Drosophila nervy protein. Structural protein analyses led us to test the hypothesis that specific MTG domains may mediate RNA binding.The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the DNA-binding domain of AML1, a transcriptional activator crucial for hematopoiesis. The AML1-MTG fusion proteins, as the wild type MTGs, display four conserved homology regions (NHR1-4) related to the By using an RNA-binding assay based on synthetic RNA homopolymers and a panel of MTG deletion mutants, here we show that all the MTG proteins can bind RNA. The RNA-binding properties can be traced to two regions: the Zinc finger domains in the NHR4, which mediate Zinc-dependent RNA binding, and a novel short basic region (SBR) upstream of the NHR2, which mediates Zinc-independent RNA binding. The two AML1-MTG fusion proteins, retaining both the Zinc fingers domains and the SBR, also display RNA-binding properties.in vitro RNA-binding properties, thus opening new perspectives on the possible involvement of an RNA component in MTG-mediated chromatin regulation.Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and chimeric AML1-MTG proteins display  Drosophila protein nervy, and therefore called nervy homology regions (NHR1-4) [The myeloid translocation gene (MTG) protein family includes three human members: MTG8 (ETO/CBFA2T1) , MTGR1 ((NHR1-4) . These d(NHR1-4) ,9-11. Th(NHR1-4) ,13; inst(NHR1-4) ,15. We a(NHR1-4) ,11,16,17(NHR1-4) ,15,18. F(NHR1-4) ,15,17,19(NHR1-4)  and refeIn vitro studies suggest that epigenetic downregulation/silencing of these target genes may be a key step in leukemogenesis [As a consequence of the leukemia-associated chromosomal translocations t8;21) and t, MTG8 and MTG16 are fused to AML1 (RUNX1), a transcription factor crucial for hematopoiesis. The resulting fusion proteins AML1-MTG8 (AML1-ETO) and AML1-MTG16 retain the DNA-binding domain of AML1  and all the four functional NHR domains of the MTG proteins .  and t, can play an important role in the epigenetic control of chromatin -30. The We investigated the RNA-binding properties of MTG8, MTG16 and MTGR1 by analyzing their ability to interact with four synthetic RNA homopolymers, poly(A), poly(C), poly(G) and poly(U), coupled to Sepharose beads. This method has been previously proven to be suitable for studying RNA-binding properties of RNA-binding proteins, including the Fragile × mental retardation protein FMRP, which we used in this study as a positive control -35. The Next, we determined the strength of MTGs binding to poly(U) beads in the presence of 150, 250, 500 and 1000 mM NaCl. All MTGs bound the poly(U) homopolymer at the physiological salt concentration of 150 mM Figure . The binin vitro transcription/translation system, in which posttranslational modifications do not occur, retained the ability to interact with poly(U) RNA  or the N-terminus (MTG8Δ2)  domains . ZF doma program . This an program  and depo program ,42. The  program , it is c) Figure , left. T) Figure , left al) Figure , right, 2 Figure , right, 2 Figure , right, in vitro. By deleting the SBR from MTG8Δ1, we obtained the deletion mutant MTG8Δ3, which lacks a.a. 1–329, but retains the ZF domains in the C-terminus  and t, almost the entire MTG8 and MTG16 protein moieties are fused to the RHD domain of AML1, leading to the chimeric AML1-MTG8 and AML1-MTG16 proteins, respectively [ and t16. Here we2 Figure , thus inDrosophila protein Nervy. Despite the presence of two zinc finger (ZF) motifs in one of the domains (NHR4), the MTG proteins do not exert their transcriptional regulatory function by direct DNA binding [The MTG proteins are transcriptional regulators capable of networking with their own family protein members and a variety of transcriptional regulatory proteins. Apparently, the major MTGs' action relies on the ability of these adaptor proteins to establish multiple interactions, on one hand with canonical DNA-binding transcription factors and, on the other hand, with chromatin regulatory proteins, including repressor proteins and histone modifying enzymes (reviewed in ). The MT binding ,13.in vitro assay based on RNA homopolymers binding [in silico protein analyses and development of MTG deletion mutants, we found that the RNA binding is not only mediated by the ZF domains in the NHR4 region, but entails also a novel, Zinc-independent, RNA-binding region proximal to NHR2, the SBR region. The SBR domain seems to be highly conserved across the three human MTGs and the MTGs of different species, and it is rich in basic amino acid residues, a feature frequently observed in RNA binding domains [bona fide novel RNA binding domain, whose primary and secondary structures do not resemble canonical RNA-binding domains [Based on the observation that ZF motifs can mediate not only DNA-protein interactions, but also RNA-protein interactions , we prev binding , we show domains . To our  domains .in vitro RNA-binding properties.The aberrant AML1-MTG fusion proteins retain the ability of interplaying with both repressor proteins and/or histone modifying enzymes, and can induce an altered epigenetic status at the chromatin of both coding and non-coding AML1-target genes ,27,45. Ain vivo remains to be established. The MTG proteins, like the well-known RNA-binding protein FMRP [Whether the wild type and chimeric MTG proteins bind RNA ein FMRP , bind prein FMRP  or long ein FMRP  and refeein FMRP .Interestingly, the two domains that we found to be involved in RNA binding in either a Zinc-dependent or a Zinc-independent fashion are also capable of interacting with specific chromatin regulatory proteins. Specifically, the ZF-containing NHR4 domain is known to interact with both the N-CoR/SMRT proteins and HDACs ,14,15,18in vitro RNA-binding properties. These findings lend support to the hypothesis of the involvement of an RNA component in MTG-mediated chromatin regulation.Evidence has been accumulating that RNA plays a role in transcriptional control. Both wild type MTGs and leukemia-associated AML1-MTG fusion proteins display novel Prediction of RNA-binding residues in the primary structure of the MTG8b protein (Acc. # NP_783552) was performed by using the both the BindN  and the 2. Cells were transiently transfected with 1 μg of plasmid DNA and Lipofectamine Plus  according to the manufacturer's instructions and harvested after 48 h.COS-7 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 1% antibiotics (penicillin and streptomycin) at 37°C and 5% COFMR1 cDNA was previously described [MTG16a cDNA was kindly provided by Drs Kosoda and Ohki  and amplified with primers P44 (5'-ACC ATG CCG GCT TCA AGA CT-3') and P10 (5'-CAG GGG CCA GTG GGG TCA-3'). MTGR1a cDNA was kindly provided by Dr I. Kitabayashi  and amplified with primers P12 (5'-AAC CAT GCC TGG ATC GCC TG-3') and P13 (5'-AGC AGA GTC CGG GGC TCA G-3'). The cDNA of wild type MTG8b was amplified from pCMV-MTG8b [MTG8b cDNA with the following primers: MTG8Δ1, lacking the region coding for amino acids 449–604, with primers P122 and P123 (5'-CTC CTC AGC TTA CTT CCA GAT C-3'); MTG8Δ2, lacking the region coding for amino acids 1–304, with primers P125 (5'-ACC ATG GCC ATT GCC CAC CAC TAC-3') and P7; MTG8Δ3, lacking the region coding for amino acids 1–329, with primers P157 (5'-GTT ATG GGG TTG CAT GGC ACA CG-3') and P7; MTG8Δ4, lacking the region coding for amino acids 1–329 and 514–604, with primers P157 and P124 (5'-CCA GCA ACT CTA GCT TGA ATC C-3'). The cDNAs of AML1-MTG8 and AML1-MTG16 type1 were kindly provided by Dr I. Kitabayashi and amplified with primers P51 (5'-ACC ATG CGT ATC CCC GTA GAT G-3') and P7, and P51–P10, respectively.The psf2 construct containing escribed . The cDNIn vitro transcription/translation was performed with TnT Quick Coupled Transcription/Translation System  according to the manufacturer's instructions. 1 μg of plasmid DNA containing a T7 promoter was used in each reaction. The samples were directly used for the RNA-binding assay.6 transfected cells were homogenized in binding buffer  containing NaCl in the indicated concentrations (150 mM when not mentioned otherwise) and 50 μM ZnCl2 when not otherwise indicated. The samples were sonicated 2–3 times for 15 sec. and spun down for 5' at 13000 rpm at 4°C. A small part of the homogenate was saved as total sample and the rest was split and used for binding to 60 μl of Sepharose 4B-poly(U), -poly(A) , -poly (C) or -poly(G) (both from Sigma) beads previously washed and diluted 1:1 in binding buffer. As a negative control we either used beads treated with 50 U/ml micrococcal nuclease for 1 h 30°C or ECH Sepharose 4B . After incubation at 4°C for 1 h on a rocking platform, the beads were washed 5 times with 1 ml binding buffer and finally eluted with NuPage LDS Sample Buffer (Invitrogen). Samples were analyzed on SDS-PAGE followed by Western blotting. Immunodetection was performed with antibodies that we developed against MTG8 , MTG16 , MTGR1  [The RNA-binding assay was based on the affinity for different RNA homopolymers, as previously described . Approxi 1:2000) . Commercin silico analyses, designed and carried out the experiments, and drafted the manuscript. LV contributed to perform the RNA-binding experiments. NS contributed to conceive the hypothesis, co-ordinated the overall project, and critically revised the manuscript. ATH contributed to conceive the hypothesis, provided experimental advice, and reviewed the manuscript.SR contributed to conceive the hypothesis, performed the


Understanding the origin of protein synthesis has been notoriously difficult. We have taken as a starting premise Wolf and Koonin's view that "evolution of the translation system is envisaged to occur in a compartmentalized ensemble of replicating, co-selected RNA segments, i.e., in an RNA world containing ribozymes with versatile activities".We propose that coded protein synthesis arose from a noncoded process in an RNA world as a natural consequence of the accumulation of a range of early tRNAs and their serendipitous RNA binding partners. We propose that, initially, RNA molecules with 3' CCA termini that could be aminoacylated by ribozymes, together with an ancestral peptidyl transferase ribozyme, produced small peptides with random or repetitive sequences. Our concept is that the first tRNA arose in this context from the ligation of two RNA hairpins and could be similarly aminoacylated at its 3' end to become a substrate for peptidyl transfer catalyzed by the ancestral ribozyme. Within this RNA world we hypothesize that proto-mRNAs appeared first simply as serendipitous binding partners, forming complementary base pair interactions with the anticodon loops of tRNA pairs. Initially this may have enhanced stability of the paired tRNA molecules so they were held together in close proximity, better positioning the 3' CCA termini for peptidyl transfer and enhancing the rate of peptide synthesis. If there were a selective advantage for the ensemble through the peptide products synthesized, it would provide a natural pathway for the evolution of a coding system with the expansion of a cohort of different tRNAs and their binding partners. The whole process could have occurred quite unremarkably for such a profound acquisition.in vitro together with a minimal set of ribosomal proteins that are required today to support protein synthesis.It should be possible to test the different parts of our model using the isolated contemporary 50S ribosomal subunit initially, and then with RNAs transcribed de novo from complementary base pair interactions between tRNAs and single-stranded RNAs present in the immediate environment.This model proposes that genetic coding arose This article was reviewed by Eugene Koonin, Rob Knight and Berthold Kastner (nominated by Laura Landweber). E. coli anticodon nuclease which is able to cleave tRNA immediately 5' to the anticodon  contacts predominantly the 50S, as opposed to the 30S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30S binding [italics in the original]" [using the anticodon loops of the existing tRNA's [sic] molecules as partial templates [italics in the original]" "  argues tiginal]" ). The opiginal]" , and Criiginal]" .In presenting this model, we take as a starting premise this statement from Wolf and Koonin (2007): "Our main guide in constructing the models is the Darwinian Continuity Principle whereby a scenario for the evolution of a complex system must consist of plausible elementary steps, each conferring a distinct advantage on the evolving ensemble of genetic elements. Evolution of the translation system is envisaged to occur in a compartmentalized ensemble of replicating, co-selected RNA segments, i.e., in a RNA world containing ribozymes with versatile activities". In what follows we seek to demonstrate selective advantage at each step, and, in line with the Continuity Principle, a continuous pathway where information is preserved from the ancestral system to the present. As logic dictates, the evolution of coded peptide synthesis must have occurred in the absence of complex proteins.Schimmel has proposed an ancestral system of noncoded protein synthesis utilizing hairpin precursors of tRNA ,18. Prio2+ ions " " .In our earlier paper  we suggeThe above considerations provide a plausible pathway for the evolution of genetic coding that is outlined below and in Figure Initially, we assume the presence of a small number of specifically-aminoacylated RNA hairpins  giving rise to a number of homo- or hetero-dipeptides. If we make the assumption that only two of these were useful, gly-gly and gly-ser, then these two peptides produced by a noncoded process had their production under positive selection because of a net benefit to the compartment or cell.in vivo).The original aminoacylated RNAs, described up to this point as 'hairpins', may have actually constituted a mixed population of molecules, having in common only the presence of an acceptor stem-like helix  and a 3' CCA terminus allowing specific aminoacylation.  