Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins. Ro ribonucleoprotein particles (Ro RNPs) are soluble complexes in vertebrate cells which are detected by autoimmune antibodies from patients suffering from systemic lupus erythematosis or Sjögren's syndrome Y RNAs are RNA polymerase III transcripts of about 100 nucleotides, which fold into characteristic stem-loop structures Ro60 protein binds to a highly conserved site on the lower stem of the Y RNAs La protein is required for the accurate and efficient termination of RNA polymerase III transcription and binds the 3′ polyuridine tail of newly synthesised RNAs in the nucleus Here, we investigate the functional consequences of RNP formation between Ro60, La and nucleolin with hY RNAs for chromosomal DNA replication. Our data show that RNPs containing Y RNAs, Ro60, La or nucleolin are not required for chromosomal DNA replication in a human cell-free system, and that that Ro60 and La proteins do not inhibit this function.To confirm that Ro60, La and nucleolin stably interact with hY RNAs, we first immunoprecipitated these proteins from HeLa cytosolic cell extract, and then analysed the protein and RNA compositions of these immunoprecipitates . Anti-RoNext, we analysed the RNA content of the immunoprecipitates by quantitative reverse transcription-PCR (qRT-PCR). We determined the relative amounts of the four hY RNAs, and of 5S rRNA as a specificity control . All fouTaken together, these data indicate that hY RNAs are present in several distinct types of RNP complexes in the cytosolic HeLa cell extract. First, all four hY RNAs are present in [Ro60/La/hY RNA] RNP complexes. Second, hY RNAs and 5S rRNA are also present in distinct [nucleolin/hY RNA] RNP complexes, which form preferentially with hY1 RNA. Thus, in the next experiments, we investigated the functional requirement of these hY-RNP complexes for chromosomal DNA replication.Y RNAs are essential to functionally reconstitute the initiation of DNA replication in a human cell-free system First, we immunodepleted these complexes from the cytosolic extract and then tested the ability of these depleted extracts to reconstitute DNA replication in vitro . Ro60 anWe then tested the ability of these depleted extracts to reconstitute the initiation of DNA replication in vitro. When non-depleted cytosolic extract was used as a positive control, 63±4% of nuclei replicated in this extract, compared with 5±4% in buffer alone , in agreWe have previously reported that Y RNAs act redundantly with each other in the initiation of chromosomal DNA replication We then went on to test the ability of this hY RNP-depleted extract to reconstitute the initiation of DNA replication in vitro . NeitherThe experiments described above exclude an essential stimulatory function for the major Y RNA-binding proteins Ro60 and La in chromosomal DNA replication in vitro. It nevertheless remains a possibility that these proteins may negatively regulate DNA replication. Ro60 and/or La may sequester Y RNAs and prevent them from functioning in chromosomal DNA replication. To address this possibility, in the last set of experiments, we purified recombinant human Ro60 and La proteins and then tested if they inhibit hY RNA-dependent initiation of chromosomal DNA replication in vitro.Recombinant human Ro60 was expressed as a poly-histidine tagged protein in E. coli 32P-labelled hY1 RNA in the presence of a ten-fold molar excess of unlabelled competitor tRNA to inhibit unspecific binding. Results were analysed on native polyacrylamide gels, followed by autoradiography. Ro60 and La both resulted both in a pronounced upwards shift of the hY1 RNA band in the gel. A four-fold molar excess of Ro60 protein resulted in a shift of about 30–40% of the input hY1 RNA , whereas addition of >100 µg led to the saturation of the system with the maximal number of nuclei replicating 54±5%; , in agreY RNAs are essential for the initiation of chromosomal DNA replication in mammalian somatic cell nuclei Antibodies against Ro60 or La specifically precipitated Ro60, La and all four hY RNAs, indicating the presence of [Ro60/La/hY RNA] RNP complexes in the HeLa cytosolic extract and confirming previous reports While anti-nucleolin antibodies precipitated nucleolin and all four hY RNAs, they did not co-precipitate Ro60 or La. Conversely, anti-Ro60 and anti-La antibodies did not co-precipitate nucleolin. Furthermore, our results show that nucleolin is in a complex with 5S rRNA, suggesting that it associates with additional RNAs, as well as with hY RNAs. In contrast to our results, Fouraux and colleagues have reported that anti-nucleolin antibodies precipitated only hY1 and hY3 RNA, but not hY4 or hY5 RNA, and that nucleolin co-precipitates with Ro60 and La Nucleolin interacts with pyrimidine-rich stretches in the loop domain of hY RNAs As result of heat shock or DNA damage, nucleolin rapidly re-localises from the nucleolus to the nucleoplasm and forms a complex with the DNA replication initiation factor RPA The main observation of our immunoprecipitation experiments and in vitro DNA replication assays is that RNP formation between hY RNAs, Ro60 and La is not required for Y RNA function in DNA replication. A potential caveat of these in vitro results is the lack of confirmation by an independent experimental approach . However, our data do directly confirm predictions derived from deletion experiments of the respective protein binding sites from Y RNAs. Ro60 binds to a highly conserved site on the lower stem of Y RNAs This separation of function allows the reconciliation of controversial reports in the literature. Consistent with our Ro60 immunodepletion data, Wolin and colleagues have reported that Ro60 knockout mice are viable and that derived embryonic stem cells show no major proliferation defects Here we have determined by quantitative RT-PCR that about 50% of the hY RNAs in the HeLa cytosolic extract are present in [Ro60/La/hY RNA] or [nucleolin/hY RNA] RNPs and about 50% are therefore outside of these RNPs. This is in contrast to an earlier report We do not currently know much about the association of these functionally active Y RNAs with other proteins outside of [Ro60/La/Y RNA] or [nucleolin/Y RNA] RNPs. As it is highly unlikely that the Y RNAs exist as naked RNAs without associated proteins, we therefore speculate that these Y RNAs can associate with proteins involved in the initiation of chromosomal DNA replication. We are currently testing this hypothesis by identifying and characterising novel Y RNA-protein associations in our laboratory and preliminary results show that this is indeed the case. The results of this study will be reported elsewhere.We found that the addition of more than a 10-fold molar excess of Ro60 and La proteins over the endogenous Y RNA pools did not inhibit the initiation of DNA replication. If one assumes that this extra protein is able to associate with hY RNAs in the extract, one can thus not rule out entirely that Y RNAs may support DNA replication even when associated with Ro60 or La. This scenario is, however, unlikely because La or Ro60 are already present in higher or equimolar amounts to endogenous Y RNAs in the extract, respectively, whilst immunodepletion of more than 99% of total La and Ro60 proteins only removes half of the soluble Y RNAs. It is therefore likely that excess Ro60 and La would be either outcompeted on the Y RNAs in the extract by other Y RNA-binding factors involved in DNA replication, or be bound by additional RNAs with no function in DNA replication, or remain uncomplexed.An independent precedence for selective targeting of Y RNAs to a cellular pathway independently of Ro RNP formation has recently been reported for retrovirus assembly Cytosolic extract of asynchronously proliferating human HeLa cells was obtained from Cilbiotech . Extract was diluted in replication buffer  and pre-depleted with protein G-agarose or protein A-agarose (both from Roche). .) Pre-depleted extracts were incubated with the following primary antibodies: anti-60 (Euro-Diagnostica), anti-La (Santa Cruz sc-80655), and anti-nucleolin . Pre-immune rabbit serum and unspecific IgG2a antibodies from murine myeloma (Sigma M5409) were used as negative controls. Antibodies were then precipitated from extracts with protein G-agarose or protein A-agarose (both from Sigma). Protein content of immunoprecipitates was determined via SDS-PAGE followed by Western blot analysis using the same primary antibodies as used for immunoprecipitation. RNA composition was determined by quantitative reverse transcription-PCR (qRT-PCR) following phenol/chloroform extraction and ethanol precipitation.Primary antibodies as used for immunoprecipitation were coupled to protein G-agarose or