common knowledge and a quantitative analysis of the relationship between sequence and structure conservation in RNA. Here we have addressed this knowledge gap by applying our RNA alignment method  to a setOur study provides an initial assessment of the current limits of comparative modeling of RNA structures. We anticipate that our work will aid the development of new methods for RNA structure prediction from sequence. In the near future, it is expected that large-scale comparative modeling of RNA structures will extend opportunities for answering key questions about RNA evolution, such as the origin of RNA as functional molecules . We haveInfernal program [Two types of RNA alignments were obtained in our experiment. First, 3D structure-based alignments, which were produced by the SARA program  and used program  and usedN nucleotides, for each i = 1,...,N-1 extract the vector from the i-th to the (i+1)-st selected atoms; (ii) normalize all vectors to a unit-distance, and place the tails of all normalized vectors at the origin of a unit-sphere; the resulting collection of normalized vectors is the unit-vector representation of the input RNA. In this work, SARA aligned two RNA structures by selecting only C3' atoms involved in base-pairing as computed by the 3DNA program [Pairs of RNA structures were aligned using the SARA program, which is based on the unit vector approach . A simil program ; (iii) c program ; (iv) th program ; (v) the program ,60; and The three alignment scores calculated by SARA are: first, percentage structure identity (PSI):nal is the number of aligned C3' atoms within a threshold distance of 4.0 Å and N is the length of the shortest of the two RNA structures. Second, percentage of aligned secondary structure (PSS):where pal is the number of aligned base pairs within a threshold distance of 4.0 Å and NP is the smallest number of base pairs of the two aligned RNA structures. Two base pairs are aligned when both C3' atoms of the interacting nucleotides in the first structure are below 4.0 Å to the two C3' of the interacting nucleotides in the second structure. Third, percentage of sequence identity (PID):where nid is the number of identical nucleotide types aligned in the structural alignment and N is the length of the shortest of the two RNA structures.where i.e., PSI, PSS and PID) for alignments between unrelated RNA structures follows an extreme value distribution and the probability for a given alignment to obtain a score x larger than z can be calculated by integrating the Gumbel distribution:After an alignment is produced, SARA calculates the PSI, PSS and PID scores as well as their associated p-values for estimating the probability of obtaining an equal- or better-scored alignment by chance. The distribution of the accuracy scores , -ln(PPSS), and -ln(PPID) higher than or equal to 4.5) with a crystallographic resolution higher than 5 Å and removing all alignments between a RNA structure and its sub-structures. The ability of the negative logarithm p-values scores for identifying biologically relevant alignments was previously tested by means of functional annotation of the SCOR database [related non-identical high-resolution RNA structures. Therefore, the HA-RNA09 dataset is a good compromise between the need of a large set of alignments and the appropriate sampling of the sequence and structure spaces. The entire datasets of RNA structures and alignments used in this work are available as Additional files as well as for downloading at http://sgu.bioinfo.cipf.es/datasets.As of March 2009, the PDB database stored a total of 1,534 structure files containing at least one RNA chain. This initial dataset of structures was filtered by removing RNA structures that: (i) were less than 20 nucleotides long and had less than 4 base pairs; and (ii) were 100% identical in sequence to another RNA structure in the dataset. The final dataset (RNA09) included 451 RNA chains from 417 PDB entries (Table database ,38. The EC carried out the computational analysis. EC and MAM-R conceived and designed the study as well as drafted the manuscript. All authors read and approved the final manuscript.P-value parameter optimization. Fitting of the μ and σ values for the calculation p-values for PID, PSS and PSI.Click here for fileRNA09 dataset. Structural alignments in the NR-RNA09 dataset used in the all-against-all structural comparison.Click here for fileNR-RNA09 dataset. Structural alignments in the NR-RNA09 dataset used for the calculation of the background distributions for PID PSS and PSI.Click here for fileHA-RNA09 dataset. Structural alignments in the HA-RNA09 dataset used to quantify the relationship between sequence and structure in RNA.Click here for file


Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is confounded. This can irreversibly reduce the robustness, resolution, or expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible.We developed a single embryo RNA extraction method based on sample homogenization in liquid nitrogen, RNA extraction with phenol and column purification. Evaluation of this method showed that: the quality of the RNA was very good with an average RIN value of 8.3-8.9; the yield was always ≥ 200 ng RNA per embryo; the method was applicable to all stages of zebrafish embryogenesis; the success rate was almost 100%; and the extracted RNA performed excellent in microarray experiments in that the technical variation was much lower than the biological variation.Presented is a high-quality, robust RNA isolation method. Obtaining sufficient RNA from single embryos eliminates the necessity of sample pooling and its associated drawbacks. Although our RNA isolation method has been setup for transcriptome analysis in zebrafish, it can also be used for other model systems and other applications like (q)PCR and transcriptome sequencing. Mus musculus [Drosophila melanogaster [Caenorhabditis elegans [Danio rerio) has joined these experimental model organisms in many domains of biological and biomedical research [Transcriptome studies of model organisms during development such as musculus , Drosophnogaster  and Caen elegans  are exciresearch -6. This research . Additioe.g. pathogen infection [In studies of genetic networks in zebrafish development , responsnfection ,10, or tnfection -14, biolnfection -17. PoolThe zebrafish book [Available methods for embryonic zebrafish RNA isolation commonly need to utilize 20 embryos or more to obtain sufficient RNA  is indispensable for maximum yield and limiting Qiazol carry-over. To validate and demonstrate the value of our method, several dedicated experiments were performed.One of the challenges of isolating RNA from zebrafish is the presence of a rigid and insoluble chorion. Enzymatic degradation of the chorion is possible, but will most likely affect the transcriptome. Therefore, we chose for a sample preparation method involving mechanical disruption of the chorion under frozen conditions. RNA isolations itself, can be performed by several well-established methods based on phenol-chloroform extraction and precipitation or column-based nucleic acid purification with the aid of  methods  until itDanio rerio, strain AB) at the germ ring stage. Because embryonic staging is quite difficult, the embryos will be in a marginally different stage and will because of that, show biological variation. From four embryos, RNA was individually isolated . To eliminate the biological variation, the other four embryos were first individually homogenized, then the homogenized material was pooled, and finally split into four samples for separate RNA isolations (Semi-single 1 to 4). Hence, the Semi-single samples should hold the same RNA content and differences could be attributed solely to technical variation of the RNA isolation method. The quality of the isolated RNA was very good, as all RIN values were ≥ 8.3  between the samples was investigated with a principle component analysis (PCA)  Figure , which c) Figure . The corTo show the robustness of our RNA isolation methods, two experiments including larger numbers of samples were executed: 30 unfertilized eggs and 186 individual embryos, ranging from dome stage to 90% epiboly. The average RIN value of all samples was 8.9 for the unfertilized eggs and 8.4 for the epibolic samples Figure . Only on2 ratios (test/reference) showed a developmentally ordered separation of all samples PCR or transcriptome sequencing. Our method could also be made applicable for embryos and small samples of dissected tissues from other model systems, as we already have done for small human skin biopsies, especially when sample material is limited or pooling is unwanted.http://zfin.org[Danio rerio (strain AB) were kept at 30°C in egg water . Individual embryos were imaged with stereo microscopy (Leica MZ16 FA) and transferred to 1.5 ml tubes. Remaining egg water was removed and embryos were quickly snap frozen in liquid nitrogen and stored at -80°C. Females were anesthetized briefly in egg water containing 0.02% buffered ethyl 3-aminobenzoate methanesulfonate . They were transferred to petri-dishes and unfertilized eggs were harvested by gentle squeezing. Afterwards, they were allowed to recover for one month.Zebrafish were handled in compliance with local animal welfare regulations and maintained according to standard protocols /zfin.org. EmbryosMicroarrays have been custom designed and