T. thermophilus and E. coli, supporting the idea of a core made by both RNA and proteins, conserved by evolution. Recent work showed also that ribosomal RNA is modularly composed. Motifs are generally single-stranded sequences of consecutive nucleotides (ssRNA) with characteristic folding. The role of these motifs in protein-RNA interactions has been so far only sparsely investigated.One important goal of structural bioinformatics is to recognize and predict the interactions between protein binding sites and RNA. Recently, a comprehensive analysis of ribosomal proteins and their interactions with rRNA has been done. Interesting results emerged from the comparison of r-proteins within the small subunit in tripod.This work explores the role of RNA structural motifs in the interaction of proteins with ribosomal RNA (rRNA). We analyze composition, local geometries and conformation of interface regions involving motifs such as tetraloops, kink turns and single extruded nucleotides. We construct an interaction map of protein binding sites that allows us to identify the common types of shared 3-D physicochemical binding patterns for tetraloops. Furthermore, we investigate the protein binding pockets that accommodate single extruded nucleotides either involved in kink-turns or in arbitrary RNA strands. This analysis reveals a new structural motif, called It corresponds to small pockets consisting of three aminoacids arranged at the vertices of an almost equilateral triangle. We developed a search procedure for the recognition of tripods, based on an empirical tripod fingerprint.A comparative analysis with the overall RNA surface and interfaces shows that contact surfaces involving RNA motifs have distinctive features that may be useful for the recognition and prediction of interactions. Protein-RNA interactions occur frequently within living cells as part of crucial processes, such as gene expression and regulation, protein synthesis and viral replication, as well as stabilizers of ribosomal RNA molecules within the ribosome. RNA recognition mechanisms raised much interest within the RNA community as soon as new structures became available. Several statistical studies were conducted on growing datasets of interacting structures -7, thougH. marismortui, HM, 50S [T. thermophilus, TM, 30S [T. thermophilus and E. coli, supporting the idea of a core made by both RNA and proteins, conserved by evolution. Though fascinating, the hypothesis is still difficult to validate due to the lack of high resolution data; indeed so far only four ribosomal high resolution crystals are available. On the other hand, in [A comprehensive analysis of ribosomal proteins and their interactions with rRNA has been done by  for the  HM, 50S ) and by  HM, 50S  for the  TM, 30S ). In [11hand, in  authors hand, in ,2.Recent work showed that ribosomal RNA is modularly composed -17. ModuThe role of these motifs in protein-RNA interactions has been so far only sparsely explored. The only related work is in  where thThe goal of this study is to provide an insight into the role of RNA motifs on r-protein interactions, with a particular interest for tetraloops, kink-turns, and single extruded nucleotides. The analysis is conducted on two large ribosomal subunits, HM 50S (PDB no. 1S72) and the 50S subunit of E.coli  and on the small subunit of TM 30S (PDB no. 1FJF). We will show how RNA motifs significantly interact with r-proteins, and analyze both composition and 3D conformation of interfaces made by motifs and proteins interacting with each other.RNA contact surfaces (R-CS) and to the protein regions that interact with the RNA as protein contact surfaces (P-CS). We focus on the RNA interface regions consisting of atoms that participate in structural motifs. We denote such regions by SMR-CS. Similarly, protein contact surfaces restricted to those atoms that make contact with structural motifs are called SMP -CS. For the analysis, the RNA contact surfaces are extracted using the tool ENTANGLE [In the following, we will refer to RNA surface regions that are in contact with r-proteins as ENTANGLE .The analysis on available crystal structures of ribosomes aims at uncovering relatioships, if any, between protein-RNA interaction mechanisms and RNA structural motifs. The goal is pursued first by quantifying the presence of RNA motifs in regions interacting with proteins, then by evaluating the composition of contact surfaces in the presence of motifs, and finally comparing and analyzing the 3D conformation of these surfaces.We are interested in establishing whether RNA structural motifs tend to appear more frequently at RNA interfaces than on the entire surface of the ribosome.π-turn, Ω-turn, and S2-turn. Definitions and methods to search for these structures are given in [k ≥ 3 helices and composed by k distinct ssRNA sequences are called RNA junctions. Junctions do not share conserved structures, but they show recurrent conformation in 3D [We start by briefly recalling the definitions of RNA motifs. A motif is composed by sequences, generally 1 or 2, of single-stranded consecutive nucleotides characterized by a distinctive 3D structure and, sometimes, sequence pattern. An RNA motifs is either a hairpin loop, i.e. connecting the two anti-parallel chains of one helix, or internal loop, i.e. connecting two helices. Known RNA motifs are: tetraloop, kink-turn, loop-E, given in ,22,17. Con in 3D .The importance of motifs in protein-RNA interactions is testified by the high presence of these substructures within R-CS. In our analysis, each surface atom of the ribosomes is labeled as belonging to one of the following: bulge-free helix, known RNA motif, RNA junction, or other non-helical region. The frequency of atoms of different structural elements, presented in Table Major contribution to the high presence of motifs in R-CS is given by tetraloops and kink-turns: tetraloops are 4 residues long hairpin loops characterized by well conserved structure and consensus sequence patterns given by GNRA, UNCG, and CUUG, where N can be any nucleotide and R can be either G or A; on the other hand kink-turns consist of approximately 15 nucleotides from two distinct segments which base pair to form two helices and an internal loop.However, the frequency of atoms of the different structural elements is not uniform across RNA interfaces with r-proteins For instance L18 extensively interacts with rRNA in both HM 50S and EC 50S, making ~50% of his interactions with structural motifs; by contrast L44e in HM 50S, L3 in EC 50S, and S4 in TT 30S, while forming large contact surfaces with rRNA, make no interaction with motifs.In this section we concentrate on the chemical composition of interfaces of H. marismortui, T. thermophilus and E. coli. In particular we will show that it exhibits remarkable differences depending on whether complete surfaces, interfaces, or interfaces involving structural motifs are considered. Variations are significant both at the RNA side, where we compare the distribution of phosphate-ribose-base (P-R-B) atoms, and at the protein side, where we analyze amino acid composition. These results are reported in Table It is interesting to highlight how the proportions of phosphate, ribose, and base atoms vary in the protein-RNA complexes within the ribosome when they include structural motifs.R-CS  that was not relevant within the whole contact surfaces. For TLP -CS Phe is significantly present in contact surfaces of both HM 50S and EC 50S, while less relevant in TT 30S. However in general the composition of these surfaces is diverse and the few atoms involved make the statistics highly dependent on single cases. This is showed by the relative high standard error in correspondence of most represented amino acids. Phe is for instance highly present in contacts made by L32e and L18 with HM 50S tetraloops, as well as those made by L2, L15, L18, and L34 in EC 50S; but scarcely present on the other contact surfaces with tetraloops.The composition of protein interfaces reflects known tendencies such as the high presence of arginine (Arg) and lysine (Lys) ,9. Both In this section we focus on the 3D conformation, localization and geometry of the protein interfaces with tetraloops, kink-turns and single extruded nucleotides. It has been observed  that strA significant fraction of r-proteins has a large number of contacts with tetraloops. In this section we examine the subset of r-proteins interacting with standard tetraloops in HM 50S as case study; thus the proteins considered are L2, L10e, L13, L14, L15, L15e, L18, L19e, L32e, L37e.P -CS is first assessed qualitatively by visual inspection of their localization with respect