protein A-agarose beads by incubation in replication buffer. Unspecific IgG2a antibodies from murine myeloma and/or pre-immune serum were used for mock depletions. These antibody-coupled beads were then incubated with cytosolic extract in replication buffer to specifically deplete proteins from the extract. Following three rounds of depletion, protein content of extracts was determined via SDS-PAGE followed by Western blot analysis. RNA composition was determined by qRT-PCR following phenol/chloroform extraction and ethanol precipitation.cDNA libraries were synthesised from RNA preparations of immunoprecipitates or cell extracts using random primers (Promega) with the SuperScript II Reverse Transcriptase kit according to the manufacturer's protocol (Invitrogen). These cDNA libraries were then used as the templates for qRT-PCR on the iCycler iQ platform as described R(IP)) of each hY RNA and 5S rRNA in immunoprecipitates compared with mock immunoprecipitates was calculated from the threshold cycle (CT) of each cDNA amplification by using the following equation: AR(IP) = 2(CT of mock immunoprecipitate – CT of specific immunoprecipitate). Identical amounts of starting extract and of protein A or G agarose were used.The relative amount (AR(E)) of each RNA was first calculated from the threshold cycle (CT) of each cDNA amplification by using the following equation: AR(E) = 2−ΔCT  =  2−(CT of test RNA – CT of reference RNA). The reference RNA was HPRT mRNA. After immunodepletion of cytosolic extract, proportions of the relative amounts of each RNA were normalised against the mock depleted extract using the following equation: proportion of AR(E) = 2−(ΔCT of immunodepleted extract – ΔCT of mock depleted extract) X 100%.In cell extracts, the relative amount  was added to a final concentration of 0.4 mM, the temperature was lowered to 25°C and growth continued overnight. All purification steps were conducted at 4°C. Bacteria were harvested by centrifugation, washed in PBS, and resuspended in 30 ml protein purification buffer  with 0.2% Triton X-100 and lysed by sonication. The lysate was clarified by centifugation for 20 minutes at 25,000×g. Imidazole was added to the supernatant to a final concentration of 10 mM, which was then loaded onto Ni-NTA-agarose (Qiagen), pre-equilibrated in purification buffer containing 10 mM imidazole. The column was washed with 40 ml purification buffer containing 150 mM imidazole and Ro60 protein was then eluted with purification buffer containing 600 mM imidazole. The buffer of the eluted protein fractions was exchanged for protein storage buffer  using a PD10 column according to the manufacturers protocol , before flash freezing in liquid nitrogen and storing at −80°C.For expression of Ro60, the E. coli strain C43 was transformed with the pET28a/his-Ro60 expression vector 600 of 0.05. ITPG was added to a final concentration of 1 mM, the temperature was lowered to 26°C and growth continued for 20 h. All purification steps were conducted at 4°C. Cells were washed and lysed in 20 mM Tris-Cl pH 7.0, 500 mM NaCl, 1 mM EDTA, 0.1%Tween 20, 20 µM PMSF by sonication on ice. The lysate was clarified by centifugation for 30 minutes at 20,000×g and loaded onto chitin beads (NEB) and washed in 50 mM Tris-Cl pH 8.8, 1 M NaCl, 1 mM EDTA, 20 µM PMSF. On-column cleavage was induced by addition of elution buffer . Eluted protein fractions containing La were pooled, exchanged to protein storage buffer, flash frozen in liquid nitrogen and stored at −80°C.For expression of La, E. coli C43 were transformed with the pTWIN1/La expression vector Concentrations of Ro60 and La proteins in human cytosolic extract were determined by serial dilutions of pure proteins alongside of cytosolic extract, followed by Western blotting.32P UTP as described 2, 1 mM DTT) with recombinant Ro60 and/or recombinant La at molar RNA to protein ratios of 1∶0, 1∶1, 1∶2 or 1∶4, and a molar RNA to protein to protein ratio of 1∶4∶4. Competitor tRNA (Roche) was also added to reactions at 10 fold molar excess of the hY1 RNA to inhibit unspecific binding. Reactions were incubated for 15 minutes at 37°C before being loaded onto native 8% polyacrylamide gels (acrylamide:bisacylamide ratio of 180∶1), which were run at 4°C. Following electrophoresis, gels were dried and radiolabelled hY1 RNA was visualised by autoradiography.Human Y1 RNA was synthesised with SP6 RNA polymerase from recombinant DNA templates in the presence of α-Culture of human EJ30 bladder carcinoma cells, cell synchronisation in late G1 phase by mimosine, and preparation of template nuclei were performed as described


Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare.  Caenorhabditis elegans. We have started to characterise the nicotinic receptors of Ascaris suum, and find that the genetics and pharmacology of the A. suum receptors differ from C. elegans. In both species, nicotine and levamisole preferentially activate different forms of the nicotinic receptor, the N- and L-type, respectively. In C. elegans, the L-type receptor is made up of five subunits, whereas the N-type is a homomer of a sixth subunit. We can recapitulate many of the properties of the A. suum N- and L-type receptors, including their sensitivity to two other important anthelmintics, pyrantel and oxantel, by expressing just two subunits at varying ratios. This has implications for the use of drug combinations and for cross-resistance between nicotinic anthelmintics. It may start to give an explanation for the varying effectiveness of nicotinic drugs against different parasites.Ascarid nematodes are major pathogens of humans and livestock. The major method of control is by the use of anthelmintic drugs, many of which target the nervous system. Drugs such as levamisole, pyrantel and oxantel target the nicotinic acetylcholine receptors present on muscle. Nematodes have several such receptors, and until now these have been best understood in the model species  Ascaris are large (∼30 cm) gastrointestinal parasites of swine (Ascaris suum) and humans (Ascaris lumbricoides). Ascaris lumbricoides infects ∼1.4 billion people globally, and is particularly prevalent in conditions of poor sanitation and poverty A. lumbricoides infection also exacerbates other diseases prevalent in impoverished communities such as malaria, tuberculosis and AIDS Ascaris suum is a major parasite of pigs, causing serious economic losses for farmers Nematodes of the genus A. suum can be manipulated has permitted electrophysiological characterisation of the native nAChRs. This has shown that 3 distinct pharmacological nAChR subtypes are present on Ascaris muscle cells, with different agonist and antagonist sensitivities: an L-subtype most sensitive to the agonists levamisole and pyrantel, an N-subtype most sensitive to nicotine, oxantel and methyridine, and a B-subtype most sensitive to bephenium C. elegans, where the genes unc-38, unc-29, unc-63, lev-1 and lev-8 encode nAChR subunits involved in levamisole sensitivity unc-38, unc-29 and lev-1 in the Xenopus oocyte system produces a functional levamisole-sensitive nAChR, but the currents have a small amplitude suggesting the need for additional components C. elegans. This receptor is completely insensitive to nicotine, but nicotine-sensitive receptors are present in C. elegans and are formed by a pentamer of ACR-16 subunits, which express robustly in Xenopus oocytes Ascaris spp. and several other medically important parasites  belong to a phylogenetic group (clade III) which is only distantly related to C. elegans, which is a member of clade V C. elegans are absent from the B. malayi genome lev-1 and lev-8, which encode subunits of the C. elegans L-type receptor, and a greatly reduced number of genes encoding subunits similar to ACR-16, which makes up the N-type receptor C. elegans. However, there have been very few molecular studies on parasitic nematode nicotinic receptors, and their subunit composition has not been determined to date.At present, control of helminth infections in both animals and humans relies on administration of anthelmintic chemotherapeutic agents. The nematode nicotinic acetylcholine receptors (nAChRs) on muscle are the target of the cholinergic anthelmintics levamisole, pyrantel and oxantel; such compounds cause these ligand-gated ion channels to open, leading to prolonged muscle contraction and spastic paralysis of the parasite A. suum nAChR in vitro. Here we report the identification of A. suum orthologues of unc-38 and unc-29, and confirm, using antibody labelling, their presence and co-localisation in A. suum muscle cells. When expressed in Xenopus oocytes they form functional levamisole-sensitive nAChR. By altering the ratio at which the two cRNAs encoding these subunits are