eventually became specified by a different sequence , then each combination of tRNAs would have bound a unique complementary proto-mRNA, giving a one-to-one relationship between proto-mRNA and peptide. This advantage could have driven the evolution of coding specificity - each codon sequence interacted with only a single tRNA, coding for only a single amino acid  possessed an NCC anticodon.One constraint on the first anticodon sequences was that they must form stable anticodon-codon interactions in the absence of modified anticodon loop nucleotides, if, as suggested by Crick , there wIn overview, the principle selective pressure was for the production of novel useful peptides enhancing biological function, and this in turn resulted in selection for the diversification of anticodon and proto-mRNA sequences. Wolf and Koonin have expressed a parallel concept as part of their proposal for the origin of protein synthesis .Maizels and Weiner  have sugdecrease in binding of oligolysyl-tRNA to the small subunit, reminding us that in conjunction with the small subunit the polynucleotide is playing a selective role in the interaction. As previously discussed, this idea is supported by the demonstration [non-programmed 70S ribosomes [i.e. in the absence of mRNA] contacts predominantly the 50S, as opposed to the 30S subunit, indicating that codon-anticodon interaction at the P site is a prerequisite for 30S binding [italics in the original]."Translocation provides an evolutionary route to coding, even in the absence of the small ribosomal subunit. While in the first instance merely passively binding to the anticodon loops of pairs of tRNAs in the A and P sites, upon movement of the tRNA CCA-termini from the A to P and P to E sites , the downstream codon of the proto-mRNA might well have played a role in favouring the selection of the tRNA entering the newly vacated A site. Another way of viewing the experiments of Jonák and Rychlík  and Gnirstration  that, "aPhe anticodon stem/loop in a similar fashion to the entire small subunit, suggesting such a hairpin-type structure could be an ancestral decoding fragment  anticodon triplet together with the two purines on its 3' side may yield sufficient stability..."   . We woulNA code)  or from NA code)  to the gWolf and Koonin  have sugin vitro transcribed RNA, in practice this may not be possible, as promising reports of protein synthesis by in vitro transcripts of the large ribosomal subunit RNA  of E. coli were subsequently retracted [et al. [in vitro selection aimed to enrich for peptidyl transferase activity produced a sequence able to ligate A and P site substrates; the ligated product however did not contain a peptide bond. Moreover, product formation was not sensitive to chloramphenicol, indicating that the active site in the RNA, improved by in vitro selection, is different from the classical peptidyl transferase centre [While it would be ideal to test our hypothesis using only etracted -91. The etracted . More re [et al.  have atte centre .2+ ions; also that "functional experiments, exploring the peptidyl transferase activity of a large variety of the RNA dimers are in accord with the structural analysis" [Yonath's group has used small stem-elbow-stem (SES) RNA structures in an attempt to demonstrate peptide synthesis. They report that some sequences form dimers, dependent on sequence and the presence of Mgnalysis" . HoweverDespite much effort, it has not been possible to produce 50S ribosomal subunits that are able to catalyze peptide synthesis in the complete absence of ribosomal proteins . As discThe experiments that have demonstrated peptide synthesis in the absence of mRNA and enhanced binding of tRNAs in the presence of cognate oligonucleotides have used the whole 50S subunit, including ribosomal proteins. In view of this, a realistic experimental approach would be as follows:in vitro transcribed tRNAs and complementary oligonucleotides to define the parameters for the stabilization of tRNA binding by single-stranded RNAs and enhancement of peptide synthesis, as per the experiments of Jonák and Rychlík [1. Initially, use the isolated 50S ribosomal subunit together with  Rychlík  and Monr Rychlík .in vitro transcribed 23S rRNA together with a minimal set of ribosomal proteins essential for peptidyl transferase activity [in vitro transcribed 23S rRNA fragment corresponding to the proposed ancestral peptidyl transferase [2. Repeat these experiments using (a) activity ; and (b)nsferase  in the a3. Test the ability of polyglycine (and other peptides composed of proposed evolutionarily 'early' amino acids ) to stabin vitro transcribed) is able to function in trans to control the specificity of binding of tRNAs to the 50S subunit in the presence of cognate RNA, and thus direct the synthesis of specific peptides.4. Investigate whether a proposed ancestral decoding hairpin derived from the small ribosomal subunit RNA   to bind Cu2+ and exhibit superoxide dismutase activity that the original selection pressure was to produce poly(Gly) or similar peptides, that this ability was then extended to the ability to make simple copolymers of other amino acids, that proto-mRNAs initially evolved as RNA effectors that assisted in the orientation of hairpins for non-coded peptide formation and only later evolved coding potential.The problem of how the translation apparatus evolved is an important one, especially because it is important that proposed mechanisms provide some sort of continuity of function (so that each subsequent step is an improvement). As the authors note, the steps in the process they have proposed have largely been proposed before  so the question becomes (i) whether the pathway is sufficiently interesting to be publishable as a starting point for discussion, and (ii) whether the pathway is sufficiently compelling that we should accept it as a likely account of how the code evolved.My impression is that the model passes the first test but not the second. As the authors note, one attraction of this model is that several steps are empirically testable, although the experiments are not clearly described in the present version of the manuscript. The manuscript is also relatively long in relation to its news value and could benefit from a substantial reorganization  and highlight the main differences among them.- Separate the speculations about the selective pressures that might have driven the original nontemplated peptide synthesis function into one section, rather than repeating variations on this material in several places.- Present the model earlier: the model and the methods for testing it appear on p 30 and take 2 pages out of the 45 in this version of the manuscript. Since the new model is what is central in this paper, it should appear earlier and then the steps should be justified with citation of specific, appropriate literature.- In general, shorten both the introduction and the discussion considerably to focus more on the proposals that are directly relevant to this proposal.- In the discussion, clearly enumerate what experiments you would do in order to test each step of the model, how the outcome of each experiment could discriminate between this model and the alternatives mentioned in the introduction, etc.We agree with the reviewer's suggestions and have revised the manuscript accordingly.- I was surprised not to see the original Yarus DiRT paper, or the Knight and Landweber 2000 piece on alternative models for getting from triplet/site associations to the present, or the original Szathmary paper on coding coenzyme handles, in the reference list although I acknowledge there could be reasonable motivations for omitting these and the reference list is long already.One additional question I have is whether the Cu2+/superoxide dismutase selection pressure is a reasonable candidate: since these events presumably took place before the oxygenation of the atmosphere, how strong a selective advantage would SOD activity provide? Interestingly, Schwedinger & Rode reported in 1989 that Cu2+ could catalyze peptide bond formation at high temperature/salt , so the copper binding activity could be more directly relevant to peptide evolution. However, this weakens the argument that Cu2+ sequestration would be necessary 2+ was not well developed in the original manuscript , and this has been revised. The examples of glycine-Cu2+ chemistry, while interesting from our point of view - having proposed tRNAGly was the first tRNA [We acknowledge the discussion regarding potential interactions of glycine peptides with Curst tRNA  - were gI think the model presented here is interesting and hope that the manuscript can be rewritten in such a way as to inspire the empirical testing that it deserves. In particular, the demonstration that a generic proto-mRNA could enhance the rate of nontemplated protein synthesis using aminoacylated hairpins would be an interesting finding.Reviewer 3: Berthold Kastner, Max Planck Institute, GöttingenBernhardt and Tate present in their manuscript "The transition from noncoded to coded protein synthesis: did coding mRNAs arise from serendipitous binding partners to paired tRNAs that enhanced peptide synthesis on an ancestral peptidyl transferase?" a very compelling scenario of evolution of coded protein synthesis. In addition, the line-up of the various steps leading from the RNA- to the RNP-world presented at the end of the manuscript is very attractive, as each step could lead to an advantage of the system. For readers from outside the field it might be helpful if this concept is presented in a short version already in the results section. The many possible individual steps of the evolution scenario formulated already previously require thorough discussion that leads to a quite lengthy manuscript. Nevertheless, it might be possible to compact it a bit more. For one of the steps, the first appearance of di-/oligo- peptides, the reviewer sees the focusing on the Cu2+ binding argument for the evolutionary advantage as being rather narrow. As we have very limited perception on the chemical environment that might have existed in the RNA-world "cell", the availability of short peptides could have given the system an advantage for various chemical pathways. It might have even been as simple as the prime benefit of amino acids have been the ability of buffering the pH of the system and di-peptides would have then the advantage of being more confined to the containment (the "cell"). The pH buffer function of proteins is still important in modern life. Then, the coded protein synthesis might have brought about the more specific functions of the peptides and mark the starting point of the RNP world. Still, detailed scenarios of such early stages of evolution remain mostly speculation, but the scenario presented here for evolution of the coded protein synthesis is highly plausible within the framework of current thinking. With the presented new perspectives and the careful discussions the manuscript of Bernhardt and Tate is well suited for publication in Biology Direct.2+ chemistry were given to illustrate the catalytic possibilities of simple peptides. The reviewer's suggestion of early peptides having a role in the maintenance of pH is an interesting one, and worthy of further investigation.As discussed in our response to the last reviewer, the examples of glycine-Cu