to their associated tetraloops. It is known [P -CS we first superimposed the tetraloops and then transformed the interfaces accordingly. More specifically, all tetraloops are superimposed to single one, chosen as reference, using Horn's algorithm [The overall variation exhibited by SMis known ,16 that lgorithm  applied 3V web tool by [http://www.molmovdb.org/cgi-bin/3v.cgi, by rolling a probe sphere (r = 1.5 Å) on the contact surface. Table For a more quantitative comparison, we determined the size of the interfaces measured both by the number of superficial atoms that compose them, and by their area. The contact surface area has been computed using the  tool by http://wwinteraction map. Interaction maps basically map in a discretized 2D space the positions of the atoms of all TLP -CS after they are brought into a common reference frame (see Methods). Figure P -CS in HM 50S. Figure For ease of visualization and analysis we built a two-dimensional representation of contact surfaces, that we called To better study the localization of interactions between r-proteins and tetraloops, in the interaction map we mark the regions corresponding to the discretized coordinates of all tetraloops atoms after superposition with the reference. We identify four connected non overlapping regions corresponding to the backbone atoms of the 4 nucleotides of all tetraloops; they are labeled with numbers 1,..,4 and are shown by depicting their perimeter (in black) in Figure The interaction map allows to appreciate the common tendency of the proteins to interact with tetraloop backbone atoms: the vast majority of protein interface atoms is located within the perimeters of the four regions and only a few cells outside the four regions are populated. Note that a preference is shown towards the area between the first and the second nucleotide and between the third and the fourth nucleotide, where the density of the atoms is higher.pi, than the cell is attributed to property pi and colored according to the code; else if a majority is not detectable, the cell is considered as a gap in the pattern. Figure Next, the interaction map is used to visually identify patterns of interactions in terms of physico-chemical properties of the residues. A color code of such properties has been applied to the interaction map. Since a cell of the interaction map may contain several atoms, a majority rule has been used. Precisely if most of the atoms within a cell have the property The resulting pattern shows a concentration of positive residues, mainly Arg, in the dense area between the first and the second nucleotide where the rRNA chain has a turn, changing the base orientation, to form the loop. High presence of Arg is also detectable between the third and the forth nucleotide, in correspondence of the other dense area. The same patches also contain several polar residues , while hydrophobic residues are more uniformly located within the surfaces.The authors of  were thetripod. A tripod conformation is observed for the following interaction sites: G1315 (KT1311/1338) with L32e, A1150 (KT1146/1212) with L10, and A96 (KT92/77) with L29. In Figure Our analysis of these interactions revealed an interesting conformation common to 3 of them: the extruded nucleotide mainly interacts with three aminoacids of the protein; these aminoacids form a binding site characterized by three knobs disposed as vertices of a triangle with similar angles and sides. In the following we refer to this conformation as 1150 KT116/1212 wiThe characteristic shape of these binding sites suggested a possible recurrent conformation also on protein interfaces with single extruded nucleotides not belonging to kink-turns. Thus we searched for tripods on the entire surfaces using as query a fingerprint derived from the observed tripods in kink-turns. The search method is described in the Methods section. The results of the automatic search reveal a major presence of this site among protein interactions with single extruded nucleotides, belonging to either bulges, internal loops, junctions, or other non-helical regions.Figure The tripods show conserved three-dimensional structure, nucleotide preference, and similar interaction pattern; these considerations together suggest this binding site as a recurrent RNA recognition motif. This novel motif gives a good starting point to provide a better structural characterization and hopefully an effective prediction method for protein-RNA interactions.Aim of this paper was to dissect protein-RNA interaction mechanisms within the ribosome. The high conformational diversity of protein interfaces with rRNA, following the high diversity in folds of r-proteins, is confirmed by this study. However, when focusing on restricted areas some geometric and physico-chemical patterns could be detected.Specifically, we explored the role of RNA structural motifs in protein-RNA interactions. We quantitatively showed the preference for protein interfaces to interact with structural RNA motifs by comparing the composition in terms of motifs and helical regions of RNA interfaces with that of the complete ribosomal surface.We detected similar binding sites for extruded RNA bases leading to the definition of a novel protein structural motif, the tripod, characterized by the presence of three amino acids forming three knobs on the surface at the vertices of triangle, with approximately same sides.Among RNA motifs tetraloops show the highest percentage of interactions with ribosomal proteins. Protein contact surfaces with tetraloops showed high conformational diversity making binding site characterization tough. By studying the localization of the contacts, preferred contact areas were detected; furthermore, a consensus interaction pattern was identified based on physico-chemical properties of the amino acids composing the interfaces.Future work will exploit the results of this work to provide accurate computational methods to compare protein-RNA interactions in large datasets and possibly tackle the challenging problem of predicting protein-RNA interactions.P -CS after they are brought into a common reference frame. The reference frame is defined by a chosen tetraloop; all interfaces are transformed after the superposition of the tetraloops to the chosen reference. For the interaction map, we resorted to a polar representation of protein interfaces. Since the backbone of a tetraloop typically forms a curve resembling a semi circumference, the whole structure has a semi spherical shape. Thus, we projected the protein surface points on a sphere with fixed radius and associated to each point two coordinates, the zenit, θ, and azimut, φ; angles. The interaction map is a discretized representation of these points on a grid. The granularity of the cells of the grid depends on the quantization step applied to  values. The step has been determined as the highest value so that each atom of a single contact surface falls on a different cell. This polar coordinates based representation gives the ideal framework to design an interaction pattern easily understandable and, possibly, comparable.Interaction maps are basically a map in 2D space of the positions of the atoms of all TLl1, l2, l3 ∈  Å and angles α1, α2, α3 ∈ . The fingerprint can thus be seen as a triplet of knobs, whose coordinates are the averages of coordinates of the knobs of the true positive tripods.The first instances of tripods have been derived by visual inspection of extruded nucleotides, in particular within RNA kink-turns. From this limited set of observed instances, we derived a geometric fingerprint defined by a triplet of points and their coordinates in the 3D space. The fingerprint defines a triangle with sides B ≥ R, R ≥ P, B >P then the candidate is accepted in the final list. These relations, which are empirically derived from the observed instances proved to be a good criterion to filter out a large number of false positives, while preserving the correct instances. Figure We use this fingerprint to search for tripods on the interfaces of r-proteins. The search method takes in input a protein contact surface and extracts the atoms belonging to its convex patches using the method presented in . The extThe authors declare that they have no competing interests.All authors contributed in the planning of the study and statistical analysis of the data. GC and Cl.G extracted the data and performed the analysis of 3D interfaces with tetraloops. GC analyzed tripod conformations and design the search procedure. Co.G coordinated the study and drafted the manuscript. GC and Co.G contributed to finalize the manuscript. All authors read and approved the final manuscript.


