By directly monitoring protein binding inside virions, we identify the RNA recognition motif for the viral nucleocapsid protein. Seven structurally homologous binding sites occur in a well-defined domain in the genome, consistent with a role in directing specific packaging of genomic RNA into nascent virions. In addition, we identify two distinct motifs that are targets for the duplex destabilizing activity of this same protein. The nucleocapsid protein destabilizes local HIV-1 RNA structure in ways likely to facilitate initial movement both of the retroviral reverse transcriptase from its tRNA primer and of the ribosome in coding regions. Each of the three nucleocapsid interaction motifs falls in a specific genome domain, indicating that local protein interactions can be organized by the long-range architecture of an RNA. High-throughput SHAPE reveals a comprehensive view of HIV-1 RNA genome structure, and further application of this technology will make possible newly informative analysis of any RNA in a cellular transcriptome.Replication and pathogenesis of the human immunodeficiency virus (HIV) is tightly linked to the structure of its RNA genome, but genome structure in infectious virions is poorly understood. We invent high-throughput SHAPE  technology, which uses many of the same tools as DNA sequencing, to quantify RNA backbone flexibility at single-nucleotide resolution and from which robust structural information can be immediately derived. We analyze the structure of HIV-1 genomic RNA in four biologically instructive states, including the authentic viral genome inside native particles. Remarkably, given the large number of plausible local structures, the first 10% of the HIV-1 genome exists in a single, predominant conformation in all four states. We also discover that noncoding regions functioning in a regulatory role have significantly lower ( The function of the RNA genome of the human immunodeficiency virus (HIV) is determined both by its sequence and by its ability to fold back on itself to form specific higher-order structures. In order to describe physical structures in a region of the HIV RNA genome known to play multiple, critical roles in viral replication and pathogenesis, we invent a high-throughput, quantitative, and comprehensive structure-mapping approach that locates flexible (unpaired) nucleotides within a folded RNA, assaying hundreds of nucleotides at a time. We find that the first 10% of the HIV-1 genome has a single predominant structure and that regulatory motifs have significantly greater structure than do protein-coding segments. The HIV genome interacts with numerous proteins, including multiple copies of the nucleocapsid protein. We directly map RNA–protein interactions inside virions and discover that the nucleocapsid prottein interacts with viral RNA in at least three distinct ways, depending on the context within the overall genome structure. Further application of the high-throughput RNA-structure analysis tools described here will make it possible to address diverse structure–function relationships in intact cellular and viral RNAs. Development of novel, quantitative, high-throughput RNA structure analysis tools allows the outline of structure-function relationships for the first 10% of an HIV genome, discovery of structural differences between regulatory and coding regions, and analysis of protein-RNA interactions inside authentic virions. As is the case with many natural RNAs, the function of the HIV RNA genome is strongly linked to its ability to form higher-order structure and to interact with protein effectors in each stage of its replication cycle. The 5′ end of the HIV genome alone contains a noncoding, highly structured region that is involved in viral packaging, dimerization, pairing with the cellular tRNA primer for cDNA synthesis, and binding numerous viral proteins ,2.Immediately downstream of this regulatory region, the HIV genome contains nine open reading frames. The first protein encoded by the HIV-1 genome is Gag, which is ultimately cleaved into a complex set of proteins, including matrix, capsid, and nucleocapsid . The nucBecause of its importance in regulating replication and for governing interactions with protein cofactors, extensive efforts have been directed towards developing structural models for the HIV-1 genome and for identifying candidate interaction sites for the nucleocapsid protein. Multiple models based on phylogenetic, chemical mapping, and mutagenesis approaches have been proposed –17. In gOne of the most successful methods for determining an RNA secondary structure has been phylogenetic covariation analysis ,19. HoweAlternatively, RNA structural information can be inferred by treatment with chemicals or enzymes that discriminate between paired and unpaired nucleotides ,21. ThisTherefore, comprehensive and accurate analysis of any cellular or viral RNA in a relevant biological context requires a new technology that may be used for any RNA sequence under a wide variety of biologically relevant conditions. Our approach is based on the observation that electrophiles, such as N-methylisatoic anhydride (NMIA), react selectively with flexible RNA nucleotides at the ribose 2′-hydroxyl group  22]..22].O-adduct per 300 nucleotides (nts). Adducts are detected by their ability to inhibit primer extension by reverse transcriptase. A control extension reaction omitting NMIA to assess background, along with dideoxy sequencing extensions to assign nucleotide positions, are performed in parallel. These combined steps are called selective 2′-hydroxyl acylation analyzed by primer extension, or SHAPE  and reverse [5′-CTATC CCATT CTGCA GCTTC C] primers, approximately 1 μg of plasmid template containing a partial sequence of the HIV-1 pNL4–3 molecular clone [obtained from the National Institutes of Health AIDS Research and Reference Reagent Program], 200 μM each dNTP, and 25 units Taq polymerase; 34 cycles). The PCR product was recovered by ethanol precipitation and resuspended in 300 μl of TE . Transcription reactions  were initiated by adding 300 μl of 1 mg/ml T7 RNA polymerase  in the gel.RNA transcript  was combined with internally [RNA (2 pmol) in 14.4 μl of 1/2× TE was refolded by heating at 95 °C, placing on ice, adding 3.6 μl of folding buffer, and incubating at 37 °C for 60 min. The folded RNA was divided equally between two tubes and treated with either NMIA ,23  and Superscript III . Extension continued at 52 °C for 15 min. Sequencing reactions used to identify peaks in the (+) and (−) reagent experiments contained transcript RNA , 3 μl of primer (2 μM WellRED D2 or 1.2 μM LICOR IR 800), enzyme mix (6 μl), ddNTP solution , and Superscript III (1 μl). Four sets of primers were used that were complementary to positions 342–363 (5′-CGCTT AATAC CGACG CTCTC GC), 535–555 (5′-CTTCT GATCC TGTCT GAAGG G), 743–762 (5′-CCATT TGCCC CTGGA GGTT C), or 956–976 (CTATC CCATT CTGCA GCTTC C). Depending on the quality of synthesis, primers were purified by denaturing gel electrophoresis  and passively eluted into 1/2× TE overnight. The four reactions corresponding to a complete hSHAPE analysis ((+) NMIA, (−) NMIA, and two sequencing reactions) were combined and precipitated with ethanol in the presence of acetate, EDTA, and glycogen. Pellets were washed twice with 70% ethanol, dried under vacuum, and resuspended in deionized formamide. cDNA samples were separated on a 33-cm × 75-μm capillary using a Beckman CEQ 2000XL DNA sequencer.In vitro transcript or authentic genomic RNA  corresponding to either the (+) or (−) NMIA reactions was heated to 95 °C for 3 min and cooled on ice for 1 min. Fluorescently labeled primer (3 μl) was added to the (+) (0.2 μM Cy5) and (−) (0.4 μM WellRED D3) NMIA reactions, respectively, and primer-template solutions were incubated at 65 °C for 5 min and 35 °C for 10 min. Primer extension was initiated by addition of enzyme mix  baseline correction, (2) color separation to correct for spectral overlap of the fluorescent dyes, and (3) mobility shift correction to align corresponding peaks in the four channels. Areas under each peak in the (+) and (−) NMIA traces were obtained by (1) peak detection and interpolation to align peaks in each channel with the RNA sequence and (2) performing a whole-trace Gaussian-fit integration. Integrated peak intensities were corrected for signal decay [Raw fluorescence intensity versus elution time profiles were analyzed using a draft software suite called ShapeFinder. ShapeFinder is derived from BaseFinder , which ial decay  as a funD is the signal decay adjustment factor, A and C are scaling factors that reflect the arbitrary initial and final intensities of the trace, and p is the probability of extension at each nucleotide. Typical values for p spanned 0.995–0.999 for elution times in units of 2 Hz. Each peak intensity calculated at the same elution time was divided by D. SHAPE intensities at each individual nucleotide were examined manually to identify positions where high background was present in the (−) reagent control experiment. A very small number of positions fell into this category (<6%) and were marked as containing no data. Quantitative SHAPE reactivities for individual datasets were normalized to a scale in which 0 indicates an unreactive site and the average intensity at highly reactive sites is set to 1.0. The normalization factor for each dataset was determined by first excluding the most-reactive 2% of peak intensities and then calculating the average for the next 8% of peak intensities. All reactivities were then divided by this average. This simple normalization procedure places all absolute reactivities on a scale of 0 to approximately 1.5 . The maximum allowed distance between paired bases was restrained to be 300 nts or less. Increasing the maximum pairing distance to 600 nts yielded a series of short, poorly predicted, and transient pairings. The reactivity of the nucleotides participating in these interactions could be explained by shorter distance pairings. To determine the pairing persistence, structures were also computed for larger values of the b and m parameters, which has the effect of increasing the contribution of the SHAPE reactivity information on the secondary structure calculation. Helices considered to be highly predicted persisted even when these parameters were set to values as high as b = 0 and m > 4. RNAstructure is available for download at http://rd.plos.org/pbio.0060096.2.SHAPE intensities were converted into a pseudo-free energy change term in the RNAstructure program   [To analyze differences in SHAPE reactivities between groups of nucleotides, such as comparing the 5′ regulatory versus 3′ protein coding regions, several tests were applied to determine the statistical characteristics of the subgroups and to determine the appropriate statistical measures of differences. We applied Levene's test to analyze homoscedasticity, which confirmed that the SHAPE reactivity variances between analyzed groups of nucleotides are equivalent. However, quantile-quantile (Q-Q) plots of these data indicated departures from normality for the