Through its association with trans-acting effector proteins, the m7G cap participates in multiple aspects of RNA metabolism including localization, translation and decay. However, at present relatively few eukaryotic proteins have been identified as factors capable of direct association with m7G.A unique attribute of RNA molecules synthesized by RNA polymerase II is the presence of a 7-methylguanosine  complex, as a factor capable of direct and specific interaction with the m7G recognition. The findings presented here will facilitate understanding of gemin5's role in the metabolism of non-coding snRNAs and perhaps other RNA pol II transcripts.This study rigorously identifies gemin5 as a novel cap-binding protein and describes an unprecedented role for WD repeat domains in m In metazoans a two-component enzyme complex engendering triphosphatase, guanylyltransferase, and methyltransferase enzymatic activities is responsible for modifying RNAs at the 5′ end with m7G Saccharomyces cerevisiaeA salient feature of RNA transcripts produced by eukaryotic RNA polymerase II is the presence of a 7-methylguanosine (m7G. In the nucleus a bipartite cap-binding complex (CBC) composed of CBP20 and CBP80 associates with nascent pre-mRNAs to promote splicing and export 7G cap moiety participates in every major aspect of mRNA metabolism.Functions attributed to the cap depend upon specific trans-acting factors that have been shown to directly bind matac, U5, U7, U11, and U12 7G cap, but 3′ end processing is carried out by distinct machinery that generates mature non-polyadenylated termini. Similar to mRNAs, newly synthesized Sm class snRNAs are bound by CBC which, along with the adaptor protein PHAX In addition to mRNAs, RNA pol II mediates the synthesis of multiple non-coding RNAs including the Sm class of small nuclear (sn) RNAs composed of eight unique species: U1, U2, U4, U45′-AUUU/CUUG-3′) within snRNAs Eukaryotes have evolved macromolecular protein complexes of varying complexity Subsequent to Sm core assembly, the snRNA cap is hypermethylated by the TGS1 methyltransferase to form 2,2,7-trimethylguanosine (TMG) 7G is critical for their metabolism, it is not clear whether the full complement of cellular proteins capable of binding the cap structure has been defined. Here, we present evidence that gemin5 is capable of direct and specific interaction with m7G in a manner that depends upon integrity of its WD repeat domains. These findings expand the repertoire of identified cellular m7G cap-binding proteins and raise the possibility that gemin5 is a novel regulator of gene expression at the post-transcriptional level.While it is well established that capping of RNA pol II transcripts with m7GTP immobilized to sepharose by covalent linkage 7GpppG, RNase A) indicated robust stringency of the purification method  had no effect, demonstrating strict binding specificity of eIF4E to methylated guanosine. As with GpppG, supplementation of reactions with ATP or treatment of lysate with RNase did not affect eIF4E precipitation  that cleaves eIF4G downstream of the eIF4E interaction site pro harbors eIF4A-binding sites The pattern of proteins purified by cap-affinity was also examined using lysates from cells acutely stressed with arsenite or infected with coxsackievirus B3 (CBV3). CBV3 encodes a protease  of gemin5 that are required for association with cap-sepharose by introducing serial deletions into the FLAG-tagged gemin5 cDNA construct. Gemin5 is 1508 amino acids in length and contains 13 WD repeats in its N-terminal half. Characteristic β-propeller structures are known to be formed by 6 to 7 WD repeat domains We next questioned whether the minimal N-terminal deletion, hereafter referred to as gemin5(Δ25), affected interaction with binding partners gemin3 and gemin4. 293T cells were transfected with plasmids expressing tagged gemin5, gemin5(Δ25), or vector DNA. For additional comparison, we also expressed an N-terminally FLAG-tagged version of human argonaute2 (Ago2), a component of the RNA-induced silencing complex (RISC) that has been reported to interact with gemin3 and gemin4 7G cap-sepharose. One possibility is that gemin5 binds to the prototype cap-binding protein, eIF4E. Indeed, gemin5 has been recently suggested to be a novel eIF4E-interaction partner . To investigate this possibility we co-expressed tagged gemin5 and eIF4E in 293T cells and performed co-IP assays using pre-immune, α-myc tag, and α-FLAG tag antibodies (We sought to identify the molecular basis underlying the observation that gemin5 binds to mtibodies . Each an7GpppG) or nonspecific (GpppG) cap analog competitors  deletion mutant in the presence or absence of free cap analog competitor. As an additional control, eluate from FLAG-IP of mock-transfected cells was also used. After incubation, reactions were irradiated with UV light, treated with an RNase cocktail, and then subjected to SDS-PAGE. In reactions using total cytoplasmic lysate, most crosslinked proteins bound the capped RNA nonspecifically since addition of free cap analog had no effect on their crosslinking efficiency  and quantitative RT-PCR (RT-qPCR) methods were employed to measure levels of endogenous U1 snRNA co-precipitating with FLAG-tagged gemin5 over-expressed in 293T cells . The sam 35-fold . This wa 35-fold  despite  35-fold . Taken t7G cap-structure added co-transcriptionally to all RNA pol II transcripts execute critical functions in mRNA and snRNA metabolism. Here, we identify gemin5, a peripheral component of the SMN complex, as a novel m7G cap-binding protein. Gemin5 was specifically purified by cap-affinity chromatography  that may directly mediate cap interaction similar to previously characterized cap-binding proteins  represents one scenario where mRNAs may be transiently remodeled into silenced particles prior to decay or resumption of active translation. Though mechanisms of miRNA function remain obscure 7GTP-sepharose or sepharose-4B  in NT2 buffer  for one hour. Binding reactions were performed in the presence or absence or 0.1 mM cap analogs (GpppG or m7GpppG). Sepharose beads were washed 3 times with 1 ml NT2 buffer and then resuspended in sample buffer for SDS-PAGE. Antibodies to eIF4E (Abcam), PABP HeLa and 293T cell lysates were prepared using polysome lysis buffer (PLB) as described Xenopus elongation factor 1α gene was in vitro transcribed  and capped using vaccinia virus guanylyltransferase and [α-32P]-GTP according to the manufacturer's protocol (Ambion). Immunoprecipitated FLAG-tagged gemin5 and gemin5(Δ25) were concentrated using Microcon filter devices (Millipore). Crosslinking reactions (20 µl) contained 60 ng labeled RNA, 40 ng immunopurified protein or 120 µg cytoplasmic lysate, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 1 mM MgCl2. Reactions were incubated 10 minutes at RT and then irradiated 20 minutes on ice with a UV Stratalinker (Stratagene). After crosslinking, reactions were treated with 1 µl RNase cocktail (Ambion) for 10 minutes at RT and then subjected to SDS-PAGE.Buffers used for lysate preparation (PLB) and binding reactions (NT2) were identical to those used in cap-binding assays. IP reactions were incubated one hour and contained 40 µl protein G-sepharose  bound to 10 µg antibody and 2 mg protein lysate. Washed immunoprecipitates were resuspended in either sample buffer for SDS-PAGE, Trizol reagent (Invitrogen) for RNA extraction, or NT2 buffer with 0.1 mg/ml FLAG peptide (Sigma) for elution of FLAG-tagged proteins. For UV-crosslinking experiments, RNA containing the KpnI and NotI. This clone was modified with a C-terminal FLAG tag by PCR. The N- and C-terminal deletion constructs were derived in a similar fashion. Point mutations were generated by overlap fusion PCR Gemin5 cDNA clone was obtained from ATCC (RefSeq: NM_015465) and the coding region was inserted into pcDNA 3.1 (Invitrogen) using The PHYRE program 5′-GGGAGATACCATGATCACGAAGGT-3′; (U1 snRNA-reverse) 5′-ATGCAGTCGAGTTTCCCACA-3′; (U6 snRNA-forward) 5′-CTCGCTTCGGCAGCACATATACTA-3′; (U6 snRNA-reverse) 5′-ACGAATTTGCGTGTCATCCTTGCG-3′. Real-time PCR was conducted using a Roche Light Cycler. Reactions were performed in triplicate on at least two separate experiments for each gemin5 variant and the ΔΔCt method was used to calculate fold changes in positive versus negative IPs. A representative experiment is shown in One step RT-qPCR reactions were performed on co-precipitating RNA from IP reactions using SYBR green reaction mix (Roche) and the following primers specific for target RNAs: (U1 snRNA-forward)


Schistosoma mansoni and Schistosoma japonicum. Non-coding RNA (ncRNA) plays a crucial role in gene expression regulation, cellular function and defense, homeostasis, and pathogenesis. The genome-wide annotation of ncRNAs is a non-trivial task unless well-annotated genomes of closely related species are already available.Schistosomes are trematode parasites of the phylum Platyhelminthes. They are considered the most important of the human helminth parasites in terms of morbidity and mortality. Draft genome sequences are now available for S. mansoni resulted in 23 types of ncRNAs with conserved primary and secondary structure. Among these, we identified rRNA, snRNA, SL RNA, SRP, tRNAs and RNase P, and also possibly MRP and 7SK RNAs. In addition, we confirmed five miRNAs that have recently been reported in S. japonicum and found two additional homologs of known miRNAs. The tRNA complement of S. mansoni is comparable to that of the free-living planarian Schmidtea mediterranea, although for some amino acids differences of more than a factor of two are observed: Leu, Ser, and His are overrepresented, while Cys, Meth, and Ile are underrepresented in S. mansoni. On the other hand, the number of tRNAs in the genome of S. japonicum is reduced by more than a factor of four. Both schistosomes have a complete set of minor spliceosomal snRNAs. Several ncRNAs that are expected to exist in the S. mansoni genome were not found, among them the telomerase RNA, vault RNAs, and Y RNAs.A homology search for structured ncRNA in the genome of The ncRNA sequences and structures presented here represent the most complete dataset of ncRNA from any lophotrochozoan reported so far. This data set provides an important reference for further analysis of the genomes of schistosomes and indeed eukaryotic genomes at large. Non-coding RNA (ncRNA) plays a crucial role in gene expression regulation, cellular function and defense, and disease. Indeed, in higher eukaryotes, most of the genomic DNA sequence encodes non-protein-coding transcripts . The initial search was conducted by blastn with E < 0.01 with the mature and mature* miRNAs as query sequences. The resulting candidates were then extended to the length of the precursor sequence of the search query and aligned to the precursors using ClustalW[RNAfold[RNAalifold[S. japonicum and S. mediterranea genomes to examine whether these sequences are conserved in Schistosoma and/or Platyhelminthes.We followed the general protocol outlined in  to ident miRBase  [Release ClustalW. SecondaW[RNAfold for singNAalifold for aligsnoRNAbase[S. mansoni genome using BLAST[GotohScan[S. mansoni assembly. The SnoReport program [snoscan[RNAsnoop[S. japonicum and S. mediterranea using BLAST. To estimate the number of false predictions we compared the candidate snoRNAs with common ncRNA databases, in particular Rfam[NONCODE[We compared all the known human and yeast snoRNAs that are annotated in the noRNAbase to the Sing BLAST and GotoGotohScan. The sea program  was used [snoscan for box [RNAsnoop for box ular Rfam and NONCm[NONCODE. All seqFor other families, we employed the following five steps:BLAST searches with E < 0.001 using the known ncRNA genes from the NCBI and Rfam databases. For the snRNA set, see [X04249, for 5S and 5.8S rRNAs we used the complete set of Rfam entries, for the SSU and LSU rRNAs, we used Z11976 and NR_003287, respectively. The SL RNAs were searched using SL RNA entries from Rfam and the sequences reported in [GotohScan[rnabob http://selab.janelia.org/software.html.