The hair of all mammals consists of terminally differentiated cells that undergo a specialized form of apoptosis called cornification. While DNA is destroyed during cornification, the extent to which RNA is lost is unknown. Here we find that multiple types of RNA are incompletely degraded after hair shaft formation in both mouse and human. Notably, mRNAs and short regulatory microRNAs (miRNAs) are stable in the hair as far as 10 cm from the scalp. To better characterize the post-apoptotic RNAs that escape degradation in the hair, we performed sequencing (RNA-seq) on RNA isolated from hair shafts pooled from several individuals. This hair shaft RNA library, which encompasses different hair types, genders, and populations, revealed 7,193 mRNAs, 449 miRNAs and thousands of unannotated transcripts that remain in the post-apoptotic hair. A comparison of the hair shaft RNA library to that of viable keratinocytes revealed surprisingly similar patterns of gene coverage and indicates that degradation of RNA is highly inefficient during apoptosis of hair lineages. The generation of a hair shaft RNA library could be used as months of accumulated transcriptional history useful for retrospective detection of disease, drug response and environmental exposure. Apoptosis is a cellular program utilized by multicellular organisms to eliminate cells during development or in surveillance of foreign or abnormal cells altered by viral infection or neoplastic transformation DNase1L2, nuclear DNA persists in the hair and causes hair fragility The hair follicle is a model of apoptosis While DNA degradation is a common hallmark of apoptosis, the targeting of RNA for degradation during apoptosis is unclear. The removal of RNA following apoptosis may be of importance as released endogenous RNA appears to activate inflammation and the innate immune response Here we investigate the extent to which RNAs survive apoptosis by studying the external hair shaft of mice and humans. We hypothesized that pools of small RNAs, in particular, regulatory microRNAs (miRNAs), might preferentially survive destruction by nucleases during the process of cornification, because of their inherent stability snoRNA-251, we found evidence for lineage specificity using miRNA expression (mir-203), which is considered to be epidermal-specific In mice, the transcriptional profiles of living hair follicle-specific miRNAs and mRNAs have been previously examined pression . SignifiKeratin 32 (Krt32), Krt34, and Krt35 were detected at 8.6, 16.1 and 1.5-fold higher in the external hair. Sh3d19, a medulla-specific gene, was also relatively more abundant in the hair shaft. The enrichment of these RNAs in hair shaft relative to whole skin occurs as hair follicle transcripts are diluted by other tissues present in whole skin. As a negative control, genes expressed by tissues that do not contribute cells to the hair shaft were examined. One such tissue, called the dermal papilla, is a closely associated mesenchymal population that supports hair growth but does not itself contribute cells to the growing hair shaft. We found that two dermal papilla-specific genes, Sox2Bmp6As hair keratins are abundantly expressed during hair development, we next investigated whether their transcripts can be detected in the external hair shaft. In addition, hair keratin genes are also expressed by specific cell types and identify the cell type of origin for RNAs extracted from external hair . SeveralWe considered the possibility that residual RNAs in the mouse hair might reflect its rapid growth and potential persistence of cells rather than the low ribonuclease activity during and after cornification. In humans, hair does not contain viable cells From this approach, 13.5 million high-quality reads and 1.2 million unique sequences were obtained. Subsequently, the reads were aligned to the human genome (hg18) and to dedicated small RNA libraries (miRBase and snoBase). A large portion of reads aligned to more than 10 loci or no loci, due to low complexity of sequence, highly repetitive targets or the presence of non-human RNA . We alsoLET-7 family, which plays a role in regulation of proliferation, differentiation, and stem cell maintenance KRTAP family comprise 58.3% of all mRNAs in the hair shaft. Like keratins, KRTAP genes function as support proteins, which become highly crosslinked and participate in forming the shape of the hair shaft The vast majority of miRNAs detected in the hair shaft belonged to the −6 to 1.0×10−11) belonging to exposure, disease or medication-induced gene expression showed detection in hair shaft RNA but not NHEK RNA . Human samples were unidentified and pooled. The demographic profiles are shown in Supplemental Methods. Normal human epidermal keratinocytes were grown in EpiLife culture media  containing 0.06 mM calcium and defined growth supplement. All experiments and informed consents were performed and approved according to the institutional guidelines established by the University of California, San Diego, Institutional Animal Care and Use Committee and the University of California, San Diego, Human Research Protection programs, Protocol ID #091646. Written informed consents were obtained from each participant of the study.For mouse studies, hair from 3-week old animals was trimmed and stored in RNAlater (QIAGEN) solution at −80°C. Hair was visually inspected to verify absence of hair bulbs, which contain viable cells. For human studies, hair was trimmed at a distance of 5 mm from the scalp. Hair was washed in 70% ethanol and water. Two methods were used for RNA extraction. During initial studies in mouse hair and human hair shaft RNA-sequencing, Trizol reagent (Invitrogen) was utilized in combination with mechanical disruption with 1.0 mm zirconia beads . Subsequently, improved extraction was obtained with the addition of 0.1 M dithiothreitol (DTT) to Trizol reagent or a urea-based RNA extraction buffer 2 miRNA First Strand Kit (QIAGEN) and amplified with stem-loop primers from SABiosciences/QIAGEN (2 SYBR® Green qPCR mastermix (QIAGEN). Real-time PCRs were performed on an ABI 7300 Real Time PCR system and a Roche Lightcycler 480. A comparative CT method was used for quantitative analysis using snoRNA-251 in mouse microRNAs samples, Gapdh for mouse mRNAs. Half-life of mRNAs and miRNAs determined using 2∧deltaCT values normalized to levels at site of origin and Prism  non-linear fit first-order decay modeling.Primer sequences and miRNA probes are detailed in Supplemental Methods. Primers for mRNAs were designed using Primer3 s/QIAGEN  using RTFifty hair shafts, equivalent to 30 mg, were utilized to prepare total RNA using the above methods. One microgram of total RNA isolated from hair was used to generate a small RNA library. Adapter ligation, reverse transcription, amplification, gel extraction and sequencing were performed, according to Illumina protocols for miRNA-Seq (v1.5.0) (Illumina). Sequencing was carried out on an Illumina Genome Analyzer IIx. Genome Analyzer Pipeline was used to generate a FASTQ file from raw RNA-seq data, and quality scores were offset by 64 following Solexa-1.3+ standards. FASTX-Toolkit package, hg18 genome alignment with BOWTIE, was used for pre-processing and alignment. Parameters for alignment are detailed in Supplemental methods, and a list of human hair shaft transcripts are provided in Table S1Annotation and reads counts of mRNA, miRNA, snoRNAs (>200 reads).(PDF)Click here for additional data file.Table S2Representation of Genetic Association Database transcripts in hair.(PDF)Click here for additional data file.Table S3Representation of Novoseek compound-genetic pathways in hair.(PDF)Click here for additional data file.Table S4Primers and reagents used in PCR analysis.(PDF)Click here for additional data file.Table S5Annotated reads with >10 reads.(XLS)Click here for additional data file.Methods S1Supplemental methods.(DOC)Click here for additional data file.References S1Supplemental references.(DOC)Click here for additional data file.


Sabin3-like mutation (U473 → C) introduced in the domain V sequence of the CVB3 IRES led to a defective mutant with a serious reduction in translation efficiency and ribosomal initiation complex assembly, besides an impaired RNA-protein binding pattern. With the aim to identify proteins interacting with both CVB3 wild-type and Sabin3-like domain V RNAs and to assess the effect of the Sabin3-like mutation on these potential interactions, we have used a proteomic approach. This procedure allowed the identification of three RNA-binding proteins interacting with the domain V: eIF4G (p220), eIF3b (p116) and eIF4B (p80). Moreover, we report that this single-nucleotide exchange impairs the interaction pattern and the binding affinity of these standard translation initiation factors within the IRES domain V of the mutant strain. Taken together, these data indicate how this decisive Sabin3-like mutation mediates viral translation attenuation; playing a key role in the understanding of the cardiovirulence attenuation within this construct. Hence, these data provide further evidence for the crucial role of RNA structure for the IRES activity, and reinforce the idea of a distribution of function between the different IRES structural domains.Internal ribosome entry site (IRES) elements fold into highly organized conserved secondary and probably tertiary structures that guide the ribosome to an internal site of the RNA at the IRES 3′end. The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. In each poliovirus Sabin vaccine strain, a single point mutation in the IRES secondary-structure domain V is a major determinant of neurovirulence and translation attenuation. Here we are extrapolating poliovirus findings to a genomic related virus named coxsackievirus B3 