The RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can specifically recognize RNA targets with diverse sequences and structures. Understanding the principles governing this specificity will be important for the rational modification and design of RRM-RNA complexes.In this paper we have investigated the origins of specificity of the N terminal RRM of the U1A protein for stem loop 2 (SL2) of U1 snRNA by substituting modified bases for essential purines in SL2 RNA. In one series of modified bases, hydrogen bond donors and acceptors were replaced by aliphatic groups to probe the importance of these functional groups to binding. In a second series of modified bases, hydrogen bond donors and acceptors were incorrectly placed on the purine bases to analyze the origins of discrimination between cognate and non-cognate RNA. The results of these experiments show that three different approaches are used by the U1A protein to gain specificity for purines. Specificity for the first base in the loop, A1, is based primarily on discrimination against RNA containing the incorrect base, specificity for the fourth base in the loop, G4, is based largely on recognition of the donors and acceptors of G4, while specificity for the sixth base in the loop, A6, results from a combination of direct recognition of the base and discrimination against incorrectly placed functional groups.These investigations identify different roles that hydrogen bond donors and acceptors on bases in both cognate and non-cognate RNA play in the specific recognition of RNA by the U1A protein. Taken together with investigations of other RNA-RRM complexes, the results contribute to a general understanding of the origins of RNA-RRM specificity and highlight, in particular, the contribution of steric and electrostatic repulsion to binding specificity. The RRM is one of the most common RNA-binding domains -3 and isExtensive biophysical and biochemical investigations have made U1A a paradigm of RRM-RNA recognition -22. U1A KD of the complex and a 6.4–6.8 kcal/mol destabilization of the complex  × 10-6 M) as A1G SL2 RNA to U1A. The C10A substitution alone had little effect on binding affinity (KD = 6 ( ± 1) × 10-10 M). Thus, these experiments suggest that specific recognition of A1 does not require an extensive hydrogen bond network with the base.A1 stacks between U2 and the CG base pair that closes the loop in the U1A-SL2 RNA complex Figure  16]. A1. A116]. 2 group is replaced with hydrogen, and with c1A in which N1 is replaced with C-H. Both substitutions resulted in very small destabilizations of the complex  even though a hydrogen bond is formed between N1 and the side chain of Arg52 in the X-ray structure. In contrast, base substitutions that altered the pattern of hydrogen bond donors and acceptors, such as A1I, or added hydrogen bond donors or acceptors, such as A1DAP or A1-2AP, resulted in a much larger destabilization of the complex of 2.4–2.9 kcal/mol. Within the wild type structure the 2AP and DAP substitutions would introduce unfavorable steric interactions between the side chain of Leu49 and the 2-NH2 group, while the substitution of inosine for A1 would introduce an electrostatic repulsion between N1-H and the side chain of Arg52. These unfavorable interactions may contribute to the destabilization of the complexes containing A1-2AP and A1-DAP, which could contribute to alternate structures being formed in these complexes. These data suggest that the poor affinity of A1G SL2 RNA for U1A is due to the altered pattern of hydrogen bond donors and acceptors on G compared to A, rather than a loss of specific hydrogen bonding interactions. Thus, at this position, specificity is a consequence primarily of the destabilization of complexes containing non-cognate bases.To probe the role of base functional groups without introducing new functional groups A1 was substituted with purine, in which the 6-NH2 and O2 of U2, 2-NH2 and the side chain of Glu19, and N1-H and the side chain of Glu19. Substitution of A for G4 resulted in a 6.4 kcal/mol loss of binding affinity. This value is comparable to that reported previously [G4 is packed between U3 and a number of amino acids in the U1A protein Figure  16]. G4. G416]. eviously .2 (G4I) or the substitution of N7 with C-H (G4c7G) resulted in destabilizations of the complex of 1.5 and 3.3 kcal/mol, respectively. The greater loss in binding free energy upon substitution of G4 with c7G than with I suggests that N7 is a more important contributor to binding than is the 2-NH2 group even though two hydrogen bonds are observed between the 2-NH2 group and the U1A protein in the X-ray structure, compared to one hydrogen bond between N7 and the U1A protein. \These results are consistent with previously performed protein substitutions. The substitution of Asn15, which forms a hydrogen bond between the amide side chain and N7 of G4, with Val abolished binding ), where [P] is the total protein concentration. In gel mobility shift assays that did not saturate at approximately 100% bound, this equation was modified to: fraction bound = A/(1+KD/[P]), where A was allowed to vary between 0.7 and 1. This modification was necessary to estimate the KD's of some of the least stable complexes. Representative gel mobility shift assays and plots illustrating fraction RNA bound as a function of U1A concentration are shown in Figure KD's obtained from these experiments are listed in Tables Protein-RNA equilibrium dissociation constants were measured by gel mobility shift assays. Reaction mixtures containing 25 pM UV melting curves were performed on a Shimadzu UV-2401PC spectrophotometer using 2–10 μM RNA samples in 100 mM NaCl, 0.5 mM EDTA, and 10 mM sodium phosphate at pH 7. The samples were heated from 10 to 95 °C with a heating rate of 1 °C/min, while monitoring absorbance at 280 nm. The melting curves were fit using the program Meltwin 3.5.YB carried out the all of the experiments presented here and participated in the design of the study. AMB conceived of the study, participated in its design and coordination, and drafted the manuscript. Both authors have read and approved the final manuscript.


Sweet potato mild mottle virus , the role of silencing suppressor is played by the P1 protein  despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC.RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in  Sweet potato mild mottle virus, the role of silencing suppressor is played by P1 protein and it works by inhibiting si/miRNA-loaded RISC through targeting Argonaute 1 (AGO1). We confirmed using immunoprecipitation and in vitro binding assays that the interaction between P1 and small RNA loaded AGO1 is specific and direct. The suppression activity mapped to the N-terminal part of P1 containing three WG/GW motifs that resemble the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are essential for both binding and suppression of AGO1 function. P1 protein is the only silencing suppressor identified so far that inhibits active RISC and this is the first demonstration of a WG/GW protein having negative effect on RNA silencing.RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as a major antiviral mechanism in higher plants and insects. Viral RNAs are processed by Dicer-like proteins into small interfering (si) RNAs, which trigger the RNA-induced silencing complex (RISC) assembly. Then siRNA loaded RISC inactivates cognate viral RNA. However, viral silencing suppressors evolved to counteract with RNA silencing targeting one or more key points in the silencing machinery. Here we show that in  Most eukaryotes, including plants, make use of a well-conserved RNA silencing mechanism to regulate many essential biological processes, ranging from development and control of physiological activities, to responses to abiotic and biotic stress, in particular antiviral defense Antiviral defense in plants begins with the activity of RNase III type Dicer-Like (DCL) enzymes, which target viral RNAs Polerovirus P0 suppressor protein has been suggested to target PAZ domain of AGO1 and directing its degradation Viruses have evolved suppressors to counteract the RNA-silencing defense of the host Potyviridae is the largest family of plant RNA viruses; in most members, the single-stranded RNA genome is about 10 kb in size and encodes a single polyprotein that is processed into at least 9 mature proteins Potyvirus, the multifunctional HC-Pro (helper component-proteinase) was the first viral product to be recognized as a silencing suppressor Cucumber vein yellowing virus (CVYV), genus Ipomovirus, family Potyviridae, lacks HC-Pro but contains two P1-type proteases Ipomovirus, Sweet potato mild mottle virus (SPMMV), possesses an HC-Pro region and a single large P1 serine protease The The peculiarities of the SPMMV genome that incorporates the largest P1 region among all known members of the family together with a typical HC-Pro region , promptein vivo P1 inhibits target RNA cleavage mediated by RISC complexes loaded with either endogenous miRNA or with virus-derived siRNA. Moreover, suppression activity mapped to the N-terminal half of P1, a region containing three WG/GW motifs that mimics AGO-binding linear peptide motif conserved both in metazoans and plants In the present study, we show that the large P1 protein of SPMMV possesses silencing-suppressor activity, while its HC-Pro protein does not, on its own. Using various reporter systems, we show that Potyviridae, including other ipomoviruses  as RNA silencing suppressor for SPMMV, we used the standard Agrobacterium coinfiltration assay ilencing . Weak flTobacco etch virus (TEV) were performed next. First, we checked in vitro if SPMMV proteins could bind either typical 21 nt ds siRNAs, or longer dsRNAs. Extracts of N. benthamiana leaves infiltrated