(a) For candidate sequences of ribosomal RNAs, spliceosomal RNAs, the spliced leader (SL) and the SRP RNA, we performed set, see . For 7SLorted in . For morClustalW[ClustalX[RNAfold, RNAalifold, and RNAcofold[stockholm-format alignment files in the emacs editor utilizing ralee mode [(b) In a second step, known and predicted sequences were aligned using ClustalW and visu[ClustalX. To idenRNAcofold were uselee mode . AlignmeMEME[(c) Putatively functional sequences were distinguished from likely pseudogenes by analysis of flanking genomic sequence. To this end, the flanking sequences of snRNA and SL RNA copies were extracted and analyzed for conserved sequence elements using MEME. Only snRNAduplex http://www.tbi. univie.ac.at/RNA/RNAduplex.html was used to find the pseudoknot structure. In order to confirm that the SL RNA candidate is indeed trans-spliced to mRNA transcripts, we searched the FAPESP Genoma Schistosoma mansoni website for ESTs including fragments of the predicted SL RNA. We found 52 ESTs with blastnE < 0.001 that span the predicted region of the SL RNA (nt 8-38), indicating that this RNA does indeed function as a spliced leader.(d) Additional consistency checks were employed for individual RNA families, including phylogenetic analysis by neighbor-joining  to checkBLAST queries against the S. mansoni genome to determine their copy number in the genome assembly.(e) Accepted candidate sequences were used as http://www.bioinf.uni-leipzig.de/Publications/SUPPLEMENTS/08-014 provides extensive machine readable information, including sequence files, alignments, and genomic coordinates.The website CSC, PB, and PFS designed the study. CSC, MM, DR, JH, CBS, SK, CSA, and PFS performed the computational analyses. CSC wrote the first draft of the manuscript. All authors contributed to the final assessment of the data as well as the writing of the final version of the manuscript. CSC, MM, DR, JH should be considered as joint first authors.Supplemental figures and captions. contains supplemental Figures S1 - S4 mentioned in the main text.Click here for file


The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region. Although the genomes of RNA viruses occur in a variety of conformations, all must be efficiently copied within the infected cell. These copies are essential to the production of messenger RNA (mRNA) that can be translated by host ribosomes, and as a source of genomic RNA for packaging into mature progeny virions. The alphaviruses are a group of enveloped, positive (+) strand RNA viruses in the family Togaviridae Members of the alphavirus genus pose a serious or potential threat to public health in many areas of the world. Nearly all alphaviruses are maintained in nature by transmission cycles that involve alternating replication in a susceptible vertebrate and invertebrate host. Because infection of the vertebrate host is acute and often associated with disease, continual transmission depends on life-long persistent infection of the invertebrate vector host, for many alphaviruses a mosquito. It is presently unclear how persistent alphavirus infections are maintained in the vector host, after (−) strand synthesis terminates in the infected cells. One of the difficulties in addressing this question has been the limitations of methodologies for detecting and measuring (−) strand RNA in infected cells. Competition between viral (+) strands and labeled probe makes nuclease protection assays problematic for the detection of (−) strand RNA, particularly late in the infection when (+) strands are much more abundant Although a variety of strand-specific quantitative real-time PCR (ssqPCR) assays utilizing different designs, detection strategies and chemistries have been reported Aedes albopictus C6/36 cell monolayers were grown to 80% confluency in 12-well plates, washed twice with PBS, and infected with ONNV at a multiplicity of infection (MOI) of 5. Virus was diluted with Dulbecco's Modified Eagle Medium (DMEM)  to a total volume of 0.5 mL/well and placed on the cells at 4°C for one hour. After one hour, fresh medium was added to the wells bringing the total volume to 1 mL, and cells were placed at 28°C for 1 hour. Total RNA was isolated from ONNV-infected cells with TRI Reagent RT®  at 1 hour post infection.in vitro from a plasmid containing a portion of the nsP1 gene from either ONNV or CHIKV. The plasmids pblue-nsP1 (ONNV) and pblue-nsP1 (CHIKV) were produced by cloning the 5′ terminal 853 and 669 nucleotides of the respective viral nsP1 gene into pBluescript II SK (−) (Stratagene). Minus strand RNA was synthesized with T7 RNA polymerase from HindIII-digested plasmid templates in a standard in vitro transcription reaction. Positive strand RNA was synthesized with SP6 RNA polymerase from KpnI-digested plasmid templates in a standard in vitro transcription reaction. The RNA generated during the in vitro transcription reactions was isolated with TRI Reagent RT®, as per manufacturer's instructions. The absence of template DNA was confirmed through PCR. The concentration of RNA transcripts was determined with a NanoDrop spectrophotometer (Thermo Scientific). The cloned ONNV nsP1 gene fragment has a molecular weight of 273,545 g/mol, while the cloned CHIKV nsP1 gene fragment has a molecular weight of 214,574 g/mol. One µg of RNA transcribed from pblue-nsP1 (ONNV) equals approximately 2.2×1012 molecules, while one µg of RNA transcribed from pblue-nsP1 (CHIKV) equals approximately 2.8×1012 molecules.To generate strand-specific standard curves for ssqPCR, (+) and (−) strand RNA was transcribed 10–102 gene copies/µl) and stored at −20°C until further use.cDNAs of both polarities were transcribed with primers containing a 5′ tag sequence To increase fluorescent signal strength during ssqPCR reactions, an AT-rich 12-nucleotide flap sequence (5′AATAAATCATAA 3′) was added to the 5′ end of tagged primers ssqPCR was performed with the appropriate forward or reverse and tag-specific primer pair . When a 2R) of 0.994, and amplification efficiency (Eff%) of 96.8%. The standard curve for the assay using a combination of tag-specific and ONNV-specific primers had a slope of −3.4, an 2R of 0.999, and amplification efficiency of 96.8%. Both assays gave values for the quantity of ONNV (−) strand RNA that were within the acceptable ranges of their respective standard curves: 8.0×105 copies of (−) strand RNA/µg of total RNA for the ssqPCR assay using unmodified primers, and 1.1×105 copies of (−) strand RNA/µg of total RNA for the ssqPCR assay using a tagged primer system , suggesting that one or both of the ssqPCR assays was prone to error.The ability of ssqPCR assays that employ a tagged primer system to detect and quantify viral RNA of a specific polarity has been well demonstrated r system . However6 copies of ONNV RNA/µg of total RNA (the polarity of the RNA is unclear) was determined by standard curve when the two unmodified ONNV-specific primers were used together  strand cDNA, the slope was −1.4, the 2R value was 0.809, and the amplification efficiency was 417.9%. Only at dilutions in which ONNV (−) strand cDNA was present in excess of ONNV (+) strand cDNA were CT values comparable between the two standard curves  strand, suggesting amplification of cDNA from both the intended target strand and the competing (+) strands. Standard curves generated with the tag-specific primer set were similar in the absence or presence of ONNV (+) strand cDNA: the slopes were −3.6 and −3.7, the 2R values were 0.995 and 0.990, and amplification efficiencies were 89.6% and 86.3%, respectively  strand RNA ssqPCR assays in the presence or absence of a fixed amount of cDNA corresponding to ONNV (+) strand RNA. Standard curves produced with the unmodified primer set were very different depending on whether or not ONNV (+) strand cDNA was present during qPCR . In the d curves . Higher ectively . OverallSYBR® Green emits a strong fluorescent signal upon binding to dsDNA. Because the intensity of this fluorescent signal increases with the amount of dsDNA present, the dye can be used to detect and measure the accumulation of qPCR amplicons. However, amplification of nonspecific templates after many PCR cycles can result in additional fluorescence unrelated to any specific target. This nonspecific fluorescence has been shown to limit the sensitivity of dsDNA specific dyes for the detection and quantification of low-copy number targets 3 copies of ONNV cDNA/reaction  or (−) strands were synthesized in an in vitro transcription reaction. Following reverse transcription with a tagged forward or reverse primer , ONNV +. New tagreaction . Howeverreaction . These r3 copies of CHIKV cDNA/reaction  strand cDNA, and a slope of −3.5,an 2R value of 0.973, and amplification efficiency of 93.07% in the presence of CHIKV (+) strand cDNA  strand RNA is inhibited by the presence of relatively higher levels of cDNA generated from the competing (+) strand RNA, when standard unmodified ONNV-specific primers are used for reverse transcription and qPCR . FalselyThe sensitivity of our ONNV (−) strand ssqPCR tag-specific assays was determined with serial 10-fold dilutions of cDNA generated from in vitro transcribed ONNV RNAs. With our SYBR® Green assay we were able to detect 8000 copies per reaction but could not detect 800 copies per reaction, indicating a lower limit of detection somewhere within this range. However, the limit of low-copy number detection decreased to somewhere between 800 (which could be detected) and 80 (which could not be detected) copies per reaction when a TaqMan® DNA hydrolysis probe was used to monitor amplification. The increased sensitivity likely resulted from eliminating the non-specific amplification of dsDNA products that is common when monitoring the generic fluorescence emitted by dsDNA-binding dyes. The sensitivities of our ONNV (+) strand ssqPCR tag-specific assays were determined to be identical to those of the (−) strand assays, with both detection strategies and chemistries. In the case of alphaviruses, the increased sensitivity of assays incorporating DNA hydrolysis probes should be useful, particularly when quantifying much less abundant (−) strands.Finally, using the information obtained by directly comparing various parameters of multiple ONNV-specific ssqPCR assays, we developed and validated assays to detect and quantify CHIKV (−) and (+) strand RNAs. Serial dilutions of cDNA generated from in vitro transcribed CHIKV RNAs were used to confirm strand-specificity and determine sensitivity. Amplification of dsDNA was undetectable when the (+) strand primer set was used with cDNA derived from the (−) strand at all concentrations tested. The reverse was also found to be true using the (−) strand primer set and cDNA generated from (+) strand RNA. The accuracy of the CHIKV (−) strand assay was confirmed in the presence of cDNAs transcribed from competing (+) strand RNAs . The senIn summary, we have developed and validated two new ssqPCR assays for the medically important alphaviruses, CHIKV and ONNV. These assays will be useful in studies to determine how persistent alphavirus infections are maintained in the vector host, and in the detection and quantification of replicating virus from clinical specimens and potential reservoir hosts. In the course of developing these assays, we have shown that accurate quantification of cDNAs generated from specific strands of viral RNA, in the presence of higher levels of falsely-primed cDNA products generated from competing RNA strands, requires incorporation of a unique tag sequence during reverse transcription, which in combination with a tag-specific primer can be used to specifically amplify cDNAs corresponding to the intended target strand during qPCR. While it was also possible to quantify specific strands of viral RNA with assays employing unmodified virus-specific primers, the accuracy of these measurements depends on conditions in which lower relative levels of cDNA generated from the competing strand are present during qPCR. Therefore, previously reported results obtained with assays of this type should be interpreted with caution, particularly when the assay in question has been used to measure amounts of anti-genomic strands, which are typically less abundant than genomic RNAs in cells and tissues infected with RNA viruses.


