Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality and could be inhibited by divalent ions, such as Mg2+, Mn2+, Ca2+ or Zn2+, to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5′-3′ cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5′-3′ panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis.For double-stranded RNA (dsRNA) viruses in the family  Reoviridae) that contain segmented double-stranded RNA (dsRNA) genome packaged by a single- or double-layered inner capsid and include several important pathogens that are responsible for diseases in humans, livestock animals, insects and plants , which contains 10 genomic dsRNA segments, is one of the 15 genera within the family Reoviridae and is further classified into 16 types (cypovirus 1–16) based on the electrophoretic migration profiles of their genome segments (http://www.ictvonline.org/virusTaxonomy.asp?version=2012). And the CPV particles are embedded in polyhedra for surviving unfavorable external environment  was initially isolated by our laboratory in 2006 from a mixture of HaCPVs and can quickly cause lethal disease in H. armigera, which is one of the most serious agricultural pests in China  through a CPV panhandle-structured RNA template, thereby strongly suggesting a direct role of the RNA chaperone activity of VP5 in the initiation of cypoviral dsRNA replication.The type 5 in China . Thus fain China ,25. HaCPin China ,12,26. ISupplementary Table S1. The constructed plasmids were subjected to the Bac-to-Bac system (Invitrogen) to express wild type and mutant MBP fusion VP5 (MBP-VP5) proteins.Standard procedures were used for extraction of viral genome RNA and reverse transcription (RT)-polymerase chain reaction . A cDNA g to remove debris. The protein in the supernatant was purified using amylose affinity chromatography , according to the manufacturer’s protocol. Fractions containing the recombinant protein were combined, followed by concentration using an Amicon Ultra-15 membrane column . After that, the buffer was exchanged to 25 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)-KOH (pH 7.5), 50 mM NaCl, and the protein was stored at −70°C in aliquots. All proteins were quantified by the Bradford method.The expression and purification of recombinant MBP-VP5 and its derivatives as well as the negative-control protein MBP were carried out as previously described ,32. BrieSodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot assays were performed as described previously . The anthttp://robetta.bakerlab.org/)] (http://swissmodel.expasy.org)]. The figure of the modeled HaCPV-5 VP5 3D structure was drawn by PyMOL program 1.1  from coordinate file. The surface representation of modeled VP5 on CPV particle are drawn by PyMOL 1.1 based on the atomic cryoEM structure of BmCPV capsid (PDB number 3IZX) by replacing BmCPV VP5 with HaCPV-5 VP5.The 3D structure of HaCPV-5 VP5 was modeled by submitting its amino acid sequence to the HMMSTR/Rosetta server  . Five moin vitro transcription using T7 RNA polymerase . The transcribed RNA strands were purified by Poly-Gel RNA Extraction Kit  according to the manufacturer’s instruction.RNA or DNA helix substrates were prepared by annealing two complementary nucleic acid strands. One strand was labeled at 5′-end with hexachloro fluorescein (HEX) , and the other strand was unlabeled. The two strands were mixed in a proper ratio, and annealed through heating and gradually cooling. All unlabeled DNA strands were synthesized by Invitrogen, and HEX-labeled DNA and RNA strands were purchased from Takara. Unlabeled RNA strands were synthesized by us from the Supplementary Table S2.Standard RNA helix substrate was annealed with RNA1 and RNA3, D*/R substrate was annealed with DNA1 and RNA3, R*/D substrate was annealed with RNA1 and DNA2, D*/D substrate was annealed with DNA1and DNA2, 3′-tailed substrate was annealed with RNA1 and RNA4, 5′-tailed substrate was annealed with RNA1 and RNA5, and blunt-ended substrate was annealed with RNA1 and RNA2. The 3′-tailed RNA helix substrates with different lengths of 3′-tails were prepared by annealing RNA1 with and RNA4, RNA6, RNA7, RNA8 and RNA9. The sequences of all DNA and RNA strands are listed in Gel mobility shift was performed in 25 mM HEPES-KOH (pH 7.5), 100 mM NaCl, in a volume of 10 μl with indicated amount of protein and 0.1 pmol of ssRNA (RNA1) or blunt-ended dsRNA (RNA1/RNA2). RNA1 is HEX-labeled. Reactions were incubated for 30 min at room temperature. For the competition experiments, unlabeled ssRNA or dsRNA competitor was added together with the HEX-labeled RNA probe (RNA1) to the binding reaction. The reactions were terminated by the addition of 2.5 μl 5× sample buffer . The nucleic acid–protein complexes were separated by electrophoresis on 2% agarose gels, and gels were scanned by a Typhoon 9200 imager . The values of Hill coefficient, an indicator of the cooperativity of RNA binding, were calculated by applying Hill transformation to the RNA binding data obtained in repeated experiments.Chemical cross-linking assays were performed as previously described . MBP-tag2, 5 mM Dithiothreitol (DTT) and 5 U RNasin (Promega) and incubated at 37°C for 60 min unless otherwise indicated. The reactions were terminated by adding 5 U proteinase K and 2.5 μl 5× loading buffer . Mixtures were electrophoresed on 12% native-PAGE gels. Gels were scanned with a Typhoon 9200 imager . The ratio of released single strands versus the total substrates was quantified with ImageQuant software.The standard helix destabilizing assay was performed as previously described ,32 with 2, 2 mM DTT, 0.01% BSA and 5 U RNasin. The reaction was terminated and analyzed as described above. For the hybridization assay of the stem-loop structured RNA strands, the sequences of the two stem-loop RNA strands were indicated in http://mfold.rna.albany.edu/?q=mfold). For the hybridization of the HEX-labeled 46-nt and unlabeled 146-nt RNA strands, their sequences are listed in Supplementary Table S2. Then mixtures were also resolved on 12% native-PAGE gels  in a eukaryotic (baculovirus) expression system and then purified the protein B and C. Supplementary Figure S1).Moreover, we incubated 0.01 μM of HEX-labeled RNA1 with increasing concentrations of MBP-VP5 (0.05–0.48 μM), and our result showed that MBP-VP5 binds to ssRNA in a dose-response manner C. The RNin vitro chemical cross-linking assay was performed, followed by SDS-PAGE and western blot analysis. Our result showed that MBP-VP5 has both monomeric and dimeric forms in solution . Then, it is intriguing to determine which form of MBP-VP5 binds to RNA. To this end, MBP-VP5 was incubated with HEX-labeled RNA1 probe and then subjected to UV cross-linking. The purpose of this step is to label the VP5 proteins that are binding to RNA. After that, the samples were subjected to chemical cross-linking with 0.01% [vol/vol] glutaraldehyde, and analyzed via SDS-PAGE and scanned by a Typhoon 9200 to visualize the presence of HEX-labeled RNA. Our results showed that both monomeric and dimeric forms of MBP-VP5 can bind to ssRNA in vitro  and hantavirus N do ,35. For Supplementary Figure S1), suggesting that the protein binding to nucleic acids is the prerequisite but not the only determinant for helix destabilization.Because some viral proteins with RNA helix-destabilizing activities can also unwind DNA helices or RNA–DNA hybrids, we sought to determine whether VP5 can destabilize nucleic acid helices containing DNA. To this end, we constructed three different helix substrates: D*/D B by anneTo further confirm the helix-destabilizing activity of VP5, we generated point mutations  at the highly conserved residues within the consensus regions of CPV VP5 A. These To characterize the helix-destabilizing activity of VP5, we designed three different RNA helix substrates: one containing a 3′ single-stranded tail (15 bases), one containing 5′ single-stranded tail (15 bases) and one with blunt ends A and B, Ectropis obliqua picorna-like virus (EoV) nonstructural protein 2C, which also displays RNA chaperone activity  of 3′ single-stranded tails were generated A, and unactivity .Figure Previously, we established the RNA helix-destabilizing activity of HaCPV-5 VP5 in the absence of ATP. Thus, we sought to determine whether the presence of ATP has any effect on the unwinding activity of VP5. For this purpose, the standard RNA helix substrate with both 3′ and 5′ tails A was incAfter determining that ATP has an inhibitory effect on helix destabilization by VP5, we sought to determine whether other nucleoside triphosphates (NTPs) or dNTPs have similar or different effects. To this end, the destabilizing activity of VP5 was assessed in the presence of ATP, GTP, CTP, UTP, dATP, dGTP, dCTP or dTTP at a final concentration of 0.5 mM. As shown in To characterize the activity of VP5 in destabilizing RNA secondary structures, we adapted a canonical assay, which was initially developed by DeStefano and colleagues for measuring the helix-destabilizing and annealing acceleration activities of vRNA chaperones , for thiTo further determine the strand-annealing stimulation activity of VP5, we generated a shorter HEX-labeled RNA strand (46 nt in length) and a longer nonlabeled complementary RNA strand , and the inhibitory effect of Zn2+ was more dramatic (lane 6).To further characterize the biochemical properties of VP5, we evaluated its RNA helix-destabilizing activity under various conditions with different divalent metallic ions, ion concentrations and pH values. First, we assessed the requirement of divalent metallic ions, including Mg2+, Mn2+, Ca2+ or Zn2+ concentrations on the helix-destabilizing activity of MBP-VP5. Of note, because the RNA duplex substrate was completely unwound by VP5 in the presence of Mg2+ or Ca2+ when incubated for 60 min, a shorter incubation time (30 min) was used in the subsequent experiments to measure the effects of varying ion concentrations. Our results showed that the destabilizing activity was optimal at 0 mM for Mg2+ or Mn2+  on VP5-mediated helix destabilization  through the cypoviral panhandle structure. To this end, a structured RNA mimicking the cypoviral panhandle structure was constructed using 36 nt from the 5′-end and 30 nt from the 3′-end of HaCPV-5 RNA segment 8, and then used as the template for RT RNA should occur before or when the (−)RNA synthesis proceeds on the cyclized (+)RNA template , it is pTo further confirm the capacity of VP5 to facilitate RT through the panhandle structure, we conducted the reactions at varying molar ratios of VP5 to the panhandle-structured template. Our data showed that the stimulating effect of VP5 on the RTs was optimal at molar ratios of 10:1–50:1 RNA template for replication initiation.As one of the three major CPV capsid proteins, VP5 is also named as clamp protein or large protrusion protein. Previous studies have demonstrated that 120 copies of VP5 are located on the surface of the CPV capsid, exist in two conformations and function as molecular clamps to interact with and tie together neighboring VP1 shell proteins, thus enhancing the stability of the CPV capsid ,33. MoreIt is believed that the vRNA molecules of many RNA viruses could be kinetically trapped in incorrect/inactive intermediate structures, thereby probably requiring RNA chaperones to facilitate proper RNA folding and efficient vRNA replication/translation ,43,44. SReoviridae, CPV VP5 may not be the only RNA chaperone, as rotavirus nonstructural protein 2 (NSP2), which is a multifunctional enzyme involved in rotaviral dsRNA replication, was previously shown to contain ATP-independent nucleic acid helix-destabilizing activity -vRNAs (mRNAs) are encapsidated into the nascently assembled inner capsids and are then used by reoviral RdRPs as replication templates to synthesize genomic dsRNA segments within the inner capsids ,2,4. As As a heterogeneous group of proteins that share no consensus sequences or motifs, RNA or nucleic acid chaperones are poorly understood in regard to the mechanism(s) governing their ATP-independent helix-destabilizing and annealing stimulation activities. To explain the RNA chaperone activities, an ‘entropy transfer’ model has been proposed. According to this model, RNA chaperones contain intrinsically disordered (unstructured), highly flexible regions that can transfer their disorder or entropy to misfolded RNA molecules on binding to RNA. Such an entropy transfer can destabilize kinetically trapped misfolded RNA molecules, leading to the rearrangement of RNA folding in an ATP-independent manner ,14. So fReoviridae, CPV is well recognized as an ideal and simplified model for studying the mechanisms of vRNA replication and mRNA capping of the Reoviridae and probably other dsRNA Viridae. In this study, we found that a CPV capsid clamp protein VP5 possesses a novel RNA chaperone activity, which is thought to be directly involved in the initiation of CPV dsRNA replication. These findings show that CPV capsid proteins not only include RNA polymerase and mRNA capping enzyme, but also RNA chaperone that is important for vRNA replication and/or translation. This study should extend our understanding of RNA replication of CPV, Reoviridae and dsRNA Viridae, and also enrich our knowledge about virus-encoded RNA chaperones. Furthermore, since the (+)-vRNA cyclization and panhandle structures commonly exist in the family Reoviridae, encoding an RNA chaperone or other RNA remodeling protein, like helicase, might be a common strategy for all reoviruses. Future studies by our group and others should reveal whether rotavirus NSP2, the clamp proteins of other turreted reoviruses, as well as other reoviral structural or nonstructural proteins can also function in 5′-3′ panhandle unwinding and RNA replication initiation.As a single-shelled member of the family Supplementary Data are available at NAR Online.National Basic Research Program of China ; the National Natural Science Foundation of China ; the Fundamental Research Funds for the Central Universities [204274391 to J.Y.]; the Chinese 111 Project [B06018]. Funding for open access charge: Wuhan UniversityThis work was supported by Conflict of interest statement. None declared.