SHAPE reactivity data, so the standard Student n 2.6.1) .Differences in structure between regulatory and coding domains. SHAPE reactivity data for the ex virio state were divided into two groups corresponding to the 5′ regulatory (nucleotides 1–335) and Gag coding sequences (nucleotides 336–906) and analyzed by the Wilcoxon rank sum test. Differences between the 5′ regulatory and 3′ coding regions were highly significant (p < 0.0001).Absence of differences between TAR and DIS SHAPE reactivities for in vitro transcript, ex virio, and AT-2–treated states. Reactivity data were analyzed for the TAR (nucleotides 1–57) and DIS (nucleotides 236–282) regions. Three groups of reactivities were collected, reflecting each RNA state  with 141 and 129 nts for the TAR and DIS regions, respectively. We applied a one-way analysis of variance (ANOVA) to the three groups for each of the regions, to test whether they displayed statistically significant differences among groups for either region. The F ratio was calculated to determine whether the means of individual groups, corresponding to the three states, represent significant variation from the overall mean. The initial F ratio values calculated showed no significant difference between groups, with p-values of 0.63 for TAR and 0.98 for DIS, respectively. However, because SHAPE reactivities are not normally distributed, the F ratio can be inaccurate. To assess whether the initial F ratio was representative of the underlying distribution, we performed bootstrap resampling of the SHAPE data from the groups, with replacement [p-values by determining the number of times F ratios with larger (more extreme) values were obtained. The bootstrap–re-estimated p-values were 0.625 for TAR and 0.974 for DIS, in close agreement with the initial calculation. This indicates that the original F ratio, which showed no statistical difference between these groups, is representative of the lack of difference between the TAR and DIS structures in these three HIV-1 RNA genome states.lacement . We recaStatistical significance of nucleocapsid binding sites. SHAPE reactivity data were divided into two groups corresponding to the in virio and AT-2–treated states. For each state, the nucleotides were divided into the three nucleocapsid protein binding classes Click here for additional data file.Figure S1In this figure, structures are illustrated schematically at the left. Representative nucleotides are colored by their experimental SHAPE reactivity using the scale shown in 22CU24 bulge in TAR. Native and ΔU22CU24 histograms are black and purple, respectively.(A) Deletion of the U(B) SHAPE analysis of the 79–85/443–339 pseudoknot. Pseudoknot structure was analyzed for the 976 nt in vitro transcript (black histogram) as compared to a 362 nt RNA that lacks sequences required to form the pseudoknot (cyan).(C) Ex virio genomic RNA bound to tRNA(lys) (red) undergoes a local rearrangement to maximize intra-molecular pairs (orange) upon heating and refolding.(294 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession number for the pNL4–3 molecular clone is AF324493.The GenBank (


Here we describe experiments that show that replication of the important picornavirus pathogen foot-and-mouth disease virus (FMDV) in the presence of increasing concentrations of ribavirin results in the sequential incorporation of three amino acid substitutions  in the viral polymerase (3D). The main biological effect of these substitutions is to attenuate the consequences of the mutagenic activity of ribavirin —by avoiding the biased repertoire of transition mutations produced by this purine analogue—and to maintain the replicative fitness of the virus which is able to escape extinction by ribavirin. This is achieved through alteration of the pairing behavior of ribavirin-triphosphate (RTP), as evidenced by in vitro polymerization assays with purified mutant 3Ds. Comparison of the three-dimensional structure of wild type and mutant polymerases suggests that the amino acid substitutions alter the position of the template RNA in the entry channel of the enzyme, thereby affecting nucleotide recognition. The results provide evidence of a new mechanism of resistance to a mutagenic nucleoside analogue which allows the virus to maintain a balance among mutation types introduced into progeny genomes during replication under strong mutagenic pressure.Resistance of viruses to mutagenic agents is an important problem for the development of lethal mutagenesis as an antiviral strategy. Previous studies with RNA viruses have documented that resistance to the mutagenic nucleoside analogue ribavirin  can become resistant to ribavirin. Amino acid changes in the viral polymerase, selected by ribavirin, are able to modify the types of mutations produced in the presence of ribavirin. Biochemical data indicate that the alteration of the enzyme changes the pairing behavior of ribavirin, avoiding the production of an excess of some types of mutations, supporting the hypothesis that an unbalanced mutation repertoire is detrimental to the virus. Thus, this new mechanism of resistance to ribavirin is based not as much in limiting the number of mutations in the virus genetic material but in ensuring an equilibrium among different types of mutations that favors viral survival. This problem has been manifested with the selection of picornavirus mutants with decreased sensitivity to the mutagenic base analogue ribavirin (1-Picornaviruses have contributed to the understanding of the molecular basis of resistance to R. A poliovirus (PV) mutant with decreased sensitivity to R included substitution G64S in its RNA-dependent RNA polymerase (termed 3D). This substitution confers resistance to R through a general increase in template copying fidelity, at the cost of producing mutant spectra of lower complexity than wt PV. Limited mutant spectrum complexity resulted in PV populations which were less adaptable to a complex environment, a direct proof of the essential contribution of high mutation rates to RNA virus adaptability Replacement M296I was sufficient to prevent extinction of FMDV by high concentrations of R, but the virus was extinguished by an alternative mutagenic treatment that included 5-fluorouracil (FU) A biological clone of FMDV termed C-S8c1 is the standard virus used in our studies of molecular evolution and lethal mutagenesis of FMDV To test whether population of R-Ap60 was better adapted to R than population R-Ap35, the relative fitness of the two populations was determined in growth-competition experiments in the presence and absence of R, using as reference the virus population Ap35  were constructed as described in To investigate the effect of the 3D substitutions in the sequence context of pMT28  failed to reproduce the fitness difference between populations R-Ap60 and R-Ap35 in the presence of R compare  and 4. TThe mutant spectra of FMDV populations passaged in the absence of drugs display a balance among the four types of transition mutations with a slight dominance of U→C and A→G versus C→U and G→A 2 = 0.02, P>0.1; χ2 test), with a slight dominance of U→C and A→G, as previously found in FMDV populations that had replicated in the absence of R 2 = 0.49, P>0.1) . These results suggest that adaptation of FMDV to high R concentrations was related to modulation of the types of transitions imposed by the pairing behavior of RMP, preventing a highly biased mutation pattern in progeny genomes.To explore possible variations in mutation frequency and in the types of mutations as a result of R treatment, FMDV wild type (Wt) (rescued from plasmid pMT28) and FMDV 3D(SSI) were subjected either to five passages in the absence of R  or to four passages in the presence of 5000 µM R . The comparison of mutation frequencies in the mutant spectrum of the different populations showed a 3.5-fold increase in both viruses after passage in the presence of R, as expected , P>0.1) . This re, P>0.1) . Thus, RTo investigate whether FMDV 3D(SSI) was resistant to extinction by R, FMDV Wt and FMDV 3D(SSI) were subjected to serial cytolytic passages in BHK-21 cells in the presence or absence of 5000 µM R. The wild type population was extinguished by passage 7, as expected To further substantiate the hypothesis that substitutions P44S, P169S and M296I confer a selective advantage in the presence of R but not in the presence of another mutagen that induces a different mutational repertoire, growth-competition experiments between FMDV Wt and FMDV 3D(SSI) were carried out in the presence of either R or FU , the polymerases that include the individual substitutions  and the polymerase with the three substitutions [termed 3D(SSI)] were purified as detailed in in vitro, is consistent with the proportion of transition types observed during replication of FMDV 3D(SSI) in the presence of R during infections of BHK-21 cells (compare Previous studies documented that 3D(M296I) displayed a defect in the incorporation of RTP opposite either U and C, in comparison with 3D Wt  compare . FMDV 3D compare , suggest5′GCAUGGGCCC3′, crystallized, and analyzed by X-ray diffraction. Sym/sub- U indicates that U is the template residue which directs the incorporation of A to produce a +1 elongation product. This is the same RNA used in our previous structural studies with FMDV 3D To identify possible structural modifications of the viral polymerase associated with the important alterations of the mutational spectrum in progeny RNA, and to investigate how these modifications can affect RNA binding and polymerase activity, the different mutant 3Ds were incubated with the heteropolymeric sym/sub-U RNA of sequence 2212 space group. The RNA molecule appeared mostly disordered in the two structures. In contrast, 3D(SSI) crystallized in the trigonal P3221 space group with the sym/sub-U RNA incorporated in the structure. Since the biochemical results indicate that P44S plays a critical role in the misincorporation of RMP into RNA by 3D, and a 3D(P44S)-RNA complex was not obtained, we attempted the crystallization of the double mutant 3D in complex with RNA. 3D also crystallized in the space group P3221 space group, with the sym/sub-U RNA incorporated in the structure.Two different crystal forms were obtained ; the sinFurther attempts to obtain the structures of ternary complexes, using ATP or RTP were unsuccessful, despite using different substrate concentrations and incubation times. The X-ray structures were determined to 2.2 Å and 2.6 Å resolution for 3D(P44S) and 3D(P169S), respectively, and to 2.6 Å and 2.5 Å for 3D and 3D(SSI), respectively . The quaNo major structural changes were observed in the polymerase active site when the structures of the different polymerases (either unbound or bound to RNA) were compared. The structural superimpositions of all 476 amino acids residues of 3D(P44S) and 3D(P169S) and of 3D onto the 3D(SSI) showed root mean square deviation (rmsd) values of 0.46Å, 0.35Å and 0.22Å, respectively.Subtle domain movements, in particular a ∼1° rotation of the thumb domains relative to the fingers, were observed between the unbound, tetragonal, and