with Agrobacterium strains expressing different suppressors were tested for siRNA binding with labeled 21 nt ds siRNA, and the complexes were resolved on a native gel. As expected, TEV HC-Pro bound ds siRNA, while SPMMV P1 and HC-Pro did not show any siRNA binding activity  construct. To this end, patches infiltrated with constructs expressing SPMMV P1, SPMMV HC-Pro , or SPMMV P1HC-Pro (a construct containing both proteins in cis), or with TEV HC-Pro, were analyzed after 3 days for the presence of GFP mRNA and siRNAs by Northern blotting. No reductions in siRNA processing from the GFP-IR were observed in all expressed proteins  19 Stop mutant (Cym19S) was used Pothos latent virus, a virus unrelated to CymRSV, cannot be cleaved, and was used as a negative control Next, we investigated if SPMMV P1 inhibits viral siRNA-loaded active RISC complexes. A previously described system which exploits When the agroinfiltration was performed only with sensors, Northern analysis using a GFP probe detected a shorter hybridizing band, diagnostic for RISC cleavage of the mRNA expressed from the GFP-Cym construct mediated by viral siRNA, while the hybridizing band remained intact in the case of the GFP-PoLV sensor. As expected, the control TEV HC-Pro was not competent to inhibit ss viral siRNA-loaded active RISC complexes, so the GFP-Cym sensor RNA was cleaved N. benthamiana, transiently expressed or endogenous AGO1 protein co-fractionates in extracts with small RNAs, and can be found in at least two distinct complexes of above 669 kD and 158 kD Arabidopsis thaliana (myc-AGO1) N. benthamiana leaves. At 3 dpi, extracts prepared from infiltrated leaves were fractionated on a Superdex 200HR column. Small RNAs were extracted from each fraction and analyzed by Northern blotting, and the AGO1 and P1 protein contents of fractions were monitored by Western blotting using antibodies raised against the HA and myc tags. Consistent with previous results, GFP siRNAs and miR159 were fractionated in two distinct complexes peaking at >669 and 158 kD, although they appeared in all fractions which also contained AGO1 (The RISC complex is of high molecular weight (>669 kD) in animals ned AGO1 . We expened AGO1 .Next, we investigated if co-fractionation of P1 with myc-AGO1 and small RNAs was due to physical interaction. To test this, we agroinfiltrated HA-P1 with myc-AGO1 and GFP-IR. As a negative control, myc-AGO1 and GFP-IR were agroinfiltrated with HA-UPF1, which is known not to be involved in RISC formation. At 3 dpi, extracts were prepared from infiltrated leaves, and HA-tagged proteins were immunoprecipitated (IP) with an anti-HA antibody. Inputs and eluates of IPs were tested for proteins by Western blotting and for GFP siRNA in Northern blots. The results showed that HA-P1 and HA-UPF1 were expressed at comparable levels and could be efficiently immunoprecipitated from extracts. Importantly, we found that myc-AGO1 coimmunoprecipitated with HA-P1, but not with HA-UPF1, confirming that the interaction between HA-P1 and myc-AGO1 is specific . MoreoveTaken together, we showed that siRNAs derived from GFP-IR became incorporated into myc-AGO1, and that the P1 silencing suppressor specifically interacted with GFP siRNA-loaded myc-AGO1. These results, along with earlier data proving that P1 inhibits si- and miRNA programmed RISC, suggest that the large complex (669 kD) containing AGO1 and small RNAs corresponds to the plant RISC complex.1-383 . Furthermore, analysis of amino acid composition revealed that the regions neighboring the WG/GW residues in P1 are rich in alanine, serine, glutamic acid, asparagine and aspartic acid, providing a context similar to that described for Tas3 and RNA Pol V proteins Our results showed that the N-terminal end of the P1 is required for Ago binding. Further inspection of the N-terminal end of P1 revealed repeating tryptophan-glycine/glycine-tryptophan residues (WG/GW) , which aThis observation prompted us to investigate the significance of the tryptophan residues at the N-terminal end of P1 in silencing suppressor activity. For this, we generated single, double and triple mutants of P1 by replacing tryptophan (W) by alanine (A) residue(s) at positions 15, 101 and 131, individually and in all double (3) and triple (1) combinations, by site-directed mutagenesis. Silencing-suppressor activity of the HA-tagged P1 single mutants was compared to the HA-P1wt, co-infiltrated with the GFP-171.1 sensor construct. Expression analysis of the GFP marker gene reflecting the strength of suppression of RNA silencing showed that suppressor activity of any of the single mutants was not reduced significantly, suggesting that presence of the remaining two tryptophan residues were sufficient to maintain the silencing suppressor activity of P1 (data not shown). In contrast, the suppressor activities of double and triple mutants were greatly reduced. Consistently, wt P1 and the mutants were expressed at comparable level .N. benthamiana plants. As positive control, we used HA-P1wt and as negative control, myc-AGO1 and GFP-IR were infiltrated without P1 wt. At 3 dpi, extracts of infiltrated leaves were used to immunoprecipitate HA-tagged P1 wt and mutant proteins. Western analysis showed that HA-tagged proteins were expressed at comparable levels and were successfully immunoprecipitated. However, probing Western and Northern blots to detect myc-AGO1 protein and GFP siRNA derived from GFP-IR revealed that myc-AGO1 protein and GFP siRNAs were specifically co-immunoprecipitated with P1 wt, but not with any of the double or triple mutants of P1 -infiltrated leaf extracts. RNA samples from inputs and immunoprecipitates were probed for presence of two endogenous miRNAs  and P1s miRNAs . The resd miRNAs . To test protein . This reAvailable data suggest that virtually all plant viruses encode at least one suppressor and more than 35 individual viral silencing suppressor families have been identified to date. However, the mechanisms of action have been explored only for a few In this work we explored the mechanism of silencing suppression mediated by the SPMMV P1 silencing suppressor protein, which interferes with miRNA and siRNA driven RISC activity by binding to the AGO1 subunit of RISC complexes through its WG/GW motifs conserved also in Argonaute binding cellular proteins.SPMMV is the only ipomovirus that has a typical potyvirid genome structure with P1 and HC-Pro regions in the C-terminal part of the polyprotein To better understand the molecular basis of P1-mediated silencing suppression, we analyzed the effect of P1 on different steps of the RNA silencing pathway. We found that P1 does not seem to interfere with the biogenesis of either transgene-derived siRNAs or endogenous miRNAs, since we observed that the accumulation of GFP-IR-derived siRNAs was mostly unaltered in the presence or absence of P1 . Similarin vivo. Moreover, we showed that transiently expressed AGO1 protein was found in a large (>667kD) and an approximately 158 kD protein complexes in size. Interestingly, P1 was found co-fractionating only with the large AGO1 containing complex with GFP siRNAs. This results distinguishes P1 from previously studied silencing suppressors, because it does not inhibit RISC assembly, like small RNA binding suppressors, nor inhibits RISC assembly by promoting degradation of AGO proteins, as it was found in the case of P0 protein of poleoviruses in vivo and in vitro and to inhibit RISC activity in vitroTomato aspermy virus (TAV) and the FNY strain of CMV bind 21nt ds small RNAs We tested the effect of P1 on miRNA- and viral siRNA-activated RISC complexes using GFP sensor constructs  and it tlarge >66kD and anThe WG/GW motifs located at N-terminal part of P1 strongly resemble the evolutionarily conserved GW linear peptide motifs shared by different silencing-related proteins used as “Ago hooks” to interact with Argonaute proteins Recent results showed that the AGO-binding domains and the effector domain of GW182 paralogs map in different parts of the proteins We can also postulate alternative explanations for the action of P1. Since small RNA-dependent target cleavage by RISC requires base-pairing between the small RNA and the target RNA, the presence of P1 as an AGO1 interactor might result in covering the small RNA-binding groove of AGO1, thus interfering with base-pairing between the small RNA and the target RNA. This latter possibility is really plausible, because precluding base-pairing between the target RNA and the small RNA would inhibit translation as well, and indeed the importance of translation inhibition in plants has recently been highlighted The efficient binding of AGO1, and inhibition of its function by P1, shown by our experiments suggest that this suppressor might have evolved to bind AGO1 protein with high affinity to inhibit its function. Independently of its final mode of action during suppression, P1 is another example of the extraordinary adaptation of viruses, which are able to target highly conserved key elements of the antiviral silencing response, to be able to complete their infectious cycle. In our study we analyzed only AGO1 as P1 interactor, and we don't know whether P1 is able to interact with other plant AGO proteins. Interestingly, P1 failed to show any effect  when assayed in an R gene-induced anti-viral response test that is dependent on AGO4-like but not AGO1-like activity de novo assembled RISCs, including miRNA- and viral siRNA-loaded RISCs, should be considered as potential targets for P1. At early stages of infection, the concentration of existing active RISC might be much greater than that of the de novo viral siRNA-loaded active RISC, and this would lead to the sequestration of P1 mainly by existing active RISC complexes. Consequently, we