injected into the oocytes, receptors with different pharmacological profiles can be produced that respond to different nicotinic anthelmintics. The pharmacology of these receptors resembles the L- and N-subtypes present in native A. suum muscle cells.We have therefore been following up this bioinformatic analysis with molecular studies, with the intention of recapitulating the L- and N-subtypes of the A. suum, we first aligned the amino-acid sequence of the C. elegans UNC-38 and UNC-29 subunits with their predicted orthologues from B. malayi. A partial cDNA sequence from A. suum similar to C. elegans unc-38 was found in the database (Accession number AJ011382) and used to design specific primers to extend the sequence. Degenerate primers based on well conserved regions of amino-acid sequence were used to amplify a partial cDNA clone of the unc-29-like gene from A. suum  are shown in C. elegans and predicted B. malayi subunits. The residues within the predicted loops that make up the ligand-binding site C. elegans equivalent, also possesses the amino-acid residues reported to be necessary for activation by levamisole at positions 153 (glutamate) and 191 (proline) of the mature polypeptide A. suum equivalents of UNC-29 and UNC-38 is based on the results of BLASTP searches and pair-wise sequence comparisons with the C. elegans nicotinic receptor subunits  response to 100 µM acetylcholine (Ach). The receptors also responded to 100 µM levamisole and 100 µM nicotine, and all of these responses could be reversibly blocked by application of 10 µM mecamylamine, a broad-spectrum antagonist of nAChRs known to inhibit ACh-induced contraction of Ascaris muscle A. suum muscle tissue 50 for the agonists, was being produced. There is precedence for the second explanation from studies on mammalian neuronal nicotinic receptors composed of α4β2 subunits Since the two subunits seemed to co-localise on muscle cells and may thus be predicted to co-assemble in the same receptors, we injected cRNAs encoding the two polypeptides into 5, (Asu-UNC-38)4∶(Asu-UNC-29)1, (Asu-UNC-38)3∶(Asu-UNC-29)2, (Asu-UNC-38)2∶(AsuUNC-29)3, (UNC-38)1∶(UNC-29)4 , (UNC-38)0∶(UNC-29)5). We have already shown that molecules containing the subunits in the ratios 5∶0 and 0∶5 Asu-UNC-38∶Asu-UNC-29 do not form functional receptors 3∶(UNC29)2, (UNC38)2∶(UNC29)3 stoichiometries. Oocytes injected with the two cRNAs at ratios of 10∶1 and 1∶10 produced only very small currents in response to acetylcholine, but injection at ratios of 5∶1 and 1∶5 resulted in robust currents in response to agonist. Injection of the cRNAs in both ratios (5∶1 and 1∶5) produced receptors with comparable responses to ACh, but a striking difference in the agonist efficacy of levamisole and nicotine was observed between the two ratios (Asu-unc38∶Asu-unc-29) cRNA ratio experiments. When the ratio was 5∶1 Asu-unc-38∶Asu-unc-29 nicotine was a full agonist, whereas levamisole was only a partial agonist, with a maximal response of only 60% of that of ACh , and nicotine was now only a partial agonist with a maximal response of only 45% eceptors . The shao ratios . Figure t of ACh . When cRonly 45% .Asu-unc-38∶Asu-unc-29 cRNAs. Pyrantel and oxantel, in addition to their respective specificity for the L- and N- subtypes native nAChR found in A. suumA. suum nAChRs at lower concentrations, but open channel blockers at higher concentrations, producing an asymmetric bell-shaped dose-response relationship Asu-unc-38∶Asu-unc-29 cRNAs responded to oxantel, giving a maximal response at ∼10 µM, but gave very little response to pyrantel. The oocytes injected with a 1∶5 ratio of Asu-unc-38∶Asu-unc-29 cRNAs were more sensitive to pyrantel, giving a maximal response at ∼1 µM, but gave a negligible response to oxantel. Maximal responses in both cases were comparable to the maximal response to ACh  were injected; injection of the two cRNAs at ratios of 1∶10 and 10∶1 produced very small currents which were impossible to study further. Though several different populations could theoretically be generated in these experiments A. suum nAChRs can be altered simply by generating different combinations of two subunits, this may help to explain how sufficient pharmacological and neurological complexity can be found in parasites with remarkably few nAChR genes compared to C. elegans. Pharmacological diversity generated by alternate stoichiometries has not previously been observed in invertebrates, but is well described for heterologous expression of the mammalian neuronal nAChR subunits α4 and β2. Receptors with the stoichiometry (α4)3(β2)2 have lower agonist affinity and a different pharmacological profile to receptors with the stoichiometry (α4)2(β2)3, and addition of an accessory subunit to give (for example) an (α4)2(β2)2(α5) combination confers additional pharmacological differences 2(Asu-UNC-29)3, and (Asu-UNC-38)3(Asu-UNC-29)2.We could express cRNAs encoding the two A. suum muscle 2(Asu-UNC-29)3 constitutes the L-subtype nAChR and that (Asu-UNC-38)3(Asu-UNC-29)2 forms the N-subtype. The complex mixture of receptors present in the in vivo preparation makes a quantitative comparison with our in vitro data difficult, but there are clear qualitative similarities. The L-subtype of the native receptor is preferentially activated by levamisole, though nicotine does also open L-type channels, and vice versa for the N-subtype; our oocyte data show that nicotine is only a partial agonist at the putative (Asu-UNC-38)2(Asu-UNC-29)3 receptor and levamisole is, similarly, only a partial agonist at the putative (Asu-UNC-38)3(Asu-UNC-29)2 receptor. There are also clear similarities in the effects of the anthelmintics, pyrantel and oxantel, between our results and the single channel measurements; pyrantel acts at the same sub-type that levamisole prefers and oxantel at the one preferred by nicotine. We believe, therefore, that we have recapitulated many of the properties of the L- and N-subtypes by varying the expression levels of the two A. suum subunits within the oocyte system. These results have important implications for the development and management of parasite resistance to cholinergic anthelmintics; it has previously been assumed that parasites resistant to levamisole or pyrantel may still be susceptible to oxantel as different receptor sub-types are involved If our results are compared with those obtained from single channel recordings on A. suum UNC-63 subunit. Despite exhaustive efforts, so far we have been unable to generate a cDNA clone encoding an obviously full-length UNC-63 subunit. This may be due to purely technical difficulties, or it may that, in A. suum, unc-63 encodes only a truncated mRNA. It is tempting to speculate that the B-subtype nAChR has the composition (Asu-UNC-38)2(Asu-UNC-29)2(Asu-UNC-63)1, by analogy with the (α4)2(β2)2(α5)1 receptors found in mammals An important open question concerns the function of the A. suum homologues of UNC-38 and UNC-29 co-express in Xenopus oocytes to form a functional nAChR, which is the first successful expression in vitro of a nAChR from parasitic nematodes. This will create opportunities for future work to be performed, where for the first time candidate drug resistance mutations in parasite nAChR subunits can be assessed directly for their effect on anthelmintic efficacy and receptor function. This work also paves the way to an accelerated understanding of anthelmintic drug targets and the potential development of parasite-specific target-based screens for new compounds.In summary, we have demonstrated that the All animals used for production of antisera were handled in strict accordance with good animal practice and the conditions defined by the United Kingdom Home Office  or the United States Department of Agriculture (Sigma Genosys).Ascaris suum unc-38 and unc-29 were amplified using primers designed on a partial sequence of Asu-unc-38 present in GenBank (AJ011382) (Primer sequences GTCGCGCTTACCGTTTTCTTCC and CCATCGCCACATATTTCCAGTCTT) and using degenerate primers based on an alignment of the unc-29 sequences from C. elegans and Brugia malayi (primer sequences ATCAAYGTNGAYGARAARGAYCA and ATYTCRTTYTCRTTRTANGTCCA). The sequences were extended to full length using 5′ and 3′ RACE GGTTTAATTACCCAAGTTTGAG) was used in conjunction with specific internal primers . For 3′ RACE an ‘anchor’ primer sequence incorporated into the oligo(dT) primer used for reverse transcription (GACCACGCGTATCGATGTCGAC) was together with an internal primer specific for Asu-unc-29 (GCACTAAGAGCTATTGACGCG)). Full length cDNAs were amplified using specific primers . Full length cDNAs were cloned into pGEM-T (Promega) and transformed into XL-1 blue competent cells (Stratagene).Partial sequences of A. suum collected from a local