the RNA-bound, trigonal structures when the individual domains were superimposed. When the unbound and RNA-bound structures were compared for 3DWt a similar small rotation (∼2°) was also observed. As a consequence of this rotation, the active site appeared more closed in the unbound state. Thus, the changes observed seem to be a consequence of either RNA-binding, or of the different packing constraints in the tetragonal and trigonal space groups or both, but they do not seem to be related to the presence of substitutions P44S or P169S.The 3D-RNA and 3D(SSI)-RNA structures are almost identical . These structures are also similar to the structure of 3D(M296I)-RNA mutant complex determined previously  passaged in the absence of R or in the presence of FU or 5-azacytidine in vivo. Do the high concentrations of R used in our experiments weaken the relevance of the conclusions? We think not for two reasons: (i) the actual concentration of RTP in the replication complexes of viruses is unknown, and it cannot be excluded that methods of targetted delivery could be developed that result in high local RTP concentrations; (ii) extreme environmental conditions  have previously been used to unveil either evolutionary responses or the sensitivity of biochemical processes to subtle genetic change in vivo.It may be argued that selection of the multi-substituted polymerase occurred as a result of subjecting the virus to extremely large ribavirin concentrations, unlikely to be reached during any standard antiviral treatment with R. Certainly, the concentrations used were not intended to reproduce actual R concentrations in the course of treatments with R in clinical practice. In the case of direct aerosol application of R to the upper respiratory tract the drug may reach intracellular concentrations of around 800 µM The virological and biochemical evidence presented here support the hypothesis that the polymerase substitutions, whose effect was to avoid a highly biased distribution of mutation types normally induced by a mutagenic agent, contributed to viral survival and escape from extinction , implyinDespite the clear virological and biochemical effects of substitutions P44S, P169S and M296I in 3D, the comparison of fitness values for clones and populations indicates that it is unlikely that the replacements in 3D are the only determinants of high level resistance to R. Indeed, the fitness of the uncloned FMDV population R-Ap60 was 15-fold higher than the fitness of control population Ap35, when measured in the presence of 5000 µM R, while the fitness of the cloned FMDV 3D(SSI) was 2-fold higher than the fitness of the cloned FMDV Wt, measured under the same conditions  and 4. TP44 is conserved among known picornaviral polymerases, and it lies in a loop that connects strand β2 and helix α2 in the fingers domain . This loThe structures of the mutant polymerases determined in the present study do not show large domain movements. However, the crystal structures of 3D and 3D(SSI) in complex with the RNA template-primer reveal a rearrangement in the template channel with important effects in template binding, in particular, at position n+1 (nucleotide A3). The conformational changes in the main and side chains of residues M16 and R17 allow the opening of a hydrophobic pocket formed by residues of motifs G and F and by M16 that facilitates the entrance of nucleotide A3 . The polFinally, P169 is a non-conserved residue located in motif F of 3D  that has6 BHK-21 cells were passaged with supernatant of virus from the previous passage (0.2 ml), and the infection allowed to proceed for about 24h. Values of PFU for each passage can be estimated from infectivities given in The origin of BHK-21 cells, procedures for cell growth and for infection with FMDV in the presence or absence of ribavirin , 5-fluorouracil , or guanidine hydrochloride  have been previously described Pfu ultra DNA polymerase (Stratagene). Amplification protocols, nucleotide sequencing and primers used for amplification and sequencing have been previously described RNA was extracted from the supernatants of infected cells using described procedures FMDV RNA was quantified by real-time RT-PCR amplification using the Light Cycler instrument (Roche) and the RNA Master SYBR green I kit (Roche) as previously described Pfu ultra DNA polymerase and pMT28 DNA as template. A first amplification with 3AR3  and 3′3DP169S ; and a second amplification with 5′3DP169S  and 3D1  were performed (bold-face letters indicate modifications of the genomic sequence introduced to express 3D with substitution P169S). (Nucleotide positions correspond to the numbering of FMDV genomic residues described in RsrII (position 5839) and ClaI (position 7004) (New England Biolabs) and ligated to pMT28 DNA linearized with the same enzymes. A similar procedure was used to construct pMT28-3D(P44S) and pMT28-3D. To prepare pMT28-3D (an infectious clone expressing 3D harboring substitution P44S and M296I in the context of the C-S8c1 genome), pMT28-3D(M296I) was subjected to the same procedure described above for pMT28, except that the two pairs of primers used for the PCR amplification were 3AR3 (described above) with 3′3DP44S , and 5′3DP44S  with A3 . The two amplicons were shuffled, digested with RsrII and ClaI, and ligated to pMT28 or pMT28-3D(M296I) DNAs linearized with the same enzymes, rendering pMT28-3D(P44S) and pMT28-3D, respectively. Finally, to construct pMT28-3D , procedures were carried out as those described for pMT28-3D(P169S) except that the parental plasmid used both as template for DNA amplifications and for cloning was pMT28-3D instead of pMT28. For simplicity, the plasmid that includes the three amino acid substitutions in 3D has been termed pMT28-3D(SSI) and the rescued virus FMDV 3D(SSI).Plasmid pMT28 encodes an infectious transcript of FMDV C-S8c1 coli DH5α, colony screening, nucleotide sequencing, preparation of infectious RNA transcripts, and RNA transfections were carried out as previously described Ligation, transformation of E. GGTACCCACTCTGCTGGAGGCGTT; sense, 5′ position 6502) and Pol1-XbaI . cDNA was digested by KpnI and XbaI enzymes (New England Biolabs), and ligated to plasmid pGEM-3Z Vector (Promega) previously digested with the same enzymes. Transformation, colony screening and nucleotide sequencing were carried out as previously described To determine the complexity of mutant spectra, FMDV RNA was extracted as described above and subjected to RT-PCR using primers PolC-KpnI  in PBS was prepared at a concentration of 100 mM, sterilized by filtration, and stored at −70°C. Prior to use, the stock solution was diluted in DMEM to reach the desired R concentration. To prepare culture medium containing 5-fluorouracil (FU) (Sigma), the analogue was dissolved in DMEM to yield a 5 mg/ml solution, and diluted in DMEM, as needed for the experiments. For infections in the presence of R (5000 µM) and FU (2000 µM), cell monolayers were treated during 7 h and 10 h, respectively, prior to infection. The relative fitness of FMDV SSI was determined by growth competition with the Wt virus in BHK-21 cells in the presence of R, FU or a mixture of both drugs. Briefly, the viral population to be assayed was mixed with the same number of PFU of FMDV Wt, used as reference. For each determination, four serial infections were carried out at moi 0.3 PFU/cell. The proportion of the two competing genomes at each passage was determined by measuring the area of the three peaks corresponding to the residues that distinguish 3D Wt from 3D(SSI). Each mutation was confirmed by two independent sequencing assays using primers of different orientation. The average of triplicate measurements and standard deviations are given.FMDV 3D with substitutions M296I [termed 3D(M296I)], P169S [termed 3D(P169S)], P44S [termed 3D(P44S)], with P44S and M296I [termed 3D], or with the three of them [3D which is abbreviated as 3D(SSI)] were obtained from plasmid pET-28a 3Dpol  ATP and polynucleotide kinase (New England biolabs), and annealed using standard protocols 3COO)2, and 50 µM UTP (Amersham) in the case of sym/sub-AU, for either 10 min  or 30 min [for 3D(P44S) and 3D(SSI)] at 37°C, or 33°C when mentioned; 3D(SSI) and 3D(P44S) were incubated for longer periods of time because they display a defect in RNA binding (see Incorporation of standard nucleoside-5′-triphosphates or ribavirin-5′-triphosphate (RTP) by wild type and mutant 3Ds was measured in self-complementary RNAs that form double stranded RNA in which each strand can act both as template and primer ding see . After f15 as template-primer molecule, VPg uridylylation with poly(A) as template and Mn++ as ion, and RNA binding assays were carried out as previously described Poly(rU) synthesis using poly(A)-oligo(dT)5′GCAUGGGCCC 3′ (NWG-Biotech) (sym/sub-U) was annealed following the described procedure 2. The mutant 3Ds and their complexes were crystallized as previously described Purified FMDV mutant polymerases 3D(P44S), 3D(P169S), 3D and 3D(SSI) were stored in a buffer containing Tris-HCl , NaCl (0.5M), DTT (0.8mM), EDTA (0.8mM), and glycerol (8%), at a concentration of ∼4.6 mg/ml. The oligonucleotide Four different data sets were collected at 100 K: 3D(P44S) (2.2 Å), 3D(P169S) (2.6 Å), 3D-RNA (2.6 Å) and 3D(SSI)-RNA (2.5 Å), using synchrotron radiation at the ESRF beamlines ID14 EH1 and EH2 (λ = 0.93 Å). All data were processed and reduced using DENZO/SCALEPACK package 2212 space group (PDB:1U09) 221 crystals) were obtained following the same procedure but using the trigonal P3221 coordinates of 3D (PDB:1WNE) 221 space group. Several cycles of automatic refinement, performed with program REFMAC, were alternated with manual model rebuilding using the graphic programs TURBO and Coot The initial maps for the 3D(P44S) and 3D(P169S)  were obtained after a rigid body fitting of the coordinates of isolated 3D protein that was crystallized in the tetragonal p4Figure S1Incorporation of nucleotides into sym/sub-AC by mutant FMDV polymerases. (A) Kinetics of incorporation of GMP into sym/sub-AC (sequence shown at the top) by the indicated FMDV polymerases [3DWt and 3D(SSI)], performed at 37°C. The reactions were initiated by addition of 1 µM GTP after the formation of 3D-RNA(n+1) complex, as described in (7.49 MB TIF)Click here for additional data file.Figure S2Incorporation of nucleotides into sym/sub-AU by mutant FMDV polymerases. (A) Kinetics of incorporation of AMP into sym/sub-AU (sequence shown at the top) by the indicated FMDV polymerases [3DWt and 3D(SSI)], performed at 37°C. The reactions were initiated by addition of 1 µM ATP after the formation of 3D-RNA(n+1) complex, as described in (7.46 MB TIF)Click here for additional data file.Figure S3Incorporation of nucleotides into sym/sub-C and sym/sub-U by mutant FMDV polymerases. (A) Kinetics of incorporation of GMP into sym/sub-C (sequence shown at the top) by the indicated FMDV polymerases [3DWt and 3D(SSI)], performed at 37°C. The reactions were initiated by addition of 1 µM GTP after the formation of 3D-RNA complex, as described in (6.37 MB TIF)Click here for additional data file.