may hypothesize that the newly formed viral siRNA-loaded RISC could escape from suppression, resulting in low SPMMV titre, mild transient symptoms, and recovery of the plant. However, in cases where plants have additionally been infected with other viruses such as SPCSV, the initial antiviral response might be suppressed by the two-component silencing suppressor system of SPCSV Our results can also help to explain the pathology of SPMMV, either alone or in synergism with other viruses. Generally speaking, defeat of the host RNA silencing response by a virus equipped with a silencing suppressor requires a high concentration of the suppressor in infected cells, above both the dissociation constant of the suppressor with its target and the intracellular concentration of the target molecule N. tabacum Xanthi plants. The complete P1 and HC-Pro regions of the virus were RT-PCR amplified from total nucleic acid extracts using primers TCTAGAATGGGGAAATCCAAACTCACTTAC3′5′CC and CCCGGGTCAATAGAATTGTATCTGTTTAAGTTTACTAG3′5′GT for P1, and TCTAGAATGGCAAGTTCTGTTGTACCCAATTTC3′5′CC and CCCGGGTCAACCAACCTTATAGGTTAACATCTCAC3′5′GT for HC-Pro, and cloned, using the restriction sites highlighted in bold, into competent plasmids for sequencing. The two viral genes were cloned into pBIN-derived constructs for transient expression in N. benthamiana leaves. Variants incorporating the tagging element HA were also prepared. Plants were kept in a greenhouse at 22°C under a photoperiod of 12 h/12 h light/dark. Infiltration assays were performed on expanded N. benthamiana leaves of plants about 21 days old.The African isolate SPMMV-130 was kindly provided by Jari Valkonen  in a sweet potato plant, and maintained in N. benthamiana leaves were infiltrated essentially as previously described 600 = 0.1. SPMMV 35S-HC-Pro, HA-UPF1 600 = 0.2–0.3. Infiltrated patches were used for total RNA and protein isolation, then analyzed by Northern and western blotting.Total RNA was isolated using TRIZOL reagent. RNA was analyzed on 37% formaldehyde containing agarose gels as described 32P labeled LNA oligonucleitide probes Total RNA samples were oxidized, ß-eliminated and detected as described in 2, 5mM DTT and 10% glycerol, then incubated for 1 hour at 4°C with beads containing anti-HA (Roche) or anti-myc antibody (Sigma). The beads were then washed with IP buffer. Half of the eluates were used for RNA isolation as described N. benthamiana AGO1 Extracts for immunoprecipitation were prepared in IP buffer containing 30mM TRIS (pH 7.5), 150mM NaCl, 5 mM MgClPlant extracts for gel filtration were prepared in IP buffer and fractionation was carried out similarly as described earlier 5′GGGGATCCCTAGAATGGGGAAATCCAAACTC3′, and P1-383 5′GCGGATCCTCAATCATCAACTTGTGCGTTTAGGGA3′. All mutants were verified by sequencing.Site-directed mutagenesis was performed using the Quickchange site-directed mutagenesis kit (Stratagene) according the manufacturer's instructions to generate single, double and triple mutants with oligonucleotides listed in GenBank accession numbers for new viral nucleotide sequence: GQ353374 and for complete SPMMV sequence: NC_003797.Text S1Alignments of sequences of P1 proteins(0.07 MB DOC)Click here for additional data file.Figure S1Amino acid alignment of ipomoviral P1 proteins, including sequences corresponding to SPMMV P1 (isolate 130), CBSV P1, CVYV P1a+P1b, and SqVYV P1a+P1b. Black background indicates identical residues in at least two sequences, with two shades of gray to account cases where identity exists between two pairs of sequences. Positions of the catalytic triads  are highlighted in red lettering and labelled with asterisks, and the cleavage sites are indicated with vertical lines and inverted delta symbols at the end of the sequences for P1 and P1b, while the cleavage between P1a and P1b in CVYV and SqVYV are also shown with similar marks at the corresponding internal sites. WG and GW motifs found in P1 proteins are indicated by orange letterings and yellow boxes. Positions of conserved cysteine residues are indicated with green and blue boxes, respectively for cysteine rich domains near the N-terminus or in a region found to be implicated in the RNA silencing suppression activity of CVYV P1b (1.92 MB EPS)Click here for additional data file.Figure S2Amino acid alignment of the N-terminal region corresponding to one ipomoviral and one potyviral P1 proteins, up to the previously identified region of putative intergeneric recombination (0.80 MB EPS)Click here for additional data file.


Escherichia coli. Here, we present the crystal structure of the E. coli RNase E catalytic domain in the apo-state at 3.3 Å. This structure indicates that, upon catalytic activation, RNase E undergoes a marked conformational change characterized by the coupled movement of two RNA-binding domains to organize the active site. The structural data suggest a mechanism of RNA recognition and cleavage that explains the enzyme's preference for substrates possessing a 5′-monophosphate and accounts for the protective effect of a triphosphate cap for most transcripts. Internal flexibility within the quaternary structure is also observed, a finding that has implications for recognition of structured RNA substrates and for the mechanism of internal entry for a subset of substrates that are cleaved without 5′-end requirements.RNase E is an essential bacterial endoribonuclease involved in the turnover of messenger RNA and the maturation of structured RNA precursors in  E. coli . A bent tetramer may explain this observed 1:1 ratio of stoichiometry of tetramer to RNA. Further experiments are needed to determine the role of RNase E tetramer organization in the maturation of structured RNA.In each of the four apoprotein protomers, the combined S1 subdomain and 5′ sensor (from here on referred to collectively as the “5/S1 subdomain”) has moved as a single unit through an angle of about 60° relative to the holoprotein configuration B. This cIn their report of the holoprotein structure of RNase E, The holoprotein model identified a magnesium ion in the catalytic site coordinated by D303 and D346 with the support of N305. Each of these residues has been implicated from mutation studies as required for catalytic activity . In the We favor this model because it accounts for the preference of the enzyme for substrates with a 5′-monophosphate terminus over those with either a triphosphate or hydroxy cap. It predicts that with only a terminal hydroxy cap, binding of an RNA substrate by RNase E will be substantially weaker and cleavage will be impeded under nonsaturating conditions. If a triphosphate cap is present on substrates, then the 5′ sensing site may still be able to bind them with moderate affinity because there is sufficient space in the open configuration of the enzyme to accommodate the three phosphate groups. However, RNA cleavage will again be greatly impeded because the extra phosphates are likely to sterically clash with the rest of the structure during the transformation to the closed configuration, acting as a wedge at the base of the fulcrum. Furthermore, the movement of these charged groups into a hydrophobic environment represents an additional thermodynamic barrier to domain closure.Our crystal structure suggests that the enzyme will bind isolated phosphate and sulfate groups within the 5′ terminal recognition site. Thus, it seems likely that the recognition site residues make a key contribution to the RNA binding affinity. In this respect, our model conflicts with reports that the 5′ end of the substrate provides no preference for RNA binding affinity and that the effect of the 5′-monophosphate is primarily due to its role in catalytic activation . HoweverAlthough the model for substrate interaction effectively describes global, nonspecific RNA degradation by RNase E, the mechanism used to process complex substrates through restricted cleavage at only specific sites is still unknown. The open configuration of the apoprotein may allow these more complex RNAs to be accommodated within the active site and bound at the 5′ end without the requirement for the same structural change that maneuvers small single-stranded RNA into the cleavage orientation. We would envisage that a single-stranded segment would be accommodated into the shallow channel that leads to the active site. The secondary structure of complex substrates may be sufficient to bring a defined RNA segment into close proximity to the catalytic site. In such cases, the substrates may not depend on RNase E undergoing a conformational change and may be effectively cleaved in the presence of either a 5′ mono or triphosphate cap. Likewise, it is also possible that the observed open configuration may allow a complex substrate to bind to a single protomer while positioning another segment of the RNA to be cleaved by a second protomer. Such a model would help to explain the functional value of the enzyme's tetrameric organization and is particularly attractive because of the observed flexibility of the structure.A recent report indicates that the conversion of a 5′-triphosphate cap to a monophosphate represents the rate-limiting step in bacterial mRNA decay . Recent 2BX2)  over their equivalents in the first apoprotein model, running MOLREP rigid-body refinement on these, and manual rebuilding using Coot. The model was then refined again using TLS plus restrained refinement and structure idealization in Refmac. The positions of the zinc ions were again confirmed by an anomalous Fourier synthesis. This later model has electron density and stereochemistry significantly improved over the first and is presented here. The structure was examined and validated using PROCHECK   were use2BX2)  and mult2BX2) . The “ZnPROCHECK  and SFCHPROCHECK  and was PROCHECK .