abattoir were kindly supplied by Professor Aaron Maule and Emma Kidd from Queen's University, Belfast. Muscle cells were prepared by collagenase digestion Antisera were raised, in rabbit and goat respectively, against subunit-specific N-terminal peptides from Asu-UNC-38 and Asu-UNC-29  then antibodies were affinity purified as described A. suum unc-38 and unc-29 cDNAs were sub-cloned into the expression vector pT7TS , containing 5′ and 3′ untranslated regions from Xenopus β-globin. Plasmids were linearised, and then used as template for cRNA synthesis using the T7 mMessage mMachine kit . Oocytes were obtained from EcoCyte Biosciences, Germany. Oocytes were injected with nicotinic receptor cRNAs in RNAse-free water in a total volume of 50 nl, which was the same for all experiments. When a single cRNA species was injected, 25 ng was used; when both Asu-unc-29 and Asu-unc-38 cRNAs were injected, a total of 50 ng was used. Oocyte injection was carried out using a Drummond ‘Nanoject’ microinjector and electrophysiological experiments were carried out maxI/I = (maxI−minI/[1+10(logEC50−[ag]*nH)])×maxI. For pyrantel and oxantel, the equation was fitted to the data obtained for the rising phase of the response only.C. elegans: UNC-38 – Q23022, UNC-29 –P48181, UNC-63 – Q9N587, ACR-12 – Q9GQU9, ACR-8 – Q23355, ACR-6 – Q9N4M3, LEV-1 – P48181, ACR-3 – Q93149, ACR-2 – P48182, ACR-16 – P48180. B. malayi: BM_UNC-29 – Bm1_35890.The Accession numbers for proteins mentioned in the text are:


Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5′-bromouridine 5′-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection. Positive-strand and double-strand RNA viruses compartmentalize their replication machinery in infected cells. This compartmentalization is thought to favor the catalysis of RNA synthesis, and sequester viral RNA molecules from detection by innate immune sensors. For the negative-strand RNA viruses that replicate in the cytoplasm, the site of RNA synthesis is less clear. Here, using a prototype non-segmented negative-strand (NNS) RNA virus, vesicular stomatitis virus (VSV), we investigated whether viral derived inclusions are sites of RNA synthesis in infected cells. Our work shows that prior to viral protein synthesis the invading viral cores synthesize mRNA throughout the host cell cytoplasm. Viral protein expression leads to the formation of intracytoplasmic inclusions that contain the viral machinery necessary for RNA synthesis and become the predominant sites of transcription. The newly synthesized viral mRNAs escape the inclusions by transport along microtubules and this facilitates their translation. Our work demonstrates that in contrast to the positive-strand and double-strand RNA viruses, VSV does not require the establishment of specialized compartments in the cytoplasm of the cell for RNA synthesis. Our findings suggest that the confinement of RNA synthesis to inclusions once infection is established may reflect a host response to infection. RNA viruses that replicate within the cytoplasm often form specialized structures that are the sites of RNA replication Cis-acting signals required for RNA replication and for each step of mRNA synthesis, including cap addition and polyadenylation have been defined  RNA viruses is less well characterized. Vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, has provided many mechanistic insights into RNA synthesis for NNS RNA viruses Rhabdoviridae, pathologic specimens of infected neuronal cells identified inclusion-like structures termed Negri bodies that contain viral nucleocapsids. This led to the suggestion that such inclusions might be sites of RNA synthesis. Subsequent studies showed that Negri body-like inclusions appear to be bona fide sites of RNA synthesis as they contain the viral N, P and L proteins necessary for RNA synthesis as well as the mRNA products of transcription The site(s) within the cytoplasm at which VSV RNA synthesis occurs and the cellular requirements for RNA synthesis remain uncertain. For rabies virus, a related member of the in vitro as well as in cells. Imaging the location of the viral RNA synthesis machinery and the viral RNA in infected cells by fluorescent microscopy revealed that the infecting RNP can synthesize mRNA throughout the cytoplasm. Following protein synthesis, however, viral RNA synthesis appears to be restricted to inclusions. The viral mRNAs are subsequently transported away from those inclusions in a microtubule-dependent manner to facilitate translation. Our experiments show that VSV does not require a specialized site for RNA synthesis, but the viral RNA synthesis machinery is redirected to inclusions following protein synthesis.In the present study, working with VSV, we further probed the relationship between inclusion formation and RNA synthesis. To do this, we used recombinant viruses in which P was fused to eGFP 5′-GAAAAAAACTAACAGATATCATGGCCTCCTCCGAGGACG-3′ and 5′-CTTTTGTGAGATTATCGGCGCCGGTGGAGTGGC-3′ were used to amplify the mRFP gene from pRFP-N1 . Recombinant virus was recovered as described previously Recombinant VSV expressing eGFP fused to P was previously described Spodoptera frugiperda (Sf21) cells from a recombinant baculovirus generated by cloning the relevant portions of the L gene under the control of the polyhedrin promoter using pFASTBAC-DUAL . An N-terminal hexa-histidine tag was introduced to facilitate L protein purification. The L protein fragment was purified by affinity chromatography on Ni-nitrilotriacetic acid-agarose  followed by MonoQ then MonoS ion exchange chromatography . A polyclonal antiserum was obtained following immunization of a single rabbit with purified protein . The rabbit antiserum detects full-length VSV L in infected cell lysates by Western blot (data not shown).Amino acids 1594–2109 of VSV L were expressed in 2HPO4, 2mM KH2PO4) and treated with ice-cold 100% methanol for 3 min. Cells were rinsed twice with PBS, incubated in PBSAT , followed by PBSA  each for 10 minutes. For RNA detection, we used a monoclonal antibody against bromodeoxyuridine conjugated to Alexa Fluor-488 (Invitrogen) at a 1∶50 dilution in PBSAT . Cells were incubated for 1 hour at RT or 16h at 4°C, prior to detection of immune complexes using a 1∶2000 dilution of a secondary anti-mouse antibody conjugated to Alexa Fluor-488 (Invitrogen). VSV N and M proteins were detected using monoclonal antibodies 10G4 and 23H12 Imaging experiments were performed in BSR-T7, CV-1 or Vero cells. Cells were fixed with 2% paraformaldehyde for 15 min, washed twice with phosphate buffered saline (PBS)  equipped with a 63× (NA 1.4) objective. Samples were excited with a Xenon lamp, and filtered emission photons were collected with a Hamamatsu Orca-HR (C4742-94) camera . Confocal images were acquired using a Zeiss observer Z1 microscope (Carl Zeiss MicroImaging) fitted with a confocal spinning disk unit  and a 63× (NA 1.4) objective. Excitation wavelengths were 473 nm for Alexa Fluor-488, 561 nm for Alexa Fluor-594 or DyeLight-549 and 660 nm for DyeLight-649. For 3-D acquisitions, images were captured at intervals of 0.26 µm. The X, Y, Z positions of the stage were controlled using a PZ-2000 automated stage . Microscope hardware was controlled with Slidebook 4.2 Software .4)/1.5% potassiumferrocyanide (KFeCN6) (Electron Microscopy Sciences), washed 3 times in H2O and incubated in 1% aqueous uranyl acetate (Sigma). This was followed by 2 washes in H2O and subsequent dehydration in grades of alcohol for 5 min each .Vero cells were infected with VSV at an MOI of 3 and fixed 6 hpi with 2.5% glutaraldehyde  to preserve membrane integrity and 2% paraformaldehyde (Sigma) in 0.1 M sodium cacodylate buffer (pH 7.4) (Sigma) for 1h. The cells were then postfixed for 30 min in 1% osmium tetroxide  and labeled with primary antibodies against viral L (1∶100 dilution) and N (1∶50 dilution) as above. To detect P, we infected cells instead with VSV-eGFP-P and visualized the location of P with a rabbit anti-GFP antibody (1∶50 dilution) (Sigma). Secondary labeling was performed with anti-rabbit or anti-mouse nanogold-1.4 nm (1∶50 dilution) in 1% BSA for 1 h at RT. Samples were washed 5× in 1× PBS/1% BSA for 1h and postfixed in 1% glutaraldehyde (Electron Microscopy Sciences) in 1× PBS for 10 min. Cells were then washed 3 times for 5 min in PBS, followed by 2 washes for 5 min in deionized water and 1 wash for 5 min in 0.02 M citrate buffer. The 1.4 nm gold particles were silver enhanced (giving ∼15–40 nm particles) by incubating the samples for 4 min in freshly mixed developer using the HQ Silver Enhancement kit  and rinsed 3 times in deionized water for 1 min. Cells were treated with 0.5% osmium tetroxide before dehydration.2 Spirit BioTWIN. Images were