CVB3); a causative agent of viral myocarditis. We have previously reported that http://www.diagnosticpathology.diagnomx.eu/vs/6160165131045880.The virtual slide(s) for this article can be found here:  Picornaviridae family[Herpesviridae family has been reported[Internal Ribosome Entry Site (IRES) elements are specialized RNA regulatory sequences governing cap-independent translation initiation in eukaryotic mRNAs that are translated during cellular stress, that is, when cap-dependent translation is compromised-4. IRES e family-8. Soon e family,10, or re family-13, as we family,15, plane family, and proe family. Recentlreported.7GpppN structure (termed cap) located at the 5′-end of most mRNAs[via the IRES using a cap-independent mechanism, bypassing proteins bound to the 5′ UTR as well as strong RNA structures in front of the start site[Initiation of translation in eukaryotes, which is the rate-limiting step in protein synthesis, involves a set of specialized proteins that recruit the small ribosome subunit to the mst mRNAs,20. The st mRNAs. In markart site.Because IRESs differ in nucleotide sequence, RNA secondary structure and trans-acting factors requirement-25, deciMyocarditis or inflammatory cardiomyopathy is inflammation of heart muscle which can be caused by several agents. Myocardial fibrosis occurs in a number of pathological processes, most commonly hypertension. Other disease states capable of producing cardiac fibrosis include hypereosinophilia, scleroderma, viral myocarditis and inherited genetic mutations. In the The detection of viral genomes in endomyocardial biopsies by molecular techniques has greatly expanded the list of viruses implicated in myocarditis. Initially, the molecular diagnosis of the viral etiology of myocarditis focused on the enteroviruses. More recently, Tavora and collaborators reportedPicornaviridae[Coxsackievirus B3 (CVB3) is a picornavirus that can also cause viral myocarditis in humans. It has a single-stranded plus sense RNA. The 7.4 kb long genomic RNA is naturally uncapped and contains a viral encoded oligopeptide (VPg), covalently linked at the 5′ end. The highly structured 5′UTR is 741 nt long and has been shown to contain a type I IRES element which helps in mediating internal initiation of translation of CVB3 RNA,35. Typenaviridae,36. Theynaviridae,36. Instnaviridae.591) about 25 nucleotides downstream of the pyrimidine-rich tract[Internal initiation of translation in CVB3 has been shown to be influenced by various cis-acting elements present in the 5′UTR. The corch tract. It was ch tract and thatch tract.Sabin3-like mutation (U473 → C), obtained by direct mutagenesis of the CVB3 genome, and precisely in the domain V sequence of the CVB3 IRES led to a defective mutant with a serious reduction in translation efficiency compared to the wild-type strain. Prediction of the secondary structure by MFOLD program indicated a structural perturbation of the stem containing the Sabin3-like mutation, suggesting that specific protein-viral RNA interactions were disrupted, preventing efficient viral translation. The poor translation efficiency of the Sabin3-like IRES was then explicated by its inability to correctly bind some essential non-canonical translation factors during the initiation of translation. To fill this gap, we have used a proteomic approach that has allowed the identification of some RNA-binding proteins interacting with both wild-type and mutant CVB3 IRESes[Sabin3-like mutation induces a partial destabilization of the RNA secondary structure of the domain V, leading to a reduced recognition of this region by protein factors necessary for CVB3 translation initiation.Importantly, we have previously addressed the question of whether the attenuating mutations of domain V of the PV IRES were specific for a given genomic context or whether they could be transposed and extrapolated to a genomic related virus, i.e. CVB3. In this context, Ben M’hadheb-Gharbi and collaborators,42 have 3 IRESes. Interes3 IRESes and a re3 IRESes comparedSabin3-like CVB3 RNAs, a proteomic approach was carried out. Over the years, RNA-protein interactions have been determined using different approaches, such as Electrophoretic mobility-shift assays, UV cross-linking or Filter-binding assays. Along this idea, riboproteomic approaches have facilitated the identification of various proteins interacting with different IRES elements. Accordingly, in the present study, we performed UV-crosslinking assays to compare the RNA-protein interaction pattern between CVB3 wild-type and mutant domain V RNAs in the presence of HeLa and BHK21 cell extract proteins. Filter-binding experiments were then carried out in order to assess the binding affinity of some initiation factors: eIF3, eIF4G and eIF4B to both wild-type and Sabin3-like domain V RNAs. Interestingly, the mutant RNA showed a reduced protein-binding profile compared to the wild-type domain V. A number of proteins binding to the domain V were identified: p220 (eIF4G), p116 (eIF3) and p80 (eIF4B). Filter-binding assays showed a better binding affinity of eIF3, eIF4G and eIF4B to the wild-type CVB3 domain V. These results perfectly correlate with the impaired protein-binding and the reduced translation efficiency previously reported with the Sabin3-like construct[Thus, in order to confirm this hypothesis and with the aim of identifying translation initiation factors specifically binding to domain V of the wild-type and onstruct-44.Sabin3-like mutant of CVB3, used as a “vaccine candidate” and obtained by direct mutagenesis (U473 → C)[The Coxsackievirus B3 (CVB3) Nancy prototype strain and the 473 → C) were use(Wt)-F (5”- TAT gAA TTC TAA TAC gAC TCA CTA TAg gTC CTC Cgg CCC CTg AAT gCg gCT AAT CCT AAC TgC ggA gCA CAC ACC CTC AAg CCA gAg ggC AgT gTg TCg TAA -3”)/DVS3)(-F (5′- TAT gAA TTC TAA TAC gAC TCA CTA TAg gTC CTC Cgg CCC CTg AAT gCg gCT AAT TCT AAC TgC ggA gCA CAC ACC CTC AAg CCA gAg ggC AgT gTg TCg TAA -3′) and DV-R (5′- TAT ggA TCC ATg AAA CAC ggA CAC CCA AAg TAg TCg gTT CCg CTg CAg AgT TgC CCg TTA CgA CAC ACT gCC CTC Tgg CTT gAg ggT gT -3′) were designed for the synthesis of domain V for both CVB3 wild-type (Wt) and Sabin3-like (S3) strains by primers- hybridization and extension method. EcoRI restriction site/T7 promoter upstream and BamHI restriction site downstream were introduced into primer’s sequences.Based on the sequence of the CVB3 domain V, primers DV(Wt)-F/DV-R or DVS3) and the final volume (50 μL) was adjusted by adding sterile distilled water. The reaction was then incubated in a thermocycler (BioRad) according to the following thermal profile: 4 min at 72°C, 6 min at 44°C and 60 min at 70°C. Amplification products were revealed by electrophoretic migration on 2% agarose gel containing Ethidium Bromide.Primers- hybridization and extension technique was used as previously described to synthEscherichia coli DH5α cells. Transformants were selected in Luria–Bertani (LB) agar supplemented with 100 μg/ml Ampicillin, 64 μg/ml 5-bromo-4-chloro-3-indolyl-beta-D galactopyranoside, and 0.2 mM isopropyl beta-D-thiogalactopyranoside. Blue–white selection was used to identify white clones containing inserts, while blue clones contained undigested cloning vector. White colonies were individually cultured in LB broth.Amplified CVB3 wild-type and mutant domains V were digested with EcoRI and BamHI (Roche Applied Science). Digestion products were, then, purified using the “QIA quick PCR Purification Kit” (Qiagen) and inserted into the pUC19 plasmid (Invitrogen) digested with the appropriate enzymes and purified using the “Wizard SV Gel and PCR Clean-Up system kit” (Promega). Ligation products were, then, transformed in chemocompetent In order to analyze the cloned sequences, randomly chosen clones were tested by PCR-colony as previously described. Positiv6 cpm/pmol using (α32P)-CTP (400 Ci/mmol). Reactions were incubated for 10 min at 37°C with 1 U of RQ1 DNase (Promega), and unincorporated (α32P)-CTP was eliminated by exclusion chromatography in TE-equilibrated columns . RNA was extracted with phenol–chloroform, ethanol precipitated, and resuspended in RNase-free water (1–2 × 105 cpm/μL). Prior to further use, the integrity of probes was verified in 6% acrylamide, 7 M urea denaturing gel. Dried gels were exposed for autoradiography.Plasmids were linearized to obtain transcripts corresponding to wild-type and mutant CVB3 domains V. Following digestion, DNAs were phenol extracted and ethanol precipitated. Transcription was performed for 1 h at 37°C using 50 U of T7 RNA polymerase (New England Biolabs) in the presence of 0.5–1 μg of linearized DNA template, 50 mM DTT, 0.5 mM each rNTP, and 20 U of RNasin (Promega). Transcripts were uniformly labeled to a specific activity of 0.5–1 × 10For a comparative analysis of the binding affinity of cellular proteins to domain V of the CVB3 IRES, two cell lines were used: HeLa (Human) and BHK-21 (Hamster) cells, respectively known as prominent and lacking expression of the coxsackie-adenovirus receptor (CAR). These cell lines had been chosen since they represent well established systems for studying viral replication and infectivity. In addition, cells lysates contain host proteins that support internal translation initiation of the viral RNA.g for 5 min. The clear lysate was centrifuged at 10,000 × g for 5 min, adjusting the supernatant to 3% glycerol[BHK-21 and HeLa cells, grown to 100% confluence in 10 cm dishes in 5% FCS-supplemented DMEM, were washed twice with cold PBS, scraped and collected by centrifugation. The cellular pellet was resuspended in 1–2 volumes of 10 mM HEPES (pH 7.4), 10 mM KAc, 1.5 mM MgAc, and 2.5 mM DTT, and homogenized by 30 strokes in a glass Dounce. Cellular debris was eliminated by centrifugation at 5,000 × glycerol. The conglycerol. Followi5 