S1 and OriS2 RNAs and in case of α4 null mutant additional transcript that spans the junction between L and S component of the HSV-1 genome. Current data do not suggest that a peptide is translated from these RNAs.The genome of herpes simplex virus 1 encodes at least 84 transcripts from which proteins are translated and several additional RNAs whose status as mRNAs is unknown. These RNAs include latency-associated transcript, OriS5-1 that spans 4.5 kb of the unique-short (US) region. The RNA initiates in US5 and terminates in the α22 open reading frame. It is expressed antisense to US5, US4, US3 and ICP22 mRNAs. This transcript is expressed with γ2 kinetics and has a half-life of 80 minutes.We describe here a novel RNA designated UThese results identify a novel transcript encoded within HSV-1 genome. Since no major hypothetical open-reading frames are present in this transcript it is feasible that this RNA exerts its function as a non-coding RNA. S) DNA sequences of the herpes simplex virus 1 (HSV-1) listed 12 open reading frames (ORFs) designated US1 (α22) through US12 (α47) [S8.5 mRNA co-terminal with US8 and US9 [S1.5 co-terminal with the α22 transcript [S3.5 co-terminal with the US3 mRNA [S1 initiated in α22 or α47 ORFs, run across the origins of DNA synthesis and terminated at the transcription termination site of the α4 gene. OriS2 terminated at or near the transcription initiation site of the α22 or α47 gene [S transcripts or Δα4-specific transcript are unknown. In this report we describe an additional long transcript designated US5-1 RNA. As the designation implies, this transcript originates in the US5 ORF and terminates in the α22 ORF.The original report of the organization of the unique short  were exposed to 10 PFU of HSV-1(F) per cell, RNA was extracted 18 h later and subjected to RT-PCR or Northern blot analysis. For RT-PCR analysis cDNA was generated from total RNA using primer 51(R), specific for RNAs antisense to US3. PCR reaction was performed using the 51(F) as forward and 51(R) as reverse primer. While reverse transcription with 51(R) primer would also generate cDNA from US2 transcript, this cDNA would not be amplified under PCR reaction used in this experiment since 51(F) primer lies upstream from US2 transcript . Knowing the size of this RNA (~1.6 kb), we estimated the location of its 5'-end on the basis of the assumption that the sequences upstream of the insertion site would be identical in both R7802 and wild-type virus. In order to fine map the location of the 5'-end of the US5-1 transcript, we performed a series of northern blot analyses that helped us place the 5'-end within the sequences covered by the probe 3 (data not shown). To determine more precisely the location of the 5'-end of the US5-1 transcript we performed an RNase-protection assay using probe 3. The results of this experiment reveal the presence of a protected fragment with size of about 270 bases  per cell, RNA extracted 18 h later and subjected to RNase protection assay using a probe that spanned the predicted termination site identified within ICP22 ORF (probe 1). The probe 1 protected fragment of approximately 180 bases long . In addition to the 180 bases long fragment we also observed another protected fragment that was equal in length to the full-length probe. This fragment does not correspond to the US5-1 transcript, considering that the US5-1 transcript is ~4.5 kb long. It is possible that a different RNA, expressed antisense to ICP22, is present in infected cells.On the basis of the size of the Uong Fig. , arrow. Figure S5-1 RNA. In the first series of experiments, RSC were exposed to 10 PFU of HSV-1(F) per cell, RNA extracted 1, 3, 6, or 9 h later and subjected to northern blot analysis using probe 2 for detection of the US5-1 transcript. The results  per cell in the presence or absence of 100 μg/ml cycloheximide. Total RNA was extracted 9 h later and subjected to northern blot analysis. Addition of cycloheximide completely abrogated the expression of the US5-1 transcript  per cell in the presence or absence of 300 μg of PAA per ml. Total RNA was extracted 9 h later and subjected to northern blot analysis. PAA completely abrogated the expression of the US5-1 transcript  per cell. At 6 h after infection the cells were exposed to 100 μg of actinomycin D per ml of medium. The RNA was extracted at 0, 1, 2, 3 or 4 h after the addition of the actinomycin D and subjected to northern blot analysis  One possible explanation for the failure to detect the 4.5 kb RNA in earlier studies is the failure of transfer of high molecular weight RNA from gels containing high concentrations of agarose. In the studies reported here, we detected the 4.5 kb RNA on transfer from gels containing 1%, or less, of agarose Fig. , but notS5-1 RNA is dependent on viral DNA synthesis for its accumulation. In contrast, the OriS1 RNA does not require de novo viral protein or DNA synthesis for its accumulation [(ii). The Umulation . The meamulation .S5-1 RNA is not known. The US5-1 RNA contains only two large ORFs of 300+ codons. An ORF of approximately the same size is present in the corresponding sequences of HSV-2 DNA. However, In HSV-1 DNA the first large ORF initiates with the fifth methionine codon and its predicted amino acid sequences is not conserved in the corresponding HSV-2 ORF. We cannot exclude the possibility that the very small ORFs upstream of the large one are expressed. It seems it would be very unlikely that an RNA 4.5 kb long would encode a peptide with less than 100 amino acids, as it would be highly energy inefficient. However, it would not be unprecedented, since Drosophila's tal gene expresses 1.5 kb long transcript that codes for several 11 amino acid long peptides [S5-1 directs protein synthesis.(iii). In principle, viruses evolve continuously and do not retain DNA sequences that have no functions related to their survival in nature. The function of Upeptides . OverallS5-1 RNA.(iv) As noted earlier, the accumulation of HSV-1 non coding RNAs is not unprecedented. There is considerable current interest in non coding RNAs inasmuch as they frequently serve as precursors of microRNAs or directly regulate the expression of genes located antisense to the RNA. We should note however that none of the studies published to date reported microRNA derived from the domain of the US5 to the α22 genes and is transcribed antisense to US5, US4, US3 and α22 mRNAs, with γ2 kinetics. Our data suggest that US5-1 is a long non-coding RNA. The role of this transcript is currently unknown, but it is plausible that, similarly to other long non-coding RNAs, it is involved in the regulation of expression of viral genes.In this report we identified a novel HSV-1 transcript, expressed from the unique-short region of the viral genome. It spans the region from the URabbit skin cells (RSC) were originally obtained from J. McClaren. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal bovine serum. HSV-1(F) is the prototype HSV-1 strain used in the laboratory . Mutant 2 flasks were either mock infected or infected with 10 PFU of virus per cell and maintained at 37°C in medium 199 V consisting of a mixture of 199 supplemented with 1% calf serum. The cells were harvested at time points after infection specified for each experiment. In some experiments cells were infected in the presence of 100 μg/ml cycloheximide or 300 μg/ml phosphonoacetate (PAA). Total RNA was extracted by using Trizol reagent . Aliquots of 10 μg of total RNA were separated on 1,2%, 1% or 0.7% formaldehyde agarose gels, transferred to Biodyne B nylon membranes  and hybridized with single-stranded RNA probe transcribed in vitro and labeled with biotin using MAXIscript kit  from DNA template driven under the T7 promoter. DNA template was generated by PCR from the HSV-1 genomic DNA. Primers used for the generation of the DNA template were the following:Cultures of RSC in 25-cmProbe 2(F): TAATACGACTCACTATAGGCATATACTAGCGCGGATGCCGCGGACProbe 2(R): CATGCCAGTCACCAGCTTGGCCATGGTCGAPCR reaction was performed using Pfu DNA polymerase  under the following conditions: 2 min. at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, 1 min at 68°C, with final 7 min at 68°C. The location of the probe 2 within the unique-short region of the HSV-1 genome and the direction of its transcription is shown in figure 4 cpm of probe labeled with 32P-UTP using MAXIscript protocol  according to manufacturer's instructions and precipitated. RNA samples were dissolved in hybridization buffer, incubated at 56°C over night and treated with RNaseA/RnaseT1 mix (1:150 dilution) for 30 minutes. RNA was precipitated, dissolved in loading buffer, separated on 5% acrylamide/8 M urea gel and exposed to Kodak blue-bio autoradiography film . DNA templates from which the probe 1 for identification of the 3'-end or probe 3 for identification of the 5'-end of the US5-1 RNA were transcribed were generated by PCR from the HSV-1 genomic DNA using primers for probe 1:RNA samples were prepared as described for northern blot experiments. RNase protection assay was performed using RPA III kit  according to manufacturer's instructions. Briefly, aliquots of 10 μg of total RNA were mixed with 2 × 10P1(F): TAATACGACTCACTATAGGCATATACTTTCTGTGTGTCGCGATGTP1(R): AGTTTGTACACGGATTTCCTGGTTAGand for probe 3:P3(F): TAATACGACTCACTATAGGCATATACATGTCGGCGGAATCCACCP3(R): AGTTTGTAATTGATACACTGGCGCAT for probe 3For the PCR reactions Pfu DNA polymerase was used  under the following conditions: 2 min. at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, 1 min at 68°C, with final 7 min at 68°C. The location of the probe 1 and probe 3 within the unique-short region of the HSV-1 genome and the direction of their transcription is shown in figure RNA samples were prepared as described for northern blot experiments. Reverse transcription was performed on 1 μg of total RNA using Reverse Transcription System  according to manufacturer's instructions. Primer used for reverse transcription was 51(R): ATGTACGGAAACCAGGACTACC. As a control, the same amount of RNA was subjected to the same treatment, except that reverse transcriptase was omitted. For PCR amplification the following primers were used: 51(R) and 51(F): TCCCGGCTGCGTCGTCGTATACAll PCR reactions were performed using Pfu DNA polymerase  under the following conditions: 2 min. at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 60°C, 1 min at 72°C, with final 7 min at 72°C. PCR products were separated on 1% agarose gel and visualized by ethidium-bromide staining. The location of primers used for RT-PCR within the unique-short region of the HSV-1 genome is shown in figure 2 flasks were either mock infected or infected with 10 PFU of virus per cell and 6 h after infection actinomycin D (AcD) was added to the concentration of 100 μg/ml. RNA was extracted 1, 2, 3 or 4 h after the addition of AcD as processed for northern blot analysis. The intensity of the bands on the film was measured by UN-SCAN-IT densitometer  and relative amount calculated as the percentage of RNA present at the time of the addition of AcD was plotted as the function of time.Cultures of RSC in 25-cmThe authors declare that they have no competing interests.VJ performed the experiments and drafted the manuscript. BR reviewed and revised the manuscript.