recorded with an AMT 2k CCD camera.For embedding, unlabeled and immunogold labeled cells were removed from dishes using propyleneoxide (Sigma), pelleted at 3000 rpm for 3 min and infiltrated for 2 h in an equal mixture of propyleneoxide and TAAB Epon . The samples were subsequently embedded in TAAB Epon and polymerized at 60 degrees C for 48 h. Ultrathin sections (about 60nm) were cut on a Reichert Ultracut-S microtome, picked up on to copper grids stained with lead citrate and examined in a TecnaiGin vitro as previously described 32P]-GTP . As a control, transcripts were also synthesized by T7 RNA polymerase  using the previously described VSV expression plasmid pN Viral RNAs were transcribed −1 actinomycin D (ActD) to inhibit cellular transcription, 100 mM nocodazole (Noc) to disrupt microtubules or 10 µg ml−1 puromycin (Pur) to inhibit protein synthesis. For pulse-chase analyses, the cell culture medium was supplemented with 50 mM uridine (Sigma) throughout the chase period. In some experiments, RNAs were simultaneously metabolically labeled by the incorporation of 33 µCi ml−1 [3H]-uridine (Perkin Elmer) from 4–9 hpi.Approximately 30,000 BSR-T7 cells grown on cover slips in 24 well plates were infected with VSV at the specified MOI (3–500). At the indicated times post infection, cells were depleted of uridine by low glucose DMEM (Invitrogen) supplemented with 20 mM glucosamine (Sigma), and transfected 1h later with 5 mM BrUTP in 250 µl of DMEM supplemented with 6 µl of lipofectamine 2000 (Invitrogen). In some experiments, cells were treated 15–40 minutes prior to BrUTP labeling with a variety of chemical inhibitors (Sigma). Specifically, we used 10 µg mlin vitro synthesis reactions were purified using an RNeasy kit (Qiagen). For cellular RNA analysis, cytoplasmic extracts were prepared and RNA was purified by phenol-chloroform extraction as described previously The products of −1 ActD. Where indicated, cells were exposed to 10 µg ml−1 Pur for 1 h or 100 mM Noc during the last 15 min of starvation. Proteins were labeled by addition of 17.5 µCi [35S]EasyTag express (Perkin Elmer) in DMEM lacking L-methionine and L-cysteine (Invitrogen). Where indicated, nocodazole was washed out to permit repolymerization of microtubules. Total cytoplasmic proteins were analyzed by 10% SDS-PAGE and detected by phosphoimage analysis. Quantitative analyses were performed using ImageQuant Software .At the indicated times post infection, cells were starved of L-methionine and L-cysteine for 1h in the presence of 10 µg mlPreviously we described a recombinant VSV in which eGFP was fused to the N terminus of P To examine the cellular location of the inclusion-like structures, we performed electron microscopy of cells infected with VSV. As previously de novo synthesis of viral RNA, we tested the ability of purified VSV L protein to incorporate fluorescent nucleotides in vitro. Viral RNA synthesis was inhibited in reactions containing fluorescein-12-UTP, -GTP or -ATP, Alexa Fluor-488-UTP or Cy3-17-UTP or the RNA products were not fluorescent (data not shown). This result indicates that L cannot incorporate nucleotides that contain such large modifications. To test whether nucleotides with smaller modifications can be incorporated into viral RNA, we supplemented in vitro transcription reactions performed in the presence of [32P]-GTP with 5-BrUTP, and monitored the products of RNA synthesis by electrophoresis on acid-agarose gels  with nocodazole. Under those conditions viral RNA was confined to specific regions of the cytoplasm  The viral protein requirements for RNA synthesis are N, P and L. To determine whether inclusions containing N, P and L are active sites of RNA synthesis, we infected cells with either rVSV or rVSV-RFP-P and visualized RNA and protein using confocal microscopy. In these experiments, we restricted RNA to its site of synthesis by treating cells with nocodazole prior to transfection of BrUTP. The viral RNA was observed as granular structures around inclusions that were visualized by RFP-P expression or following staining with antibodies against N or L . All visThe above experiments show that viral RNA is synthesized at, and actively transported away from inclusions. To establish infection however, the input RNP must synthesize mRNA presumably in the absence of such inclusions. To determine where such primary transcription occurs, we infected BSR-T7 cells with rVSV-RFP-P at an MOI of 500 in the presence of the protein synthesis inhibitor puromycin and monitored RNA synthesis by BrUTP incorporation. Genome replication requires the ongoing synthesis of N protein 35S]-methionine  show that viral genomes are targeted into similar cytoplasmic inclusion bodies that contain the viral replication machinery Rhabdoviridae, rabies and VSV had reached different conclusions regarding the intracellular site of RNA synthesis. For rabies virus, RNA synthesis occurs at intracytoplasmic inclusions Prior to the present study, experiments with two members of the The mechanism by which mRNA moves away from the inclusions involves transport along microtubules. Three pieces of evidence support this conclusion. First, the viral mRNAs are restricted to the inclusions following chemical depolymerization of microtubules with nocodazole. Second, the restriction of viral RNA to the inclusions is quickly released upon wash-out of nocodazole; and third, the viral mRNA are visualized along microtubules. The finding that viral RNA undergoes such directed transport is perhaps unsurprising as cellular and other viral RNA is frequently transported within the cytoplasm 35S]-met, we also show that the depolymerization of microtubules diminished the rate of viral protein synthesis by 40%  at an MOI of 3, fixed at 6 hpi and markers of the endoplasmic reticulum , Golgi (GM130) and early endosomes  detected by immune fluorescence microscopy (green). Lysosomes and mitochondria were detected by incorporation of fluorescent lysotracker and mitotracker (green) into cells for 30 minutes prior to fixation. Cell nuclei were counterstained with DAPI (blue). Size bars = 10 µm.(2.39 MB TIF)Click here for additional data file.Figure S2Visualization of primary mRNA transcripts and input RNPs. BSR-T7 cells were treated with puromycin 15 minutes before infection with rVSV at an MOI of 500. At 2 hpi, cells were depleted of intracellular UTP and exposed to ActD and nocodazole. After 1 hour, cells were transfected with BrUTP. Viral RNA (green) as well as N protein (red) were detected 1h later by immuno staining prior to imaging by fluorescence microscopy. Two representative cells are shown. Size bar = 5 µm.(0.51 MB TIF)Click here for additional data file.Video S1A Z-series showing RFP-P protein localization at viral inclusions surrounded by RNA. BSR-T7 cells were infected with rVSV-RFP-P at an MOI of 3, and exposed to nocodazole at 4 hpi. Following a 1h incubation, cells were transfected with BrUTP, fixed 40 minutes later and the viral RNA (green) and RFP-P (red) visualized by confocal microscopy as described in methods. The video of a single representative cell shows combined Z-stacks (0.26 µm thickness) of images taken through the cell shown in (0.05 MB MOV)Click here for additional data file.Video S22.A three dimensional projection of viral RNA surrounding RFP-P inclusions. A 3D view of the cell shown in (0.26 MB MOV)Click here for additional data file.Video S3A Z-series showing N protein localization at viral inclusions surrounded by RNA. BSR-T7 cells were infected with rVSV at an MOI of 3 and exposed to nocodazole at 4 hpi. Following a 1h incubation, cells were transfected with BrUTP, fixed 40 minutes later and the viral RNA (green) and N (red) visualized by confocal microscopy as described in methods. The video of the two adjacent representative cells depicted in (0.12 MB MOV)Click here for additional data file.Video S42.A three dimensional projection of viral RNA surrounding inclusions containing N protein. A 3D view of the cells shown in (0.35 MB MOV)Click here for additional data file.Video S5A Z-series showing L protein localization at viral inclusions surrounded by RNA. BSR-T7 cells were infected with rVSV at an MOI of 3 and exposed to nocodazole at 4 hpi. Following a 1h incubation, cells were transfected with BrUTP, fixed 40 minutes later and the viral RNA (green) and L (blue) visualized by confocal microscopy as described in methods. The video of a single representative cell shows combined Z-stacks (0.26 µm thickness) of images taken through the cell shown in (0.35 MB MOV)Click here for additional data file.Video S62.A three dimensional projection of viral RNA surrounding inclusions containing L protein. A 3D view of the cell shown in (0.35 MB MOV)Click here for additional data file.