cpm) in 15 μL of 10 mM HEPES–KOH, pH 7.9, 35 mM KCl, 2 mM MgCl2, 10% glycerol, 0.05% NP40, 0.5 mM DTT, 1 μg/μL yeast tRNA (as non-specific competitor). After 15 min of incubation at room temperature, RNA–protein mixtures were exposed to UV light  for 30 min on ice, at 10 cm from the lamp. The non-protected probe in UV-irradiated extracts was digested with RNase A (0.3 μg/μL) during 30 min at 37°C. In parallel, competitive UV-cross-linking assays were performed. Specific competitors (self RNA competitors) were added, five min before adding radioactive probes, at specified molar excess ratios. Yeast tRNA was also used as a non-specific competitor as described above.Extracts from HeLa and BHK-21 cells (40 μg of proteins) were incubated with uniformly radiolabeled probes  binds proteins and RNA–protein complexes, and the bottom membrane (which is charged) collects free RNA. After washing and drying, the membranes are exposed for quantification.Sabin3-like domain V RNAs within the initiation factors eIF3, eIF4G and eIF4B, filter-binding assays were carried out.Accordingly, in the present study, in order to assess the affinity and the specificity of interaction of CVB3 wild-type and Initiation factors  were prepared following previously established procedures,48.2, 1 mM DTT), and incubated 5 min at room temperature. Initiation factors eIF3, eIF4G and eIF4B were serially diluted immediately before use and then added to reactions. These were incubated at 4°C for 15–30 min before application to the filter. Measurements were performed in parallel within wild-type and mutant domain V RNAs.Labeled RNAs encoding domain V were annealed by heating to 80°C for 2 min in water then cooled at room temperature for 7 min. RNA (10 nM) was then added to a tube containing a folding/binding buffer , assembled in a dot blot apparatus or on glass filter funnel and the reactions were applied and directly vacuum filtered. Filters were then rinsed, removed, dried, and radioactivity was quantified using a storm phosphorImager . The percentage of protein-RNA bound was then calculated and graphs: % binding RNA – protein = f (protein concentration) were drawn.p < 0.05.To assess the binding affinity of CVB3 wild-type and mutant domain V RNAs to eIF3, eIF4G and eIF4B, binding assays were performed and the percentage of protein-RNA bound was calculated. ANOVA test using STATVIEW statistical software package was performed. Normality and homogeneity of data were confirmed before the test and values were considered statistically significant at Sabin3-like strains. The attenuation of the Sabin3-like strain was mainly conferred by a single point mutation in the IRES domain V sequence[Sabin3-like domains V were amplified using the primers- hybridization and extension technique as described in the “Materials and Methods” section. Amplified wild-type and mutant domains V were analyzed on 2% agarose gel  and a non-specific (tRNA) competitors for both radiolabeled domain V RNAs (data not shown). Formation of RNA-proteins complexes was reduced by the addition of increasing amounts of unlabeled self RNA. This demonstrates the specificity of the cell protein interactions. In contrast, formation of complexes between cell proteins and yeast tRNA was minimal, as the addition of tRNA did not present significant competition effects.In fact, a reduction in the band intensity of In order to assess cellular proteins binding to domain V of the CVB3 IRES, UV- crosslinking assays were performed. Domain V of the FMDV IRES was used as a control probe. The comparative analysis of autoradiogram profiles Figures  and3 shSabin3-like RNA competitors. RNAs competed efficiently with the binding of these proteins and to radiolabeled wild-type and mutant RNAs. Thus, the loss of detection of RNA-protein complexes observed with the mutant domain V was due to the loss of protein binding. Taken together, the above results indicate that the domain V contains a major determinant for protein-binding to the CVB3 IRES.Interestingly, the mutant RNA showed a reduced protein binding profile compared to the wild-type domain V in both cell extracts. To confirm that the reduction of detection of RNA-protein complexes formed within the mutant CVB3 domain V RNA was due to the reduction in the protein binding efficiency to domain V and not to the loss of label transfer from the radioactive RNA to the protein, competitive assays were performed using unlabeled wild-type and In order to identify proteins that bind to the CVB3 IRES domain V, UV-crosslinking experiments were carried out. RNase A was then added to digest non-interacting regions of the labeled probes, and thus allows realistic molecular weight estimates. Domain V of FMDV IRES was used as a control probe in parallel to CVB3 RNAs. Figures Sabin3-like point mutation in domain V of the CVB3 IRES seriously affected ribosome association[Sabin3-like domain V RNA, the binding of p80 (eIF4B), p116 (eIF3) and p220 (eIF4G) was indeed impaired by this mutation compared to the wild-type RNA; thus correlating with the reduced translation efficiency of the Sabin3-like construct[Sabin3-like mutation. Competition experiments confirmed that the observed reduction in band intensities was due to a reduced binding of these factors and not to a reduced label transfer only, while other bands were less strongly affected by the competitions.Our previous observations that the ociation and protociation within tonstruct. In thisAccording to the data obtained by UV-crosslinking experiments, RNA-protein complexes obtained with both CVB3 domain V RNAs were differently susceptible to competitions. The differences in susceptibility to competition can be a consequence of the binding specificities of proteins. Specific interactions were concluded when band intensity corresponds to the decrease in competitor ratios. All proteins appeared to specifically interact with the domain V sequence as they were all susceptible to competitions. To confirm this specificity of RNA–protein interactions, filter-binding assays were carried out. We assayed whether the CVB3 domain V RNA could directly interact with individual initiation factors eIF3, eIF4B and eIF4G. Putative interactions were analyzed by filter-binding assays and the amount of resulting RNA–protein complexes was determined. Values obtained were extrapolated to draw graphs: % binding RNA–protein = f (protein concentration)  than the Sabin3-like RNA for all studied initiation factors; suggesting a better binding affinity of the initiation factors eIF3, eIF4G and eIF4B to the CVB3 wild-type domain V compared with the mutant RNA.According to ANOVA statistical data, the percentage of RNA protein binding was significantly higher for the CVB3 wild-type RNA ((i) the identification of essential regions in the cis-acting elements (likely involved in the interaction with other regions of the RNA that composes the IRES as well as with cellular proteins), and (ii) the identification of translation initiation factors that bring the ribosome into contact with the mRNA. During the last decade, many efforts have been devoted to understand the mechanistic basis of IRES function and how these elements interact with host-cell components in order to recruit the translation machinery[Gene expression control largely depends on ribonucleoprotein complexes regulating mRNA translation. Initiation of translation in mRNAs that overcome cap-dependent translation inhibition is often driven by internal ribosome entry site (IRES) elements, whose activity is regulated by multifunctional RNA binding factors. Understanding the mechanism used by IRES elements to promote internal initiation of translation requires: achinery,44,49-54RNA structure plays a fundamental role in viral IRES dependent translation initiation. In suppIn the best-known example, each of the three attenuated Sabin vaccine strains for poliovirus, considered the prototype picornavirus, contains nucleotide substitutions in domain V of the 5′UTR that are responsible for attenuation,61. ThesSabin3-like mutant (U473 → C) of CVB3[Sabin3-like point mutation in domain V of the CVB3 IRES seriously affected ribosome association[S10 extracts prepared from BHK-21 or HeLa cells that support efficient IRES activity. We have observed a number of proteins binding to the CVB3 domain V RNA. Some of these binding proteins may be identical proteins that have multiple binding sites in the domain V; some of them may be different proteins with similar molecular weights. In fact, three major polypeptides of 220, 116 and 80 kDa, previously identified as eIF4G, eIF3 and eIF4B, respectively, were observed in both cell extracts.Similarly, we have previously reported the limited efficiency of the translation of  of CVB3. Additioociation and protociation within tAlthough molecular weight determinations were not definitive in our UV-cross-linking assays, RNA-protein complexes that we have detected appear to be within reasonable range of these above-mentioned proteins, suggesting possible identities of those CVB3 domain V-binding proteins. To confirm the identity of these proteins, immunoprecipitation assays were carried out. Smear-like patterns represent multiple RNA-protein complexes which could not be distinguished as discrete bands. This suggests that multiple proteins interact with the same RNA molecule and that some of these proteins are in a relative higher content than other specific binding proteins. The RNA–protein interaction pattern could vary depending on the protein source used for the UV-cross-linking assay. This suggests two possible processes: the presence of multiple host proteins that bound the probes at differential affinities and quantities; or the co-operative binding of multiple copies of the same protein toward the probe.Sabin3-like strain is