Difficult problems in structural bioinformatics are often studied in simple exact models to gain insights and to derive general principles. Protein folding, for example, has long been studied in the lattice model. Recently, researchers have also begun to apply the lattice model to the study of RNA folding.http://www.cs.usu.edu/~mjiang/rna/DeltaIS/.We present a novel method for predicting RNA secondary structures with pseudoknots: first simulate the folding dynamics of the RNA sequence on the 3D triangular lattice, next extract and select a set of disjoint base pairs from the best lattice conformation found by the folding simulation. Experiments on sequences from PseudoBase show that our prediction method outperforms the HotKnot algorithm of Ren, Rastegari, Condon and Hoos, a leading method for RNA pseudoknot prediction. Our method for RNA secondary structure prediction can be adapted into an efficient reconstruction method that, given an RNA sequence and an associated secondary structure, finds a conformation of the sequence on the 3D triangular lattice that realizes the base pairs in the secondary structure. We implemented a suite of computer programs for the simulation and visualization of RNA folding on the 3D triangular lattice. These programs come with detailed documentation and are accessible from the companion website of this paper at Folding simulation on the 3D triangular lattice is effective method for RNA secondary structure prediction and lattice conformation reconstruction. The visualization software for the lattice conformations of RNA structures is a valuable tool for the study of RNA folding and is a great pedagogic device. Difficult problems in structural bioinformatics are often studied in simple exact models to gain insights and to derive general principles. Protein folding, for example, has long been studied in the lattice model -4. RecenIn this paper, we present a method for predicting RNA secondary structures with pseudoknots by folding simulation on the 3D triangular lattice. A drawback prevalent in the existing models for RNA secondary structure prediction is that they are static: the predicted secondary structures are selected only because they have the lowest free energies. But RNA folding is a dynamic process. RNA pseudoknots that are too complex are difficult to realize naturally because of the barriers in the energy landscapes due to the steric constraints of excluded volume. To cope with this problem, various restrictions may be imposed on the predicted secondary structures; for example, the hairpin constraint requires two bases forming a base pair be separated by at least three other bases in the sequence. The steric constraints of excluded volume, however, are inherently geometric; it is impossible to capture them all with only a few combinatorial restrictions. Our method overcomes this difficulty by simulating the folding of RNA tertiary structures on the 3D triangular lattice directly, as a dynamic process; the secondary structures are then extracted from the tertiary structures. Folding simulation can be computationally intensive  because of its exaustive-search nature, especially for complex models that include considerable details. Thus the simulation of RNA folding dynamics has only been used in mapping and analyzing the energy landscapes of certain small RNAs ,13. NeveRNA secondary structures can be visually represented in many different ways. Besides the simple representations of the dot-bracket notation, the Feynman diagram, and the dot plot, most RNA software packages such as Mfold ,22 and VUGUGCGUUUCCAUUUGAGUAUCCUGAA(((((((:(((((::::::::[[[[::AACGGGCUUUUCAGCAUGGAACGUACA::[[[[::::{{{{:))))))))))))UAUUAAAUAGUAGGAGUGCAUAGUGGC::::::::::::::::::::::::::]CCGUAUAGCAGGCAUUAACAUUCCUGA]]]:::::]]]]:::::::::::}}}}Despite the best effort, flat drawings of such complicated secondary structures will inevitably contain crossing edges or edges of different lengths that distort the true geometric structures. The simulation of RNA folding on the 3D triangular lattice provides an alternative method for the visualization of RNA secondary structures. Given a secondary structure of an RNA sequence, we can first reconstruct a lattice conformation of the sequence that realizes the base pairs in the secondary structure, then display the lattice conformation using a 3D visualization software. The lattice conformation of an RNA secondary structure may not represent the true tertiary structure, but at least it preserves geometric properties such as proportion and distance and hence looks more realistic.RNA folding on the 3D triangular lattice http://www.cs.usu.edu/~mjiang/rna/DeltaIS/.We implemented a suite of computer programs for the simulation and visualization of RNA folding on the 3D triangular lattice. These programs come with detailed documentation and are accessible from the companion website of this paper: In this section, we introduce the 3D triangular lattice and explain our choice of this particular lattice over other alternatives.xy diagonal, then skew the square lattice until the angle between x, y) has six neighbors , , , , , and . Using the auxiliary axis We first examine the 2D triangular lattice as a warm-up exercise. Refer to Figures xy plane at an angle of 120° from both S of n bases can be represented as a turn sequence T of n - 1 directions over the alphabet Refer to Figure Many types of lattices have been proposed by researchers in structural bioinformatics. Besides the 2D and 3D triangular lattices ,10 and tRegularity: On the 3D triangular lattice, all lattice points have the same number of neighbors, and all pairs of adjacent lattice points have the same distance. This allows a uniform handling of geometric proximity in base pairing.Density: The coordination number of a lattice is the number of neighbors of each lattice point. The 3D triangular lattice has a coordination number of 12; the cubic lattice has a coordination number of only 6. Thus the 3D triangular lattice is better than the cubic lattice in approximating the 3D space. Indeed the 3D triangular lattice is the densest regular lattice in 3D. There exist, admittedly, alternative 3D lattices with even higher coordination numbers. The  lattice (which models chess moves of a knight in 3D) and other extensions of the cubic lattice by Skolnick et al. ]]))))))Although the two structures are very different, they have roughly the same number of base pair stackings and hence very close scores in the folding simulation. We recommend that the users of our program always run the prediction program multiple times on each sequence and consider each predicted structure as a possible candidate.As we observed from the scatter plot in Figure Folding simulation on the 3D triangular lattice is effective method for RNA secondary structure prediction and tertiary structure reconstruction. The visualization software for the lattice conformations of RNA structures is a valuable tool for the study of RNA folding and is a great pedagogic device.http://www.cs.usu.edu/~mjiang/rna/DeltaIS/.Our RNA folding programs have been tested on three major platforms: Microsoft Windows (Cygwin), Linux, and Mac OS X. The complete source code, documentation, data set, and experimental results can be downloaded from the companion website of this paper: RNA folding on the 3D triangular lattice MJ conceived of the study and designed the algorithms, the software, and the experiments. MM and MJ implemented a basic version of the software and performed initial experiments in a preliminary study. JG and MJ improved the accuracy of the RNA secondary structure prediction method, optimized the source code, revised the documentation, and created the companion website. All authors read and approved the final manuscript.


Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors.The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA.Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism. In eukrRNA 27S ,40,44. TrRNA 27S ,45. Accoy-U RNAs . Here weP. horikoshii RNase P is formed by the RNA component and five proteins, including Rrp30 [in vivo.Pa1135 was shown here to bind poly-rAU RNA but contrary to PaNip7 does not strongly affect the exosome RNase activity, although Pa1135 inhibits RNA binding by the RNase PH ring and PaCsl4-exosome complexes. The ng Rrp30 . Since PAlthough eukaryotic SBDS/Sdo1p has not been shown to interact with the exosome, it has been shown to be required for pre-rRNA processing, for 60S ribosomal subunit translational activation and Tif6p recycling . In addiArcheoglobus fulgidus SBDS has shown that this protein contains an RNA binding domain [in vitro in a length-dependent manner, and competes with the PaRrp4-exosome for binding to A-rich RNAs. These results indicate that PaSBDS is also involved in RNA processing and may regulate one of the archaeal exosome complexes in vivo. Further indication of exosome regulation was obtained in RNA degradation assays, in which the RNase activity of the RNase PH ring and of the PaRrp4-exosome was slightly inhibited by PaSBDS. It is therefore possible that PaSBDS interacts with RNA in vivo and controls its processing by the exosome.Structural analysis of g domain . In thisin vivo. The evolutionary conserved structures and RNA affinities of Nip7 and SBDS raise the hypothesis that their eukaryotic orthologues also control the exosome function.We show in this work that two archaeal proteins that bind RNA can affect the Pa-exosome activity, making them candidates to be exosome regulatory factors. PaSBDS binds A- and AU-rich RNAs and inhibits mainly the PaRrp4-exosome. PaNip7 binds preferably AU-rich RNAs and strongly inhibits PaCsl4-exosome. Furthermore, similar to the eukaryotic counterparts, PaNip7 interacts with the Pa-exosome. Based on the results shown here that PaSBDS and PaNip7 inhibit preferentially specific exosome complexes, it is possible that these proteins control the Pa-exosome Escherichia coli strains used in this study were DH5α and BL21-CodonPlus (DE3)-RIL (Stratagene). Genomic DNA of P. abyssi GE5 was kindly provided by Dr. Patrick Forterre . Plasmid DNA was extracted using Qiagen plasmid purification systems. Restriction enzymes and other DNA-modifying enzymes were used as recommended by the manufacturer (New England Biolabs).The E. coli expression of PaRrp4 [R151A, R152A and PaNip7K155A, K158A [P. abyssi genomic DNA using primers PaSBDSfor (5'-AGGATCCCATATGCCTATTAGCGTTG-3') and PaSBDSrev (5'-CGGCCTCGAGTCATAGCCCCTT-3'), digested with BamHI-XhoI and inserted into vector pET-28a (Novagen), also digested with BamHI-XhoI. Pa1135 coding sequence was PCR-amplified from P. abyssi genomic DNA using primers Pa1135for (5'-GTTAGGGGGGATCCATGGCAG-3') and Pa1135rev (5'-CGGCCTCGAGTCAATCCTCCC-3'), and inserted into vector pET28a, digested with BamHI-XhoI. The PaRRP41 coding sequence was amplified from P. abyssi genomic DNA using primers PaRRP41for (5'-TACTCGAGCATATGATGGAGAAACCAGAAG-3') and PaRRP41rev (5'-ACGAATTCATCTCATTATCACTCACTTTC-3') and inserted into the PCR product cloning vector pGEM-T (Promega) prior to subcloning into the NdeI and SalI restriction sites of the expression vector pET29a (Novagen). PaRRP42 was amplified using primers PaRRP42for (5'-AGGGATCCCATATGAGTGATAATGAGATCG-3') and PaRRP42rev (5'-TACGCGTATCGATGTTATATCATTGCTTTGC-3'), inserted into pGEM-T vector (Promega) and subsequently subcloned into the BamHI and EcoRI sites of the expression vector pAE [NdeI and EcoRI sites of the expression vector pET28a (Novagen).Plasmids for f PaRrp4 , wild tyf PaRrp4  and mutaA, K158A  have beector pAE . PaCSL4 E. coli BL21-CodonPlus (DE3)-RIL strain, transformed with the correspondent plasmids. Expression was induced by addition of 10 mM lactose or, in the case of PaSBDS, 0.5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). Cells expressing PaRrp41 and PaRrp42 were harvested and suspended in buffer A . Cells expressing PaSBDS were suspended in buffer B . Cells expressing Pa1135 were suspended in buffer C . All cells were lysed in a French press. The lysate was heated at 85°C for 30 min and cooled on ice for 15 min. After centrifugation at 20,000 × g for 30 min, the supernatant was fractionated by affinity chromatography in Ni-NTA-agarose (Qiagen), followed by gel filtration in superdex 75 .Recombinant proteins were expressed in the R151A, R152A and PaNip7K155A, K158A were purified as described previously [E. coli, transformed with the plasmids pET29-PaRrp41 (KanR) and pAE-PaRrp42 (AmpR). For PaRrp4-exo assembly, cells expressing PaRrp4 were mixed with cells expressing PaRrp41 and PaRrp42, prior to lysis. PaCsl4 was obtained by co-expression with PaRrp42. PaCsl4+PaRrp42-expressing cells were mixed to PaRrp41+PaRrp42-expressing cells to obtain the PaCsl4-exo. Protein samples were concentrated by centrifugation using Centricon microconcentrators (Millipore). Protein content was determined by bicinchoninic acid (BCA) assay for protein quantitation (Sigma).PaNip7, PaNip7eviously ,48. To r32P-labeled RNAs in Tris-50 buffer , and 10 mM NaH2PO4, in 10 μl at 65°C for 15 minutes. RNA polymerization was accomplished under the same conditions, but using ADP instead of NaH2PO4, in 20 μl at 37°C for 30 minutes. RNA degradation and polymerization products were resolved on 8% or 10% denaturing polyacrylamide gel and visualized on a Phosphorimager (MolecularDynamics).RNA phosphorolysis was assayed by incubating the complexes with 1 pmol 32P 5'-labeled 14-mer poly-rA, poly-rU, 13-mer poly-rC, and 21-mer poly-rAU (5'-UUAUUAUUUAUUUAUUAUUUA-3') oligoribonucleotides (IDT). The assays were performed under the same conditions as described previously [2 1 mM DTT, 100 μg/ml BSA, 0.8 U RNasin. Different concentrations of proteins were incubated with the substrate RNA in 20 μl at 37°C for 30 minutes. The samples were resolved on 8% native polyacrylamide gels and visualized on a Phosphorimager (MolecularDynamics).RNA binding assays were carried out with 1 pmol 32P 5'-labeled poly(rAU) oligoribonucleotides. Protein cofactors were incubated with the RNAs for 30 min at 37°C, after which time the exosome complexes and NaH2PO4  were added to the reactions, which were incubated at 65°C for further 15 min. The products were resolved on native or denaturing 8% polyacrylamide gels and visualized on a Phosphorimager (MolecularDynamics).RNA protection assays were carried out with 1 pmol A Phosphorimager (MolecularDynamics) was used for quantitation of the bands obtained from RNA degradation, polyadenylation and binding assays.PaNip7 was labeled with FITC (fluorescein isothiocyanate), by following the instructions of the manufacturer's protocol (Molecular Probes). 12.5 μg of PaNip7-FITC were incubated with 25 μg of the exosome complexes at 25°C for 30 minutes. The samples were subsequently subjected to electrophoresis on native 6% polyacrylamide gels, and the fluorescence was analyzed on a Phosphorimager (MolecularDynamics). Concomitantly, samples were subjected to size exclusion chromatography and the absorbance was monitored at 280 nm and the fluorescence was monitored at 520 nm.JSL purified the recombinant proteins and carried out the activity assays, protein interaction assays and drafted portions of the manuscript. CRRR cloned the archaea exosome genes, stablished the protein purification protocols, and drafted portions of the manuscript. MCTS helped with the purification of proteins. PPC cloned PaNip7 gene and performed mutagenesis to obtain PaNip7 mutants. FLP helped with PaNip7 labeling and analysis of protein complexes. NITZ and DF coordinated parts of the work, participated in the interpretation of data and drafted portions of the manuscript. CCO designed, organized and coordinated the experiments, drafted the manuscript and edited the final text. All authors read and approved the final manuscript.in vitroFigure S1: Analysis of PaSBDS, PaNip7 and Pa1135 interaction with RNA oligonucleotides . Electrophoretic mobility shift assays with different radiolabeled RNA probes incubated with the indicated amounts of purified proteins. Proteins were incubated with 1 pmol of 10-mer, 12-mer or 14-mer poly-rA, 13-mer poly-rC, 14-mer poly-rU, or 21-mer poly-rAU RNA oligos at 37°C for 30 min. RNA-protein complexes were fractionated on 8% native polyacrylamide gels and visualized by phosphorimaging. (A) Increasing amounts of PaSBDS were added to the reactions: 50, 100, 200 or 400 pmol. PaSBDS binds poly-rA, poly-rU and poly-rAU. (B) 10, 50 or 100 pmol of PaNip7 were incubated with poly-rC or poly-rAU. PaNip7 binds poly-rAU efficiently. (C) 50, 100, 200 or 400 pmol of Pa1135 were incubated with poly-rA, poly-rU, poly-rC or poly-rAU. Pa1135 binds poly-rAU efficiently. -, No protein was added to the reaction. Free structured and unstructured RNA oligos and protein-RNA complexes are indicated on the right hand side.Click here for fileFigure S2: Purification of Pa-exosome complexes and regulatory proteins. Coomassie-stained polyacrylamide gels showing proteins purified through size exclusion chromatography. (A) Purification of RNase PH ring. (B) Purification of PaRrp4-exosome. (C) Purification of PaCsl4-exosome. (D) Purification of PaNip7. (E) Purification of PaSBDS. (F) Purification of Pa1135. (G) Purification of PaNip7R151A, R152A. (H) Purification of PaNip7K155A, K158A. M, molecular weight marker; TE, total extracts; FT, flow through; W, wash.Click here for file


The endothelium plays a key role in the development of atherogenesis and its inflammatory and proliferative status influences the progression of atherosclerosis. The aim of this study is to compare the effects of two beta blockers such as nebivolol and atenolol on gene expression in human umbilical vein endothelial cells (HUVECs) following an oxidant stimulus.HUVECs were incubated with nebivolol or atenolol (10 micromol/L) for 24 hours and oxidative stress was induced by the addition of oxidized (ox)-LDL. Ox-LDL upregulated adhesion molecules ; proteins linked to inflammation , thrombotic state , hypertension such as endothelin-1 (ET-1), and vascular remodeling such as metalloproteinases  and protease inhibitor (TIMP-1). The exposure of HUVECs to nebivolol, but not to atenolol, reduced these genes upregulated by oxidative stress both in terms of protein and RNA expression. The known antioxidant properties of the third generation beta blocker nebivolol seem to account to the observed differences seen when compared to atenolol and support the specific potential protective role of this beta blocker on the expression of a number of genes involved in the initiation and progression of atherosclerosis. It seoceptors , a key eoceptors , and alsoceptors , 4, 6, 9κB, AP-1) and the consequent induction of genes involved in the endothelialfunctions  and in the change of theextracellular matrix that is involved in the formation and progression of the atheroscleroticdisease.The oxidative status of cells of thecardiovascular system is dependent on the balance between ROS and endogenousantioxidants and recent studies indicate that increase in ROS is theintracellular signal and marker of their condition , 11. ExtIn view of the previous observation of thenebivolol noticeable inhibitory activity on ROS formation –6, the a2culture flasks  filled with 10 mL of M-199  containing 10% fetal calf serum , 2 mM glutamine , 30 μg ml−1 endothelial cell growth supplement , 100 μg ml−1 heparin , 100 U mL−1 penicillin-streptomycin , 100 μg mL−1 streptomycin , and 2.5 μg mL−1 amphotericin . The flasks were incubated at37°C, 100% humidity, and 5% of CO2. Themedium was refreshed every 2 days. At the beginning of each experiment, thecells were harvested by trypsinization, using 0.05% trypsin  and 0.537 mM ethylenediamine tetraaceticacid (EDTA) in phosphate buffered saline without calcium and magnesium . Thetrypsin was inactivated