Depending on their specific structures, noncoding RNAs (ncRNAs) play important roles in many biological processes. Interest in developing new topological indices based on RNA graphs has been revived in recent years, as such indices can be used to compare, identify and classify RNAs. Although the topological indices presented before characterize the main topological features of RNA secondary structures, information on RNA structural details is ignored to some degree. Therefore, it is necessity to identify topological features with low degeneracy based on complete and fine-grained RNA graphical representations.In this study, we present a complete and fine scheme for RNA graph representation as a new basis for constructing RNA topological indices. We propose a combination of three vertex-weighted element-contact graphs (ECGs) to describe the RNA element details and their adjacent patterns in RNA secondary structure. Both the stem and loop topologies are encoded completely in the ECGs. The relationship among the three typical topological index families defined by their ECGs and RNA secondary structures was investigated from a dataset of 6,305 ncRNAs. The applicability of topological indices is illustrated by three application case studies. Based on the applied small dataset, we find that the topological indices can distinguish true pre-miRNAs from pseudo pre-miRNAs with about 96% accuracy, and can cluster known types of ncRNAs with about 98% accuracy, respectively.The results indicate that the topological indices can characterize the details of RNA structures and may have a potential role in identifying and classifying ncRNAs. Moreover, these indices may lead to a new approach for discovering novel ncRNAs. However, further research is needed to fully resolve the challenging problem of predicting and classifying noncoding RNAs. Recent years have witnessed an explosive growth in RNA research, as numerous new noncoding RNAs (ncRNAs) have been discovered ,2, and rAs there remain many difficulties in predicting three-dimensional RNA structure, secondary structures are typically used as a basis for researching RNA conformation. RNA secondary structure can be viewed as a combination of basic structural elements, also known as stems, hairpin loops, bulge loops, interior loops, multiple loops and external loops (the latter five categories are referred to collectively as 'loops'). Mathematical representations of RNA secondary structure are of great importance. Some approaches for deducing these structures have been proposed as planar graphs -9. Amonget al successfully used topological indices from tree and dual graphs to explore the repertoire of RNA secondary motifs , i.e.As the first non-trivial topological index, the Wiener-type indices can be defined for all molecular graph matrices with the Wiener operator. Suggested by Merris [Wiener index has been introduced into a fine-grained RNA graph, in which each nucleotide becomes a node of the graph [Wiener indices increase rapidly with the magnitude of a graph, especially for the weighted Wiener indices. This may be the main reason why Avihoo and Barash limit their Wiener index to fine-grained RNA graphs that characterize only small RNAs (≤ 50 nt) [Wiener indices by assigning α = -0.5 to the exponent of each item in the equation (1) to reduce their range.y Merris ,65 and ty Merris ,18, the he graph ,67. Thus≤ 50 nt) , we geneBalaban index of a graph G also is a distance-based graph connectivity index, defined asThe Di and Dj denote the distance sums of the vertices vi and vj, and can be easily computed by summarizing corresponding rows or columns in the distance matrix, q is the number of edges in the molecular graph, μ is the cyclomatic number and the summation goes over all edges in the graph.where Randić indices of ECGs encode aspects of element connectivity for RNA secondary structure. The m th order Randić index of a graph G is given asThe δv is the degree of vertex v and the summation is over the total number of sub-graphs of order m. The first two order Randić indices, 0χ and 1χ, are employed in this study.where Wiener index, and the Balaban index and Randić indices of a graph are given as:As vertex-weighted RNA graphs, ECGs offer convenience for constructing weighted numerical descriptors aimed at detailed structure characterization. The method presented by Zmazek and Zrovnik  is emplowi and wj are the weights of vertex vi and vj, respectively.where α = -0.5, β = -0.5, and γ = -0.5 to reduce their ranges, respectively. The symbols representing these indices are listed in Table Both the weighted and the unweighted topological indices are examined in this study to evaluate their utility and potential in structure determination applications. The exponents of each item in equations (1) ~ (6) are assigned to To explore the relationship among the topological indices and RNA secondary structures, we have selected a dataset of 6,305 ncRNAs as representatives of the human RNA population and have evaluated their topological indices. We divided these 6,305 ncRNAs into two classes. One class covers five ncRNA types with known structures, obtained from the Comparative RNA Web Site , RNase PThe K-means algorithm  is one oK, clustering accuracy r is defined asWe used the clustering accuracy as a measure of a clustering result. Given the final number of clusters, n is the number of instances in the data set and ri is the number of instances partitioned into the correct cluster i. For miRNA identification, we use receiver operating characteristic (ROC) curves to evaluate and compare the classification performance. The ROC curve provides a convenient graphical display of the trade-off between true- and false-positive rates. Additional terms associated with ROC curves are sensitivity and specificity [where cificity .This paper presents a complete and fine-grained topological description for representing RNA graphs, and establishes a new basis for constructing RNA topological indices. Distinct from other methods, RNA secondary structure is represented by a combination of three vertex-weighted element-contact graphs. Based on the opinion that the stem and loop regions in RNA molecules have similar importance in biochemical processes, the stem and loop topologies are described in stem-contact and loop-contact graphs, respectively, while the overall pattern of the structure is abstracted into a stem-loop-contact graph. In addition, these graphs can be selected according to the needs of a particular application. Three typical topological index families defined with ECGs are described.To investigate the relationship between the topological indices and RNA secondary structures, we constructed a detailed analysis on a dataset of 6,305 ncRNA sequences downloaded from different databases, and explored the numerical features of these indices. We then employed the topological indices to quantify the structural aspects of the selected RNAs, and utilized them to identify miRNAs, classify ncRNAs and conduct deleterious mutation analyses. Based on the applied small dataset, we find that the topological indices can distinguish true from pseudo pre-miRNAs with about 96% accuracy, and cluster known types of ncRNAs with about 98% accuracy. The results indicate that the topological indices can characterize RNA structure details, and show high potential for identifying and classifying ncRNAs. Importantly, while difficult, the successful identification and classification of ncRNAs may provide a new approach for discovering new ncRNAs. The difficulty of correctly identifying and classifying these molecules is underscored by the fact that the predictions of both Evofold  and RNAzThe utility test and the application examples of typical topological indices defined on the ECGs illustrate their latent utility for RNA structure analysis. With the aid of topological indices, it is now possible for biologists to explore 'RNA spaces' visually, as exemplified by the three case studies presented herein. Characterizing RNA molecules using topological indices may open a door to studying the structure-function relationships of RNA molecules by combining many application algorithms for pattern recognition and classification, most of which are based on feature space. Further applications of these topological indices are represented by our studies on robustness analysis of RNA secondary structure ,79, wherThe author(s) declare that they have no competing interests.WS wrote the programs, analyzed the results and drafted the manuscript. XB and ZZ helped in analysis and discussion, and gave useful comments. SW and XB guided the project. All authors read and approved the final manuscript.Wiener, Balaban, Randić indices based on the dataset of 6,305 ncRNAs.Supplementary table S1 – S3. Calculation of Click here for fileWeiner index based on SLCG representation. (B) Probability distribution curves (left) and Cumulative distribution curves (right) of weighted Balaban index based on SLCG representation. (C) Probability distribution curves (left) and Cumulative distribution curves (right) of weighted zero order Randić index based on SLCG representation.Distribution Fitting results for three representatives of topological indices. The distribution fitting results for three representatives of topological indices based on the dataset of 6,305 ncRNAs are shown. Four typical distribution models  are employed here to model the statistical distributions of the three topological index families. (A) Probability distribution curves (left) and Cumulative distribution curves (right) of weighted Click here for fileSupplementary table S4 – S7. Statistical properties of topological indices.Click here for fileWiener indices and the free energy of RNA. Correlations between Wiener indices and free energy for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileBalaban indices and the free energy of RNA. Correlations between Balaban indices and free energy for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileRandić indices and the free energy of RNA. Correlations between Randić indices and free energy for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileWiener indices and the length of RNA. Correlations between Wiener indices and length for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileBalaban indices and the length of RNA. Correlations between Balaban indices and length for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileRandić indices and the length of RNA. Correlations between Randić indices and length for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileWiener indices and the GC content of RNA. Correlations between Wiener indices and GC content for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileBalaban indices and the GC content of RNA. Correlations between Balaban indices and GC content for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileRandić indices and the GC content of RNA. Correlations between Randić indices and GC content for the dataset of 6,305 ncRNAs are shown. For convenience of visualization, both X and Y axes are scaled logarithmically.Correlations between Click here for fileWiener indices. Correlations between Wiener indices for the dataset of 6,305 ncRNAs are shown. The diagonal figures show the distributions of the Wiener indices.Correlations between Click here for fileBalaban indices. Correlations between Balaban indices for the dataset of 6,305 ncRNAs are shown. The diagonal figures show the distributions of the Balaban indices.Correlations between Click here for fileRandić indices. Correlations between Randić indices for the dataset of 6,305 ncRNAs are shown. The diagonal figures show the distributions of the Randić indices.Correlations between Click here for file


The transfer of tumour-specific cytotoxicity against a murine fibrosar-coma has been demonstrated in vitro using xenogeneic RNA extracted from tumour-cell-immune animals. Poly(A)-tailed messenger RNA from immunogenic RNA was isolated by passage through an oligo(dT)-cellulose column, and evaluated to determine whether the same tumour-specific cytotoxicity could be transferred. Aliquots of normal C3H mouse lymphocytes were treated with poly(A)-containing immune RNA, whole-cell immune RNA lacking poly(A) and total cellular immune RNA. Treated cells were tested in vitro using an adaptation of the Takasugi and Klein microcytotoxicity assay. Percent cytotoxicity was calculted using cells treated with fractions of normal RNA as control. An increase in tumour cytotoxicity was found with poly(A)-containing immune RNA. The optimum dose of poly(A)-tailed immune RNA was estimated as 6.5 microgram of RNA per 4 x 10(6) lymphocytes. Populations of lymphocytes were separated using glass and nylon wool. T- and B-enriched populations were treated with various RNA components. The adherent cell population showed no significant cytotoxicity, whilst treatment of the nonadherent population with poly(A)-tailed immune RNA produced high levels of cytotoxicity.