We here have developed a simplified, general, nondenaturing purification protocol for RNA to ask whether avoiding denaturation of a co-transcriptionally folded RNA can reduce commonly observed in vitro folding heterogeneity. Our protocol bypasses the need for large-scale auxiliary protein purification and expensive chromatographic equipment and involves rapid affinity capture with magnetic beads and removal of chemical heterogeneity by cleavage of the target RNA from the beads using the ligand-induced glmS ribozyme. For two disparate model systems, the Varkud satellite (VS) and hepatitis delta virus (HDV) ribozymes, we achieve >95% conformational purity within one hour of enzymatic transcription, without the need for any folding chaperones. We further demonstrate that in vitro refolding introduces severe conformational heterogeneity into the natively-purified VS ribozyme but not into the compact, double-nested pseudoknot fold of the HDV ribozyme. We conclude that conformational heterogeneity in complex RNAs can be avoided by co-transcriptional folding followed by nondenaturing purification, providing rapid access to chemically and conformationally pure RNA for biologically relevant biochemical and biophysical studies.Due to the energetic frustration of RNA folding, tertiary structured RNA is typically characterized by a rugged folding free energy landscape where deep kinetic barriers separate numerous misfolded states from one or more native states. While most  By contrast, heterogeneity is not observed upon refolding of the compact double-nested pseudoknot of the hepatitis delta virus (HDV) ribozyme. We conclude that, depending on the particular RNA of interest, significant differences can exist between natively- and non-natively purified RNA populations. Our nondenaturing purification approach therefore paves the way for biologically relevant biochemical and biophysical studies of the vast array of emergent, often structurally complex, non-coding RNAs.Shortcomings in previous native purification methods necessitated the development of a simplified and generalizable nondenaturing purification methodology. Our method purifies co-transcriptionally folded RNAs with homogenous 3′ ends to >95% conformational purity and avoids both denaturation and large-scale auxiliary protein purification. We use this method to demonstrate that refolding glmS ribozyme In vitro transcription using RNAP yields a transcript containing the target RNA fused to the glmS ribozyme and the binding sequence. The transcription reaction , releasing the co-transcriptionally folded target RNA with homogeneous 3′ end from the solid support  leads to collection of self-cleaved (and thus active) target ribozyme at >99% purity (as judged by denaturing polyacrylamide gel electrophoresis (PAGE); Application of this protocol to an 78-nucleotide (nt) antigenomic HDV ribozyme associated with the human pathogen HDV and S2) when included in the 100-µl transcription reaction; this amount has to be optimized on a case-by-case basis. Exactly saturating the RNA with capture strand will maximize the RNA yield since any additional capture strand will not bind RNA but compete with the RNA-bound capture strand for binding sites on the streptavidin-coated beads. Second, robust activity of the glmS ribozyme is critical to the release of target RNA. We therefore measured the extent of glmS ribozyme cleavage over time at varying concentrations of GlcN6P (and S3). The maximal extent of cleavage (which is ∼40%) requires only 200 µM GlcN6P and an incubation time of ∼1 min . The same cleavage extent can be achieved at a 40-fold lower concentration of 5 µM GlcN6P if the reaction proceeds for 10 min (and S4). HDV ribozyme purity was maintained at >96% throughout and no significant decrease was observed in the amount of RNA purified per cycle.First, excess capture strand will compete for the streptavidin available on the magnetic beads, while using too little capture strand will prevent some transcribed RNA from attaching to the beads. We therefore varied the amount of capture strand and found that ∼350 picomoles of capture strand was just about saturating . Samples of natively purified, self-cleaved HDV and VS ribozymes were heated to either 70°C or 90°C, annealed by slow cooling to room temperature, then analyzed by non-denaturing electrophoretic mobility shift assay (EMSA) in comparison with the unheated RNA. The RNA concentrations were ∼0.5 µM, in accordance with a study showing that concentrations below 3 µM more effectively regenerate RNA secondary structures during heat annealing Development of this native purification protocol allowed us to test the impact of heat denaturation and refolding on co-transcriptionally folded RNA. We examined this effect on two RNAs of varying sizes: the 78-nt HDV ribozyme as discussed above, as well as the 164-nt Varkud Satellite (VS) ribozyme  group I introns showed no structural differences upon artificial refolding 2+-agarose chromatography column and subsequent self-cleavage of the affinity tag Several protocols for native purification of RNA have been previously described glmS ribozyme. Site-specific self-cleavage results in a target RNA that is chemically pure in terms of its 3′ end homogeneity. Overall, our method only requires the purchase of commercially available magnetic beads, the accompanying magnet, and standard biochemical tools for in vitro transcription.We describe here a protocol that achieves >95% chemical and conformational purity within an hour of transcription for two disparate model RNAs. The chemical purity, as evident from the observation of a single band in lanes G1–G2 compared to the other lanes of Our method is straightforward to scale up, allowing for a wide range of applications. The protocol was developed on a scale that yields ∼50 picomoles of pure target RNA per 100 µl transcription reaction. We have also scaled it up to yield ∼500 picomoles of target RNA from a 1-ml transcription reaction (data not shown), and the necessary equipment is readily available for scaling it up to a 100-ml transcription reaction to yield ∼50 nanomoles of nondenatured target RNA. Several rounds of purification using the same magnetic beads would yield enough (∼500 nanomoles) RNA for high-resolution structural characterization by X-ray crystallography or NMR.Figure S6a). An alternate version of this plasmid that contains an inactive T7 promoter  is also available upon request and is intended to be used in case an RNA of a specific 5′ sequence is desired, which will require cloning of a T7 promoter immediately upstream of the RNA gene. There is no requirement for the target RNA to be catalytically active or to have enhanced folding when transcribed in proximity to the glmS ribozyme. The only role of the glmS ribozyme in this procedure is to specifically select for the target RNA by GlcN6P-dependent elution. Since denaturation is entirely avoided, the purified RNA sample will in turn reflect the distribution of structures formed during transcription.Our methodology was here successfully exploited to co-transcriptionally purify two different catalytic RNAs, and the plasmid used in this procedure Figure 1 Neurosporain vitro transcription thus suggests that some general principles are at work among diverse RNA polymerases under varying conditions.We did not investigate here the precise mechanism of co-transcriptional folding and why it would yield in case of the VS ribozyme a different fold than heat denaturation followed by reannealing. Wong et al. have suggested that nature utilizes transcriptional pause sites between the upstream and downstream segments of native long-range helices to kinetically guide their formation through the transient adoption of labile nonnative structures in vitro avoids folding heterogeneities otherwise observed in RNA that is heat denatured and subsequently refolded in its entirety, as is currently the standard protocol in the field. Apparently, segmental folding from the 5′ end during transcription yields a fundamentally different conformational ensemble for certain RNAs than denaturation/refolding, even in the absence of any chaperones postulated to play an important role for RNA folding in vivoin vitro, featuring multiple local and global minima that can represent either functionally active or inactive states In conclusion, we have developed an improved nondenaturing purification protocol that demonstrates significant conformational differences between natively- and non-natively purified RNA. We show conclusively that purification of RNA co-transcriptionally folded glmS ribozyme gene, and a U1A-protein binding site Figure S5).The pVS plasmid encodes for the VS ribozyme. A PCR product was generated using a stepwise preparation and purification procedure based on four nested primers with 5′ overhangs and the plasmid topWT The pVS plasmid served as parental plasmid for the construction of the HDV ribozyme encoding plasmid pHDV. The pVS plasmid was simultaneously digested with HindIII and AflII, the enzymes heat inactivated at 70°C for 20 min, the DNA phenol chloroform extracted, and the product cut out after electrophoresis on a 1% agarose gel using a commercial extraction kit . A PCR product  was simultaneously digested with HindIII and AflII, the enzymes heat inactivated at 70°C for 20 min, and the DNA phenol chloroform extracted prior to insertion into the prepared doubly digested pVS plasmid using T4 DNA ligase as suggested by the manufacturer (Invitrogen) to generate the pHDV plasmid.Figure S6). The pMCGLΔT7 plasmid  was generated through site-directed mutagenesis of pMCGL. DNA sequencing was used to confirm all plasmid sequences. All synthetic DNA oligonucleotides used in this study were purchased from Invitrogen and purified by denaturing PAGE.The same HindIII and AflII digested pVS plasmid was used to generate pMCGL through inserting into the overlap region two synthetic, partially complementary oligodeoxynucleotides with sticky 5′ phosphorylated ends and the multiple cloning site sequence  of 32P-α-GTP was incubated at 37°C for 2 h. The capture strand oligonucleotide was biotinylated on the 5′ end and had the following sequence: 5′-AAAAAAAAAAGAATTCCGCAGGCCTGCTCG-3′. Phenol/chloroform extraction yielded ∼100 µl final volume , to which 25 µl of 5 M NaCl was added for a final Na+ concentration of ∼1 M. This reaction mixture was added to 100 µl (1 mg) of Streptavidin C1 Dynabeads  equilibrated in wash buffer .A 150-µl transcription reaction with 4 mM of each NTP, 40 mM HEPES-KOH, pH 8.0, 5 mM DTT, 25 mM MgClThis bead suspension was rotated at room temperature for 30 min to form the complex between the biotinylated RNA:ssDNA hybrid and the streptavidin-coated magnetic beads. A magnetic particle concentrator (MPC) was applied to remove the supernatant and yield a solid bead pellet  to the bead pellet. The sample was incubated for 1 min, upon which the supernatant was collected. This CB wash was performed a total of twice . This EB wash was performed a total of three times, and the supernatants were discarded. The EB wash was then performed a 4All collected supernatants were placed on the MPC a second time to remove any residual magnetic beads; a 20µl aliquot of each supernatant was removed and added to an equal volume of denaturing loading buffer (80% (v/v) formamide, 0.025% (w/v) xylenecyanol blue, 0.025% (w/v) bromophenol blue and 10 mM EDTA) and analyzed by denaturing, 8 M urea, 10% PAGE, run in 1× TBE at 20 W for 2 h. The gel was exposed to a PhosphorImager screen overnight and quantified using a Typhoon 9410 Variable Mode Imager with ImageQuant software . The band intensities were corrected for the number of guanines in each sequence so that the intensity values report on the relative counts of RNA molecules within each band. These counts were used to calculate the fractional purities of specific molecule populations detected in the supernatants.2, and GlcN6P concentrations of 5, 50, 100, 200 or 500 µM. Progression of the reaction was monitored by removing 10 µl aliquots that included the suspended beads after 0.25, 0.5, 0.75, 1, 2, 5, and 10 min and mixing them with 30 µl of stop buffer (SB: 90% (v/v) formamide in 1× TBE). The different RNA species present were separated by denaturing, 8 M urea, 10% PAGE, run in 1× TBE at 20 W for 2 h. The zero time point was taken just prior to removal of WB so that the CB buffer could be added to the beads to initiate the reaction. The gel again was exposed overnight to a PhosphorImager screen, the intensities of the HDV ribozyme band were quantified using a Typhoon 9410 Variable Mode Imager with ImageQuant software  and corrected for the number of guanines as described above. The fraction cleaved was then calculated as Hcounts/(Hcounts+HGcounts), where H represents the self-cleaved HDV ribozyme species and HG represents the HDV+glmS ribozyme species. The 200 µM GlcN6P data  were fit with the single-exponential first-order rate equation obs.Self-cleavage activity of the glmS ribozyme was assayed following the native purification protocol described above, with slight modifications only to the cleavage step. The CB added to the beads was composed of 40 mM HEPES-KOH, pH 7.4, 1 M NaCl, 10 mM MgClFigure S2) consisted of transcriptions with 100 or 250 pmol capture strand prepared identically as above except that the linearized pHDV transcription template was omitted. 5-µl aliquots were removed and analyzed by EMSA on a nondenaturing 6% PAGE in 1× TBE at 5 W for 4 h. The gel was stained using a 1∶100,000 dilution of SYBR Gold  in 1× TBE for 5 min. The fluorescence intensity in the gel was measured using a Typhoon 9410 Variable Mode Imager  with an excitation of 488 nm, an emission bandpass of 530±10 nm, and a PMT setting of 500 V. The results were quantified using ImageQuant software . The fluorescence intensities of the free capture strand were converted into pmol amounts using the 100 and 250 pmol control standards of free capture strand in side lanes of the gel. The calculated free picomoles of capture strand were subtracted from the known amounts included at the beginning of the transcription reaction to give the bound pmol of capture strand shown in In ten independent 50-µl transcription reactions varying amounts of ssDNA capture strand  were included. Control reactions  and cyan , the glmS ribozyme in shown in green, and restriction enzyme sites used for cloning the plasmid transcription template are highlighted in red. The sites of self-cleavage of the two ribozymes are indicated by arrows; the color coded legend describes the nucleotide (nt) lengths of the resulting self-cleaved transcript fragments, with that of the target RNA boxed. The binding sequence is the boxed segment at the 3′ end of the glmS ribozyme that forms a hybrid with the biotinylated ssDNA capture strand (orange).(0.84 MB TIF)Click here for additional data file.Figure S2Capture strand titration. Samples from the capture strand binding assay, including varying concentrations of ssDNA capture strand added to the transcription reaction as indicated, were analyzed by EMSA using non-denaturing 6% PAGE in 1× TBE, followed by SYBR Gold staining and visualization with a Typhoon 9410 Variable Mode Imager. Lanes 1 and 12 are control lanes representing 200 and 500 pmol of capture strand, respectively, that were used to calibrate the amount of free capture strand in the titration lanes.(1.05 MB TIF)Click here for additional data file.Figure S3Monitoring glmS ribozyme self-cleavage over time in the presence of 200 µM GlcN6P in CB at 25 oC. (a) Aliquots with the suspended beads were removed at various time points and analyzed by denaturing, 8 M urea, 10% PAGE and subsequent autoradiography. Increases in density of the glmS and HDV ribozyme populations over time indicate self-cleavage of the glmS ribozyme. (b) Plot of the fraction of the HDV+glmS band converted into the HDV band over time after correcting for the number of guanines in both sequences.(0.28 MB TIF)Click here for additional data file.Figure S4Regenerating streptavidin-coated magnetic beads for reuse. 20-µl aliquots from the 4th WB wash (W), the 1st CB elution , and the 1st removal elution with heated EB (R) were taken from each cycle and analyzed by denaturing 10% PAGE. The lower band is cleaved HDV ribozyme, while the upper bands are the HDV+glmS and self-cleaved glmS ribozyme species, respectively, as indicated.(0.62 MB TIF)Click here for additional data file.Figure S5Application of the purification protocol to the VS ribozyme. (a) The VS-glmS-binding sequence construct used in this study. The self-cleaving VS ribozyme is indicated in grey (5′ sequence) and cyan , the glmS ribozyme in shown in green, and restriction enzyme sites used for cloning the plasmid transcription template are highlighted in red. The sites of self-cleavage of the two ribozymes are indicated by arrows; the color coded legend describes the nucleotide (nt) lengths of the resulting self-cleaved transcript fragments, with that of the target RNA boxed. The binding sequence is the boxed segment at the 3′ end of the glmS ribozyme that forms a hybrid with the biotinylated ssDNA capture strand (orange). (b) Supernatants collected at various steps of the protocol and analyzed by denaturing, 8 M urea, 10% PAGE. “VS” refers to the targeted self-cleaved VS ribozyme, “glmS” to the self-cleaved glmS ribozyme with attached binding sequence, and “VS+glmS” to the transcript containing the VS ribozyme with attached uncleaved glmS ribozyme and binding sequence.(1.34 MB TIF)Click here for additional data file.Figure S6Sequences of the available plasmids with multiple cloning sites. The nucleotides and dotted line in green denote the sequence that encodes the glmS ribozyme. (a) The pMCGL plasmid contains an active T7 promoter upstream of the multiple cloning site. (b) In the pMCGLΔT7 plasmid the T7 promoter is inactivated by four point mutations (cyan).(0.61 MB TIF)Click here for additional data file.