mediated by an impaired binding of the standard translation initiation factors eIF3, eIF4B and eIF4G to the mutant RNA. This in turn causes impaired association of ribosomes within the viral RNA as was previously reported[Filter-binding assays results showed a better affinity of binding of the initiation factors eIF3 (p116), eIF4G (p220) and eIF4B (p80) to the wild-type CVB3 IRES domain V compared with the mutant RNA. This finding clearly correlates with the data obtained by UV-cross-linking assays. Taken together, this can be explicated by the inability of the mutant domain V RNA to interact with some trans-acting factors critical for enhanced IRES function. Consequently, here we demonstrate that the reduction in the coxsackievirus B3 translation efficiency caused by the single nucleotide exchange in the IRES domain V sequence of the reported. In concIndeed, our present findings correlate with the data published by De Breyne and collaborators who repoSince there are no more previous reports on protein-RNA interactions of the CVB3, we compare our findings to a close genomic related virus, the poliovirus. Our present results are in a perfect correlation with the previously published data of Ochs and collaborators,65 who rImportantly, the introduction of the attenuating Sabin 3 mutation into domain V of the wild-type PV IRES at nucleotide 469 reduced cross-linking of eIF4G and eIF4B to this domain in RRL. These deIF4G and eIF4A together induced strong toe prints at the 3′ border of type I IRESs (in domain VI and its immediate vicinity) that likely indicate conformational changes in the IRES, because no direct interaction of eIF4G or eIF4A with this region of the IRES was detected. The finIt was also shown that the binding site for eIF4B in the poliovirus IRES resides mainly in the IRES domain V, whereas domain VI sequences, including the silent AUG at nucleotide 586, contribute to the binding of eIF4B to a lesser extent, which is a finding consistent with the observation that domain VI is not absolutely essential for poliovirus translation,70. In dProbably due to the possible complexity of protein-RNA interactions involved in the activity of the poliovirus IRES, the initiation factor requirements have been investigated so far mainly with the distantly related picornavirus type II IRES elements. The IRES element of FMDV is organized in structural domains, termed 2–5 in 5′- to 3′-end, which appear to have a division of functions. Domainsin vitro selection with random sequences. The high affinity ligands showed a conserved set of nucleotides located in a structural motif consisting of a bulge stem-loop[eIF4B contains an RNA recognition motif (RRM) near the N-terminus and a second RNA-binding region in the carboxy-terminal half of the protein. Although the carboxy-terminal region binds RNA non specifically with high affinity, the RRM binds such RNAs inefficiently. RNA-binding specificity of the eIF4B RRM has been studied using tem-loop.It was reported that eIF3 bound to the FMDV IRES at multiple sites. A preferential eIF3 binding site resided in domain 5. However domain 4 also interacted with this factor and, although to a lower extent, they observed a crosslink of p116 to domains 1–2 and 3. The sequence of conserved residues in domains 4 and 5 is not an essential determinant for eIF3 interaction with FMDV IRES. It remaExtending these results to the type I IRES of poliovirus, Ochs and collaborators showed t(i) those that do not need proteins to assemble the initiation complex and (ii) those that do need factors to recruit the ribosome [Although there are similarities among IRES elements in terms of structures and ITAF requirements, there are also specific differences between IRESes even among those of the same classification. The lack of complete conservation of structural elements among viral IRESes and the differences among the viral IRESes in ITAF requirements have made stringent classification difficult. Attending to the essential requirements for internal initiation, IRES elements can be grouped in two main categories: viruses). Within viruses),49. Typeviruses).In addition to eIFs, auxiliary factors termed IRES transacting factors (ITAFs) contribute to modulate (either stimulate or repress) picornavirus IRES activity. In support of the relevance of factors different than eIFs for internal initiation, transcripts encompassing the region interacting with eIFs do not possess IRES activity, indicatSabin3-like mutation that contributes to (i) the attenuation of the cardiovirulence, (ii) the reduction efficiency of translation, (iii) the impaired ribosomal initiation complex 48S and 80S assembly and (iv) the reduction of RNA-protein binding pattern within the full IRES sequence is the impaired binding of the translation initiation factors eIF3, eIF4G and eIF4B to the IRES domain V mutant RNA. Indeed, the Sabin3-like mutation could induce a partial destabilization of the secondary structure of domain V, leading to reduced recognition of this region by protein factors necessary for CVB3 translation initiation. This may cause slower translation of the viral RNA and thus may contribute to the attenuated phenotype of the Sabin3-like strain.In summary, the study presented here is an attempt toward the identification of standard initiation factors binding to the domain V of CVB3 IRES and involved in the internal translation initiation of the CVB3 genomic RNA. Interestingly, our present findings implicate that the main effect of the Sabin3-like mutant is reduced, and if their possibly reduced binding contributes to the attenuated phenotype of the Sabin3-like strain. Hence, mapping the exact binding sites of eIF3, eIF4G and eIF4B to domain V of the CVB3 IRES and the identification of other proteins involved in the enhancement of CVB3 IRES-mediated translation would be clearly a challenge in the near future to confirm our results and to increase our understanding of the translation initiation, the 5′UTR-related tissue tropism and the cardiovirulence mechanisms.Nevertheless, the possible involvement of other trans-acting factors (ITAFs) is not ruled out. It can only be speculated if these ITAFs additionally modulate the CVB3 IRES activity, if their binding to Translation initiation mechanisms affecting the efficiency of protein synthesis of a given mRNA are diverse and, importantly, more frequent than anticipated, sometimes giving rise to the expression of different polypeptides from a single transcriptional unit. Therefore, presence of any of these regulatory elements can seriously complicate efforts to accurately define the sites of translation initiation at the genome wide scale.The authors declare that they have no competing interests.JG and MBMG: participated in the design of the study. AS carried out the experiments and wrote the manuscript. All authors red and approved the final manuscript.


Each mature T cell or B cell has an allele with a productively rearranged T cell receptor (TCR) or B cell receptor (BCR) gene, respectively. The repertoire of TCRs and BCRs in the blood of a healthy person is diverse, with no single specific rearrangement found in more than one percent of cells, and most rearrangements representing a minute fraction of total B cells or T cells-3. When High-throughput sequencing has the potential to significantly improve sensitivity and reduce cost for clonal detection and post-treatment monitoring of B and T cell leukemias and lymphomas. For leukemia, direct sequencing of BCR and TCR genes from blood cells is straightforward. However, in the case of lymphoma, the primary tumor resides in the lymph node, with the fraction of cancerous cells found in blood variable. A recent paper by He et al. presents the interesting idea that free DNA in blood plasma is potentially a better source of lymphoma specific DNA than white blood cells. In the He et al. employ a targeted capture and sequencing approach that they call IgCap. It is similar to a shotgun sequencing approach, yet they include an enrichment step between fragmentation and sequencing which enriches for sequences derived from the IgH locus. Fragmented DNA sequences from plasma have adapters attached to each end, and are then hybridized to a set of probes that bind to the germline encoded fragments of the IgH locus. The subset of plasma DNA fragments which hybridize to the IgH derived probes are then sequenced using Illumina's high-throughput sequencing technology.This methodology has some pros and cons. On the positive side, the method is capable of detecting the majority of sequences in the IgH locus, including any possible non-templated rearrangement. Importantly, IgCap was effectively able to detect and identify lymphoma specific IgH sequences in plasma. There are also a few negative aspects of this technology. First, the hybridization step does not distinguish between rearranged and non-rearranged IgH sequences. So, an unknown portion of the DNA fragments sequenced will truly be derived from the rearranged IgH loci. Second, fragmenting sequences and then reconstructing the pieces requires sequencing each receptor multiple times. For a diverse, non-templated region of DNA, such as the rearranged IgH locus, to detect an specific IgH sequence, many copies would need to be in the plasma and sequenced.Direct methods for sequencing rearranged BCR and TCR genes have been developed over the past few years, 3, 6, 7The work of He et al. is an important step in showing that high-throughput sequencing of the adaptive immune receptor repertoire has direct clinical application. The idea of focusing on plasma DNA instead of cells for monitoring lymphoma is intriguing. Hopefully, the larger scale experiments required to prove utility will be accomplished soon and these or similar methods will be moved into the clinic.