The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle. Plus-strand RNA viruses, a major group of plant and animal pathogens, employ a variety of gene expression strategies. In some groups, the genome is translated into a single polyprotein precursor comprising all viral proteins, while the expression of genomes containing multiple open reading frames commonly depends on the production of additional, subgenomic mRNAs. These serve to translate the open reading frames that are inaccessible to host cell ribosomes engaged in genome translation. Arteriviruses and coronaviruses secure the expression of their structural protein genes by generating an extensive nested set of subgenomic mRNAs, which are copied from a set of complementary minus-strand templates. The production of these subgenome-length minus strands involves a unique mechanism of discontinuous RNA synthesis that essentially competes with the production of the full-length minus strand, the template for genome replication. We describe here that arterivirus non-structural protein 1 (nsp1) modulates the accumulation of minus-strand RNAs to control the relative abundance of both genome-length and subgenomic mRNAs, thereby ensuring efficient production of new virus particles. We found that specific nsp1 mutants with imbalanced mRNA levels and low virus production rapidly acquire additional nsp1 mutations that rescue these defects. Thus, a single arterivirus protein plays a decisive role in the integral control of replication, sg mRNA synthesis, and virus production. Plus-strand RNA (+RNA) viruses are ubiquitous pathogens of plants, animals, and humans. The translation of their messenger-sense RNA genome yields the core viral enzymes that always include an RNA-dependent RNA polymerase (RdRp) and assemble into a cytoplasmic machinery for viral RNA synthesis. Many +RNA virus groups employ polycistronic genomes and different mechanisms to express genes located downstream of the 5′-proximal open reading frame (ORF). One of these mechanisms involves the synthesis of subgenomic (sg) mRNAs (referred to as “transcription” in this paper). Although the sg mRNAs of +RNA viruses are invariably 3′-coterminal with the viral genome, diverse +RNA viruses have evolved different mechanisms for their production Nidovirales comprises several clades of distantly related enveloped +RNA viruses, including the arteri- and coronavirus families, which infect a wide variety of hosts, ranging from invertebrates to humans. Human coronaviruses are associated with respiratory disease (including severe acute respiratory syndrome (SARS), reviewed in The order Despite recent advances in the structural and functional characterization of individual replicase subunits, the molecular details of nidovirus replication and gene expression remain poorly understood. Studies with nidovirus model systems such as equine arteritis virus (EAV), the arterivirus prototype, have provided some critical insights about viral replicase functions and the regulation of RNA synthesis in infected cells. EAV replicase pp1a and pp1ab are co- and post-translationally cleaved into 13 nsps by viral proteases residing in nsp1, nsp2, and nsp4. The seven viral structural proteins, which are all dispensable for replication and transcription 5′ UCAACU 3′ in EAV) precede each structural protein ORF (body TRSs). The same sequence motif is also present at the 3′-end of the genomic leader sequence (leader TRS). Minus-strand RNA synthesis, initiated at the 3′-end of the viral genome, is presumably attenuated at one of the body TRS regions  and sg mRNAs (RNA2–RNA7) accumulate in specific molar ratios see  that areWe have now explored the key regulatory roles of nsp1 in the EAV replicative cycle in unprecedented detail. Our results indicate that in addition to the ZF region, both PCP subdomains of nsp1 are essential for transcription, and suggest an additional role of PCPα in virus production. We also established that nsp1 modulates viral RNA accumulation in an mRNA-specific manner, and thus maintains the balance among the seven viral mRNAs, including the genome. Our data suggests that nsp1 does so by controlling the levels of the full-length and subgenome-length minus-strand templates required for viral mRNA synthesis. The results we obtained from detailed characterization of nsp1 mutants and pseudorevertants provided compelling evidence for a close link between the regulation of individual nidovirus mRNA levels and the efficient production of infectious progeny.Previous studies of the role of nsp1 in the EAV replicative cycle focused on the conserved amino acids presumed to be essential either for zinc binding by the ZF domain or for the catalytic activity of the PCPβ autoprotease The ZF domain of nsp1 is essential for sg mRNA production We previously reported that certain substitutions of proposed nsp1 zinc-coordinating residues considerably reduced the yield of infectious progeny without noticeably affecting viral RNA accumulation Two mutant phenotypes were previously described upon examination of the role of EAV nsp1 in transcription: one in which sg mRNA accumulation was selectively abolished, and another in which the levels of all sg mRNAs were uniformly reduced relative to that of the genomic RNA Since EAV mRNAs accumulate to different levels in molar ratios that are maintained through most of the replicative cycle see , we will32P-labeled transcripts of positive polarity, which were derived either from a region unique to the EAV genome, or from the leader-body junction regions of RNA6 and RNA7. The samples were then subjected to a second round of RNA denaturation, hybridization, and digestion with RNase A and T1, after which the protected fragments were analyzed by electrophoresis.According to the current consensus in the field, minus-strand RNA synthesis in arteri- and coronaviruses can operate in either continuous or discontinuous mode, generating genome- or subgenome-length templates respectively The minus-strand templates of the most abundant viral mRNAs – RNA1, 6, and 7, were selected for quantitative analysis. Accumulation levels of genome-length [(−)RNA1] and subgenome-length minus strands corresponding to RNA6 and RNA7 [(−)RNA6 and (−)RNA7] were quantified in total intracellular RNA extracted at 11 h post-transfection with the ZCH, A1, and A4 mutants, and a wt control. Subgenomic minus- and plus-strand levels were similarly affected by the nsp1 mutations in a sg RNA-specific manner . GenomicThe relative abundance of nidovirus mRNAs likely serves to regulate the relative concentration of their respective translation products during infection. It was therefore important to determine whether viral protein levels indeed mirrored the specific changes in viral mRNA levels caused by mutations in nsp1. To this end, we examined the intracellular accumulation of the EAV replicase subunit nsp3, and the structural proteins M and N, in cell lysates harvested 11 h after transfection . When co5 and M 5 and M protein levels, as well as the levels of their mRNA templates, RNA5 and RNA6, were strongly affected in the ZCH and A1 mutants  at 39.5°C or at a reduced temperature of 35°C, or after using these supernatants to infect fresh BHK-21 cells (data not shown). By contrast, the rapid appearance of large plaque variants among the prevailing small plaques produced by the ZCH, A1, and A4 mutants see  suggesteTo ascertain these second-site substitutions conferred a replicative advantage, they were introduced into their respective parental (mutant) full-length cDNA clones, yielding a set of viruses collectively referred to as “nsp1 pseudorevertants”. Cell culture supernatants harvested 24 h after transfection with RNA from nsp1 pseudorevertants indeed contained between 2 and ∼4 logs more infectious progeny than those of the original mutants , confirmIn order to progress through their replicative cycle, +RNA viruses need to orchestrate key macromolecular processes that often overlap in time and possibly also in space. Multifunctional viral proteins, which can regulate and interlink multiple of these steps, may therefore have evolved in order to facilitate this spatio-temporal coordination. EAV nsp1 was first identified as a general transcription factor that is also critical for replicase polyprotein processing and efficient production of infectious progeny cis-cleavage of the nsp1/2 site in the nascent replicase polyproteins trans-acting, non-proteolytic activities it may have. Such secondary non-proteolytic functions have previously been reported for several +RNA viral autoproteinase domains, such as those of