I will offer some possible responses to these objections in the light of work by our and other labs. Finally, I will critically discuss an alternative theory to the RNA world hypothesis known as ‘proteins first’, which holds that proteins either preceded RNA in evolution, or – at the very least – that proteins and RNA coevolved. I will argue that, while theoretically possible, such a hypothesis is probably unprovable, and that the RNA world hypothesis, although far from perfect or complete, is the best we currently have to help understand the backstory to contemporary biology.The problems associated with the RNA world hypothesis are well known. In the following I discuss some of these difficulties, some of the alternative hypotheses that have been proposed, and some of the problems with these alternative models. From a biosynthetic – as well as, arguably, evolutionary – perspective, DNA is a modified RNA, and so the chicken-and-egg dilemma of “which came first?” boils down to a choice between RNA and protein. This is not just a question of cause and effect, but also one of statistical likelihood, as the chance of two such different types of macromolecule arising simultaneously would appear unlikely. The RNA world hypothesis is an example of a ‘top down’ (or should it be ‘present back’?) approach to early evolution: how can we simplify modern biological systems to give a plausible evolutionary pathway that preserves continuity of function? The discovery that RNA possesses catalytic ability provides a potential solution: a single macromolecule could have originally carried out both replication and catalysis. RNA – which constitutes the genome of RNA viruses, and catalyzes peptide synthesis on the ribosome – could have been both the chicken This article was reviewed by Eugene Koonin, Anthony Poole and Michael Yarus (nominated by Laura Landweber). The problems associated with the RNA world hypothesis are well known, not least to its proponents,2. In the.g. has been reduced by ritual abuse to something like a creationist mantra”, and“[The] RNA world is an expression of the infatuation of molecular biologists with base pairing in nucleic acids played out in a one-dimensional space with no reference to time or energy”.On a less emotional note, Harish and Caetano-Anollés earlier Without debating the merits or otherwise of their phylogenetic techniques, the most serious objection to these conclusions is that phylogenetic analysis has the limitation that it can only analyze the protein sequence record as it has been captured in DNA . Therefore, any information we can recover can only date from the advent of coded protein synthesis, as that is the point at which protein sequence became coded in nucleic acid. In an online report on Haris“This is a very engaging and provocative article by one of the most innovative and productive researchers in the field of protein evolution,” said University of California at San Diego research professor Russell Doolittle, who was not involved in the study. Doolittle remains puzzled, however, by “the notion that some early proteins were made before the evolution of the ribosome as a protein-manufacturing system.” He wondered how – if proteins were more ancient than the ribosomal machinery that today produces most of them –“the amino acid sequences of those early proteins were ‘remembered’ and incorporated into the new system.”.To which, Caetano-Anollés’ reported response is slightly puzzling:non-ribosomal protein synthesis – a complex and apparently universal assembly-line process of the cell that does not involve RNA molecules and can still retain high levels of specificity – are more ancient than ribosomal proteins. It is therefore likely that the ribosomes were not the first biological machines to synthesize proteins.” .Phe anticodon stem-loop in a similar fashion to the entire small subunit[1492 and A1493  that proofread the anticodon-codon helix in the modern ribosome[As mentioned above, Harish and Caetano-Anollés are not the first to suggest an RNA replicase origin of the ribosome . The idea, which was possibly first proposed by Weiss and Cherry, is that subunit. This haribosome. It woulribosome.As part of their model of early RNA replication by oligonucleotide ligation, Manrubia and colleagues propose that an increase in the catalytic rate of the replicase/ligase would have occurred with an increase in sequence length through a process of bootstrapping,69. Furt“Highly mutagenic replication processes could have produced relatively large repertoires of short, genetically different molecules, some of them folding into secondary/tertiary structures able to perform selectable functions”.Similarly, we have proposed that, in an RNA world evolving at acidic pH, non-standard base pairing interactions due to base protonation could have provided a means of increasing RNA sequence variation through non-enzymatic replication.e.g.[Wiener and Maizels’ genomic tag hypothesis proposes that the 3′ (or ‘top’) half of tRNA originally functioned as a tag demarking the 3′-end of genomic RNAs for replication, and thus was the first part of tRNA to evolve. Sun ande.g.), but – e.g..is in fact more ancient-like, as it retains the base-paired 3′ and 5′ ends of the original hairpin from which it derives. In contrast, the bottom half, considered to be the more recently acquired, contains the ‘join’ between the two hairpins, which has altered the conformation of the original hairpin, giving this bottom half a new structure. If one accepts a hairpin duplication-ligation origin of tRNA, this explains why the top half of tRNA interacts with the peptidyl transferase region of the large ribosomal subunit: it is because this half retains the same structure (and possibly nucleotide sequence) as the hairpin from which it derives, which originally interacted with the peptidyl transferase region of the large subunit. In fact - and this point has been made by others[ As discussed above, a plausible scenario for the origin of tRNA is the duplication and subsequent ligation of an RNA hairpin approximately half the length of modern tRNA ,3, with y others – this ry others,3.is ancestral, it implies that eukaryotic nuclear tRNAs  have a more ancestral structure than eubacterial tRNAs, which usually lack tRNA introns altogether or possess self-splicing introns at a variety of different positions in the molecule. Such a finding is consistent with the introns-early hypothesis, and the proposal that eubacteria have undergone a process of intron loss[One of the strongest arguments in favour of the hairpin ligation being catalyzed by an ancestral self-cleaving intron as depi is the pron loss,85.I have argued that the RNA world hypothesis, while certainly imperfect, is the best model we currently have for the early evolution of life. While the hypothesis does not exclude a number of possibilities for what – if anything – preceded RNA, unfortunately the evolution of coded protein synthesis has drawn a veil over the previous history of proteins. The situation is different in the case of non-coding RNAs such as ribosomal RNA and tRNA, as these were able to replicate prior to the evolution of ribosomal protein synthesis.As we have noted previously, the proAlthough possible, it seems unlikely that the A-C base pair 'mismatches' found in the tRNA genes of Ferroplasma acidarmanus and Picrophilus torridus  are corrmRNA: messenger RNA; tRNA: transfer RNA.The author declares that he has no competing interests.Referee 1: Eugene KooninI basically agree with Bernhardt. The RNA World scenario is bad as a scientific hypothesis: it is hardly falsifiable and is extremely difficult to verify due to a great number of holes in the most important parts. To wit, no one has achieved bona fide self-replication of RNA which is the cornerstone of the RNA World. Nevertheless, there is a lot going for the RNA World  whereas the other hypotheses on the origin of life are outright helpless. Moreover, as argued in some detail elsewhere, the RNAMoreover, straightforward observations on modern proteins indicate that the role of RNA in the ancient translation system was much greater that it is in the modern system. Indeed, Class I aminoacyl-tRNA synthetases (aaRS) represent only a small branch on the complex evolutionary tree of Rossmann-like domains, so the common ancestor of all 10 Class I aaRS emerged after extensive diversification of this particular class of protein domains had already taken place. Accordingly, one is compelled to conclude that a high-fidelity translation system that alone would enable extensive protein evolution existed already at the late stages of the hypothetical RNA World.All this discussion is not pointless play with hypotheses. Realization of the unique status of the RNA World among the origin of life scenarios is critical for maintaining the focus of research on truly important directions such as experimental and theoretical study of the evolution of ribozymes rather than futile attempts to debunk the RNA World.Referee 2: Anthony PooleHarold Bernhardt’s review of the RNA world hypothesis is readable and timely. He presents a very open-minded review of recent results and how they impact on old ideas, and distills a large amount of material. Aside from the admirable attempt to synthesize a vast array of ideas, a valuable contribution hidden within is the critical assessment of the view that the RNA world hypothesis needs to be abandoned in favour of a peptides-first model.I have revised the abstract and introduction to include reference to my critique of the ‘proteins (or peptides) first’ hypothesis.Author’s response: While I doubt that anyone seriously excluded peptides as part of a prebiotic milieu, the primacy of peptides does need careful consideration. In this regard, the explicit explanation of why a pre-genetic code origin of proteins will not be detectable from comparative genomic analyses is an important contribution. Perhaps this is obvious to some, but in light of a growing view that non-ribosomal peptide synthesis preceded ribosomal peptide synthesis, it would seem that the community needs a reminder, and Bernhardt spells it out in a very informative manner. Another issue with arguing for non-ribosomal peptide synthesis preceding the ribosome is that there is an enormous difference in information input versus output. As discussed in[, megaenzymes like cyclosporin are ~15000 amino acids in length and produce products of 11 amino acids in length – a factor of 104is not trivial. While non-ribosomal peptide synthetases are modular and could in principle be engineered into minimal entities, the challenge of equalizing information input and output is significant regardless of one’s favoured prebiotic starting point. It is clear from reading Bernhardt’s review that the RNA community is much closer to this than those who seek to replace primordial RNA-based replication with peptide-based replication.ussed in, megaenReferee 3: Michael Yarus (nominated by Laura Landweber)Almost always, progress to new understanding is sporadic, with insights coming in separated locales. Difficulties temporarily immobilize discussion, but then are surmounted by a successful theory. This sometimes inchoate stagger toward a broader, more self-consistent argument is all that can be expected, even of an ultimately successful idea. Discussions of the RNA world sometimes forget this, and demand e.g., the ultimate replicase today! But this essay by Harold Bernhardt remembers what has happened for other successful evolutionary ideas, like the big tree. For all its successes, the tree is still being questioned under extreme prejudice in certain quarters, as is the RNA world.Contrariwise, here we have here a sympathetic review of the support for the RNA world, which specifically makes the point that it fits our descent better than other ideas . It will be useful to those who want an entry to the RNA world literature, and could easily serve as the crux of a university course.However, this is also its weakness; the text is polite and respectful, even to those whose ‘contribution’ has been otherwise. It treats even loony ideas (‘we need proteins to evolve translation!’) with deference. Or to put it in other words, it is edgeless – some attitude would be welcome. Some choice between hypotheses should go with the territory; some consequent make-or-break predictions are the responsibilities of a guide. But as a gentle introduction, you will not find better.In revising the manuscript, I have – to some degree inadvertently – added a bit more bite!Author’s response:


Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of  Campylobacter jejuni is the leading reported cause of bacterial gastroenteritis in developed countries C. jejuniC. jejuni have been linked to a restricted geographical area Multi-locus sequence typing (MLST), a genotyping system based on single-nucleotide polymorphisms (SNPs) of housekeeping genes, has emerged as the state-of-the-art method for resolving bacterial population genetics flaA gene of Campylobacter jejuni; their results demonstrated that the method is both accurate and easy to implement.The development of fluorescent DNA binding dyes with improved saturation properties has allowed a more precise assessment of sequence variation based on the analysis of DNA melting curves. This technique, referred to as high resolution melting (HRM), can distinguish single base variation and then has the potential to identify SNPs without the burden of sequencing C. jejuni isolates. Our goal was to resolve the C. jejuni sequence types as defined in the existing MLST database (http://pubmlst.org/campylobacter) at substantially lower cost than the conventional sequence-based method.In this study, we describe the novel application of an HRM protocol optimized to perform MLST of m separation, even if the SNP was near the amplification primer. Excluding uncA, about 90% of SNPs were transition mutations , but inversion mutations (G to C or C to G and A to T or T to A) were also readily detected.For all 47 isolates, successful amplifications were achieved across the 17 sub fragments spanning the seven MLST loci. C. jejuni isolates. The HRM curves for the same DNA preparations or for the same sequences (SNPs) amplified from different isolates were readily grouped together; conversely, curves for different alleles could be consistently resolved.For each sub fragment, the expected 3 to 6 alleles were resolved by HRM as distinct difference plots . The repaspA was represented by five alleles and the locus was analyzed in three fragments  contained two SNPs generating three unique sequences; but in this region, aspA-1 and -4 were identical, as were aspA-2 and -7. Finally, the right fragment (247 bp) included 3 SNPs generating 4 unique sequences, with aspA-2 and -8 being identical. Within each sub fragment, the unique sequences had distinct HRM profiles  per isolate compared with $100 for sequence-based typing (data not shown).To evaluate the relative efficiency and cost of performing MLST by HRM compared with conventional direct sequencing, we analyzed a confirmation set of 84 additional C. jejuni has been used in population studies of isolates from different geographical areas C. jejuni by identifying the most common alleles more rapidly and at lower cost.MLST has emerged as the state-of-the-art method for studying bacterial population genetics. The MLST system for unc-17, an allele which is known to come from C. coliaspA-2, 7 and aspA-1, 8 in aspA-7 and aspA-1 in HRM can resolve the SNPs that define the different alleles in the MLST system because two DNA amplicons that differ at even a single nucleotide will have different melting profiles. For the demonstration set of 47 diverse isolates, HRM resolved all 35 predicted alleles among the seven MLST genes. The differences in melting profiles among alleles varied with the number and type of SNPs as well as the gene fragment being amplified. For example, the profiles for C. jejuni isolates has many important advantages, but the single greatest benefit is the reduction in the total time and cost required. HRM requires neither agarose gel analysis, sequencing, nor sequence analysis. We estimate that the per isolate cost to perform MLST using HRM is 20–30% that of sequencing. This is achieved without compromising the portability of MLST since the existing nomenclature can be used. As of August 2010, the C. jejuni MLST database contained more than 4600 alleles among almost 10330 isolates. The 47 isolates in our study were drawn from the six major clonal complexes and included alleles whose frequency in the current database ranged from 40% and 68% . We were able to resolve most of these alleles using a single reference isolate for each of the six major sequences types. Distinguishing all known alleles might require additional reference isolates. However, an advantage of this HRM system is that any sequenced allele can be used as the reference profile. Our experience with the 84 C. jejuni isolates from chickens demonstrated that the system is particularly efficient when analyzing ecological niches with relatively few ST variations. Analyzing isolates from niches with more variation, novel niches, or from several niches simultaneously would be less efficient as it would require additional reference strains or sequencing more samples, but would still be less expensive than sequencing of all genes.This strategy for typing Obviously, an HRM system cannot replace sequence-based MLST. If a previously unidentified melting profile is encountered, it is necessary to revert to sequencing; however, once identified, the new profile can be used for reference in subsequent HRM analyses. If the sequence proves to be a new allele, it can be submitted to the database.At a technical level, HRM can be performed using cyclers that accept 384-well plates, permitting high-throughput studies. Because HRM compares amplicons from independent PCR reactions, it is essential to standardize the quantity of DNA used in order to minimize reaction-to-reaction variability. We observed that variation in DNA quantity or quality could shift amplification curves; such offsets have been previously observed to compromise the HRM groupings C. difficile belongs to MLST type 35 HRM can be readily applied to a wide range of genetic analyses that involve detection of a single SNP or a signature allele representing a specific set of SNPs. Examples in microbiology include studies requiring the identification of particular clonal complexes, sequence types or individual mutations. By selecting the locus amplified and the reference standard for the HRM system based on the objective of the study, this approach can be applied to questions in pathogenesis, ecology, epidemiology and antibiotic resistance. As just one example, the NAP1/027 epidemic strain of C. jejuni isolates from diverse geographical sources and representing diverse genotypes, the HRM results were consistent with sequencing and thus could be expressed using the existing MLST nomenclature, but were obtained with greater speed, less effort and at lower cost. HRM has the potential to complement classical sequence-based methods and facilitate a wide range of genotyping studies.In summary, we have demonstrated that by analyzing multiple loci concurrently HRM technology can resolve the SNPs that are the basis of MLST. In our studies of >120 C. jejuni isolates used in this study; all have been previously reported C. jejuni isolates were grown on 5% (vol/vol) defibrinated sheep blood TSA  in a micro aerobic atmosphere at 42°C for 24–48 h. Isolated colonies were used to inoculate Mueller-Hinton broth , grown to 0.5 McFarland standard density, 0.5 ml of the broth transferred to a microfuge tube, centrifuged at 13000 rpm for 10 minutes and the supernatant discarded. Genomic DNA was extracted from the pellet by adding 10 µl of NaOH 0.5 N. After 5 minutes, 10 µl of Tris 1 M pH 8.0 and 980 µl of sterile distilled water were added. DNA extracts were stored at −20°C. DNA concentration was measured using a NanoVue spectrophotometer .All C. jejuni MLST database website. In four cases  the primer was upstream or downstream from the comparison fragment by −8, −4, +4 and −6 nucleotides, respectively. One primer (GLY HRM F3) included the first nucleotide of the comparison fragment. Internal primers overlapped each other to cover the entire sequence. Primers were synthesized by Integrated DNA Technologies  and used without further purification.The fragments for the seven loci in the MLST system (402 to 507 bp) are longer than the maximum that can be efficiently analyzed by HRM (100 to 300 bp) 2, 0.5 µM of each primer and between 10 and 20 ng of DNA The amplification protocol consisted of a first denaturation step at 95°C [5 min], 45 cycles of denaturation at 95°C [10 s], annealing at 55°C [30 s], and extension at 72°C [30 s]. The HRM step consisted of a first denaturation step at 95°C [1 min], followed by a renaturation step at 40°C [1 min]. Melting curves were generated by ramping from 70°C to 95°C at 0.02°C/sec, 25 acquisitions/°C.Real-time PCR cycling was performed in a 96-well plate on a LightCycler® 480 II real-time PCR system (Roche). Each plate must contain at least two reference isolates for each allele that would be identified on the plate together with the unknown samples. The reaction was performed in a 15 µl PCR mix containing 1X LightCycler® 480 High Resolution Melting Master Kit (Roche), 3.5 mM MgClgly for 96 isolates. All curves reached a similar plateau height and, as per manufacture's recommendations, the mean cycle number at which fluorescence exceeded background (referred to as the crossing point or cycle threshold) was <30 with a range of less than 7 across all samples. Reactions that did not meet these criteria were discarded and the fragment amplified again in a subsequent run. The software automatically analyzed the raw melting curve data and set the pre-melt  and post-melt  signals of all samples to uniform values (During amplification, fluorescence data were normalized and then plotted using the automated grouping functionality provided by the LightCycler® 480 II Gene Scanning Software version 1.5.0.39 and by manual editing. m values ; occasiom values . For eacm values  as the dm values  the diff


Interest of the research community in the aspects of chemical biology of RNA has increased vastly over the last twenty years, primarily due to the discovery of RNAi, our deepened understanding of the role of miRNA in the subtle regulation of vital cellular processes, and the realization of the fact that the RNA world—the realm where RNA plays the key parties as a self-replicating molecule and a universal catalyst—is still pretty much with us today as the ribosomal RNA performs its catalytic solo in the formation of the peptide bond in the ribosome. To answer the needs of Biology, Chemistry had, in turn, multiplied and perfected its approaches to study the molecular mechanisms underlying RNA functions in living systems. So, the idea behind this special issue is to show our readership a screenshot of what could be, and has been, achieved recently by applying chemical methods to solve the problems of RNA biology. Ten articles have been carefully selected out of the bunch of those submitted to provide, we believe, a balanced view of different facets of RNA structure and function and, also, of the array of chemical tools to enable us to peek into them.trans translation and the role played by transfer-messenger RNA (tmRNA). A Herculean task of the identification of minor nucleotides in RNA and the use of specific chemical reagents for its accomplishment are reviewed in the contribution from a group of French researchers led by Professor Y. Motorin. Taking snapshots of RNA in live cells by means of molecular beacons technology is the subject of a review of R. Monroy-Contreras and L. Vaca.The issue is a mix of reviews and research papers and thus falls into two parts. We have purposefully incorporated a number of authoritative overviews to paint a wider background of RNA functioning in the first half. It starts with a general treatise on chemical approaches to study RNA structure-function relationships in the postgenomic era by T. S. Ro-Choi and Y. C. Choi. Dependence of RNA-protein interaction on RNA conformation is highlighted by a medicinally important example of a noncoding RNA BC1 and the Fragile X Mental Retardation protein in another paper by X. Yan and R. B. Denman. The one that follows by D. Kurita et al. amply shows how chemical probing has provided insights into the mechanisms of Then the issue goes into details of the interplay between a single chemical, resveratrol, and miRNAs during inflammatory processes and cancer, reviewed by E. Tili and J.