Multisubunit RNA polymerase, an enzyme that accomplishes transcription in all living organisms, is a potent target for antibiotics. The antibiotic streptolydigin inhibits RNA polymerase by sequestering the active center in a catalytically inactive conformation. Here, we show that binding of streptolydigin to RNA polymerase strictly depends on a noncatalytic magnesium ion which is likely chelated by the aspartate of the bridge helix of the active center. Substitutions of this aspartate may explain different sensitivities of bacterial RNA polymerases to streptolydigin. These results provide the first evidence for the role of noncatalytic magnesium ions in the functioning of RNA polymerase and suggest new routes for the modification of existing and the design of new inhibitors of transcription. DNA-dependent RNA polymerase (RNAP) is a potent target for antibiotics. At present, two specific inhibitors of bacterial RNAPs, rifampin and lipiarmycin (fidaxomicin), are in clinical use as antibiotics, and there is still great potential for other known inhibitors of bacterial RNAPs (or their derivatives) to be used in the clinic in the future.The antibiotic streptolydigin (Stl) is a derivative of 3-acetyltetramic acid , and it 1–4–8–Thermus thermophilus core RNAP  and STL2 (β557–576) and the N-terminal portion of the BH (β′769–788) (789–795) and with the ordered segment of the TL  , while tf the TL . The acef the TL , 24. HerThermus aquaticus core RNAPs were constructed and purified as described previously (Recombinant wild-type (WT) and mutant eviously .E. coli numbering]) RNAPs as described previously (32P labeled at the 5′ end by using [γ-32P]ATP (PerkinElmer). All reactions were carried out at 40°C. Stl (Sigma) with or without 10 mM MgCl2 was added before the reactions for 10 min at 40°C. Elongation reactions were initiated by addition of 1 mM GTP or 1 mM GTP with 10 mM MgCl2; endonucleolytic cleavage reactions were initiated by the addition of 10 mM MgCl2. Reactions were stopped by the addition of formamide-containing buffer, and products were analyzed as described previously .Elongation complexes (ECs) were assembled with WT and mutant (H936A/R933A and M932A [eviously  and placeviously . Fast kieviously . The kin2 in 20 mM HEPES buffer (pH 8.0).The spectra of Stl were recorded using a UV160U UV-visible (UV-Vis) recording spectrophotometer  in a fused silica measuring cell in the presence of the indicated concentrations of the MgClThermus aquaticus RNAP, fully complementary synthetic template and nontemplate DNA oligonucleotides, and an RNA oligonucleotide . These complexes are assembled with cleotide  and D. Tcleotide , 26, 27.cleotide . Note thCs. Theseand slow .iK cannot be used in this case, given that the kinetics cannot be described by a single-exponent equation) during hydrolysis was ∼8 times lower than that during NTP addition . Indeed, as seen from 2+, the half-inhibitory concentration of Stl was ∼15 times lower than that when Mg2+ was added together with NTP  are much more sensitive to Stl than the RNAPs that have asparagine at this position (including E. coli RNAP). Finally, the β′N792D substitution in E. coli RNAP leads to a significant enhancement of the enzyme sensitivity to Stl (2+ bridges β′N792D and Stl, leading to tighter binding of the latter. This scenario also raises an intriguing possibility of a role for noncatalytic Mg2+ ions in the functioning/inhibition of the enzyme.The principal finding of this study is that Stl requires Mgwith Stl  and E (2with Stl . The appperiment , is arou by them . Howeverthem (2+  (Fig. 2Bthem (2+ . Also, tthem (2+ . Furthery to Stl . Taken t


O methyl-modified residues in a series of all pyrimidine RNA TFOs, and we studied the binding to two RNA hairpin structures. The 12-base-triple major-groove pyrimidine–purine–pyrimidine triplex structures form between the duplex regions of RNA/DNA hairpins and the complementary RNA TFOs. Ultraviolet-absorbance-detected thermal melting studies reveal that the locked nucleic acid and 2-thio U modifications in TFOs strongly enhance triplex formation with both parental RNA and DNA duplex regions. In addition, we found that incorporation of 2′-O methyl-modified residues in a TFO destabilizes and stabilizes triplex formation with RNA and DNA duplex regions, respectively. The (de)stabilization of RNA triplex formation may be facilitated through modulation of van der Waals contact, base stacking, hydrogen bonding, backbone pre-organization, geometric compatibility and/or dehydration energy. Better understanding of the molecular determinants of RNA triplex structure stability lays the foundation for designing and discovering novel sequence-specific duplex-binding ligands as diagnostic and therapeutic agents targeting RNA.Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is not well understood. Here, we incorporated locked nucleic acid, 2-thio U- and 2′- The biological functions of RNAs include gene regulation, catalysis, immunomodulation and acting as templates for the synthesis of protein, RNA and DNA. Recent advances in understanding the structures, energetics and functions of RNAs provide the foundation for developing nucleic acid-based diagnostics, therapeutics and nanobiotechnologies . TriplexO methyl (2′-OMe)  B modific(2′-OMe) ,32 with (2′-OMe) B signifi(2′-OMe) . A base (2′-OMe) B in TFOs(2′-OMe) .Figure L), 2-thio U (s2U), 2′-OMe 2-thio U (s2UM), 2′-OMe U (UM) and 2′-OMe C (CM) residues into 12-nt all pyrimidine RNA TFOs, and we studied the major-groove triplex formation of the modified TFOs with RNA and DNA hairpins , RNA hairpin 1 (rHP1), RNA hairpin 2 (rHP2), RNA hairpin 3 (rHP3), biotin-labeled RNAs and unmodified RNA TFOs  were pur2U3) was recorded every 0.5°C. The samples for UV-absorbance-detected thermal melting studies contained 5 µM hairpin and 5 µM TFO in 100–1000 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 20 mM 2-morpholinoethanesulfonic acid (MES)  or 20 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) . We chose MES and HEPES buffers because their pKa values are relatively independent of temperature  in the melting (heating) curves were taken as the melting temperatures (Tm1 for triplex to hairpin transition and Tm2 for hairpin to single-strand transition). Hysteresis between heating and cooling curves was observed for triplex to hairpin transitions. No hysteresis was observed for hairpin to single-strand transition. Equilibrium thermodynamic parameters for the triplex to hairpin transitions were not obtained because of the hysteresis. Equilibrium thermodynamic parameters for the duplex and hairpin formation were obtained by fitting to a two-state model with the MeltWin program  with a Peltier temperature controller, and the absorbance at 260 or 265 nm (only for 1-Rsperature . A detai program .2 and 1× TAE (40 mM Tris–acetate and 1 mM EDTA) at pH 6. The gel electrophoresis experiments were run at 160 V for 12–16 h, and the gels were stained by ethidium bromide, and the hairpin and triplex bands were imaged by a Typhoon scanner .The native polyacrylamide gel electrophoresis experiments were performed in 16.5- × 22-cm gel containing 12% acrylamide (acrylamide/Bis–acrylamide = 19:1) at 4°C. RNA hairpin and TFOs were both 1 µM, which were incubated in 40 µl of buffer of 100 mM NaCl, 20 mM MES and 0.5 mM EDTA at pH 5.5, and left for 3 h before loading. Eight microliters of 35% glycerol was then added into 40 µl of loading buffer. The running buffer contains 10 mM NaCl, 2 mM MgClN-ethyl-N′-[3-(diethylamino)propyl]carbodiimide and 50 mM N-hydroxysuccinimide. Neutravidin was then dissolved into 10 mM sodium acetate at pH 6.0 and immobilized at 10 µl/min flow rate onto the surfaces by standard amine coupling procedure to achieve 4000 RU. The surfaces were finally blocked with 0.5 M ethanolamine–HCl at pH 8.5 for 7 min. Biotinylated RNA hairpin  residues incorporated as a linker between biotin and RNA hairpin, rHP3) and an RNA hairpin control  residues incorporated as a linker between biotin and RNA hairpin, rHP1) were captured on sensor chip surfaces to ∼740 RU. Serially diluted TFOs  were injected (10 µl/min) for 10 min across the surfaces with immobilized RNA hairpins. After a dissociation period (1200 s), a 60 s pulse of 0.1% sodium dodecyl sulfate in H2O was applied to regenerate the surfaces, followed by a 10-min running buffer flow. All the sensorgrams were corrected by subtraction of the buffer blanks and responses of TFOs on the RNA hairpin control surface. Processed data were globally analyzed and fit to a simple 1:1 interaction model with