A significant effect of Cl− ions and H2O2 molar ratios on the rate of chloro-organic product formation has been observed. Studies on oxychlorination of tert-butylethyl ether (ETBE) at pH 7, 3.5, and 2.5 have been carried out. It was found that introduction of hydronium ions into the reaction system considerably hastened the process of chloro-organic product formation. Hydronium ions contribute to the formation of the reactive tert-butyl carbocation, which undergoes secondary reactions in the presence of reactive forms of chlorine and oxygen. Moreover, the effect of temperature on ETBE (tert-butylethyl ether) and MTBE (tert-butylmethyl ether) conversions was verified. The reactions of MTBE and ETBE oxychlorination were carried out at temperatures of 5°C, 20°C, and 35°C.The effect of concentration, molar ratios of reagents, pH, and temperature on formation of chloro-organic products in reaction of  Tert-butyl ethers are added to fuels as the oxygen components since they increase the octane number of petrol. They contribute to the improvement of air quality by reducing the emission of carbon oxide and precursors of free radicals in the exhaust gases. On the other hand, out-of-control leakages of fuels containing tert-butyl ethers are the source of their presence in the environment. Ethers are identified in ground, surface, storm, and waste waters as well as in air  65%, hydrogen peroxide 30%, and sodium chloride  were used in our studies. Samples for experiments were prepared by liquid-liquid extraction technique. Carbon disulfide (Merck) was applied as a solvent. Samples were analyzed by gas chromatograph HP 5890-Hewlett Packard equipped with flame ionization detector FID. Operating parameters were as follows: column HP-1 (0.53 mm × 60 m × 0.2 μm) temperature of injector 250°C, temperature of detector 250°C, temperature program: 40°C/4 min-10°C/min-200°C.MTBE 98%, 1,2-dichloro-2-methylpropane, 3-chloro-2-chloromethylpropene (Sigma-Aldrich Germany), ETBE (Polish Petrol Concern Orlen” S.A. Płock Poland), Precision was evaluated by injection of samples (repeated six times) obtained after extraction of ETBE, 1,2-dichloro-2-methylpropen, and 3-chloro-2-chloromethylpropene solutions at concentrations of 1 mL/L, 10 μL/L, and 10 μL/L, respectively. The values of relative standard deviations were determined as follows: ETBE, 3.37%; 1,2-dichloro-2-methylpropen, 2.84%; and 3-chloro-2-chloromethylpropene, 4.41%.2) were as follows: ETBE, 0.987; 1,2-dichloro-2-methylpropan, 0.994; and 3-chloro-2-chloromethylpropene, 0.996.In the next stage, a linearity of calibration curves for ETBE 1,2-dichloro-2-methylpropan and 3-chloro-2-chloromethylpropene was determined. For that purpose, water solutions of ETBE  and 1,2-dichloro-2-methylpropen and 3-chloro-2-chloromethylpropene  were prepared. Then, liquid-liquid extraction was performed by the use of carbon dioxide and calibration curves were determined. The linear correlation coefficients . pH of solution was measured by pH-meter Elmetron CX-741.ETBE solution at a concentration of 0.07 mol/L with the addition of 0.7 mol of NaCl and 0.35 mol of Htert-butyl alcohol (TBA), which was the product of oxidation of ethers. Composition of the solution studied is presented in Studies on the effect of temperature were carried out in the presence of ETBE and MTBE as well as 2O2/Cl−/H+ system. According to the literature data, the H2O2/Cl−/H+ system is of the complex character and it is described by a series of secondary reactions. They are both ionic  with hydrochloric acid and hydrogen peroxide at a temperature of 35°C, processes of chloroproduct formation and decomposition proceeded similarly.2O2/HNO3 . At a temperature of 5°C, 3-chloro-2-chloromethylpropene was formed very slowly and in small quantities . At a te2O2/HNO3 . Howevert 4 mg/L . Similart 4 mg/L . Howevert 4 mg/L .2O2/HCl system at temperatures of 20 and 35°C were carried out. At higher temperature, the reaction runs at higher rate  of the successive portion of chloro-organic compounds that are relatively stable and can cumulate in various organisms.The stables in neutral medium ethers can migrate to considerable distances and thus, they contribute to contamination of drinking waters. In the case when they come into contact with acid sewage, they can contribute to formation of new, more reactive products, which, in turn, in the presence of reactive forms of chlorine, are a potential source of toxic chloro-organic products. On the basis of results obtained, it can be assumed that intensity of chloro-organic compound formation is higher in summer than in the winter season. Therefore, introduction of


Caenorhabditis elegans halt development and survive in this state for several weeks. We isolated mutations in unc-31, encoding a calcium-activated regulator of neural dense-core vesicle release, which conferred enhanced starvation survival. This extended survival was reminiscent of that seen in daf-2 insulin-signaling deficient mutants and was ultimately dependent on daf-16, which encodes a FOXO transcription factor whose activity is inhibited by insulin signaling. While insulin signaling modulates metabolism, adult lifespan, and dauer formation, insulin-independent mechanisms that also regulate these processes did not promote starvation survival, indicating that regulation of starvation survival is a distinct program. Cell-specific rescue experiments identified a small subset of primary sensory neurons where unc-31 reconstitution modulated starvation survival, suggesting that these neurons mediate perception of food availability. We found that OCR-2, a transient receptor potential vanilloid (TRPV) channel that localizes to the cilia of this subset of neurons, regulates peptide-hormone secretion and L1 starvation survival. Moreover, inactivation of ocr-2 caused a significant extension in adult lifespan. These findings indicate that TRPV channels, which mediate sensation of diverse noxious, thermal, osmotic, and mechanical stimuli, couple nutrient availability to larval starvation survival and adult lifespan through modulation of neural dense-core vesicle secretion.For most organisms, food is only intermittently available; therefore, molecular mechanisms that couple sensation of nutrient availability to growth and development are critical for survival. These mechanisms, however, remain poorly defined. In the absence of nutrients, newly hatched first larval (L1) stage  C. elegans that survive about 50% longer than wild-type animals when starved. For one such mutant, we found that the extended survival was due to mutation in the unc-31 gene, which functions in the nervous system to mediate release of neuroendocrine signaling molecules including insulin. Although this gene is broadly expressed in the nervous system, we found that its activity is required in a small subset of sensory neurons to regulate starvation survival. These neurons have ciliated endings that function in detection of environmental cues. Disruption of these cilia, or inactivation of a TRPV channel localized to these cilia, mimicked the perception of nutrient deprivation leading to extended starvation survival, which is dependent on an insulin-regulated transcription factor. Disruption of this channel also extended adult lifespan. Taken together, our findings reveal that TRPV channels couple nutritional cues to neuroendocrine secretion, which in turn determines adult lifespan and larval starvation survival.Starvation is a common physiological condition encountered by most organisms in their natural environments. However, the molecular mechanisms that allow organisms to accurately sense nutrient availability and match their energetic demands accordingly are not well understood. To elucidate these mechanisms, we isolated mutants in  In their natural environments, most animals are faced with fluctuating food availability. To survive in this changing environment, animals must be able to coordinate their energy demanding processes such as basal cellular functions, growth, reproduction, and physical activity to available energetic resources. Therefore, the ability to appropriately gauge food availability to initiate programs of growth or arrest is of considerable survival value particularly during starvation. In multicellular organisms, cell-autonomous nutrient sensing mechanisms as well as hormonal cues are thought to ensure coordinated responses among various tissues C. elegans provides a genetically tractable system for uncovering molecular mechanisms of nutrient sensation and starvation resistance. The ability to withstand nutrient deprivation is critical for C. elegans survival, as this organism is often found in a starved state in its natural environments C. elegans postembryonic growth and development are tightly linked to nutrient availability. C. elegans embryos are packaged with sufficient nutrients to support development into the mid-first larval stage. When hatched in favorable conditions, C. elegans hermaphrodites undergo four larval transitions (L1–L4) before becoming egg-laying adults. In the absence of food, newly hatched L1 animals arrest development and remain in this diapause state until nutrient is available. L1s in diapause are morphologically similar to well-fed siblings at the same stage. This is in contrast to animals in the hibernating dauer stage, an alternative developmental state characterized by extensive morphological rearrangements  channel that localizes to these sensory cilia, also extended larval starvation survival as well as conferring extended adult lifespan in a DAF-16/FOXO dependent manner. These findings demonstrate that the decision to grow or remain in diapause is under neural control and reveal a previously unidentified role for the polymodal TRPV channels as ancient sensory gauges that couple nutrient availability to larval starvation survival and adult lifespan.To identify starvation resistance mechanisms of L1s in diapause, we screened for mutants with enhanced capability to survive starvation. We isolated loss-of-function mutations in To identify regulators of larval starvation survival, we developed an assay to measure survival of L1 worms under nutrient deprived conditions see . We defiunc-31 locus using standard single nucleotide polymorphism mapping techniques ft1–ft4) of unc-31, with ft1, ft3 and ft4 likely to be null alleles  mutants  mutagenesis screen for mutants with enhanced starvation survival and recovered twenty-eight mutant lines. At least 50% of starving L1s from each of the mutant lines could resume growth upon reintroduction to food at a time when all starving wild-type controls had lost this capacity (data not shown). Of these twenty-eight lines, ten were severely uncoordinated and this Unc phenotype was linked to increased survival. The Unc phenotype was mapped to the  alleles . In overild type . Althougperature . Previou alleles . Additio mutants . Togetheunc-31 mutants in Finally, while background mutations could potentially contribute to differences in starvation survival, this effect appeared to be minimal. We obtained similar starvation survival curves for mutants that were outcrossed to the Ashrafi lab wild-type strain as those received from other