hepatitis C virus NS2 Previous reports have suggested that the multiple roles of nsp1 during EAV infection can (in part) be functionally separated, with the ZF domain being essential for transcription and efficient virus production, and the PCPβ protease cleaving the nsp1/2 site, irrespective of the ZF integrity The previously uncharacterized PCPα domain, a PCPβ paralog that has lost its proteolytic capacity in EAV 2 = 0.95) a negative exponential regression between infectious virus yield and mRNA imbalance, which is depicted as a negative linear regression in the semi-logarithmic plot of per se, is a chief factor affecting progeny yield. It should be noted that several factors may have affected our analysis of this relationship to a certain extent. For example, only a relatively small variety of nsp1 mutants and pseudorevertants was analyzed and although some pseudorevertants were recovered repeatedly, they were plotted only once. Also, the rapid emergence of pseudorevertants likely contributed to the virus titers measured for the ZCH, A1, and A4 mutants a 24 h post transfection, which would thus be overestimated in our analysis. Therefore, an extension of this study with new mutants and pseudorevertants may further refine the relationship illustrated by Characterization of the three nsp1 mutants with imbalanced viral mRNA accumulation profiles showed that the disruption of the balance was due to a reduction of the levels of certain viral mRNA species only for the A1 mutant see . By contThe molecular basis of perturbed infectious particle production in the mutants is probably complex, although it is likely related to the altered relative abundances of viral structural proteins resulting from the respective changes in the levels of their mRNA templates  and 6A. The onset of +RNA gene expression is marked by translation of the viral genome. In subsequent stages of infection, this molecule is utilized also as the template for replication and, in some cases, transcription, as well as packaging into progeny virus particles. Virtually nothing is known about the temporal coordination of these distinct processes in the nidovirus replicative cycle. Non-structural protein expression is extensively regulated at the translational and post-translational level, by ribosomal frame-shifting and concerted autoproteolytic processing of replicase polyproteins, which together control the production of the core viral replicative enzymes in vitro activity assays using a recombinant form of the EAV RdRp Some of the mutant phenotypes in this report, however, are poorly compatible with the above scenario. Genome RNA levels were increased 4–6 fold in the A1 and A4 mutants, for which sg mRNAs synthesis was clearly detectable and even enhanced . In addiNsp1 remains the only known arterivirus protein specifically implicated in the regulation of transcription Baby hamster kidney cells  were used for all experiments. The cells were maintained at 37°C in BHK-21 medium  supplemented with 5% fetal calf serum (FCS), 10% tryptose phosphate broth, 100 U/ml of penicillin, 100 µg/ml of streptomycin and 10 mM HEPES, pH = 7.4. Upon transfection or infection with wt or mutant EAV, BHK-21 cells were incubated at 39.5°C, since this elevated temperature shortens the replication time of the virus substantially without any adverse side effects The substitutions in the nsp1-coding sequence listed in XhoI-linearized wt or mutant EAV full-length cDNA clones was performed using the mMESSAGE mMACHINE T7 Kit (Ambion). Seven µg of in vitro-synthesized EAV RNA were electroporated into 3.5×106 BHK-21 cells using the Amaxa Cell Line Nucleofector Kit T and the program T-020 of the Amaxa Nucleofector (Lonza) according to the manufacturer's instructions. Cells were resuspended in BHK-21 medium and subsequently seeded on coverslips for immunofluorescence analysis or in 6-well clusters for analysis of intracellular protein and RNA levels, as well as virus production.In vitro RNA transcription from For EAV infection, subconfluent monolayers of BHK-21 cells were inoculated with transfected cell culture supernatant diluted in PBS-DEAE/2% FCS. Following incubation at 39.5°C for 1 h, the inoculum was removed, DMEM/2% FCS was added, and the cells were incubated at 39.5°C for 16–18 h. For virus titration, BHK-21 cells seeded in 6-well clusters were infected with serial 10-fold dilutions of supernatants harvested from transfected cells and then incubated under semi-solid overlays consisting of DMEM supplemented with 50 mM HEPES, pH = 7.4, 2% FCS and 1.2% Avicel (FMC BioPolymer) at 39.5°C for 72 h. The overlays were aspirated, cells were fixed with 8% formaldehyde in PBS, and stained with crystal violet. For plaque purification, a solid overlay of DMEM containing 50 mM HEPES, pH = 7.4, 2% FCS and 1% agarose was used.Immunofluorescence analysis of EAV-transfected cells was performed as described previously 32P-labeled probe  complementary to the 3′ end of the genome that recognizes both genome and all sg mRNAs Analysis of viral RNA accumulation was carried out before completion of the first replication cycle. Cells transfected with wt or mutant EAV derivatives were lysed at 11 h post-transfection, and total intracellular RNA was isolated by acid phenol extraction as previously described A two-cycle ribonuclease (RNase) protection assay, adapted from 32P]CTP (Perkin Elmer) using MAXIscript T7 Kit (Ambion) and purified from 5% polyacrylamide/8M urea gels by elution for 3h at 37°C in 0.5 M NH4OAc, 0.2% SDS, 1mM EDTA. The transcript generated for detection of genomic minus strands –pRNA1, was 356 nt long and contained 327 nt of (−)RNA1-specific sequence. The transcript and EAV-specific sequence length were 382 nt and 327 nt, respectively, for the probe detecting (−)RNA6 (pRNA6), and 372 nt and 319 nt for the probe specific for (−)RNA7 (pRNA7). Detection of RNA1(−) was performed using sample RNA corresponding to approximately 1.25×104 cells and 20 fmol of radiolabelled probe. Levels of (−)RNA6 and (−)RNA7 were determined in samples corresponding to 4×104 and 2.5×104 cells, respectively, using 5 fmol radiolabelled probe. These conditions ensured that the values obtained were in the linear range of the assays (data not shown).To generate probes for minus-strand detection, cDNA fragments derived from the EAV genome (nucleotides (nt) 3687–4013), RNA6 (nt 68–206 from the leader sequence and nt 11870–12057 from the body sequence) and RNA7 (nt 68–206 and nt 12252–12429) were inserted downstream of the T7 promoter in pcDNA3.1 using standard cloning procedures. Radiolabelled RNA transcripts were generated by in vitro transcription in the presence of [α-Cells transfected with wt or mutant EAV derivatives were lysed at 11 h post-transfection as described previously 5′-AGCCGCACCTTCACATTG-3′). Quantitative PCR (qPCR) was performed essentially as previously described 5′ CATCTCTTGCTTTGCTCCTTAG-3′) using HotStar Taq Polymerase (Qiagen) and SYBR Green I (Molecular Probes) in an iCycler machine (Bio-Rad). The data obtained were analyzed with iCycler software, and the specificity of the reaction was confirmed by the melting curve of the amplified products. To generate a standard curve, serial ten-fold dilutions of the virion RNA sample derived from cells transfected with the wt EAV construct were reverse-transcribed and amplified by qPCR in parallel. The resulting standard curve had an R2 = 0.99 and a 6-log linear range for the EAV ORF1a amplicon (data not shown). The relative genomic RNA contents of virions produced by EAV mutants were calculated by comparing their threshold cycle (Ct) values against the standard curve and the resulting values were normalized to the wt genomic RNA content, which was set at 1. The relative specific infectivity of each EAV mutant was then determined by dividing the respective mutant∶wt pfu ratio by the mutant∶wt relative genomic RNA content.In order to determine relative specific infectivity, supernatants from BHK-21 cells transfected with wt or mutant EAV derivatives were harvested 24 h after transfection and clarified by low-speed centrifugation. Virions from 1 ml of clarified supernatant were purified by pelleting through a 0.4 ml cushion of 20% sucrose in 20 mM Tris pH = 7.5, 100 mM NaCl, 1 mM EDTA at 55,000 rpm for 45 min at 4°C using a TLS-55 rotor in a Beckman tabletop ultracentrifuge. Virion RNA was isolated from pellet fractions by acid phenol extraction as described above. Complementary DNAs were synthesized with Thermoscript Reverse Transcriptase (Invitrogen) using a primer complementary to a region in ORF1a of the EAV genome (EAV418as,