obtained from Agilent Technologies using the 8 × 15 k slide format. The 15 k microarray (design ID: 021987) contains ~15.000 probes and has been designed on a non-rendundant set of Ensembl and Vega transcripts with a ZFIN annotation. The design for this array and microarray data discussed in this publication have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO)  and are Per RNA isolation, one 1.5 ml tube was filled beforehand with 75-100 mg phase-lock gel heavy (5-Prime) and pelleted for 30 s at 12,000 × g. Tubes with individual embryos were kept in liquid nitrogen until processing. Single embryos were grinded individually with a liquid nitrogen pre-chilled metal micro-pestle (Carl Roth). The pestle was lifted slightly and 200 μl Qiazol (Qiagen) was added. The pestle was placed back into the tube with Qiazol and the homogenate was allowed to thaw. Before removal, the pestle was washed with an additional 100 μl Qiazol to rinse of any material that might have stuck to the pestle. The homogenate was vortexed vigorously for 15 s, left at room temperature for at least 5 min, and then spun down quickly for 15 s. 60 μl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred as a whole to a pre-prepared phase-lock gel heavy containing tube and centrifuged for 15 min at 12,000 × g. The aqueous phase was transferred to a new 1.5 ml tube. The RNA was purified by column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) - Appendix D: RNA Cleanup after Lysis and Homogenization with Qiazol Lysis Reagent (Qiagen). At the end of the procedure, the RNA was eluted in 14 μl nuclease-free water.The amount of RNA per μl was measured on the NanoDrop ND-1000 (Thermo Scientific). The integrity of the RNA was investigated with the BioAnalyzer (Agilent Technolgies) using the RNA pico 6000 kit (Agilent Technologies).200 ng RNA including controls,  from the Two-Color RNA Spike-In Kit (Agilent Technologies), was taken as input for half volume reactions of the one-round mRNA amplification per zebrafish embryo . 5 μg of amplified RNA was dried in a speedvac and dissolved in 5 μl 50 mM carbonate buffer (pH 8.5) by thoroughly up and down pipetting and placed at 42°C for 5 min. They were vortexed and spun down briefly. Vials of mono-reactive CyDyes  were dissolved in 200 μl DMSO. To each reaction 10 μl of dissolved CyDye was added . They were mixed thoroughly by pipetting up and down, vortexed and spun down briefly. Subsequently, samples were incubated for 60 min at room temperature in the dark. After 30 min the samples were vortexed and spun down briefly and left the residual 30 min in the dark. The reactions were quenched by addition of 5 μl 4 M hydroxylamine for 15 min in the dark at room temperature. 80 μl of nuclease-free water was added to each sample to bring the volume to 100 μl. The RNA was purified with column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) - Protocol: RNA Cleanup and Concentration (Qiagen). Finally, the RNA was eluted twice in 14 μl nuclease-free water. The yield and CyDye incorporation were measured with the NanoDrop ND-1000 (Thermo Scientific).2 converted and the average log2 signal intensity was calculated for the foreground and background signals. Log2 converted ratios were used to perform standard PCA and median signal intensities were used for the calculation of Spearman correlations between samples.Each hybridization mixture was made up from 300 ng 'Test' and 300 ng 'Reference' sample according to the Two-Color Microarray-Based Gene Expression Analysis Manual version 5.5 (Agilent Technologies). The RNA was allowed to hybridize for 16 hours at 65°C and 10 RPM. Afterwards the slides were washed and scanned in an ozone-free room with the Agilent DNA microarray scanner G2565BA (Agilent Technologies). Slides were scanned with eXtended Dynamic Range and 5 μm resolution. Microarray data was extracted with Feature Extraction Software version 9.5.3 (Agilent Technologies). The obtained median signals were logThe authors declare that they have no competing interests.MdJ and TMB conceived the method and wrote the paper; MdJ developed the method; MdJ and JV performed the experiments; OB and HR analyzed the data; TMB and HPS supervised the project. All authors read and approved the final manuscript.RNA quality Single and Semi-single samples. This figure shows the RNA profiles of the Single and Semi-single samples as given by the Agilent 2100 BioAnalyzer together with their respective RNA Integrity Numbers (RIN).Click here for fileRNA quality Development samples. This figure shows the RNA profiles of the Development samples as given by the Agilent 2100 BioAnalyzer together with their respective RNA Integrity Numbers (RIN).Click here for file