labs without further outcrossing  required for the regulated release of dense-core vesicles, which contain biogenic amines, neuropeptides, and insulins unc-31 is broadly expressed in the C. elegans nervous system, but not in other cell types unc-31 were originally identified based on their dramatically reduced movement unc-31 mutants are pleiotropic, we sought to determine whether reduced movement, general defects in synaptic transmission, or reduced secretion of specific dense-core vesicle cargoes could account for their enhanced starvation survival phenotype.unc-34, encoding an axon guidance cue unc-36, encoding a voltage-gated Ca2+ channel unc-60, encoding a regulator of the actin cytoskeleton unc-87, encoding a muscle myofilament component unc-31 mutants, yet all had either wild-type or reduced L1 starvation survival  with either unc-31(ft1) or unc-31(ft4) completely suppressed the extended survival of these mutants  mutants  animals  mutant to wild-type levels  mutants  play an important role in determining L1 starvation survival. To examine the role of this subset of neurons, we used an ocr-2 promoter unc-31 to ADL, ASH, AWA, and ADF head sensory neurons as well as PHA and PHB tail phasmid sensory neurons and a gpa-11 promoter unc-31 using either of these promoters was sufficient to partially abrogate extended L1 starvation survival of unc-31(ft1) mutants  mutants, suggesting that defects in sensory perception might regulate starvation survival. Cilia are specialized organelles that are enriched in sensory transduction mechanisms and disruption of cilia cause defects in sensation of environmental cues (reviewed in osm-1(p808), osm-3(p802), osm-6(p811), and che-11(e1810) mutants, which have shortened and defective sensory cilia unc-31 mutants , che-3(e1124), and bbs-1(k1111); osm-12(n1606); bbs-8(nx77) triple mutants unc-31 mutants, the extended survival of osm-6 mutants was fully dependent on daf-16 (unc-31(ft1); osm-6(p811) double mutants did further enhance starvation survival, suggesting that defects in cilia function and dense-core vesicle release likely function in the same genetic pathway to regulate starvation survival (data not shown).Expression of  mutants . osm-1, n daf-16 . Furtherunc-31 with an ocr-2 promoter abrogated extended L1 starvation survival of unc-31(ft1) mutants (ocr-2(ak47) null and ocr-2(yz5) hypomorphic mutants displayed a ∼30% enhancement in starvation survival  mutant was dependent on daf-16 . Importantly, in the ocr-2 mutant, coelomocyte uptake of GFP-tagged UNC-122 neuropeptide unc-31 and ocr-2 mutants was associated with decreased neural secretion of dense-core vesicles that contain neuroendocrine signaling molecules such as insulin-like peptides.To determine whether n daf-16 . We thene levels . Double ocr-2 mutants may have diminished insulin signaling. Since insulin-signaling deficient animals have extended adult lifespan, we determined the adult lifespan of ocr-2 mutants. Relative to wild-type animals, inactivation of ocr-2 caused a substantial extension in lifespan, and as previously reported unc-31 mutations also caused extended adult lifespan  worms were indistinguishable at room temperature ocr-2 inactivation moderates insulin signaling to a threshold sufficient for bypass of dauer entry, allowing for normal growth and reproduction, yet sufficiently reduced to enhance larval starvation survival and adult lifespan.Our findings raised the possibility that lifespan . As in sn daf-16 . Unlike unc-31 enhance starvation survival. This indicated that diminished neural release of dense-core vesicles enhances starvation resistance. Using functional reconstitution studies, we identified a small subset of ciliated sensory neurons that are likely to directly participate in the perception of nutrient availability and to convey this information to the rest of the organism via regulated secretion of molecules contained in dense-core vesicles. Although these vesicles contain a number of diverse cargoes such as insulins, biogenic amines and neuropeptides, our findings indicated that modulation of insulin-regulated DAF-16/FOXO transcription factor is the principle mechanism that determines the extent of survival during periods of nutrient deprivation. Therefore, the simplest model consistent with these results is that neural assessment of nutrient availability coordinates growth and starvation survival pathways directly through release of insulin-like peptides contained in neural dense-core vesicles  or 20°C on NGM (nematode growth media) plates seeded with OP50 E. coli as a food source. Dauer constitutive strains were grown and maintained at 15°C. Strains were obtained from the C. elegans Genetics Center, CGC (www.cbs.umn.edu/CGC/), unless otherwise noted. Strains KQ35 and KQ170 were generated by backcrossing unc-31(ft1) and unc-31(ft4), respectively, 4× with wild type (N2). KQ34 and KQ108 corresponding to unc-31(ft2) and unc-31(ft3), respectively, were backcrossed 2× with wild type. KQ190: tdc-1(ok914), KQ191: tbh-1(ok1196), and KQ344: egl-3(gk238) were obtained by backcrossing RB993, RB1161, and VC461, respectively, from the OMRF Knockout Consortium (www.mutantfactory.ouhsc.edu/) 4–5× with wild type. MX288: bbs-1(ok1111); osm-12(n1606); bbs-8 (nx77) was a gift from Michel Leroux.Strains were constructed by standard daf-16(mgDf47) and each of unc-31(ft1), unc-31(ft4), ocr-2(ak47), and osm-6(p811) were constructed by mating each single mutant to wild-type males, picking heterozygous males and mating them with hermaphrodites of the second mutant and scoring F2 progeny. daf-16(mgDf47) and ocr-2(ak47) deletions were scored by PCR. unc-31(ft1) and unc-31(ft4) were scored by Unc phenotype and by RFLP with RsaI and BstB1, respectively. osm-6(p811) was scored by failure to dye-fill with DiO and by RFLP with Hpy188III Double mutants between Pshr-220::daf-28::mCherry; unc-122::GFP; Pmyo-2::GFP into wild type. KQ1041, KQ1042, KQ1044, and KQ1074 were constructed by mating KQ1016 to ocr-2(ak47), daf-16(mgDf47), unc-31(ft1), and osm-6(p811) mutants, respectively. KQ1084 was constructed by mating KQ1016 to ocr-2(ak47) and selecting for wild-type ocr-2. KQ1041, KQ1042, KQ1044, KQ1074 and KQ1084 were used for insulin uptake assay.KQ1016 was constructed by injection of unc-31(ft1) animals were injected with plasmid of interest at a concentration of 20 ng/µl or 100 ng/µl with Pmyo-2::GFP or Pmyo-3::GFP at 50 ng/µl as a co-injection marker . Note that position M133 was annotated as the start codon of unc-31 in Wormbase Release WS160 and the resulting cDNA fully rescued unc-31(ft1) mutants. Clones were sequenced and mutations fixed by Stratagene site-directed mutagenesis. For daf-28, the corresponding genomic sequence from start to the codon immediately preceding the stop codon was PCR amplified from wild-type genomic DNA and cloned into Gateway pDONR-221 vector and sequenced to verify for accuracy.Plasmids were constructed using Gateway Technology. The egl-3, osm-6, tax-4, and ceh-23 were constructed by the Vidal Lab Promoterome myo-2 and myo-3 were gifts from Marc Vidal shr-220, glr-2, glr-5, glr-8, ocr-2, gpa-11 and gpa-13 were constructed by PCR from wild-type genomic DNA using the Vidal Lab Promoterome primers and cloned into pDONR-P4-P1R vector (Invitrogen). Rescue constructs were generated using the pKA460 plasmid to obtain promoter::unc-31::intercistronic::mCherry polycistronic fusions. This resulted in the expression of mCherry from the same transcript as unc-31 without modifying unc-31. Posm-6::unc-31 was constructed by cloning the osm-6 promoter with unc-31 cDNA into pDEST-MB14, a gift from Marc Vidal Promoters for unc-119 rescue fragment and religating the vector to generate pKA296. The GFP sequence was excised from pKA296 with AgeI/EcoRI and replaced with a PCR fragment containing the mCherry sequence to generated pKA452. The intercistronic sequence between gpd-2 and gpd-3 was PCR amplified from wild-type genomic DNA and cloned into the AgeI site of pKA452. Correct orientation of the insertion was verified by sequencing. Pmyo-2::GFP and Pmyo-3::GFP were obtained by cloning myo-2 and myo-3 promoter into pKA370 destination vector. pKA370 was constructed by digesting pDEST-DD04, a gift from Marc Vidal unc-119 rescue fragment and religating the vector. Pshr-220::daf-28::mCherry was constructed by Gateway LR reaction between Pshr-220, daf-28 donor vector and pKA452 destination vector described above.pKA460 was constructed by cutting pDEST-MB14 Worms were grown for at least two generations at either ∼23°C or 20°C in an uncrowded state and treated with hypochlorite to collect embryo For transgenic rescue experiments, worms were grown at room temperature and synchronized as described above. For each comparison, transgenic and non-transgenic L1s siblings were starved in the same flask as described above. Transgenic and non-transgenic animals were scored based upon the expression of a co-injection marker using a Leica fluorescence microscope. The average from multiple independent transgenic lines for each genotype and their non-transgenic siblings are reported along with the standard error of the mean for each time point.unc-31(e169) at day 3 was 90% but at day 6 was 96%, the survival at day 3 was corrected to 96%, as the difference is likely due to an underestimation at day 3. The smoothing only affected the early time points on the survival curves before significant numbers of animals had lost viability. The noise was likely due to pipetting and counting errors and the correction did not significantly change the mean survival rates that were calculated but allowed for application of log-rank statistics. For example, the mean survival rates of unc-31(e169) in Log-rank (Mantel-Cox) test was used to analyze starvation survival curves based upon the percentage of survivors at the sampled time points. Statistics were calculated using STATA software. In cases where multiple replicates of a genotype or multiple independent transgenic lines were examined in one experiment, the average survival rate at each time point was determined and that value was used for the reported analyses. To apply log-rank statistics, survival curves were smooth to a non-increasing function to remove noise. For example in ft1 and ft3 alleles emerged from the first while ft2 and ft4 alleles emerged from the second screen.∼1000 synchronized L4 stage wild-type worms were mutagenized as described in ocr-2 mutants used in these lifespan studies were not outcrossed to the Ashrafi-lab wild-type strain. While background mutations can contribute to the lifespan of different “wild-type” strains ocr-2, which were isolated and backcrossed in two independent laboratories, gave very similar results  within that cell. The mean fluorescence for each coelomocyte along with the standard error of the mean was calculated for each strain and normalized to wild type.Table S1Log-rank statistical analyses of starvation survival rates.(0.16 MB DOC)Click here for additional data file.Table S2unc-31.Promoters used for tissue-specific reconstitution of (0.07 MB DOC)Click here for additional data file.Table S3Log-rank statistical analyses of lifespans.(0.07 MB DOC)Click here for additional data file.