We have demonstrated that urinary trypsin inhibitor (UTI) purified from human urine is able to inhibit lung metastasis of mouse Lewis lung carcinoma (3LL) cells in experimental and spontaneous metastasis models. In this study, we have investigated whether UTI in combination with an anti-cancer drug, etoposide, can prevent tumour metastasis and show an enhanced therapeutic effect. Subcutaneous (s.c.) implantation of 3LL cells (1 x 10(6) cells) in the abdominal wall of C57BL/6 female mice resulted in macroscopic lung metastasis within 21 days. Microscopic lung metastasis was established by day 14 after tumour cell inoculation, and surgical treatment alone after this time resulted in no inhibition of lung metastasis. The number of lung tumour colonies in the group of mice which received surgery at day 21 was greater than in mice which had tumours left in situ (P = 0.0017). Surgical treatment on day 7, followed by UTI administration (s.c.) for 7 days, led to a decrease in lung metastasis compared with untreated animals. A significant inhibition of the formation of pulmonary metastasis was obtained with daily s.c. injections of UTI for 7 days immediately after tumour cell inoculation. UTI administration did not affect the primary tumour size at the time of operation. In addition, etoposide treatment alone led to a smaller primary tumours and yielded reduction of the formation of lung metastasis in the group of mice which received surgery at day 14 (P = 0.0026). Even in mice which received surgical treatment on day 14, followed by the combination of UTI  with etoposide , there was significant reduction of the formation of lung metastasis (P = 0.0001). Thus, the combination of an anti-metastatic agent with an anti-cancer drug, etoposide, might provide a therapeutically promising basis for anti-metastatic therapy.


The challenge of gene expression studies is to reliably quantify levels of transcripts, but this is hindered by a number of factors including sample availability, handling and storage. The PAXgene™ Blood RNA System includes a stabilizing additive in a plastic evacuated tube, but requires 2.5 mL blood, which makes routine implementation impractical for paediatric use.The aim of this study was to modify the PAXgene™ Blood RNA System kit protocol for application to small, sick chidren, without compromising RNA integrity, and subsequently to perform quantitative analysis of ICAM and interleukin-6 gene expression.Aliquots of 0.86 mL PAXgene™ reagent were put into microtubes and 0.3 mL whole blood added to maintain the same recommended proportions as in the PAXgene™ evacuated tube system. RNA quality was assessed using the Agilent BioAnalyser 2100 and an in-house TaqMan™ assay which measures GAPDH transcript integrity by determining 3' to 5' ratios. qPCR analysis was performed on an additional panel of 7 housekeeping genes. Three reference genes  were identified using the GeNORM algorithm, which were subsequently used to normalising target gene expression levels. ICAM-1 and IL-6 gene expression were measured in 87 Malawian children with invasive pneumococcal disease.Total RNA yield was between 1,114 and 2,950 ng and the BioAnalyser 2100 demonstrated discernible 18s and 28s bands. The cycle threshold values obtained for the seven housekeeping genes were between 15 and 30 and showed good consistency. Median relative ICAM and IL-6 gene expression were significantly reduced in non-survivors compared to survivors .We have successfully modified the PAXgene™ blood collection system for use in small children and demonstrated preservation of RNA integrity and successful quantitative real-time PCR analysis. The main challenge of quantitative gene expression studies is to extract sufficient usable messenger ribonucleic acid (mRNA), to avoid degradation and permit analysis for calculation of exact numbers of transcript. The processes of sample collection, transport, processing and storage may result in significant degradation of mRNA . BecauseThe PAXgene™ Blood RNA System , includes a stabilizing additive in an evacuated blood collection tube called the PAXgene™ Blood RNA Tube, and also sample processing reagents in the PAXgene™ Blood RNA Kit. The additive in the PAXgene™ tube reduces RNA degradation of 2.5 mL of blood in the evacuated tube, and furthermore, the RNA in whole blood has been shown to be stable at room temperature for 5 days, following storage for up to 12 months at -20°C and -80°C, and also after repeated freeze-thaw cycles . The sample was gently inverted and stored at -80°C within two hours of collection.RNA was extracted from whole blood using the PAXgene™ Blood RNA System Kit employing an amended version of the manufacturer's guidelines. Briefly, the samples were removed from -80°C and incubated at room temperature for 2 hours to ensure complete lysis. Following lysis the tubes were centrifuged for 10 min at 5,000 × g (Boeco M-24 centrifuge), the supernatant decanted and 500 μL of RNase-free water added to the pellet. The tube was vortexed to thoroughly re-suspend the pellet, centrifuged for 10 min at 5000 × g and the entire supernatant discarded. The remaining pelleted lysate was re-suspended in 360 μL of buffer BR1 by vortexing and the manufacturer's protocol was followed from this step.Freshly extracted RNA was measured using a NanoDrop ND-1000 UV-visible spectrophotometer . The software displays the concentration in ng/μL. There is also a quality output, which provides 260/280 and 260/230 ratios enabling purity estimations. RNA integrity was additionally assessed using the Agilent 2100 Bioanalyser [Agilent Technologies]. Samples were loaded on to either the Eukaryote total RNA nano chip or the Eukaryote total RNA pico chip.12–18 used for priming. RT qPCR assays were designed using locked nucleic acid analogues (LNA) of fluorescence resonance energy transfer hydrolysis probes [To evaluate the utility of the RNA achieved by this method we then carried out RT qPCR analysis on a panel of 10 assays on a total of 8 housekeeper genes as detailed in Table s probes  employed12–18 (500 μg/mL) and took place in the presence of RNaseOUT™ RNA was reverse transcribed using SuperScript II™ RNase H reverse transcriptase  following the manufacturer's guidelines. Reactions were primed using 1 μL oligo (dT)The Human Universal Probe Library system  ]. The calculation of ΔΔCT involves subtraction of ΔCT value for the controls from the ΔCT value for the cases [ΔCT target gene(case) - ΔCT target gene(control)]. 2-ΔΔCt is the relative expression of the target gene in cases compared to controls.cDNA was diluted 1 in 50. Each reaction comprised of 5 μL diluted cDNA, 10 μL 2× qPCR Master Mix with UNG , 0.2 μL each primer (20 μM) , 0.2 μL Probe (10 μM) Human Universal Probe Library system  , the Mann Whitney test was used to compare two groups and Kruskal Wallis to compare three groups. Values of p < 0.005 were considered statistically significant. SPSS statistical software version 14  was used for all statistical analyses.EDC participated in the design and organisation of the study, the collection of clinical and laboratory data, laboratory work, statistical analysis and in drafting and revising the manuscript. FS participated in the design and organisation of the study, laboratory work, and in drafting and revision of the manuscript. SDP and ES participated in the laboratory work, and revision of the manuscript. LAM participated in the collection of clinical and laboratory data, and in drafting and revising the manuscript. WO and CAH participated in the design and organisation of the study, and revision of the manuscript. PD participated in the design and organisation of the study, statistical analysis and in drafting and revision of the manuscript.All authors read and approved the final manuscript.