by dilution, and the cells were washed and counted.Cells were plated at a concentration of 40000 cells −12 on amultiwell plate (9.6 cm2 well−1) , havegrown for 2 days, and then used for the incubations.Human umbilical vein endothelial cells (HUVECs) wereobtained according to the method of Jaffe et al.  and usedHUVECs were harvested and characterized asto acetylated LDL binding and factor VIII expression, according to establishedand previously described techniques . To asseμM[Dl-nebivolol and atenolol were dissolved in ethanol and then diluted at the final concentration inculture medium M199. Identical dilutions of the solvent were prepared and usedas control. Since nebivolol was previously shown to reduce in aconcentration-dependent manner, the intracellular increase of ROS, andsuperoxide and to reduce them by 50% at 10 μM, this co−1), and processed for LDL separation in 1 day by sequentialflotation in NaBr solution containing 1 mg mL−1 EDTA [2+ modified LDL (1.7 mg protein mL−1) was prepared by exposure of LDL to5 μM CuSO4 for 18 hours at 37°C, and the extent ofLDL oxidation was determined by thiobarbituric acid-reactive substances, aspreviously described [μg protein mL−1 as previously reported [Whole blood, obtained by venipuncture from healthyvolunteers (with informed consent) after 12 hours of fasting, was collectedinto vacutainer tubes  containing EDTA , PECAM1 (CD31), SELE (E-selectin), SELL(L-selectin), SELPLG (P-selectin), VCAM1.CCL2 (MCP-1), CCL5 (RANTES), CCL20 (exodus-1), CSF2 (GM-CSF), CSF3(G-CSF), CX3CL1, IFNB1, IL1B, IL3, IL6, IL7, IL8, IL11, IL14, IL15, TNF.ANXA5 (annexin V), CPB2(TAFI), F3 (tissue factor), PLAT (tPA), PLAU (u-PA), SERPINE1 (PAI-1), TFPI, TFPI2, THBD (thrombomodulin),VWF.MMP1 (collagenase-1), MMP2 (gelatinase A),MMP3 (stromelysin-1), MMP7 (matrilysin), MMP8 (neutrophil collagenase), MMP9.TIMP1, TIMP2, TIMP3.EDN1 (ET-1), EDN2 (ET-2), EDN3 (ET-3), EDNRA(ETA), EDNRB (ETB).Proteinsrelated to the genes involved in the oxidative stress have been measured in themedium by assay kits based on ELISA system in solid phase. For quantification,the following kits have been used for ICAM-1, E-selectin,P-selectin, IL6, TNF-alpha, MMP-2, MMP-9, TIMP-1, and ET-1 R&D Systems kits (USA); forICAM-2 and ICAM-3Bender MedSystems kits ; for tissuefactor, u-PA, PAI-1 American Diagnostica kits .Results are expressed as means ± SD. Statistical analysis wasperformed by analysis of variance followed by Tukey's test post-hoc analysis.All data were analyzed using the “SPSS 11” program for Macintosh.P < .001) after the exposureof HUVECs to ox-LDL as both RNA (P < .01), by 37.9 ± 4.6% for RNA (P < .01), by 30.0 ± 3.5% for RNA (P < .01), respectively.Similarly, only nebivolol significantly reduced selectins expression induced byox-LDL by 30.4 ± 3.7% and 36.1 ± 4.1% for RNA (P < .01).Among the genes examined by macroarray, only ICAM-1,ICAM-2, ICAM-3, E-selectin, P ligand-selectin, IL-6, TNF-alpha, tissue Factor,PAI-1, uPA, TIMP-1, MMP-2, MMP-9, and ET-1 were upregulated by the exposure ofHUVECs to ox-LDL and, therefore, the protein related to them was measured. The expression of the adhesionmolecules studied  significantly increased . Nebivolol,but not atenolol, was able to reduce this increase by 48.4 ± 5.6% for RNA(P < .01), by 53.7 ± 5.9%for RNA (P < .01), respectively.The expression of bothRNA  and prot for RNA, by 46.3 for RNA of IL-6 for RNA , and by for RNA  of TNF-aP < .001) by the exposure of HUVECs to ox-LDL. Nebivolol,but not atenolol, at least in part reduced these values as follows: tissue factor −42.1 ± 4.8% for RNA, −30 ± 2.7% for protein (P < .01), PAI-1 −41.7 ± 4.6% and −27.1 ± 2.5% for protein (P < .01), uPA −35.6 ± 4.1% for RNA, and −25.9 ± 2.8% for protein (P < .01).As shown in P < .001). Nebivolol, but not atenolol, was able toreduce this increase by 56.7 ± 6.3% for RNA (P < .01), by 29.6 ± 3.5% for RNA(P < .05) for protein(P < .01), respectively. Finally also the expression of ET-1 was significantly increased (P < .001) after the exposureof HUVECs to ox-LDL as both RNA (P < .01), respectively.Overexpression ofmetalloproteinases (MMP-2 and MMP-9) and protease inhibitor TIMP-1 wassignificantly induced by ox-LDL in HUVECs both as RNA  and prot for RNA , by 43.9 for RNA  of MMP-2 for RNA, by 25.5 for RNA of MMP-9 for RNA, and by  protein of TIMP-both RNA  and protboth RNA . Nebivolboth RNA  and by 4both RNA  (P < .01In the present study, we have examined theeffect of nebivolol and atenolol on gene expression in response to oxidative stress induced by ox-LDL incultured HUVECs. Ox-LDL is a proatherogenic lipoprotein that has been identified as a potent stimulus for vascular ROS and has beensuggested to modulate gene expression of vascular cells  by a speIn this study, we also found thatnebivolol decreased ICAMs, P- and E-selectin at the mRNA, and protein level expression in HUVECs exposed to oxidative stress, whereas atenolol wasineffective. Our findings, even in different experimental conditions, agreewith a very recent study showing that nebivolol reduced adhesion moleculesgenes expression in human coronary endothelial and vascular smooth muscle cells . AdhesioAnother important result of thisstudy is the ability of nebivolol, but not of atenolol, to significantly reducethe oxidative stress-induced TNF-alpha, IL-6, and ET-1 gene expression inHUVECs. This finding is in agreement with a previous study showing thatnebivolol can reduce ET-1 secretion in human coronary smooth muscle and endothelialcells . InflammIn our study, nebivolol alsosignificantly reduced the increased gene expression of tissuefactor, one of the pivotal thrombogenic stimuli, PAI-1and uPA induced by ox-LDL. Atenolol on the contrary was ineffective. In thiscontext, it has been recently demonstrated in hypertensive patients thattreatment with nebivolol wasassociated with a favorable modification of hemostatic and fibrinolytic statusin addition to its antihypertensive effects . The plaAn extensive network of additional proteases, inhibitors, receptors, andmodulators is directly associated and influenced by the PA system, the largestgroup being the MMPs and their respective inhibitors TIMPs . MMPs plOur results showing that ox-LDL increased the expression of MMPs inHUVECs confirm other experimental evidences demonstrating that oxidative stressdirectly activate MMPs posttranslationally . PresentIn conclusion theresults of our study demonstrated that in cultured endothelial cells exposed tooxidative stress nebivolol can reduce the expression of a number of genesinvolved in the initiation and progression of atherosclerosis. We and othershave already demonstrated that nebivolol decreased ROS concentration by affecting thesignalling pathways leading to NADPH oxidase activation in endothelial cells , in expeOur findings suggestthat nebivolol, in addition to its antihypertensive effect, can be useful inpreventing atherosclerotic complications related to oxidative stress.


Two hairpin structures that are present at both the 5' and 3' end of the HIV-1 RNA genome have important functions in the viral life cycle. The TAR hairpin binds the viral Tat protein and is essential for Tat-mediated activation of transcription. The adjacent polyA hairpin encompasses the polyadenylation signal AAUAAA and is important for the regulation of polyadenylation. Specifically, this RNA structure represses polyadenylation at the 5' side, and enhancer elements on the 3' side overcome this suppression. We recently described that the replication of an HIV-1 variant that does not need TAR for transcription was severely impaired by destabilization of the TAR hairpin, even though a complete TAR deletion was acceptable.In this study, we show that the TAR-destabilizing mutations result in reduced 3' polyadenylation of the viral transcripts due to an extension of the adjacent polyA hairpin. Thus, although the TAR hairpin is not directly involved in polyadenylation, mutations in TAR can affect this process.The stability of the HIV-1 TAR hairpin structure is important for the proper folding of the viral RNA transcripts. This study illustrates how mutations that are designed to study the function of a specific RNA structure can change the structural presentation of other RNA domains and thus affect viral replication in an indirect way. All retroviral RNA genomes contain a repeat (R) region at the extreme 5' and 3' end. This sequence repeat allows the first strand transfer step of the reverse transcription process, which results in the formation of long terminal repeat (LTR) regions in the proviral DNA. The 97-nt R region in HIV-1 RNA can fold two stem-loop structures, the TAR and polyA hairpins Fig. . Both moThe polyA hairpin encompasses the AAUAAA polyadenylation signal that is recognized by the cleavage polyadenylation specificity factor (CPSF), resulting in polyadenylation of the viral transcripts. Whereas TAR should exert its function in the 5' LTR promoter context, the polyadenylation signal should be recognized exclusively in the 3' LTR context. Previous studies indicated that usage of the 3' polyadenylation site is promoted by an upstream sequence element (USE) in the U3 region that is uniquely present at the 3' end of viral transcripts -22. Thisin vitro produced transcripts revealed that the 5' TAR-destabilizing mutations affect the proposed riboswitch of the leader RNA, the so called LDI-BMH equilibrium  t,33 t32,3ilibrium -38. Wherilibrium  and packm hairpin or modified TAR sequences at both the 5' and 3' LTR. After culturing the cells with dox for two days, we quantified virus production by measuring the CA-p24 level in the culture medium , single spliced (4 kb) and double spliced (2 kb) HIV-rtTA transcripts, whereas normal amounts were observed for the AB mutant Fig. . SeveralTo avoid inclusion of vector sequences in the viral RNAs, we made a novel set of HIV-rtTA constructs in which the SV40-derived polyadenylation signal is positioned downstream of the 3' LTR Fig. . Transcrm) virus. This size increase corresponds with what one would expect for read-through transcription to the SV40 polyadenylation site, and was most prominent for the shorter multi-spliced transcripts. The same RNA shift was observed for 3' mutants A and B, but again not for the AB double mutant. To confirm that the longer transcripts are the result of polyadenylation at the SV40 polyadenylation site, the Northern blot membrane was stripped and hybridized with a probe that specifically detects the SV40 sequences present downstream of the 3' LTR. This analysis revealed that the extended transcripts do indeed contain this sequence . This demonstrates that these viruses do efficiently polyadenylate at the 3' LTR. In contrast, viruses with a single-side deletion in the 3' TAR element polyadenylated predominantly at the SV40 site, which resulted in the longer PCR product  or truncated (AB) 3' TAR structure are processed at the natural 3' polyadenylation site, whereas only 30–40% of the transcripts with a single-side deletion in 3' TAR  are polyadenylated at this position  and 3' polyadenylation site in the RNA transcript . This woin vitro replication, which indicates that TAR has no other essential function in HIV-1 replication [Several studies suggested that the TAR hairpin has other functions in HIV-1 replication in addition to its role in transcription, such as in translation, dimerization, packaging and reverse transcription of the viral RNA -15,42-44lication . MoreoveAlthough the TAR hairpin is not directly involved in polyadenylation of the HIV-1 RNA transcripts, destabilization of TAR does affect this process. This study demonstrates that the stability of TAR structure is important for proper folding of the adjacent polyA hairpin.F86Y A209T-2ΔtetO [Construction of the infectious HIV-rtTA molecular clone and variants with a deletion in the 5' TAR or both the 5' and 3' TAR elements were described previously ,34. For T-2ΔtetO . The Nco2.HeLa X1/6 is a HeLa-derived cervix carcinoma cell line containing chromosomally integrated copies of the CMV-7tetO