-J. Michaille. The miRNA theme continues in the review by A. Serva et al. of the application of high-throughput functional analysis for elucidation of the multiple biological roles of miRNAs. Here we cross the boundary into the second half of our special issue composed from research papers, and the one that comes next by K. Terazawa et al. opens the RNA interference section with synthetic short hairpin RNAs (shRNAs) as potent RNAi inducers. Another article, commissioned by researchers from the Iberian Peninsula R. Eritja and coworkers, deals with branched RNA architectures for RNAi.Finally, a paper of the issue done by M. A. Zenkova et al. focuses on an applied aspect of RNA technology, namely, the development of sequence-specific artificial ribonucleases incorporating multiple imidazole residues that perform similar function to their counterparts in natural RNA-cleaving enzymes. In conclusion, the issue attempts to present a selection of reviews and research papers, which are designed to illustrate how by using various implements and gadgets from the ever-growing arsenal of Chemistry one can hopefully gain a better understanding of what is happening when RNA molecules interact with their manifold targets and perform their plethora of functions. It must be added that this is by no means a comprehensive picture but rather a momentary screenshot of a dynamic process of harnessing the power of Chemistry to serve the ends of Biology. Sapienti sat. Dmitry A. StetsenkoDmitry A. StetsenkoArthur van AerschotArthur van Aerschot


Aedes aegypti.Laser microdissection microscopy (LMM) has potential as a research tool because it allows precise excision of target tissues or cells from a complex biological specimen, and facilitates tissue-specific sample preparation. However, this method has not been used in mosquito vectors to date. To this end, we have developed an LMM method to isolate midgut RNA using Ae. aegypti midguts that were either fresh-frozen or fixed with histological fixatives. Generally, fresh-frozen tissue sections are a common source of quality LMM-derived RNA; however, our aim was to develop an LMM protocol that could inactivate pathogenic viruses by fixation, while simultaneously preserving RNA from arbovirus-infected mosquitoes. Three groups (10 - 15 mosquitoes per group) of female Ae. aegypti at 24 or 48-hours post-blood meal were intrathoracically injected with one of seven common fixatives  to evaluate their effect on RNA quality. Total RNA was isolated from the fixed abdomens using a Trizol® method. The results indicated that RNA from Carnoy's and Bouin's fixative samples was comparable to that of fresh frozen midguts (control) in duplicate experiments. When Carnoy's and Bouin's were used to fix the midguts for the LMM procedure, however, Carnoy's-fixed RNA clearly showed much less degradation than Bouin's-fixed RNA. In addition, a sample of 5 randomly chosen transcripts were amplified more efficiently using the Carnoy's treated LMM RNA than Bouin's-fixed RNA in quantitative real-time PCR (qRT-PCR) assays, suggesting there were more intact target mRNAs in the Carnoy's fixed RNA. The yields of total RNA ranged from 0.3 to 19.0 ng per ~3.0 × 106 μm2 in the LMM procedure.Total RNA was isolated from Ae. aegypti midguts while inactivating viral pathogens. Our findings suggest that LMM in conjunction with Carnoy's fixation can be applied to studies in Ae. aegypti infected with arboviruses without compromising biosafety and RNA quality. This LMM method should be applicable to other mosquito vector studies.Carnoy's fixative was found to be highly compatible with LMM, producing high quality RNA from  Drosophila melanogaster [The ability to isolate a specific type or a subpopulation of cells from tissue composed of heterogeneous cell types is highly desirable for many experimental investigations. A homogeneous sample will represent a more congruent cellular response to either biotic or abiotic stimuli compared to an admixture of different cell types. Use of homogeneous biological samples, therefore, is likely to enhance detection power of cellular changes in quantitative assays such as quantitative real-time PCR (qRT-PCR), microarrays, and proteome analyses -3. In thnogaster -11. As mAedes aegypti. In the present study, we have demonstrated the feasibility of the LMM method using Ae. aegypti as a model, and we believe that this method can be readily applied to other mosquito vectors.To perform LMM, fresh tissues may be embedded in a cryo-medium  by snap-freezing without fixation ,8. RapidAedes aegypti was reared at 25°C and 80% relative humidity under a 12 h light: 12 h dark photoperiod in the laboratory following a general mosquito rearing guide [ad libitum and fed on sheep blood meal  for one hour using a glass membrane feeder  once a week for egg production. The membrane feeder was kept at 37°C by circulating preheated water through a water bath during blood feeding. Larval mosquitoes were provided with 10% suspension of bovine liver powder  in distilled water.The Rockefeller strain of ng guide . Adult mMosquitoes were blood-fed as described above to satiation for 1 hour and kept at 25°C and 80% relative humidity under a 12 h light: 12 h dark photoperiod in an environmental chamber . At 24 or 48 hours post-blood meal, engorged female mosquitoes were immobilized and harvested by placing them in 2-3 minute cold-shock at -20°C. For fixation, each fixative was intrathoracically injected into the mosquito using a 30.5 gauge needle on a 1 ml syringe. The fixative-infused mosquitoes were kept on ice in a 1.5 ml microcentrifuge tube for 2 hours. After fixation, abdomens were dissected using a pair of forceps and immediately processed whole for RNA extraction (see below). Three groups (10 - 15 abdomens per group) were prepared for each of the following fixatives:▪ Bouin's ▪ Carnoy's ▪ Formoy's ▪ Cal-Rite ▪ 4% formalin in PBS ▪ 10% neutral buffered formalin ▪ Zinc Formalin .3). The embedded abdomens were snap-frozen by placing the cryomolds in an ethanol/dry ice bath. The embedded abdomens were prepared in triplicate and kept at -80°C until used. Laser microdissection was performed following the method described by Jones et al. [et al. [After fixation with either Bouin's or Carnoy's fixative, 5-6 mosquito abdomens were dissected and embedded in the same orientation in O.C.T. Compound  in aluminum foil cryomold . The cutting energy level of the laser beam was set to ~80% of the full output of the LMM unit. Microdissected midgut cells were captured onto PCR tube caps filled with adhesive gel  and two independent collections for Bouin's and Carnoy's fixative were prepared for further analysis. Caps with dissected cells were placed in 200 μl PCR tubes stored at -80°C until RNA isolation.Microdissection of mosquito midguts was performed using a P.A.L.M. laser microdissection system ® reagent http://www.invitrogen.com after snap-freezing without any fixation, as described above. The integrity of the RNA samples was assessed using Agilent RNA 6000 Pico Kit and an Agilent Bioanalyzer 2100 system according to the directions provided by the manufacturer http://www.chem.agilent.com.Total RNA from laser-dissected tissues was isolated using the RNAqueous™ Micro kit according to manufacture's directions . This kit has been developed to optimize RNA isolation from LMM-cut tissues and cells. Briefly, 20 μl of lysis buffer was added into the LMM cap/PCR tube assembly and incubated for 30 min at 42°C with the caps positioned upside-down and touching the heating block. After incubation, the assembly was briefly centrifuged to collect the extraction fluid in the microcentrifuge tube. Then, an additional 80 μl of lysis buffer was added to a final volume of 100 μl. To each sample, 3 μl of the LMM Additive of the RNAqueous™ Micro kit was added, briefly mixed, and centrifuged. To recover total RNA, 1.25 × volumes (129 μl) of 95% ethanol was added to the samples and mixed by brief vortexing. Then, sample lysates were loaded onto spin columns, washed several times, and the total cellular RNA eluted twice each with 10 μl of elution buffer. To remove potential genomic DNA contaminants, the eluted RNA samples were treated with DNase I by adding 1/10 elution volume 10× DNase I buffer and 1 μl DNase I to each sample. After gentle but thorough mixing, the samples were incubated for 30 min at 37°C, after which DNase I reactions were stopped by adding 2 μl DNase Inactivation Reagent to each RNA sample followed by 2 min incubation at room temperature. The final RNA samples were stored at -20°C until use. As a control, total RNA was isolated from abdomens of five mosquitoes using TRIzolDQ440299, AAEL015246, AAEL012410, AAEL017251, and AAEL001466; Table 2 and 1 mM each dNTP) were added, making a final volume of 20 μl per reaction. Subsequently, the samples were incubated at 50°C for 50 min for reverse transcription and the reaction was terminated by heat inactivation at 85°C for 5 min and chilling on ice. The yields of first-strand cDNA synthesis were determined spectrophotometrically using a NanoDrop . The cDNAs were kept at -80°C until use. Following fist-strand cDNA synthesis, real-time PCRs were performed using 0.7 μg first-strand cDNA in a total volume of 25 μl containing 12.5 μl of IQ™ SYBR Green Super Mix  and 100 nM of each primer  in Bioanalyzer analysis suggested high quality RNA  values of qRT- PCR assays  that is widely used for protein tagging, but this quenching is reversible by 30 min washing in borate buffer (pH 8.5) and subsequent overnight incubation in phosphate-buffered saline  at 4°C [We set out to test seven commonly used histological fixatives to evaluate their effects on RNA integrity of ) at 4°C ,22. ConsAe. aegypti midgut tissues was as intact as the fresh frozen control RNA. The yields of RNA prepared by LMM were estimated to range between 0.3 - 19.0 ng (median of 18.4 ng) per batch of 10 - 15 sections from control, Bouin's, or Carnoy's-fixed midguts. Based on the requirement of 5 - 200 ng of total RNA for an Agilent microarray hybridization [Ae. aegypti if several batches of LMM-prepared RNA are pooled together as reported in helminth LMM/microarray studies [Ae. aegypti midguts was exceptionally high, and target mRNA was also successfully amplified in qRT-PCR assays. For protein studies, we have yet to test the suitability of Carnoy's-fixed proteins in immunological  or other proteomic assays. Nevertheless, preparation of sufficient quantities of proteins for proteomic studies would involve substantial LMM isolation because unlike nucleic acids, proteins cannot be amplified post-isolation.Molecular integrity of Carnoy's-fixed LMM RNA was evaluated using qRT-PCR of five test transcripts. Amplification kinetics of five target transcripts using the four primer sets Table  suggestedization , the RNA studies . In addi studies ,25. ThesAe. aegypti RNA using LMM is feasible and that the RNA harvested using this method is of desirable quantity and quality for subsequent molecular assays. Development of this microdissection method should facilitate identification of candidate genes that play critical roles in infection mediation and pathogenesis of dengue virus in Ae. aegypti. This LMM method could be directly applicable to other major mosquito vectors such as Anopheles gambiae and Culex mosquitoes. Therefore, the utility of the LMM method is promising in vector biology research.In conclusion, we have demonstrated that tissue- or cell-specific isolation of The authors declare that they have no competing interests.YSH designed the experiment and performed the histology work, LMM, RNA isolation, Bioanalyzer assays, data analyses, and wrote the manuscript. SK and MH performed qRT-PCR and cryosectioning, respectively. GNG and MKJ assisted LMM and reviewed the manuscript.