Serum ferritin and hepatic iron concentrations are frequently elevated in patients who are chronically infected with the hepatitis C virus (HCV), and hepatic iron concentration has been used to predict response to interferon therapy, but these correlations are not well understood. The HCV genome contains an RNA structure resembling an iron responsive element (IRE) in its internal ribosome entry site (IRES) structural domain IV (dIV). An IRE is a stem loop structure used to control the expression of eukaryotic proteins involved in iron homeostasis by either inhibiting ribosomal binding or protecting the mRNA from nuclease degradation. The HCV structure, located within the binding site of the 40S ribosomal subunit, might function as an authentic IRE or by an IRE-like mechanism.in vitro. Systematic HCV IRES RNA mutagenesis suggested that IRP1 cannot accommodate the shape of the wild type HCV IRES dIV RNA structure.Electrophoretic mobility shift assays showed that the HCV IRES domain IV structure does not interact with the iron regulatory protein 1 (IRP1) like mechanism in response to some other cellular signal remains to be tested.The HCV IRES dIV RNA structure is not an authentic IRE. The possibility that this RNA structure is responsible for the observed correlations between intracellular iron concentration and HCV infection parameters through an IRE- Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus that infects about 1% of the world's population ,3. A corAn RNA structural element located at the junction between the open reading frame (ORF) and the 5' untranslated region (UTR) of the HCV genome bears striking structural and sequential similarities to the iron responsive element (IRE), an RNA structure found in some eukaryotic mRNA that controls gene expression in response to intracellular iron concentration . If thisHepacivirus genus of the Flaviviridae family [Flaviviridae initiate translation via virtually identical non-scanning cap-independent mechanisms that utilize an internal ribosome entry site (IRES) to recruit and assemble the ribosome directly at the initiation site [HCV belongs to the e family . The RNAe family . The hepion site -15. The ion site . It is cion site . The domion site  and is uThe authentic HCV initiation codon resides in the terminal loop of the RNA hairpin structure in the HCV IRES dIV. This structure is unlikely to be tolerated in the RNA binding cleft of the ribosome while the initiation codon occupies the ribosomal P site -19, and The IRE is an example of a translation regulatory RNA element that does not otherwise actively participate in the translation initiation mechanism . The intThe HCV IRES dIV RNA structure shares many of the IRE consensus structural characteristics,24, mostIf HCV were to utilize an IRE-like mechanism to control viral expression, the RNA structure used for this regulation must be positioned within the 40S ribosomal subunit binding site. In sharp contrast to normal eukaryotic cap-dependent translation, the HCV IRES-dependent translation initiation mechanism requires that the 40S ribosomal subunit binds directly to the HCV genomic RNA sequence flanking the 5' UTR-ORF boundary to allow the start codon to occupy the ribosomal P site . This meSince the HCV IRES dIV structure differs from the consensus IRE at only three characteristics, a mutant RNA panel was constructed to evaluate the relative binding contribution of each characteristic. The panel systematically mutated the wild type HCV IRES dIV structure to a consensus IRE structure figure .Electrophoretic mobility shift assays (EMSA) were used to detect binding interactions between hIRP1 and the RNA species shown. Although a 100 ng concentration of hIRP1 was reported to bind to the wild type IRE , only thKd) of the hIRP1-ET2+3 and hIRP1-ET1+2+3 complexes. Unsurprisingly, ET1+2+3, which adheres to the consensus definition of an IRE [Kd ~ 50 pM, data not shown). ET2+3 bound to hIRP1 with a Kd = 54 ± 13 nM  contributed the greatest relative effect on binding affinity, followed by deletion of an A residue in the terminal loop (the ET2 effect), suggesting the importance of the correct three dimensional RNA structure on hIRP1 binding. The G residue at the apex of the terminal tri-loop and C-bulge residue are conserved in the consensus IRE and are presumably required for sequence-specific interactions between the IRE and recognition motifs on the IRP . These rThe primary conclusion from this work is that the wild type HCV IRES dIV RNA structure is not an authentic IRE, as it does not bind appreciably to the recombinant IRP1 protein. The hypothesis that HCV utilizes this structure to control viral expression by an IRE-like mechanism remains viable, although a putative cellular or viral factor that fulfils the analogous IRP function must be identified to properly evaluate this hypothesis. The p85 and p100 proteins that have been recently shown to bind to both the HCV IRES and the iron responsive element with high affinity  could se2HPO4, 4 mM NaH2PO4, pH 7.3) containing 1% triton-X-100 . The cell lysate was then incubated at 4°C for 30 minutes and spun at 10,000 rpm for 30 minutes. hIRP1 was purified on a 50% glutathione sepharose resin column . The protein was eluted with 50 mM Tris and 10 mM reduced glutathione (pH 8.0), concentrated using microcon YM 50  and its concentration, 1.2 μg/μL, was determined using the Micro Lowry method . Purified and unpurified hIRP1 was visualized on an SDS-PAGE and confirmed by Western blot using rabbit anti-rat IRP1 polyclonal antibodies .Human IRP1 (hIRP1) fused to glutathione S-transferase (GST) was expressed and purified as previously described  with somin vitro transcription from double-stranded DNA oligonucleotides[2, 200 mM Tris-HCl, pH 8) at 95°C for 3 minutes, and cooled to room temperature. RNA was transcribed in vitro from 456 nM of each annealed DNA using T7 MAXIscipt  following the manufacturer's instructions except that α-32P CTP (800 Ci/mmole)  and 12.5 U of RNase Inhibitor  were added. The reactions were incubated at 37°C for 1 hour, treated with DNase for 25 minutes, quenched by the addition of 25 nM EDTA , and the RNA transcripts were purified on a 20% denaturing PAGE gel.RNA was synthesized by cleotides,30. Sevecleotides, all fusin vitro. Each RNA transcript  was folded at 75°C in 20 mM MgCl2 for 2 minutes and was cooled to room temperature. hIRP1 (with GST-tag) was activated with 2% β-mercaptoethanol prior to use. Binding reactions consisted of 0.3 ng of each folded RNA transcript and 240 ng of hIRP1 in binding buffer  to a 20 μL total volume. Reaction mixtures were incubated at room temperature for 30 minutes. Heparin (0.63 μg/μL) was then added, and the mixture was incubated at room temperature for 10 additional minutes. RNA-protein complexes were resolved on a discontinuous native polyacrylamide gel (7% top and 14% bottom) in 0.5× TBE buffer to allow both the free and IRP1-bound RNA to be visualized on the same gel. The gel was dried, exposed to a phosphor-imager plate (Molecular Dynamics) overnight. The bands were visualized on a Storm phosphorimager (Molecular Dynamics) and quantified using ImageQuaNT software (Molecular Dynamics).EMSAs were used to detect and visualize binding interactions between hIRP1 and the oligoribonucleotides Kd) of the hIRP1-ET2+3 and hIRP1-ET1+2+3 complexes. Activated hIRP1 was serially diluted in binding buffer  to give a final concentration range of 74 pM to 1.1 μM. Diluted hIRP1 was incubated with 5.2 nM α32P-CTP labeled RNA. Complexed and free RNA species were resolved on a discontinuous native gel [EMSA was used quantitatively to estimate the dissociation constants  (figure The authors declare that they have no competing interests.ET synthesized and purified the RNA and hIRP1, carried out the EMSA experiments, and helped draft the manuscript. JMP reproduced and verified the results. WBL conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript.