mass transport coefficient.All SPR experiments were run with a constant flow (10 μl/min) of running buffer  on a Biacore T200  with a carboxymethylated dextran-coated sensor chip (CM5-S) at 25°C. The surfaces were first activated for 7 min with 1:1 mixture of 0.2 M Tm2) do not change significantly with pH (Supplementary Table S1), consistent with previous studies for RNA duplex structures (Tm1) decrease significantly (by >20°C) with increasing pH (from pH 5.5 to 7.0) (Supplementary Table S1), which is consistent with a pKa of ∼7.0 for cytosine N3 atoms in TFOs of major-groove RNA triplexes , we did not obtain equilibrium thermodynamic parameters.Cytosine N3 atoms in a TFO need to be protonated to form hydrogen bonds (Hoogsteen base pairs) with guanine N7 atoms in the purine strand of a duplex  and 2. Tructures . The melriplexes . BecauseTm2) increases as expected, whereas the melting temperatures of the RNA triplexes (Tm1) decrease modestly (Supplementary Table S1). Destabilization of triplexes with increasing concentration of NaCl is probably because of the fact that increasing concentration of NaCl decreases the pKa of N3 of cytosines in the TFOs, which in turn destabilizes the RNA triplexes at pH 5.0–7.0. Our results are consistent with the previous finding that Na+ and H+ are competitive in binding to unmodified DNA triplexes containing both C+·G–C and T·A–T base triples  reveal that LNA U-modified RNA TFOs enhance triplex thermal stability with parental RNA duplex structure segment within a hairpin . The Tm1 for the triplex with unmodified RNA TFO (1-R12) is 28.1°C in 200 mM NaCl at pH 6.0. At the same buffer condition, the Tm1’s for the triplexes with modified TFOs with one, two and three LNA U residues  are 36.5, 45.4 and 52.9°C, respectively. Similar stabilizing effect is observed for the complementary TFO (3-RUL2 versus 3-R12) binding to rHP3 , consistent with previous results  in the TFOs are 40.1, 51.9 and 54.4°C, respectively, in 200 mM NaCl at pH 6.0 (Supplementary Table S1). Similar stabilizing effect is also observed for the complementary TFO (3-Rs2U2 versus 3-R12) binding to rHP3 (The 2-thio U modification  in an ol to rHP3 D.Ka of N3 (from 9.3 to 8.8) on thiolation of U . Ba. Ba36).  C and U , increas 1-Rs2U2 C. In conby ∼10°C C and D. by ∼10°C . Thus, nL1) at 1:0.5. The result suggests that a mixture of ∼50% triplex and ∼50% RNA hairpin is present in lane 6  by both ethidium bromide and Syber Green II staining (data not shown). Taken together, our thermal melting and gel results suggest that the designed RNA triplex structures . The SPR results suggest that the designed triplex structures (We confirmed triplex formation by native polyacrylamide gel electrophoresis experiment . The geln lane 6 A. Directructures B and H fructures H indeed Tm1) are <20°C at pH 6.0 (Supplementary Table S1). We tested TFOs 1-RUL1 and 1-Rs2U3 binding to rHP2 at low pH (5.5 and 5.0), and Tm1 values were found to be >30°C lower than those of sequence-complementary triplexes when Tm1 and Tm2 are not overlapped (Supplementary Tables S1). The native gel results further confirm that triplexes do not form between rHP2 and TFOs with one mismatch . The results indicate that the TFOs have good sequence specificity in binding to an RNA duplex region.We further tested the binding of the RNA TFOs to an RNA hairpin with one A–U base pair inverted compared with rHP1 rHP2, C and D. Tm1 of triplex with TFO 1-RUM3 (25.7°C), which has three 2′-OMe residues, is lower than that with RNA TFO (1-R12) (46.4°C) in 100 mM NaCl at pH 5.5 (Supplementary Table S1). All the complementary modified TFOs with more than three 2′-OMe modified residues do not bind to rHP1 (Supplementary Table S1). No obvious gel electrophoresis mobility difference was seen between rHP1 alone and rHP1 mixed with TFOs with three or more 2′-OMe residues , further suggesting that 2′-OMe TFOs do not bind to rHP1. The results indicate that 2′-OMe-modified TFOs destabilize triplex formation with RNA duplex region.We then investigated how 2′-OMe modification B in a TFIt is probable that substitution of 2′-OH with 2′-OMe disrupts the hydrogen bond between 2′-OH in the TFO and a non-bridging oxygen in the purine strand of a duplex  14,15,4,53,54 anThermodynamically stable major-groove triplex structure formation without chemical modification is limited to a pyrimidine TFO strand binding to a duplex region with purines on one strand and pyrimidines on the other ,62–64. PTm1) are always higher with a DNA duplex region than with a homologous RNA duplex region (ΔT m1 ranging from 6 to 21°C in 200 mM NaCl at pH 6.0) (Supplementary Table S1). The DNA hairpin, dHP1, has a melting temperature (Tm2) of ∼74°C in 200 mM NaCl, which is ∼10°C lower than the RNA hairpin, rHP1 (Supplementary Table S1), consistent with the prediction from nearest-neighbor models , which is in contrast to the significant destabilization effect observed in TFOs binding to the RNA hairpin, rHP1 . It is likely that the major groove of a DNA duplex is more geometrically compatible for accommodating a 2′-OMe-modified TFO. The results suggest that rules for enhancing DNA triplex formation may not be applicable for RNA triplex formation. One may simply incorporate 2′-OMe or other relatively bulky residues in a TFO to selectively target a DNA duplex over a homologous RNA duplex region.The relatively wider major groove of a DNA duplex (compared with an RNA duplex) may provide relatively easier access for TFO binding. Consistently, incorporation of 2′-OMe residues in TFOs was found to enhance binding to the DNA hairpin, dHP1 (Tm2 of rHP1 (Supplementary Table S3), the designed hairpin structure forms without appreciable bimolecular duplex . It is likely that chemically modified TFOs may displace the pyrimidine segment of the parental RNA hairpin to form parallel or antiparallel duplex structure as shown in Supplementary Figure S2A and B. To test the possibility of the formation of such alternative structures because of strand invasion, we carried out thermal melting studies for the duplexes formed between a 12-nt all purine strand  and various TFOs. The results reveal that most of the duplex formation is relatively pH independent (ΔTm is within 3°C from pH 7.0 to 5.5) and have lower Tm than that of the parental RNA hairpin, rHP1 . The pH independence of melting temperatures suggests that 12-bp parallel RNA duplex structures  do not form because the stability of parallel DNA duplex is pH dependent .As evidenced by the strand concentration independent ependent . Thus, 9Tm values of the 9-bp antiparallel duplexes  are in between Tm1 and Tm2 of triplexes  suggests that neither parallel nor antiparallel duplex structure forms due to strand invasion of rHP1 . In addition, there are only two bands observed in native gels , further suggesting no appreciable alternative structures form  to destabilize RNA base triple formation because of the loss of a hydrogen bond and steric clash. LNA sugar modification in TFOs stabilizes RNA triplex structures because of backbone pre-organization, despite the loss of a hydrogen bond. The 2-thio U base modification in TFOs stabilizes RNA triplex formation in a sequence environment-dependent manner, probably because of the combined effects of enhanced van der Waals contact, hydrogen bonding, base stacking, backbone pre-organization and reduced energy penalty of dehydration. A thio group may be selected (by nature) to enhance van der Waals interaction with the hydrogen atoms in a duplex major groove  and other interactions, and thus favor RNA base triple and other tertiary structures. Our results provide useful insights into rational design of more potent and selective triplex-forming ligands targeting biologically important RNA duplex regions. Studies of other chemical modifications in bases and/or sugar–phosphate backbone and detailed sequence-dependent thermodynamic and kinetic characterizations are in progress.Supplementary Data are available at NAR Online: Supplementary Tables 1–3, Supplementary Figures 1–6, Supplementary Method and Supplementary References .Nanyang Technological University (NTU) (to G.C.); NTU internal grant (Singapore Ministry of Education Academic Research Fund Tier 1) (to G.C.); National Institutes of Health [1R03TW008739-01 to E.K. and D.H. Turner]; National Science Center [N N301 788 440 to E.K. and UMO-2011/03/B/NZ1/00576 and UMO-2011/03/B/ST5/01098 to R.K.]. Funding for open access charge: Start-up grant of Nanyang Technological University (NTU) (to G.C.).Start-up grant of Conflict of interest statement. None declared.