In silico comparative genomics approaches have been efficiently used for functional prediction and reconstruction of metabolic and regulatory networks. Riboswitches are metabolite-sensing structures often found in bacterial mRNA leaders controlling gene expression on transcriptional or translational levels.cis-regulatory RNAs have been recently classified into numerous RNA families in the Rfam database. High conservation of these RNA motifs provides a unique advantage for their genomic identification and comparative analysis.An increasing number of riboswitches and other cis-regulatory RNAs and more than 12000 target genes in 255 microbial genomes. All predicted RNA-regulated genes were classified into specific and overall functional categories. Analysis of taxonomic distribution of these categories allowed us to establish major functional preferences for each analyzed cis-regulatory RNA motif family. Overall, most RNA motif regulons showed predictable functional content in accordance with their experimentally established effector ligands. Our results suggest that some RNA motifs  are widespread and likely originated from the last common ancestor of all bacteria. However, many more analyzed RNA motifs are restricted to a narrow taxonomic group of bacteria and likely represent more recent evolutionary innovations.A comparative genomics approach implemented in the RegPredict tool was used for reconstruction and functional annotation of regulons controlled by RNAs from 43 Rfam families in diverse taxonomic groups of Bacteria. The inferred regulons include ~5200 http://regprecise.lbl.gov/).The reconstructed regulatory networks for major known RNA motifs substantially expand the existing knowledge of transcriptional regulation in bacteria. The inferred regulons can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis. The obtained genome-wide collection of reference RNA motif regulons is available in the RegPrecise database ( Riboswitches are found mostly in bacterial genomes and represent genetic elements that frequently reside at the leader regions of mRNAs. Riboswitches achieve gene regulation by possessing two alternative structural states and are likened to an “on/off” switch . Direct cis-acting RNA regulatory motifs have recently been identified using a combination of comparative genomics and experimental approaches [cis-regulatory RNA elements called T-boxes is utilized for the control of transcription of amino acid metabolic genes in Bacillus subtilis and other Gram-positive bacteria [Escherichia coli and related Gram-negative bacteria utilize a different RNA-based mechanism of transcription attenuation for the control of amino acid biosynthesis operons, which involves translation of a portion of the leader sequence that codes for a specific amino acid-rich peptide [An increasing number of riboswitches and other proaches ,4,9,10. proaches . Severalproaches ,12. A spbacteria -15. T-bobacteria . In cont peptide . Finally peptide ,19.in silico reconstruction of their respective regulons [cis-regulatory RNA motifs identified in bacterial genomes are available in the Rfam database [Many classes of widespread riboswitches controlling coenzyme and amino acid metabolism have been analyzed in completely sequenced organisms by comparative genomics resulting in regulons -15,20-24database , where tdatabase  and Ribodatabase , were prdatabase . On the database ,30. HoweThe comparative genomic analysis of microbial regulons is an efficient method for functional gene annotation and metabolic pathway reconstruction, especially when combined with traditional approaches of genome context analysis . As mostcis-regulatory RNA motif detection to elucidate the metabolic capacities of RNA-mediated gene regulation in bacteria. We combined the inference of regulatory RNAs and their operon analysis in completely sequenced bacterial genomes to evaluate the regulatory networks for different Rfam families of cis-regulatory RNA motifs. As a result, 43 RNA families including all known and candidate riboswitches, the ribosomal and amino acid operon leaders and T-boxes were analyzed in the respresentative subset of genomes from 24 taxonomic groups of Bacteria. Our results show that relatively few RNA motifs originated in the last common ancestor of Bacteria and bring into question the notion that the riboswitch is a remnant of the RNA world. Instead, many RNA motifs probably arose recently as they are restricted to a few closely related taxonomic groups. Functional analysis of reconstructed RNA motif regulons in diverse taxonomic lineages of bacteria revealed the lineage-specific metabolic gene content preferences and identified major trends in the evolution of their regulons. A large number of regulatory interactions from the inferred RNA motif regulons contribute to genome-wide transcriptional regulatory networks and metabolic models of bacterial cells.In this study, we combine genome context analysis with cis-regulatory RNA motifs that are currently available in the Rfam database [cis-regulatory RNA motifs termed ‘leaders’ that reside in the leader regions of ribosomal and amino acid biosynthesis operons. For the comparative analysis of these RNA motif regulons, we selected a set of 255 representative genomes from 24 diverse taxonomic lineages of Bacteria , metal ions (ykoK), secondary messengers (cyclic di-GMP), and unknown substrates , as well as the PyrR binding motif controlling the pyrimidine metabolism. In group C, we include RNA motifs that are restricted to no more than three taxonomic groups. The group C contains riboswitches that are specific for amino acids and derivatives , cofactors , ions (Mg sensor), and unknown substrates . In addition, we classified the cis-regulatory RNA leaders that control the ribosomal protein operons  and the amino acid biosynthesis operons  into groups D and E, respectively. T-box RNA motifs also control the amino acid metabolism, however since these cis-regulatory RNAs utilize a unique mechanism involving uncharged tRNA, we classified them into the separate group F 1. Cobalamin  and B12 , as well as isofunctional enzymes that provide alternatives to B12-dependent enzymes, have been previously found to be subject to regulation by cobalamin riboswitches in bacterial genomes [12 biosynthesis, (ii) B12 and precursor transporters, (iii) cobalt transporters, and (iv) isozymes of B12-dependent enzymes  and one group of γ-proteobacteria , both representing pathogenic species with reduced biosynthetic capacities  that still retains the complete B12 biosynthetic pathway despite the absence of a cobalamin riboswitch. This fact suggests that the riboswitch-dependent control of cognate metabolic pathways is not ubiquitous. The cobalamin riboswitch regulons are the largest in the dataset. Altogether this dataset includes 535 RNA sites that control over 2400 genes in 197 genomes , suggesting they are either constitutive or regulated by a different mechanism.We used the SFC classification to understand functional preferences for the genes regulated by cobalamin riboswitches across all taxonomic groups Figure . There i2. TPP (thiamin pyrophosphate)Lactobacillus helveticus, Bartonella quintana, Desulfovibrio magneticus, Lawsonia intracellularis and two Chlorobium spp.). It should be noted that some of the above species  possess the thiamin biosynthesis genes but not TPP riboswitches in their genomes, again indicating that there are exceptions from almost universal regulation by coenzyme-sensing riboswitches. Altogether we annotated 564 TPP RNA sites that control near 1800 genes in 249 genomes. On average, these numbers correspond to 7 regulated genes per genome or 3.2 genes per riboswitch.The TPP riboswitch had previously been found to control various biosynthetic enzymes and known and predicted transporters involved in the synthesis and salvage of thiamin and its metabolic precursors, hydroxymethylpyrimidine and hydroxyethylthiazole . For funthiC, which encodes an essential thiamin biosynthesis enzyme. Among TPP-regulated genes, thiamin biosynthesis genes dominate the SFC categories in the majority of lineages, accounting for over 65% in most cases. The thiamin and thiamin precursor transporter genes constitute the next most abundant SFC and can be found in almost all the studied lineages except Burkholderia, Caulobacterales, Chlorobiales, Cyanobacteria, and Ralstonia. On average, the transporter genes represent 30% of TPP-regulated genes. However, the thiamin transporters dominate over the biosynthesis genes in Chloroflexi, Lactobacillaceae, Pasteurellales, and Streptococcaceae, accounting for over the half of the total regulated genes. The abundance of various TPP-regulated transporters in this species correlates with their predicted thiamin auxotrophic phenotypes, caused by the absence of thiamin biosynthetic genes.Despite the wide distribution of TPP motifs, there is a large variation in the number of regulated genes per genome Figure . Vibrion3. FMN (Flavin mononucleotide)Similarly to TPP regulons, the FMN riboswitch regulons were classified into two SFCs: (i) riboflavin biosynthesis, and (ii) riboflavin transporters, that include seven and five functional gene orthologs, respectively  represent the largest proportion of regulated genes. In contrast, most species from the Proteobacterial phylum, as well as two Chlorobia spp, have a single FMN-controlled gene involved in riboflavin biosynthesis (ribB or ribH2). In contrast to cobalamin and TPP, transporters specific for riboflavin are not as widely distributed and can only be found in 7 taxonomic groups . Interestingly, the riboflavin transporter pnuX is the only FMN-regulated gene in Corynebacteria.Based on the analysis of distribution of FMN riboswitch-regulated genes Figure , roughly4. GlycineThe glycine riboswitch-regulated genes were classified into three SFCs: (i) glycine metabolism, (ii) glycine transporters, and (iii) serine metabolism that include twelve, three and three functional gene orthologs, respectively  were introduced in this work to facilitate the functional content analysis of reconstructed RNA regulons Table . OFCs prWhen all RNA motif regulons were combined, the coenzyme metabolic category appeared to be the most abundant OFC, ranging from ~20% to over 50% across different taxonomic lineages Figure . ExceptiykkC-yxkD and mini-ykkC RNA motifs are fairly well-distributed with many taxonomic overlaps  when compared to the ykkC-yxkD regulons (17%). A similar situation was observed between the preQ1 and preQ1-II riboswitches for queuosine metabolism and uptake. preQ1 regulates a small proportion of genes related to coenzyme uptake (27%), whereas the remaining target genes are classified as the coenzyme metabolic OFC. In contrast, preQ1-II exclusively regulates genes from the coenzyme uptake OFC.When analyzed according to RNA motif family, most regulons showed predictable OFC content Figure . For inscis-regulatory sequences in bacterial genomes, there have been few attempts at analyzing their overall distributions or even the operon contents of individual RNA-regulated operons across different microbial genomes. In this study we established regulatory capacities of different cis-regulatory