Palorus ratzeburgii (Coleoptera). This satellite is characterized by preservation and extreme sequence conservation among evolutionarily distant insect species. PRAT is expressed in all three developmental stages: larvae, pupae and adults at similar level. Transcripts are abundant comprising 0.033% of total RNA and are heterogeneous in size ranging from 0.5 kb up to more than 5 kb. Transcription proceeds from both strands but with 10 fold different expression intensity and transcripts are not processed into siRNAs. Most of the transcripts (80%) are not polyadenylated and remain in the nucleus while a small portion is exported to the cytoplasm. Multiple, irregularly distributed transcription initiation sites as well as termination sites have been mapped within the PRAT sequence using primer extension and RLM-RACE. The presence of cap structure as well as poly(A) tails in a portion of the transcripts indicate RNA polymerase II–dependent transcription and a putative polymerase II promoter site overlaps the most conserved part of the PRAT sequence. The treatment of larvae with alpha-amanitin decreases the level of PRAT transcripts at concentrations that selectively inhibit pol II activity. In conclusion, stable, RNA polymerase II dependant transcripts of abundant centromeric satellite DNA, not regulated by RNAi, have been identified and characterized. This study offers a basic understanding of expression of highly abundant heterochromatic DNA which in beetle species constitutes up to 50% of the genome.Transcripts of centromeric satellite DNAs are known to play a role in heterochromatin formation as well as in establishment of the kinetochore. However, little is known about basic mechanisms of satellite DNA expression within constitutive heterochromatin and its regulation. Here we present comprehensive analysis of transcription of abundant centromeric satellite DNA, PRAT from beetle  Drosophila melanogasterDrosophila hydeiD. melanogaster and D. hydei Y chromosome associated transcripts Diadromus pulchellusAphaenogaster subterraneaDiprion piniSatellite DNAs are tandemly repeated sequences that are in the form of long, Mb size arrays located in heterochromatin. Besides being a major constituent of heterochromatin they act as a centromere-building element in many eukaryotes Studies of fission yeast revealed the processing of long double strand transcripts deriving from tandemly repeated pericentromeric DNA into small 20–25 bp long RNAs involved in RNA interference (RNAi) pathway Palorus  are preserved and widely distributed among taxonomically distant species Palorus ratzeburgii comprising 40% of the genomic DNA and is located in regions of centromeric and pericentromeric heterochromatin on all chromosomes Despite high evolutionary dynamics some satellite DNAs such as those belonging to the insect genus Schizosaccharomyces pombe it is shown that RNA pol II promotes transcription of centromeric DNA Most of current research is focused on the explanation of role of satellite DNA transcripts but little is known about the transcription itself although it is likely to be the key regulatory step in biogenesis of functional transcripts. In the case of fission yeast Total, cytoplasmic and nuclear RNAs for the purpose of RT-PCR, real-time PCR, Northern and RLM-RACE analysis were isolated using the Rneasy Mini Kit (Qiagen), whereas polyadenylated RNA was recovered on Oligotex columns (Qiagen). RNA for primer extension reaction as well as RNA to be used for detection of siRNAs on Northern blots were extracted by phenol-chloroform 5′-GAATCCGAAACAAAATCGTGCC-3′) and RATZEB2 (5′-GCTGAAATCTTGCAAACTTTAC-3′) specific to the PRAT. To investigate strand origin of transcripts, reverse transcription was performed at 50°C for 30 min with one primer only, followed by heat inactivation of reverse-transcriptase and activation of HotStarTaq DNA Polymerase for 15 min at 95°C. At this step the second primer was added to start 33 cycles of PCR under the following conditions: 1 min at 94°C, 30 s at 58°C and 1 min at 72°C. Final extension was done for 10 min at 72°C. Reactions in which reverse transcription was omitted as well as reactions with no added template were performed as controls for DNA contamination and cross-contamination between samples.20 ng of RNA was transcribed into DNA and amplified using the OneStep RT-PCR Kit (Qiagen) and primers RATZEB1 , with previously cloned PRAT dimer, was digested with NcoI or SalI and in vitro transcribed by T7 or SP6 RNA polymerase, respectively, in a reaction containing [α-32P]UTP. After hybridization, membranes were washed twice in 2× SSC, 0.1% SDS at RT, once in 0.1× SSC, 0.1% SDS at 37°C, and exposed overnight on a phosphor-imager or up to 7 days on autoradiography films. Relative amounts of PRAT transcripts were determined by densitometry, subtracted for the signal corresponding to yeast RNA.RNA was separated on 1.2% agarose-formaldehyde gels and blotted onto positively charged nylon membranes. For small RNA analysis, 30 µg of RNA enriched for low molecular weight molecules was resolved on 12% polyacrylamide gels containing 8 M urea and transferred to nylon membranes in 0.5× TBE for 45 min at 100 V using wet electroblotter (BioRad). In dot blot analysis, samples of RNA were applied to membrane together with the equal amounts of yeast RNA which served as a control for hybridization specificity. Unlabelled “run off” transcripts produced in vitro from vector containing PRAT dimer sequence were used as positive controls and for calibration after hybridization. Hybridization was performed under high stringency conditions at 50°C and 60°C on dot blots and high molecular weight RNAs, and at 22°C and 40°C on blots with small RNAs, in 10 ml solution containing 50% formamide, 5× Denhardt solution, 6× SSPE, 0.1% SDS, 100µg/ml herring sperm DNA, and 1-2×105′-GCTGTATTTAAG AGAATCCG-3′) and RATZEB4 (5′-ATACAGCTGAAACCATGC-3′) corresponding to PRAT sequence were end-labelled with [γ-32P]dATP in phosphorylation reaction with T4 polynucleotide kinase (Fermentas). Each of the labelled primers was incubated with total RNA (10–30 µg) at 65°C for 90 min and slowly cooled down to RT to allow hybridization of primer to RNA. 25 U of AMV reverse transcriptase (Roche) was added to 40 µl reaction and incubated at 42°C for 1 h. The reaction was terminated by incubation with RNase A at 37°C for 30 min, and reverse transcripts were recovered by phenol-chloroform extraction and ethanol precipitation. Primer extension products were electrophoresed through a 6% denaturing polyacrylamide gel along with the sequencing products of PRAT dimer clone obtained with the same labelled oligonucleotide used for primer extension.Four oligonucleotides, RATZEB1, RATZEB2, RATZEB3 . After phosphatase and pyrophosphatase treatment, the RNA adapter was ligated to the 5′ end of decapped target molecules. RT-PCR was performed using the OneStep RT-PCR Kit as described above, with an outer adapter-specific primer and RATZEB1 or RATZEB2 primer. Additional PCR on obtained products was conducted with either RATZEB1 or RATZEB2 satellite specific primer, and inner adapter-specific primer to increase specificity of amplification. Simultaneous experiment without pyrophosphatase treatment (-TAP) served as a negative control. The same kit was used for 3′ RACE reactions to detect transcription termination sites. Total RNA from larvae was reverse transcribed using the supplied 3′ RNA adapter which has an oligo T stretch, and subsequently amplified using adapter specific outer primer and RATZEB1 or RATZEB2 primer.All PCR products from both 5′ and 3′ RACE were cloned in the pGEM-T Easy vector (Promega), positive clones were screened by colony lift and hybridization with PRAT probe, followed by sequencing.E. coli. Eleven clones were randomly chosen and sequenced in both directions using vector-specific primers. Accession numbers of PRAT RNA monomers obtained by direct sequencing and cloning are EF209021-EF209033. Nucleotide sequences were aligned and compared using ClustalW. Putative RNA Polymerase II promoter site was annotated using Neural Network Promoter Prediction Tool .A primer extension product of 122 nt size, obtained in reactions with RATZEB2, was amplified by using RATZEB1 and RATZEB2 primers. Amplified PCR product of 131 bp was directly sequenced using RATZEB2 as a sequencing primer. The same amplification product was ligated into the pGEM-T vector and transformed into P.ratzeburgii were cooled down on ice and fixed onto a double-stick tape placed over a micro slide. Larvae were injected dorsally into the hemolymph using a simple injection apparatus 2HPO4; 0,03 mM KH2PO4; 4 mM KCl; 5% green food dye) or alpha-amanitin, 1 µg/ml and 10 µg/ml, diluted in injection buffer. After injection larvae were removed from the tape and grown on whole wheat flour at 30°C for five days. No mortality or toxic effects were noted for larvae treated for five days with such concentrations of alpha-amanitin. The low mortality was observed for larvae injected with 100 µg/ml of alpha-amanitin.Late instar larvae of T method (ΔΔ CT). cDNA from larvae injected with buffer served as calibrator and 18S rRNA gene was used to normalize PRAT quantities.0.5 µg of total RNA isolated from injected larvae was reverse transcribed by 20 U of M-MuLV Reverse Transcriptase (Fermentas) using random primers in a final volume of 20 µl. Resulting cDNA (20–40 ng per reaction) was subjected to real-time quantitative PCR analysis. Primers and TaqMan probes for PRAT as well as for 18S rRNA (endogenous control) were obtained from Applied Biosystems as TaqMan Gene Expression Assays. All reactions were performed on ABI 7300 Real-Time PCR System in four replicates in a final volume of 50 µl, under following reaction conditions: pre-incubation at 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. Non-template controls were included. The data were analyzed using the comparative CP. ratzeburgii adults, larvae and pupae  pathway and produce small interfering RNAs (siRNA). To verify whether the PRAT transcripts were processed into siRNA, Northern blot hybridization using RNA preparations enriched for small RNA was performed . We wereP. ratzeburgii larvae, and RNA was extracted from each fraction. In addition, polyadenylated RNA was separated from total RNA on Qiagen Oligotex columns and the remaining non-polyadenylated fraction was recovered. All RNA isolations were treated with DNase I to eliminate traces of genomic DNA and subjected to Northern blot hybridization using 32P strand specific RNA probes .Mapping of the transcription start sites was also performed using RLM-RACE which involves ligation of a defined RNA adapter to the 5′ ends of full length mRNA followed by RT-PCR using primers RATZEB1 and RATZEB2 for mapping the initiation sites on PRAT reverse and forward strand respectively. Products of amplification  were cloTranscription termination sites were mapped using 3′ RACE. Reverse transcription was performed using the 3′ RACE adapter which has an oligo T stretch, followed by PCR amplification with satellite specific primer RATZEB1 or RATZEB2, and primer homologous to the adapter. Amplification reactions of forward and reverse strand  were ranAs in the case of 5′ RACE assay, all positive clones detected in 3′ RACE analysis correspond to shorter fragments, up to trimer, although longer multimers have been subjected to ligation . This agThe presence of a cap structure as well as poly(A) tails in at least a portion of the transcripts indicates that RNA polymerase II could be responsible for PRAT satellite DNA transcription. In order to test this assumption we injected beetle larvae with alpha-amanitin, which is known to inhibit RNA polymerase II completely at low concentration, while RNA polymerase I activity remains unaffected. Larvae were injected with approx. 0.1 ng and 1 ng of alpha-amanitin respectively, and these concentrations of alpha-amanitin caused no mortality or toxic effects. Five days after injection total RNA was isolated and treated with DNase I to remove traces of genomic DNA. RNA quality was checked by electrophoresis and cDNA library was generated. Real-time PCR analysis evidenced approximately 29% and 42% reduction in PRAT transcript quantity in larvae injected with 0.1 ng and 1 ng of alpha-amanitin, respectively . 18S rRNP. ratzeburgii: larvae, pupae and adults, at a similar level. PRAT is predominantly transcribed from one strand and the transcripts are relatively abundant comprising 0.03% of total cellular RNA while expression of the opposite strand is 10 times lower. The transcripts are heterogeneous with the size profile ranging from approx. 0.5 kb to more than 5 kb, matching those characteristic for the centromeric, 180 bp satellite of Arabidopsis thalianaArabidopsis or rice centromeric satellites whose primary transcripts are regulated by RNAi and corresponding siRNAs are readily proved by Northern hybridization The results presented in this paper reveal that evolutionarily conserved PRAT satellite DNA is continuously expressed during all three developmental stages in the beetle Although the RNAi pathway plays a role in heterochromatin establishment in various organisms, it does not seem to be indispensable for assembly and maintenance of heterochromatin, as shown for heterochromatin initiated in mammalian cells by transgene tandem repeats Drosophila, located either in heterochromatin or euchromatin Transcription of satellite DNA might be initiated from upstream promoters provided by mobile elements inserted near satellite DNA clusters Schizosaccharomyces pombe RNA polymerase II is responsible for transcription of pericentromeric regions which is coupled with RNAi-dependent heterochromatin assembly S. pombe association of RNA Pol III as well as Pol III transcripts of tRNA genes were identified The presence of a cap structure as well as poly(A) tails in a portion of the transcripts indicate that pol II could be responsible for PRAT satellite DNA transcription. The sensitivity of PRAT transcription to alpha-amanitin at concentrations that selectively inhibit pol II confirms this assumption. However, since a considerable amount of transcripts does not contain poly(A) tail, the possibility exists that RNA polymerase(s) other than pol II might also be involved in transcription. In the yeast We did not experimentally determine the position of promoter elements but a putative RNA polymerase II promoter site was annotated by a computational approach, using a time-delay neural network method A motif exhibiting perfect homology to the TATA-box was not detected in the PRAT consensus sequence, but can be created by a single point mutation of a motif (TATTTAA) located at position 134 on the forward strand. Due to satellite sequence variability it is probably present in many PRAT monomers. Complementary sequence on opposite DNA strand (TTAAATA) also resembles TATA-box. These TATA-box like motifs are positioned 11 and 18 nt upstream of the most proximate transcription start site detected on forward and reverse strand, respectively. Overlapping of TATA-box like sequences on both DNA strands and close position of transcription initiation sites indicates bidirectional transcription from the same, central promoter.RACE analysis revealed the presence of multiple termination sites located within the PRAT sequence. The method specifically picks transcripts ending with poly(A) tails. Some of them could result from polyadenylation, while due to the presence of long A stretches in PRAT sequence it is possible that some of the analyzed transcripts have a false poly(A) tail. In any case, the length of poly(A) tail is relatively short, below 13 nt, similar to those found in rice satellite DNA transcripts The function of PRAT transcripts remains highly speculative. Their preferential location in the nucleus might indicate close association with chromosomes and possible influence on chromatin modulation and centromere function. On the other hand, a small portion of PRAT transcripts which are located in the cytoplasm could perform an additional, yet unknown role. The presence of transcription initiation sites within the PRAT sequence as well as a putative promoter for RNA polymerase II point strongly to the functional significance of this evolutionary extremely conserved sequence and transcripts deriving from it. Identification of proteins, either nuclear or cytoplasmic, which interact with long PRAT satellite transcripts, could shed more light on the role of this abundant non-coding RNA and explain its long evolutionary preservation. On the other hand, further characterization of RNA polymerases and their promoters which are responsible for transcription of satellite DNAs could contribute to the elucidation of mechanisms of expression within condensed constitutive heterochromatin.


Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 protein, we focused on the specific impact of Smad4 protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P=0.024) in the level of Smad4 protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.Smad4 protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the  Smad4 gene, which mediates the intracellular signalling pathway of transforming growth factor (TGF)-beta receptor, has been detected as one of the target genes at 18q21  group. Next, 20 cases negative for liver metastasis and negative for lymph node metastasis were assigned to the liver-metastasis (−) group. The pathological T-classification, tumour location and tumour differentiation of both groups were matched.First, these 20 cases were assigned to the liver-metastasis (+) and liver-metastasis (−) groups. Pathological staging in each case was according to the UICC/TNM classification. T-classification and tumour differentiation in each group were the same, as follows: T1, one (5%); T2, one (5%); T3, 17 (85%); T4, one (5%); well-differentiated adenocarcinoma, 19 (95%); poorly differentiated adenocarcinoma, one (5%). The distribution of tumour location was also matched in both groups: right colon, two (10%); left colon, four (20%); rectum, 14 (70%). The mean and median follow-up period were 1438 days and 1014 days for liver-metastasis (−), during which no case of liver-metastasis (−) has been confirmed not to experience recurrence.The clinical and pathological features of both groups are shown in μm for both Smad4 immunostaining and haematoxylin–eosin (H&E) staining.All the samples for immunohistochemical analysis were obtained from paraffin-embedded specimens. Serial sections were cut at a thickness of 3 M sodium citrate buffer (pH 6.0), and heated in a microwave oven for three 7-min cycles (500 W). After washing three times in PBS, endogeneous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxide in methanol for 20 min. Biotinylated rabbit anti-mouse immunoglobulin and SAB complex which were supplied commercially (Histofine SAB-PO(M) kit, Nichirei, Tokyo, Japan) were used as reagents for the next step. The sections were incubated overnight at a temperature of −4°C with anti-SMAD4 monoclonal antibody (Smad4(B-8):sc-7966, Santa Cruz Biotechnology, Inc., CA, USA) at a dilution of 1 : 1000. Colour was then developed with diaminobenzidine solution. Sections were then lightly counterstained with a cocktail of Mayer's/Lillie-Mayer's haematoxyline and mounted.The sections were deparaffinized with xylene and dehydrated with 98% ethanol, placed in 0.01 All the specimen immunostained for Smad4 protein were evaluated without knowing their clinicopathological features. Smad4 staining was predominantly observed in cytoplasm.All the normal mucosa showed immunohistochemical staining against Smad4 protein. The samples were evaluated as reported previously  no Smad4 (scored 0), (2) low Smad4 (scored 1 to 3) and (3) high Smad4 (scored 4). Representative samples of Smad4 staining are shown in liver-metastasis (+) and liver-metastasis (−) groups, 12 T-matched pairs of tumours were available from their primary frozen specimens. These specimens were used for LOH analysis. The samples were snap-frozen in liquid nitrogen immediately after being resected during routine operations and stored at −80°C until the DNA was extracted from each specimen using DNAeasy kit . From other eight T-matched pair of samples whose frozen specimens were not stored, we also obtained DNA from paraffine-embedded specimens by microdissection techniques as we described in the previous report , 2.5 mM MgCl2, 200 μl deoxynucleotide triphosphates mixture, 0.5 μM of each primer, 20–40 ng of extracted DNA and 0.4 U of AmpliTaq Gold DNA polymerase . The DNA was amplified in a thermal cycler  and PCR was performed according to the following protocol: 10 min at 95°C for polymerase activation; 40 cycles at 94°C for 30 s, 56°C for 1 min and 72°C for 1 min; then, an additional 30 min at 70°C. After denaturization at 95°C for 5 min, the PCR products were electrophoresed and analysed on an automated sequencer ), and the fluorescent signals from alleles of different size were recorded and analysed using GeneScan version 3.1 and Genotyper version 2.1 software .Polymerase chain reaction (PCR) was performed in 10 For each pair of the samples, the normal mucosal DNA was used to determine the allele size for the corresponding subject. Samples whose normal mucosa showed only a single peak were considered not informative for LOH. Samples showing two distinct peaks were considered informative and were assessed for LOH. Loss of heterozygosity was defined as a loss of one of the two peak alleles. A sample showing LOH is shown in P<0.05 was considered statistically significant.Fisher's exact test was used to determine the significance of observed difference between the groups. A value of The results of immunohistochemical staining of Smad4 protein are shown in liver-metastasis (−) group, nine cases (45%) were classified as high Smad4, eight (40%) as low Smad4 and three (15%) as no Smad4. In liver-metastasis (+) group, only one case (5%) was classified as high Smad4, while 17 cases (85%) were classified as low Smad4 and two (10%) as no Smad4. In other words, more cases showed decreased intensity for Smad4 immunohistochemical staining in liver-metastasis (+) group compared to liver-metastasis (−) group.In liver-metastasis (+) and liver-metastasis (−) groups from the standpoint of Smad4 protein immunohistochemical staining and found that Fisher's exact test showed a significant difference between both the groups (P=0.024).We statistically compared the The results for individual primers are shown in liver-metastasis (−) group and 67% (eight out of 12) in the liver-metastasis (+) group. Fisher's exact test showed that the cases in liver-metastasis (+) group showed significantly higher ratio of allelic loss at D18s474 compared with those in liver-metastasis (−) group (P=0.0054). For D18s46, 24 out of 40 cases were informative, and the LOH ratios were 30% (three out of 10) in the liver-metastasis (−) group vs 71% (10 out of 15) in the liver-metastasis (+) group, with no significant difference (P=0.095) found between the groups. For the primer D18s363, 21 out of 40 cases were informative, and 18% (two out of 11) of the cases demonstrated LOH in the liver-metastasis (−) group vs 70% (seven out of 10) in the liver-metastasis (+) group. This result showed a statistically significant difference (P=0.030).For D18s474, 28 out of 40 cases were informative. The LOH ratios were 13% (two out of 16) in the liver-metastasis (+) group showed more cases of low Smad4 compared to liver-metastasis (−) group, although statistical analysis did not show a significant difference (P=0.59).We then examined if LOH at chromosome 18q21 had a direct effect on Smad4 protein level in 40 samples . Those sliver-metastasis (+)/(−) groups, and demonstrated significant statistical differences between these two groups in Smad4 protein level and chromosome 18q deletion. This is the first report to focus on the specific impact of liver metastasis on Smad4 protein expression and chromosome 18q deletion with clinical features other than liver metastasis being matched. Decreases in Smad4 protein expression and chromosome 18q deletion are known to be characteristic risk factors of liver metastasis.In the current study, we selected only lymph node negative cases and matched for T-classification, tumour locations and tumour differentiations between P=0.05 and P=0.0156) and III disease (P=0.038 and P=0.032). We showed that microsatellite stable (MSS) patients with stage III colorectal cancer showed poor prognosis when chromosome 18q showed allelic loss (P=0.006)  (P<0.025) and disease-free (P<0.013) survival than patients with low levels by immunohistochemical staining. We, too, recently discussed the association between Smad4 level and prognosis in colorectal cancers. Previous reports suggested that Smad4 protein inactivation correlates with liver metastasis; however, the groups consisted of various T- and N-stage patients.Recently, an immunohistochemical assay of Smad4 protein has been developed and several studies indicated that Smad4 protein inactivation affected survival. Anirban Maitra P=0.024) in Smad4 protein expression level between liver-metastasis (+)/(−) groups. This result confirmed more accurately the previous report that Smad4 protein inactivation is related to the process or the risk of liver metastasis.In this study, lymph node negative cases were matched for T-classification, tumour depth and tumour location and demonstrated a statistically significant difference (In conclusion, our results showed that both chromosome 18q deletion and Smad4 protein expression are associated with liver metastasis in colorectal cancers, and that they both play an important role in the development of liver metastasis.


Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3′-phosphoadenosine-5′-phosphate pAp, inhibits the activity of the Xrn1p 5′-3′ ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3′-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on  MET22 nucleotidase suppresses viral RNA recombination. In vitro experiments with a cell-free extract from yeast revealed that the substrate of Met22p bisphosphate-3′-nucleotidase, namely pAp, could promote TBSV RNA recombination via inhibiting the activity of the Xrn1p 5′-3′ ribonuclease, a known suppressor of viral RNA recombination. Altogether, we demonstrate that the Met22/Xrn1 pathway and environmental factors affecting this pathway, namely salt-stress caused by LiCl and NaCl, play a role in viral RNA recombination. The authors also provide evidence that a similar pathway affects TBSV recombination in plants as well. The most pronounced increase in accumulation of TBSV RNA recombinants was seen in the triple-nucleotidase gene silenced plants treated with LiCl, suggesting that the combined effect of genetic and environmental factors could be critical in regulation of the rate of viral RNA recombination.Viral RNA recombination plays a major role in virus evolution. Yet, we know little about the roles of host and environmental factors in viral RNA recombination. In this work, using TBSV and yeast as a model host, we show that  Viruses with RNA genomes are abundant pathogens of plants and animals. Many RNA viruses have ultrafast replication cycles and rapid evolution, leading to the continuous emergence of new strains and variants. In addition to high frequency mutations and genome reassortments for multicomponent RNA viruses RNA recombination also functions in the repair of viral RNA molecules by utilizing truncated/damaged viral RNA molecules Viral RNA recombination leads to the joining of two or more noncontiguous segments of the same RNA or two separate RNAs together Tomato bushy stunt virus (TBSV), a tombusvirus infecting a wide range of plants, and yeast model host have revealed that several dozens of host genes could affect viral RNA recombination either directly or indirectly In spite of our increasing knowledge about viral RNA recombination over the last two decades that contributed new insights into the roles of viral proteins and the viral RNA in RNA recombination MET22/HAL2 gene, whose deletion increased TBSV RNA recombination in yeast MET22 codes for a bisphosphate-3′-nucleotidase in the sulfate assimilation pathway involved in methionine biosynthesis and it affects salt tolerance MET22 might promote TBSV RNA recombination by leading to increased cytosolic level of pAp In this work, we tested the role of the previously identified met22Δ yeast with Met22p mutants defective in bisphosphate-3′-nucleotidase function, which did not suppress TBSV recombinant RNA accumulation, suggesting that the enzymatic function of Met22p is important to inhibit TBSV recombination. In addition, inhibition of Xrn1p exoribonuclease via pAp in a cell-free TBSV replication assay demonstrated increased accumulation of TBSV recombination products as well as enhanced level of partial degradation products of TBSV replicon (rep)RNA, which are intermediates in RNA recombination To test this model, we complemented GALS promoter from its original chromosomal location in BY4741 yeast  that also carried the plasmids for launching TBSV repRNA accumulation Gals-met22 yeast cultured in a media containing glucose that represses the GALS promoter RNA accumulation, we expressed Met22p from the weak galactose-regulatable met22Δ yeast with various Met22p mutants expressed from plasmids as shown in 2+ ion (MutD) met22Δ yeast when compared with the expression of the wt Met22p  yeast supported increased level of recRNA accumulation , unlike when Met22p was expressed in the met22Δ yeast strain  assay capable of supporting the in vitro assembly of the viral replicase complex, including one full replication cycle of the TBSV repRNA Nicotiana benthamiana protoplasts electroporated with DI-ΔRI repRNA that lacks the 5′ terminal 169 nt from the wt DI-72 repRNA and can recombine efficiently in plant protoplasts and yeast To test if plants have a pathway similar to Met22/Xrn1 pathway in yeast that can affect recombination of TBSV, first we used LiCl and pAp inhibitors in MET22 gene can also affect TBSV RNA recombination, first, we expressed the Arabidopsis AHL nucleotidase/phosphatase gene met22Δ yeast. Interestingly, AtAHL reduced the accumulation of TBSV recRNA and degRNA by 5- and 10-fold, respectively , which can be used as a helper tombusvirus, and the highly recombinogenic TBSV DI-AU-FP RNA after knocking down the level of NbAHL mRNA via gene silencing (NbAHL knockdown plants (XRN4 (the homolog of the yeast XRN1) NbAHL did not affect the growth of N. benthamiana, while the XRN4 knockdown plants showed some stunting  strain behaved as the single deletion (xrn1Δ) strain in the TBSV recombination assay  regulates TBSV RNA recombination and degradation of TBSV repRNA via affecting pAp level in cells , met22Δ, gcn4Δ, and xrn1Δ were obtained from Open Biosystems. To express wt and mutated Met22p proteins in met22Δ yeast, we made pYES-Met22 plasmid expressing a 6xHis-Met22p (tag is present at the N- terminus) under the control of a GAL1 promoter. The cDNA of MET22 was amplified with primers #2177 and #2178  with the PCR product of primers #2581/#2590 and pFA6-hphNT1 MET22 gene was verified by PCR with primers #2501/#2591.Yeast strain Gals-met22 was obtained based on homologous recombination. Briefly, the BY4741 yeast strain was transformed with the PCR product made by using primers #2581/#2583 and pYM-N30 GALS sequence in strain Gals-met22 strain was verified with primers KanB (Open biosystems)/#2591.Yeast strain Yeast strains were co-transformed with indicated plasmids or PCR products by using the lithium acetate/ssDNA/polyethylene glycol method 4 until reaching cell density of 0.6 OD600. Then yeast cultures were resuspended in SC-UHL with galactose medium containing 50 µM CuSO4. Yeast was grown at 29°C for indicated timepoints before collecting for Northern analyses.The yeast strain Gals-met22 was co-transformed with pGBK-His33/DI72/CUP1 and pGAD-His92/CUP1 600. Then yeast cultures were diluted to 0.1 OD600 in SC-UHL with galactose medium containing 50 µM CuSO4 to launch TBSV repRNA replication and to co-express the Met22p mutants. Yeast was grown at 29°C for 24 hours before sample collection for analyses. The statistical analysis was performed using AVEDEV (average of absolute deviations of data points) program in Microsoft Excel (version 2008 for Mac) based on 12–24 independent samples.For the complementation study , yeast wmet22Δ were transformed with pYC2-DI72 For the RNA stability studies , yeast smet22Δxrn1Δ, xrn1Δ and met22Δ yeast strains, they were co-transformed with pGBK-His33/CUP1 600.For the analysis of TBSV repRNA replication and recombination in 600 in SC-ULH medium supplemented with 2% galactose and 50 µM CuSO4 to launch TBSV repRNA replication. After 6h at 23°C, the cultures were collected and re-suspended in SC-UHL supplemented with glucose and 50 µM CuSO4. The yeast cultures were grown for additional 18 hours at 23°C before sample collection for analyses.The pre-grown yeast cultures were diluted to 0.1 ODgcn4Δ, because it has been shown that NaCl treatment increases indirectly the activity of GCN4 transcription factor, which then upregulates the expression of MET22600. The pre-grown yeast cultures were diluted to 0.1 OD600 in SC-UHLM+A  with 2% galactose and 50 µM CuSO4 to launch TBSV repRNA replication and the indicated amount of LiCl or NaCl  assay was described previously NbAHL, #2935/#2937 for NbSAL1 and primers #2932/#2933 for NbFRY1. Nine days after the VIGS treatment  NbAHL mRNA was determined by RT-PCR with primers #2940/#2941. We used the level of tubulin mRNA as a control by RT-PCR using primers #2859/#2860. Subsequently, the silenced leaves were co-agroinfiltrated with pGD-CNV and pGD-ΔRI, or pGD-CNV and pGD-DI-AU-FP. One day after agroinfiltration, selected leaves were infiltrated with 200mM LiCl. Leave samples were collected of the agroinfiltrated leaves four days after agroinfiltration, followed by total RNA extraction and Northern blot analysis as described The VIGS vectors, pTRV2-AHLNb, pTRV2-Sal1Nb and pTRV2-FRY1Nb, were obtained by RT-PCR with primers #2191/#9192 for 32P-labeled bands were imaged with a Typhoon 9400 Imager  TAP-tagged purified Xrn1p 32P-labeled region III+IV probe with T7 transcription using PCR amplified DNA obtained with primers #2754 and #2755 and pYC-DI72 as template. Northern blots were imaged with Typhoon  and analyzed by the ImageQuant program. Quantification was performed and the recRNA2 or degRNA2 levels were calculated in comparison to the amount of repRNA in each sample. Also, the repRNA measurements were normalized based on the ribosomal RNA levels in each sample.TBSV RNA replication and recombination was analyzed using total RNA obtained from yeast or plants. Northern blot analysis were performed as described previously 32P-labeled complementary RNA probe with T7 transcription from PCR products obtained with primers #312/#22 (3′ end) or #1660/#20 (5′-3′ end). An RNA probe was obtained for detection of region I of DI-72 (using primers #20/#15 in PCR). To detect MET22 mRNA, we made a 32P-labeled complementary RNA probe obtained by T7 transcription from a PCR product using primers #2177/#2200.To detect the CNV genomic RNA, we made a Table S1Yeast strains, expression plasmids and primers.(0.12 MB PDF)Click here for additional data file.Figure S1GAL1 promoter only for the first 6 hours. (B) The percent of recRNA accumulation in comparison with the repRNA (100% in each sample) in yeast expressing DI-72 or DI-AU-FP repRNAs, respectively. (C) Northern blot analysis of total RNA samples from yeast replicating TBSV DI-AU-FP repRNA. See further details in panel A. (D) Schematic representation of the recRNA2 formed in yeast replicating DI-AU-FP. The replicase-driven template-switching event between two repRNAs is shown with an arrow. Note that the recombinants have variable sequences at the very 5′ end and at the junction sites, thus most of the recRNAs are different from one another.LiCl enhances the formation and accumulation of recRNAs and degRNAs in yeast. (A) Northern blot analysis of total RNA samples from yeast replicating TBSV DI-72 repRNA. BY4741 yeast cultures were treated with the shown concentration of LiCl. Samples were taken 24 hours after launching TBSV repRNA replication. Note that the repRNA was expressed from the (0.64 MB PDF)Click here for additional data file.Figure S2Increased level of accumulation of the nondegraded ITS1 region of pre-ribosomal RNA after LiCl treatment. The activity of cellular 5′-3′ exoribonucleases, such as Xrn1p (cytosolic) and Rat1p (nucleus), was inhibited by the shown amount of LiCl as described. As expected, LiCl treatment increased the accumulation of pre-ribosomal RNA carrying the ITS1 region by up to 7-fold, which is indicative of reduced level of Xrn1p and Rat1p nuclease activities in yeast cells.(0.22 MB PDF)Click here for additional data file.