luciferase reporter construct pUHC13-3 . HeLa X12 wells, grown to 60% confluency and transfected with 5 μg HIV-rtTA construct by calcium phosphate precipitation as previously described [g for 5 min, washed in 1 ml PBS, centrifuged at 2,750 × g for 5 min, resuspended in 0.6 ml RLT buffer (QIAGEN) and homogenized with a QIAshredder column (QIAGEN). Total RNA was isolated with the RNeasy kit (QIAGEN) procedure, and contaminating DNA was removed with RNase-free DNase (QIAGEN) that was added during the isolation procedure as described in the RNeasy protocol.C33A cells were cultured in 10-cmescribed . Cells wescribed . The cel32P-labeled with the High Prime DNA Labeling kit (Roche Diagnostics) and used as HIV-rtTA probe. To generate the SV40 probe, the HIV-rtTA-SV40 TARm molecular clone was digested with AatII and BamHI and the 276-bp fragment was isolated and labeled as described above. Prehybridization and hybridization was done in ULTRAhyb buffer (Ambion) at 55°C for 1 and 16 h, respectively. The membrane was then washed two times at room temperature for 5 min in low-stringency buffer  and two times for 10 min at 50°C in high stringency buffer . Images were obtained using the PhosphorImager (Amersham Biosciences) and data analysis was performed with the ImageQuant software package. The Northern blot was stripped by boiling the membrane at 70°C in 0.1% SDS for 3 times 1 h. The stripping efficiency was controlled by scintillation counting and the blot was hybridized with the SV40 probe after prehybridization.Gel electrophoresis of RNA was performed on a 1% agarose gel in MOPS buffer  with 7% formaldehyde at 100 Volt. The RNA was transferred overnight onto a positively charged nylon membrane (Boehringer Mannheim) by means of capillary force. RNA was attached to the membrane with a UV crosslinker (Stratagene). The 373-bp EcoRV-HinDIII fragment of HIV-rtTA encoding the U3/R region was 25 and random hexamers primers. The cDNA product was used as template in a polymerase chain reaction (PCR) with primers 1 (GAG ACC ATC AAT GAG GAA GCT GCA GAA TGG GA) and 2 (GGC CGG CCC TTG TAG GCC GGC CAG ATC TTC CC) to detect the unspliced RNAs, with primers 3 (TCA ATA AAG CTT GCC TTG AGT GC) and 4 (CTC CGC AGA TCG TCC CAG AT) to detect the single spliced RNAs, and with primers 3 and 5 (CTA TGA TTA CTA TGG ACC ACA CA) to detect the double spliced RNAs. The polyadenylated RNAs were detected with primer 6 (CTG TGT CAG CAA GGC TTC TC) and the 3'RACE adapter primer 7 (GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T) that anneals to the polyA tail. The cDNA was denatured at 94°C for 5 min and amplified in 35 cycles of 1 min 95°C, 1 min 55°C, 2 min 72°C and a final extension time of 7 min at 72°C. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide.To analyze splicing and polyadenylation of the viral transcripts, the isolated RNA was reverse transcribed with ThermoScript reverse transcriptase at 50°C (Invitrogen), using the oligo(dT)2 wells to 60% confluency and transfected with 1 μg of the HIV-rtTA constructs and 0.5 ng pRL-CMV (Promega), in which the expression of Renilla luciferase is controlled by the CMV immediate-early enhancer promoter, to allow correction for differences in transfection efficiency. The cells were cultured with dox (1 μg/ml) and the medium was refreshed after 16 h. The culture medium was collected after 48 hours for CA-p24 measurement. The cells were washed with 1 ml PBS and subsequently lysed with passive lysis buffer (Promega). Firefly and Renilla luciferase activities were determined with the dual-luciferase assay (Promega). The rtTA level was calculated as the ratio between the firefly and Renilla luciferase activities and corrected for between session variation [HeLa X1/6 cells were cultured in 2-cmThe authors declare that they have no competing interests.MMV and AH performed the experiments. MMV drafted the manuscript. ATD and BB designed the experiments and revised the manuscript.


Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA–RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA–based interaction spanning ∼3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes. i.e. messenger-sensed) RNA viruses are responsible for significant diseases in plants and animals. The single-stranded RNA genomes of these viruses serve as templates for translation of viral proteins and perform other essential functions that generally involve local RNA structures, such as RNA hairpins. Interestingly, plant tombusviruses utilize a number of long-range intra-genomic RNA–RNA interactions to regulate important events during infection of their hosts, i.e. viral translation and transcription. Here, we report that an additional essential tombusvirus process, viral RNA replication, also requires a long-range RNA–RNA interaction. Our analyses indicate a role for this RNA–based interaction in the assembly of the viral replicase, which is responsible for executing viral RNA synthesis. This information was used to generate a comprehensive higher-order RNA structural model for functional long-range interactions in the genome of this eukaryotic RNA virus. The model highlights a critical role for global RNA structure in multiple viral processes that are necessary for successful infection of hosts.Plus-strand ( Plus-strand RNA viruses infect a wide variety of organisms and are responsible for causing significant diseases in plants, animals, and humans. A key step in the reproduction of these pathogens is replication of their single-stranded RNA genomes. This process occurs in the cytosol of host cells in association with membranes and requires a virally-encoded RNA-dependent RNA polymerase (RdRp) e.g. RNA hairpins); however, this perspective is rapidly changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA–RNA interactions spanning significant distances Besides being templates for replication, plus-strand RNA genomes also serve additional functions during infections, including acting as templates for (i) translation of viral proteins, (ii) transcription of viral mRNAs, and (iii) assembly of virus particles. Accordingly, these RNA genomes are multifunctional molecules that possess regulatory mechanisms to ensure that these different processes occur accurately and at the proper time during the infectious cycle. Integral to this control is the presence of different regulatory RNA elements within viral genomes that act as signals for modulating molecular events. Traditionally, such RNA elements have been viewed as localized sequences or structures , have been excellent model systems for understanding molecular aspects of virus reproduction and, particularly, the role of both local and long-range RNA elements in regulating and coordinating the multiple processes that occur during viral infections Viral RNA replication in TBSV has been studied extensively in both plant and yeast cells and the latter system has served as a genetically-tractable surrogate host that supports authentic viral RNA replication In this report we have pursued the hypothesis that the distantly located RII and RIV require some form of communication to facilitate replicase assembly. Our results support this theory by identifying a long-range RNA–RNA interaction in the TBSV genome that unites RII and RIV and is essential for efficient replicase assembly and viral RNA replication. This finding, along with previous results, has allowed us to generate an integrated higher-order RNA structural model for functional long-range interactions in the genome of a eukaryotic RNA virus. Mechanistic and evolutionary insights provided by this model are discussed.i.e. R3.5, which includes the 3′CITE) would position the putative interaction close to RIV in the higher-order RNA structure  necessary for replicase assembly. Scheme 3: Protein factors bind to the individual non-united RII and RIV elements; the UL–DL interaction then juxtaposes the bound factors, which mediates replicase assembly. With respect to the latter scheme, studies have shown that viral p33 is able to bind in vitro to RII(+)-SL in the absence of other RNA elements e.g. preferentially facilitating the amplification of full-length RNA templates, and such advantages may also be relevant to TBSV. However, TBSV is distinct from both DenV and Q-beta phage in that it produces 3′-coterminal sg mRNAs , an RNA–based interaction spanning ∼11,000 nts in its genome has been proposed to reposition the viral RdRp, bound to its 5′-proximal promoter, close to the 3′-end of the genome; thereby allowing it to efficiently initiate minus-strand synthesis sg mRNAs . Notablyi.e. 3′CITE/5′UTR) and transcriptional (i.e. AS1/RS1 and AS2/RS2) RNA elements , and then transcription. Accordingly, each of the different types of functional long-range RNA–based interactions would need to be dynamic and able to form in parallel with this series of events. Mechanistically, some of these interactions are predicted to be inhibitory to others and, accordingly, some processes may be impeded by others. Such relationships could be integral to coordinating different events or may simply act as safe-guards to prevent two processes from occurring simultaneously. For example, the UL–DL interaction would be inhibited by translation that is mediated by the 5′-3′ SL3-SLB interaction, because ribosomes translating the read-through portion of the p92 ORF would disrupt the UL–DL interaction (as well as the RII structure)  . Conversructure) , as has TBSV represents an extreme example of long-range RNA–RNA interactions participating in three distinct viral processes. Similar types of distal RNA networks have also been reported in other positive-strand RNA viruses. For instance, the luteovirus BYDV utilizes two distinct sets of long-range RNA–based interactions for mediating translational initiation Sg mRNA transcription in the nodavirus Flock house virus involves an intra-genomic long-distance interaction For genome replication, the potexvirus Potato virus X (PVX) utilizes an extensive set of long-range RNA–RNA interactions A role for long-range intra-genomic interactions in viral replication has also been demonstrated for different flaviviruses Conceptualizing viral RNA genomes as complex higher-order RNA structures will provide valuable models for understanding their many functions. Indeed, recent computationally-based structural studies suggest that viral genome-scale ordered RNA structures (GORS) are more prevalent than previously appreciated Constructs described previously that were used in plant-based experiments include: T100, the wt TBSV genome construct 32P-labeled probes and relative isotopic levels were determined using PharosFx Plus Molecular Imager (BioRad).In vitro RNA transcripts of viral RNAs were generated using T7 RNA polymerase as described previously DI RNA stability assays were performed as described previously N. benthamiana plants were supplemented with a buffer containing NTPs (UTP being labeled), viral RNA templates and other components. The reactions were then incubated at 25°C for 120 min. After phenol/chloroform extraction and ammonium acetate/isopropanol precipitation, half the amount of the RNA products was analyzed in 5% polyacrylamide-8% urea gels followed by detection by autoradiography.Plant-derived replicase assays were carried out as described previously 32P-labeled complementary-strand product generated by 5% polyacrylamide-8% urea gel electrophoresis and autoradiography. Standard western blot analysis was used to monitor the levels of p33 expressed in the cells used for replicase purification Yeast replication assays were carried out in whole cells expressing p33, p92, and the RNA replicon transcript from cotransformed plasmids, as described previously Translation of sub-saturating amounts (0.5 pmol) of RNA transcript was carried out in nuclease-treated wheat germ extract (Promega) as described previously Figure S1Optimal RNA secondary structure predicted for full-length TBSV RNA genome as determined by mfold analysis.(1.91 MB PDF)Click here for additional data file.