RNAs and used comparative genomics to describe potential functions for many previously uncharacterized genes in RNA regulons.While much effort has been put into discovering novel RNA Analysis of RNA motif abundances and diversity allowed us to make certain predictions regarding the origins of some of these regulators. Among the most widespread group of RNA motifs (Group A), many riboswitches are specific for cofactors such as TPP, cobalamin, and FMN Figure . As thesykkC-yxkD. For instance, the SAM riboswitch  controlling methionine metabolism is widely distributed in three lineages of Firmicutes and is also present in diverse species from eight other phyla, suggesting an ancient origin. Within the Firmicutes phylum, the loss of SAM riboswitches in Streptococcaceae and almost all Lactobacillus genomes is correlated with the emergence of several novel regulatory systems for the control of cysteine and methionine metabolism including two transcription factors (CymR and MtaR), the Smk-box RNA motif, and the expansion of methionine-specific T-boxes [Clostridium-like Firmicute [The occurrences of many riboswitches from group B in taxonomically distant organisms suggest two possible scenarios for their evolutionary histories  riboswitch regulates a variety of genes in several phyla, and the difference in operon contents may be influenced by lifestyle and/or habitat of individual species. For instance, Bacteroidaceae species are part of the human intestinal microflora, and the ability to synthesis B12 may be replaced by B12 transporters or transporters with multiple specificities  and the riboflavin transporter genes, s Figure . On the de novo production of glycine. The regulatory differences observed between glycine biosynthetic and transporter genes may reflect the different metabolic needs of these organisms. Although the regulatory mechanisms for glycine and serine metabolic genes were expected to be similar, given the ease of conversion between these two amino acids, serine metabolic genes were largely absent in most of glycine riboswitch regulons. Even in those lineages in which serine metabolic genes were found, they constitute a small proportion of overall regulated genes. Our findings may reflect the ways these two amino acids are utilized inside a cell. For example, serine may be more valuable as a precursor for energy production in glycolysis than as a precursor for other amino acids, but under most circumstances, glycine may better serve as a building block.Even though only glycine transporter genes were found to be regulated by glycine riboswitches in Streptococcaceae and certain γ-proteobacteria, glycine biosynthetic genes exist elsewhere in the genomes of these organisms for iunH and folE from genomes of many Firmicutes. The ydaO-yuaA riboswitch regulates a wide variety of transporters, which may all play roles in osmoregulation; in addition, regulated cell wall remodeling enzymes like COG0791 and COG3773 from Corynebacteriaceae and Clostridiaceae may serve similar roles. ykkC-yxkD and the functionally equivalent mini-ykkC riboswitches both have been proposed previously to function in regulating efflux pumps and other detoxification mechanisms; however, our findings also yielded genes (COG3382 and COG731 from Clostridiaceae) that function in capacities other than transport. For SAH riboswitches, some novel genes (PF04020 and PF04993) were predicted in Ralstonia although their functional contribution to SAM/SAH homeostasis is unknown.In contrast to group A, group B RNA motifs are responsible for regulating a wide variety of metabolic processes including the transport of amino acids, cofactors, nucleotides and metal ions. Still, many regulate genes with unknown cellular functions. The fact that frequent horizontal transfer probably occurred in this group of RNA motifs suggests peripheral metabolic functions that, when incorporated, confer certain survival benefits. Many novel genes were also found associated with other group B motifs, and their functions can be putatively assigned based on their regulators. For example, various genes found in Bacillales, Clostridiaceae, and Chloroflexi under SAM riboswitch regulation are probably involved in cysteine and methionine metabolism or the transport of corresponding substrates. Similarly, many genes regulated by GEMM RNA motifs can be assigned to cellular motility, virulence and biofilm formation. Some preQ1 riboswitch regulated genes of unknown function may function in the queuosine biosynthesis or salvage pathways; examples of this include Group C RNA motifs are phylogenetically confined to a few closely related taxonomic groups. Their limited distribution points to their recent invention and, as a consequence, their existence in these few taxa may have been the result of vertical transmission. Their regulon contents are also highly conserved with little variation between genomes, thus providing additional evidence for vertical transmission.E. coli and B. subtilis reported that most of these RNA structures were narrowly distributed in γ-proteobacteria and Bacilli, respectively, and that many distinct ribosomal RNA motifs may yet to be identified in bacteria [For group D RNA motifs, even though ribosomal subunits are highly conserved between different bacterial lineages, their regulatory regions appear to exhibit greater variation than the coding sequences. Four studied motifs  that regulate the large ribosomal subunits appear to be confined to all lineages of Firmicutes but were also identified in Thermotogales. It is plausible that these four RNA motifs were originated in Firmicutes, and that the L20 and L21 motifs in Thermotogales were horizontally acquired from Firmicutes. The L10 and S15 motifs have wider taxonomic distributions, being identified in 13 and 10 lineages, respectively , and their substrate specificities are currently restricted to histidine, leucine, threonine and tryptophan, as only these four RNA motifs were available in the Rfam database. In addition to these four motifs, a couple of other amino acid attenuator motifs specific to isoleucine/valine and phenylalanine were previously characterized ; howeverGeobacter spp. [For group F, despite the ubiquity of tRNAs and their associated genes in bacteria, the distribution of T-box RNA motifs is rather limited to Firmicutes, Actinobacteria, Chloroflexi and the Thermus/Deinococcus group. In addition, T-boxes have been previously found in ter spp. , which bter spp. -15. Aminter spp. . Many cater spp. .In this study, we performed a comprehensive comparative genomics analysis of RNA regulons controlled by 43 Rfam motifs including metabolite-sensing riboswitches, ribosomal and amino acid operon leaders, and T-boxes. As result, 5204 RNA regulatory sites were identified in 255 complete genomes demonstrating a high variability in the genomic distributions of numbers and types of RNA sites across diverse taxonomic groups of bacteria. We further performed reconstruction and functional annotation of 3251 RNA regulons controlling over 12,000 genes. Each regulon was defined as a set of genes in a single genome controlled by RNA regulatory sites from the same Rfam family. To facilitate the comparative analysis of this large number of regulons, we used the regulog approach implemented in the RegPredict web server. All studied organisms were subdivided into 24 taxonomic groups, and the analyses of RNA regulons were performed independently in each lineage, resulting in reconstruction of 393 regulogs. Based on taxonomic distribution of the obtained regulogs, each known or putative riboswitch was assigned to one of three groups, whereas the ribosomal and amino acid leaders and T-boxes were classified independently. Further, we introduced a two-level functional classification of genes controlled by each analyzed RNA motif. Specific functional categories (SFCs) allowed us to analyze functional preferences for each individual RNA motif family across taxonomic groups. Overall functional categories (OFCs) provided an overview of biological processes controlled by RNA motifs by taxonomy and also according to RNA motif family.cis-regulatory RNA regulation and the ease of horizontal transmission for these regulators, the preservation of other regulators appears necessary for normal metabolism in other organisms. Our functional annotations of many of regulated genes will allow researchers to validate our initial predictions.Our analyses allowed inferences of possible evolutionary pathways for many of the examined RNA regulatory elements as well as functional annotations to the regulated genes. Our results suggested that some RNA motifs may have arisen in the last common ancestor of most or all bacteria, but far more may have arisen relatively recently. Additional analyses of the metabolic pathways from organisms missing the corresponding RNA motifs suggested that functionally equivalent regulators are also widespread. Despite the simplicity of The results of this study suggest that many metabolic RNA motif regulons evolve rapidly in microbial lineages to adapt to changes in habitats and lifestyles. The reconstructed RNA regulons obtained for the reference collection of genomes will provide the basis for computational propagation of these regulons to new genomes. An expanded set of genomes with mapped RNA motif regulons will enable more detailed studies on the co-evolution of these regulatory RNAs and their cognate metabolic pathways.cis-regulatory RNAs in the uploaded genomes were performed using covariance models of 43 RNA motifs uploaded from the Rfam database [http://regpredict.lbl.gov) [255 complete genomes of bacteria analyzed in this study   to perfoThe RegPredict tool allows simultaneous analysis of multiple microbial genomes and integrates information on location of regulatory sites, gene orthologs, operon predictions, and functional gene annotations. Each RNA motif regulon was independently analyzed in each of the 24 selected groups of closely-related genomes. The genome scanning procedure implemented in RegPredict utilizes the pre-computed files with genomic coordinates and scores of candidate RNA sites from each Rfam family. The genome scan parameters were set up to reduce the chance of nonfunctioning sequences from being detected. Specifically, positions of RNA sites were set between 500 nucleotides upstream and 100 nucleotides downstream of a gene start codon. The maximum intergenic distance for an operon boundary was set to 200 nucleotides under ‘Operon definition’, and the ‘coding region overlap’ option was chosen. After genome scanning and filtering, the RNA motif regulons were clustered into groups termed Clusters of co-Regulated Orthologous operoNs (CRONs) in RegPredict . This aphttp://regprecise.lbl.gov) [Biological functions of regulated genes were predicted and annotated in RegPredict by a combination of a sequence similarity search against the Swiss-Prot section of the UniProtKB database , domain lbl.gov) , where tlbl.gov) .SAM: S-adenosylmethionine; SAH: S-adenosylhomocysteine; tRNA: Transfer RNA; ECF: Energy-coupling factor; SFC: Specific functional category; OFC: Overall functional category; TPP: Thiamin pyrophosphate; FMN: Flavin mononucleotide; MOCO: Molybdenum cofactor; THF: Tetrahydrofolate; CRON: Cluster of co-regulated orthologous operons.The authors declare that they have no competing interests.DAR and PSN conceived and designed the research project. EIS and DAR wrote the manuscript. EIS and SAL performed comparative genomic analysis for reconstruction of regulons. DAR also provided the quality control as a curator. MDK computed RNA regulatory sites in microbial genomes. MHS contributed to the development of the project and writing of the manuscript. All authors read and approved the final manuscript.The inferred content of 42 RNA motif regulons in microbial genomes from 24 taxonomic groups. Each cell contains all genes and operons predicted to be regulated by a particular RNA motif in a genome. Multiple candidate operons are separated by semicolons. In the operon description, dashes separate neighboring genes but are omitted for genes sharing the same locus mnemonic. Parentheses indicate fused genes.Click here for fileThe inferred content of 19 amino acid-specific T-box regulons in microbial genomes from 9 taxonomic groups. Each cell contains all genes and operons predicted to be regulated by a particular RNA motif in a genome. Multiple candidate operons are separated by semicolons. In the operon description, dashes separate neighboring genes but are omitted for genes sharing the same locus mnemonic. Parentheses indicate fused genes.Click here for fileDistribution of RNA motifs in microbial genomes. Riboswitches are arranged in six groups according to abundance and substrate types.Click here for fileTotal number of genes controlled by predicted RNA motif regulons in 24 taxonomic groups of Bacteria. Riboswitches are arranged in six groups according to abundance and substrate types.Click here for fileClassification of identified RNA motif-regulated genes by specific and overall functional categories (SFC and OFC). Only genes with annotated functional roles in the RegPrecise database are shown under ‘gene name’; number of gene families indicates the total types of different orthologues found.Click here for fileDistribution of Specific Functional Categories regulated by each RNA motif family across 24 analyzed taxonomic groups. SFC number for individual riboswitch family is listed in each row, with averaged proportion listed in the last column; ‘per genome’ values and listed at the bottom.Click here for fileComparative genomics analysis of functional regulon content for RNA motifs from groups B and C, ribosomal and amino acid operon leaders (groups D and E), and T-boxes (group F). Additional text describes detailed analyses of the remaining RNA motif families that were not included in the main manuscript text.Click here for fileSupporting figures S1-17 for Additional file Click here for file


Enterovirus 71 (EV71) is one causative agent of hand, foot, and mouth disease (HFMD), which may lead to severe neurological disorders and mortality in children. EV71 genome is a positive single-stranded RNA containing a single open reading frame (ORF) flanked by 5′-untranslated region (5′UTR) and 3′UTR. The 5′UTR is fundamentally important for virus replication by interacting with cellular proteins. Here, we revealed that poly(C)-binding protein 1 (PCBP1) specifically binds to the 5′UTR of EV71. Detailed studies indicated that the RNA-binding K-homologous 1 (KH1) domain of PCBP1 is responsible for its binding to the stem-loop I and IV of EV71 5′UTR. Interestingly, we revealed that PCBP1 is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis showed that PCBP1 is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1 is recruited in a membrane-associated replication complex. In addition, we found that the binding of PCBP1 to 5′UTR resulted in enhancing EV71 viral protein expression and virus production so as to facilitate viral replication. Thus, we revealed a novel mechanism in which PCBP1 as a positive regulator involved in regulation of EV71 replication in the host specialized membrane-associated replication complex, which provides an insight into cellular factors involved in EV71 replication. Enterovirus 71 (EV71), a member of the genus Enterovirus of Picornaviridae family, is the causative pathogen of hand, foot, and mouth disease (HFMD) in young children cis-acting element essential for negative-strand synthesis, which requires a membrane-associated replication complex of viral and cellular proteins along with a viral RNA template. Viral proteins 3CD and VPg along with cellular proteins poly(A)-binding protein (PABP) and poly(C)-binding protein (PCBP) interact with each other and with the ends of the viral RNA to form a circular ribonucleoprotein (RNP) complex The 5′UTR of EV71 RNA is about 745 nt and consists of two secondary structures: a cloverleaf structure involving in viral RNA replication and an internal ribosome entry site (IRES) directing initiation of translation Human PCBP1, also called heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1), is a protein of 38 kDa and one of four isoforms of the hnRNP E protein family. Similar to other members of hnRNP E family, PCBP1 has three RNA-binding domains called K-homologous 1, 2, and 3 , of which KH1 and KH3 bind specifically to poly(C) homopolymers In this study, we at the first time investigated the effect of PCBP1 in control of EV71 replication and revealed the mechanism involved in such regulation. We showed that cellular PCBP1 can specifically interact with the 5′UTR of EV71 RNA. Detailed studies further revealed that PCBP1 binds to the stem-loops I and IV of EV71 5′UTR and the KH1 domain of PCBP1 is responsible for such binding.All positive-strand RNA viruses, such as PV, CAV, and hepatitis C virus (HCV), rely on host intracellular membranes for replication. Upon infection, the viral RNA can be directly translated into protein by host machinery, replicated, and assembled on modified intracellular membranes Human embryonal rhabdomyosarcoma (RD), human neuroblastoma (SK-N-SH), and HeLa cells were cultured in modified Eagle's medium (MEM) , supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin sulfate at 37°C in a 5% CO2 incubator. All cell lines were obtained from China Center for Type Culture Collection . Enterovirus 71 (EV71) virus strain (Xiangyang-Hubei-09) was isolated previously in our laboratory (GenBank accession no. JN230523.1). Cells were infected with EV71 at the indicated multiplicities of infection (MOI) and unbound virus was washed away 2 h later. Infected cells were then cultured in fresh medium with 2% FBS. Virus titration was performed using RD cells in 96-well plates and expressed as 50%-tissue culture infectious dose (TCID50) per unit volume. EV71 titers conformed to the Poisson model in that 1 TCID50/ml = 0.69 pfu/ml (plaque forming unit per milliliter), as described previously 1-fwd and KH1-rev, KH1-2-fwd and KH1-2-rev, KH2-3-fwd and KH2-3-rev, KH3-fwd and KH3-fwd, respectively. The PCR products were then inserted into the EcoRI and SalI sites of plasmid pCMV-Tag-Flag2B (Invitrogen) to generate pCMV-Flag-PCBP1, pCMV-Flag-KH1 (1-288 bp), pCMV-Flag-KH1-2 (1-510 bp), pCMV-Flag-KH2-3 (289-1071 bp) and pCMV-Flag-KH3 (510–1071 bp), respectively.PCBP1-expressing plasmids were constructed as follows. DNA fragments of full-length and truncated PCBP1 gene (Genbank accession no. NM_006196.3) were amplified from RD cell cDNA by PCR using specific primers , pCMV-PCHindIII and XbaI sites of pcDNA3.0 (Invitrogen) to generate pcDNA3.0-5′UTR and deletions pcDNA3.0-5′UTR (1–565), (1–450); (100–450), (1–230), (1–180), and (100–230), respectively. Primers used in the study are list in The full-length and truncated 5′UTR of EV71 RNA were amplified from EV71 cDNA using specific primers , 5′UTR-fThe siRNA specific to PCBP1 (siPCBP1) and the control siRNA (siCtrl) were synthesized by RiboBio  according to the sequences of siPCBP1-fwd and siPCBP1-rev or siCtrl-fwd and siCtrl-rev , respectThe primary antibodies used in this study include mouse anti-hnRNP E1 monoclonal antibody, mouse anti-β-actin monoclonal antibody, normal mouse IgG , rat anti-Flag monoclonal antibody , rabbit anti-EV71-VP1 polyclonal antibody , mouse anti-dsRNA (J2) monoclonal antibody , and rabbit anti-calnexin polyclonal antibody . The rabbit anti-EV71 2C polyclonal antibody was a gift from Professor Hanzhong Wang of Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China. The rabbit anti-EV71 3C polyclonal antibody was raised against residues 76–88 of 3C protein . The purified recombinant PCBP1 and PCBP2 proteins were purchased from ProteinTech Group.g at 4°C for 10 min. The supernatants were removed and subjected to co-immunoprecipitation assays. 100 µl of pretreated lysate was diluted with 450 µl lysis buffer, and 20 µl of hnRNP E1 antibody was added. After incubation on ice for 2 h, 100 µl of pre-wash protein A/G  was added and samples incubated on ice for 1 h. Complexes were pelleted by centrifugation at 1,000×g at 4°C for 5 min and washed five times with lysis buffer. Each pellet  was resuspended in 400 µl of proteinase K buffer  and incubated with 100 µg of predigested proteinase K for 30 min at 37°C. RNA was phenol-chloroform extracted, precipitated in isopropanol at −20°C for 30 min, washed in 70% ethanol, dried and eluted in 20 µl DEPC H2O. Reverse-transcription PCR was performed with M-MLV Reverse Transcriptase  to obtain cDNA, and specific DNA fragments were amplified using primers specific for EV71 5′UTR or ribosomal protein S16 RNA (RD cells were infected with EV71 at an MOI of 40 and harvested at 6 h post-infection (p.i.). Lysates were pre-cleared by incubation on ice for 1 h with protein A/G (Santa Cruz Biotechnology) to bind non-specific antibodies. Non-specific complexes were pelleted by centrifugation at 10,000× S16 RNA .XbaI, purified by LiAc, ethanol precipitation, and dissolved in 0.1×Tris-EDTA (TE) buffer. A total of 2 µg of restricted plasmid DNA was in vitro transcribed into RNA using the MEGAscript™ T7 kit  and purified with a MEGA clear kit (Ambion) according to the manufacturer's protocol. The RNA concentration and integrity were determined using BioSpec-nano . For RNA labeling, RNA fragments were ligated to Biotin-16-UTP (Roche) at the 3′ end with T4 RNA ligase (Promega) according to the manufacturer's instructions. For the biotinylated RNA binding assay, a reaction mixture containing 200 µg of cell extracts and 5 µg of biotinylated RNA was prepared. The mixture with a final volume of 100 µl was incubated in RNA mobility-shift buffer  for 15 min at 37°C, and then added to 400 µl of Streptavidin MagneSphere Paramagnetic Particles (Promega) and allowed to bind for 10 min at room temperature. The RNA-protein complexes were washed five times with RNA mobility-shift buffer without heparin. After the last wash, the complexes were dissolved in 30 µl of 2× SDS-PAGE sample buffer, boiled and subjected to 10–12% SDS-PAGE. Bound proteins were subjected to Western blotting using specific antibodies.Plasmids of pcDNA3.0 ligated with full length EV71 5′UTR and six truncated forms of EV71 5′UTR were linearized with 2, 50 mM KCl, 1 mM DTT, 10% glycerol, and 2 U of RNasin) at 37°C for 20 min. The samples were loaded onto a native 8% PAGE and transferred to nitrocellulose membranes . The membranes were then blocked with 10% fat-free powdered milk in PBST for 30 min, incubated with HRP-Streptavidin  for 1 h, washed, incubated with HRP substrate luminol reagent , and analyzed using a Luminescent Image Analyzer .EV71 5′-UTR RNA was denatured at 95°C for 3 min and cooled to 25°C for 10 min. The RNA and purified recombinant PCBP1 protein (rPCBP1) were then incubated together in binding buffer  and Cy3-conjugated goat anti-rabbit IgG (ProteinTech Group) for 45 min at room temperature. For live-cell ER labeling, 1 µM ER-Tracker™ Blue (Invitrogen) stained cells for 15 min at 37°C. To stain nuclei, 1 µg/ml DAPI (Roche) methanol solution was added and samples incubated for 15 min at room temperature. After washing with PBS, samples were visualized by confocal laser-scanning microscopy .2, 25 mM KCl) and overlaid with 4 ml of 55% sucrose, followed by 1.5 ml of 10% sucrose in LSB. The sucrose gradient centrifugation was performed in a Beckman SW41 Ti rotor at 38,000 rpm for 14 h at 4°C. From the top of the gradient, 1-ml fractions were taken and each one was added 1.7 ml of LSB to dilute sucrose. The fractions were concentrated by being passed through an Amicon Ultra 100 k filter (Millopore) and subjected to Western blotting using specific antibodies.The assay was performed as previous described g for 10 min. Protein concentrations were determined using the Bradford assay kit . For nuclear extraction, cells were collected, washed twice with ice-cold PBS, and scraped into 1 ml cold PBS. Cells were pelleted by centrifugation for 15 s and incubated in two packed cell volumes of buffer A  for 5 min at 4°C with flipping of the tube. The crude nuclei were collected by centrifugation for 30 s and pellets were rinsed with buffer A, resuspended in one packed cell volume of buffer B , and incubated on a shaking platform for 30 min at 4°C. Nuclei were centrifuged for 5 min and supernatants were diluted 1∶1 with buffer C . Cocktail protease inhibitor  tablets were added to each buffer. Nuclear extracts were snap-frozen in liquid nitrogen and stored at −70°C until use.RD, SK-N-SH, and HeLa cells were cultured in MEM supplemented with 10% FBS. Cells were pelleted, washed twice with cold PBS, and resuspended in RIPA buffer  on ice for 30 min. Lysates were centrifuged at 10,000×After preparation of cell extracts, the lysates (100 µg) were electrophoresed on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane . Nonspecific sites were blocked with 5% nonfat dried milk and then incubated with the primary antibody. Blots were analyzed using a Luminescent Image Analyzer (Fujifilm LAS-4000).t-test. Means are expressed as a histogram with error bars representing ± SD, and p<0.05 was considered to indicate statistical significance.All experiments were reproducible and each set was repeated at least three times. Parallel samples were analyzed for a normal distribution using the Kolmogorov-Smimov test. Abnormal values were eliminated according to a follow-up Grubbs test. Levene's test for equality of variances was performed, which provided information to determine the equality of means using Student's To evaluate the role of PCBP1 in the replication of EV71, we first investigated the binding ability of PCBP1 protein to the 5′UTR of EV71 RNA. RD cells were infected with EV71, cell extracts were prepared, and RNA was determined by RT-PCR. The association of PCBP1 with EV71 5′UTR was then determined by co-immunoprecipitation (Co-IP) assay using mouse antibody to PCBP1 and with or without mouse IgG, respectively, followed by RT-PCR. Results revealed that ribosomal protein S16 (RPS16) RNA and EV71 5′UTR RNA were detected in the total RNA extracts , lane 6,The ability of PCBP1 protein binds to the 5′UTR of EV71 was further determined by electrophoretic mobility shift assay (EMSA). Labeled EV71 5′-UTR RNA (nt 1–745) was incubated with purified PCBP1 protein  or BSA  at different concentrations. EMSA results showed that a RNA-protein complex is formed in the presence of PCBP1 protein and the intensity of the complex increased as the concentration of PCBP1 protein increased   , line 1 Since PCBP1 is widely expressed in human and mouse cells http://mfold.rna.albany.edu). In this predicted secondary structures, six domains were shown, domain I (nt 1 to 87), II (nt 111 to 179), III (nt 182 to 231), IV (nt 239 to 442), V (nt 450 to 559), and VI (nt 571 to 651) , stem-loop I is a cloverleaf (CL) structure that is important for virus replication  to 651) . The thrin vitro, which were then labeled with biotin-16-UTP. For the pull-down assay, cell extracts were mixed with biotinylated or nonbiotinylated EV71 5′UTR RNAs and PCBP1 protein was determined by western blot. Results showed that PCBP1 was detected in the presence of 5′UTR-T1 RNA, 5′UTR-T2 RNA, 5′UTR-T4 RNA, 5′UTR-T5 RNA, 5′UTR-T6 RNA, 5′UTR-T7 RNA, and 5′UTR-T8 RNA  or stem-loop IV (SL-IV) of EV71 5′UTR was further evaluated by EMSA assay. Labeled CL-I and SL-IV were incubated with purified PCBP1 protein at different concentrations or BSA as a control, respectively. Results from CL-I and PCBP1 binding assay showed that a RNA-protein complex is formed in the presence of PCBP1 protein and the intensity of the complex increased as the concentration of PCBP1 increased  homopolymers ectively . For thePCBP1 is normally distributed throughout the cells, including the nucleus and cytoplasm In addition, we investigated sub-cellular distributions of PCBP1 protein and EV71 3C protein during EV71 infection under similar conditions. Results showed that in mock-infected cells, EV71 3C protein was not detected b as expeTo further confirm the sub-cellular distribution of PCBP1 protein during EV71 infection, RD cells were infected with or without EV71. Whole cell lysate, cytoplasm extraction, and nucleus extraction were prepared from mock-infected or EV71-infected cells. PCBP1 protein was then detected by western blot analysis. Results showed that in whole cell lysates and in the cytoplasm, PCBP1 protein level was higher in EV71-infected cells than in mock-infected cells . HoweverEnteroviruses and flaviviruses can reorganize the host secretory trafficking between the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and the trans-Golgi network (Golgi/TGN) to generate membrane microenvironments suitable for viral replication Most of RNA viruses replicate in the detergent-resistant membrane (DRM) fraction, such as HCV, PV, coxsachievirus B3 (CVB3), and rhinovirus (RV) Since we revealed that PCBP1 can bind to EV71 5′UTR RNA and is located in viral replication complex, the effect of PCBP1 on EV71 replication was then investigated. RD cells were transfected with pCMV-PCBP1 or pCMV, or treated with siPCBP1 or siCrtl, respectively. Western blot results showed that PCBP1 protein was detected in cells transfected with pCMV and the level of PCBP1 was increased in cells transfected with pCMV-PCBP1 at 36 h and 48 h post-transfection . PCBP1 pRD cells were then transfected with pCMV-PCBP1 or pCMV, siPCBP1 or siCtrl at different concentrations, as indicated, and then infected with EV71. Western blot results revealed that EV71 VP1 protein was not detected in mock-infected cells, but detected in EV71-infected cells as expected. In addition, the level of VP1 protein was gradually increased as the concentration of PCBP1 increased , but graTo evaluate the effect of PCBP1 on EV71 virus production, RD cells were transfected with different concentrations of pCMV-PCBP1, pCMV, siPCBP1, or siCtrl, respectively, and then infected with EV71 for 12 h. Cell culture supernatants were prepared and subjected to plaque assays and the titers of viral infectivity of EV71 were determined in infected-RD cells. Results showed that virus production and the levels of viral titers in EV71-infected cells were enhanced by over-expression of PCBP1 , but redPositive-strand RNA viruses initiate translation upon entry of the genomic RNA to host cells and start to duplicate the RNA from the 3′ end resulting in generation of a complementary negative-strand RNA, which is transcribed into a nascent positive genomic RNA. This replication cycle can produce thousands of copies of the genome within a few hours PCBP1 binds to the 3′ non-coding region (3′ NCR) of cellular mRNAs and plays a role in the stabilization or translational control of mRNAs In this study, the binding ability of PCBP1 to the 5′UTR of EV71 RNA was first evaluated. By using biotinylated RNA/protein binding assay, we revealed that cellular PCBP1 protein can specifically bind to the 5′UTR of EV71 RNA in RD, HeLa, and SK-N-SH cells infected with the virus. The 5′UTR of EV71 RNA comprises 745 nt and forms a secondary structure. We predicted that it contains six stem-loops (I–VI) with three functional regions  and demonstrated that stem-loop I (nt 1–100) and IV (nt 230–450) in EV71 5′UTR are required for the binding of PCBP1 to the viral RNA.PCBP1 contains three RNA-binding domains, KH1, KH2, and KH3. KH1 and KH3 bind to poly(C) homopolymers and KH1 acts as a dominant-negative mutant to inhibit translation of poliovirus PCBP proteins are frequently distributed throughout the cells, including the nucleus and cytoplasm ER membrane is distributed in the cytoplasm of mock-infected cells, while the viral dsRNA, ER membrane and PCBP1 were distributed in the cytoplasm of EV71-infected cells in a co-localized fashion. The sub-cellular distribution of ER membrane was not affected by PCBP1 in mock-infected cells, however, PCBP1, and EV71 RNA were focused and mainly distributed in the cytoplasm and the three components, EV71 dsRNA, ER membrane, and PCBP1 protein, were co-localized in the cells, especially in the ER-derived membrane complex or DRM. These results demonstrated that the cellular factors and the viral RNA were co-localized in EV71-infected cells, suggesting that PCBP1 interacts with EV71 RNA in the ER-derived membrane complex.The role of PCBP1 in EV71 viral protein expression was then investigated. We revealed that over-expression of PCBP1 resulted in the stimulation of EV71 VP1 protein expression and knock-down of PCBP1 resulted in inhibition of EV71 VP1 protein expression, indicating PCBP1 plays a stimulatory role in regulation of EV71 protein expression. The effect of PCBP1 on the production of EV71 virus was also determined in this study. We showed that in EV71-infected cells, over-expression of PCBP1 enhances the viral titers and knocked-down of PCBP1 reduces the viral titers, indicating PCBP1 plays a stimulatory role in EV71 viral production. Thus, PCBP1 can be viewed as an important cellular factor in EV71 replication.It's known that many RNA virus 5′UTR is fundamentally important for virus replication by interacting with cellular proteins, including viral RNA translation and synthesis Overall, our research reveals one cellular factor, PCBP1, as being an important regulator of EV71 replication, has affinity for viral 5′UTR and is located in viral membrane-associated complex to promoter viral replication. These findings not only improve our understanding of cellular factors involved in EV71 replication, but also provide a new strategy to potential anti-viral drugs by inhibiting EV71 replication.