Recent genomic data indicate that RNA polymerase II (Pol II) function extends beyond conventional transcription of primarily protein-coding genes. Among the five snRNAs required for pre-mRNA splicing, only the U6 snRNA is synthesized by RNA polymerase III (Pol III). Here we address the question of how Pol II coordinates the expression of spliceosome components, including U6. We used chromatin immunoprecipitation (ChIP) and high-resolution mapping by PCR to localize both Pol II and Pol III to snRNA gene regions. We report the surprising finding that Pol II is highly concentrated ∼300 bp upstream of all five active human U6 genes in vivo. The U6 snRNA, an essential component of the spliceosome, is synthesized by Pol III, whereas all other spliceosomal snRNAs are Pol II transcripts. Accordingly, U6 transcripts were terminated in a Pol III-specific manner, and Pol III localized to the transcribed gene regions. However, synthesis of both U6 and U2 snRNAs was α-amanitin-sensitive, indicating a requirement for Pol II activity in the expression of both snRNAs. Moreover, both Pol II and histone tail acetylation marks were lost from U6 promoters upon α-amanitin treatment. The results indicate that Pol II is concentrated at specific genomic regions from which it can regulate Pol III activity by a general mechanism. Consequently, Pol II coordinates expression of all RNA and protein components of the spliceosome. During transcription, RNA polymerases synthesize an RNA copy of a given gene. Human genes are transcribed by either RNA polymerase I, II, or III. Here, we focus on transcription of the U6 gene that encodes a small nuclear RNA (snRNA), a non-coding RNA with unique activities in gene expression. The U6 snRNA is transcribed by RNA polymerase III (Pol III); here we report the surprising finding that RNA polymerase II (Pol II) is important for efficient expression of the U6 snRNA. Interestingly, high concentrations of Pol II have been recently observed on genomic regions that are considered outside of transcribed genes. We localized Pol II to a region upstream of the U6 snRNA gene promoters in living cells. Inhibition of Pol II activity decreased U6 snRNA synthesis and was accompanied by a decrease in Pol II accumulation as well as transcription-activating histone modifications, while Pol III remained bound at U6 genes. Thus, Pol II may promote U6 snRNA transcription by facilitating open chromatin formation. Our results provide insight into the extragenic function of Pol II, which can coordinate the expression of all components of the RNA splicing machinery, including U6 snRNA. Xenopus tropicalis oocytes, Saccharomyces cerevisiae and Schizosaccharomyces pombe  orthophosphate  for 6 h before total RNA was extracted with Trizol. After 5 min denaturation at 70 °C, RNA was allowed to hybridize with biotinylated oligos complementary to 5S rRNA together with either U6, U2 [Arg for 30 min in 60% buffer D containing 5% glycerol [Arg. RNA/DNA hybrids were immunoprecipitated with streptavidin beads for 30 min followed by 5 washes in 60% buffer D. Immunopurified RNA was extracted with phenol/chloroform and separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea. Band intensities of RNAs synthesized in the presence of α-amanitin were measured using a PhosphorImager and normalized to 5S rRNA intensities to account for IP bead loss and loading differences.HeLa cells were grown to 60%–80% confluency before CaPOmaxigene . After16glycerol . The oliFigure S1http://genome.ucsc.edu/, freeze March 2006, [U6 genes are according to [The 107 nucleotide U6 snRNA sequence was aligned to the human genome with BLAT  and rding to .(1.6 MB PDF)Click here for additional data file.Figure S2U6–1 neighbouring gene BC033162 was analyzed by reverse transcription, followed by PCR. Total RNA from HeLa cells was treated with DNaseI or RNaseA and transcripts were reverse-transcribed with oligo d(T) or gene-specific primers for PGK1 and BC033162. The abundance BC033162 mRNA was analyzed by 27–30 cycles of PCR with primers amplifying a single exon. (B) Northern Blot with total HeLa RNA with probes specific for U6 snRNA together with probes covering 782 bp of U6 upstream region on − strand (lane 1) and + strand (lane 2) as well as probes covering 923 bp of downstream regions of either − strand (lane 3) or + strand (lane 4) revealed that no transcript derives from the U6–1 locus other than U6 snRNA. Lane 5, total RNA stained with ethidium bromide; lane 6, size markers. Asterisks indicate background hybridization to 28S and 18S rRNA.(A) Transcriptional activity of the (1.3 MB PDF)Click here for additional data file.Figure S3U6–1 and U6–7/U6–8 gene regions, with black lines specifying the PCR amplicons identified by the central nucleotide. Boxes represent the position of the DSE. Crosslinked pemHeLa extracts were used for ChIP with primers amplifying fragments distributed 1 kb upstream and downstream of each gene. Antibodies specific for Pol II  and Pol III were used. X-axis values in  are relative to the U6–1 transcription start site at position +1, whereas transcription start sites in  are at +1,302 (U6–7) and +2,426 (U6–8), relative to an arbitrary point upstream of U6–7. Data points are placed according to the center positions of the PCR products along the region. The peak value for each data set was set to 100% and all data points normalized accordingly. Error bars represent the SEM of at least three independent experiments. Diagrams of the (84 KB PDF)Click here for additional data file.Figure S4Crosslinked pemHeLa extracts were used for ChIP with Pol II CTD Ser5 Antibody H14, with primers amplifying fragments distributed 1kb upstream and downstream of U6–1 snRNA gene region. All values are relative to nonimmune IgG and normalized to an intergenic control region. Error bars represent the standard deviation of two independent experiments.(42 KB PDF)Click here for additional data file.http://www.ncbi.nlm.nih.gov) are tRNALeu (X04117), tRNATyr (M55612), LDHA (NM_005566), PGK1 (NM_000291), 5S rRNA (X51545), 28S rRNA (NR_003287), U2 snRNA (X59360), U1 snRNA (J00318), U6 snRNA (X07425).Accession numbers for genes mentioned in this paper from the National Center for Biotechnology Information database (


O positions of the viral RNA cap structure (GpppA→7meGpppA→7meGpppAO-me2′-). The same NS5 domain could also have a guanylyltransferase activity (GTP+ppA-RNA→GpppA). The mechanism by which this protein domain catalyzes these three distinct functions is currently unknown. Here we report the crystallographic structure of DENV-3 MTase in complex with a 5′-capped RNA octamer (GpppAGAACCUG) at a resolution of 2.9 Å. Two RNA octamers arranged as kissing loops are encircled by four MTase monomers around a 2-fold non-crystallography symmetry axis. Only two of the four monomers make direct contact with the 5′ end of RNA. The RNA structure is stabilised by the formation of several intra and intermolecular base stacking and non-canonical base pairs. The structure may represent the product of guanylylation of the viral genome prior to the subsequent methylation events that require repositioning of the RNA substrate to reach to the methyl-donor sites. The crystal structure provides a structural explanation for the observed trans-complementation of MTases with different methylation defects.The N-terminal domain of the flavivirus NS5 protein functions as a methyltransferase (MTase). It sequentially methylates the N7 and 2′- Flaviviruses and is the arthropod-borne etiologic agent of dengue fever, dengue haemorrhagic fever and dengue shock syndrome. Other medically important flaviviruses include West Nile virus (WNV), Japanese encephalitis virus (JEV) and Yellow fever virus (YFV). Currently, there is neither a vaccine nor antiviral therapy licensed for the prevention or treatment of dengue. The flavivirus genome consists of a 11 kb positive strand RNA segment with a cap 1 structure (5′-7meGpppAO-me2′--RNA-3′), where the guanosine and adenosine are methylated at the N7 position of the base and 2′-O position of the sugar respectively. The cap structure is important for RNA stability, binding to ribosomes and efficient translation pppAG) and 3′ (CUOH-3′) sequences are strictly conserved across the flavivirus genomes Dengue virus (DENV) belongs to the genus 7meGpppA-RNA) O position of adenosine resulting in formation of the cap 1 structure (7meGpppAO-me2′--RNA) The current model for the formation of the RNA cap 1 structure assumes four sequential steps: (1) the 5′-γ-phosphate of the nascent RNA is removed by the RNA triphosphatase activity of the viral protein NS3 While structural information about the SAM and GTP binding site with short capped RNA abound, there is a need to study the structure of a flaviviral MTase bound to longer 5′-capped RNA chains, given the requirement for a minimal number of nucleotides for proper MTase activity free = 0.231 at a resolution of 2.90 Å. Clear electron density was visible for protein residues up to amino-acid 263 , for the two capped RNA octamers within the asymmetric unit (a.s.u) and also for the SAH by-products that co-purifies with the enzyme. This allowed building of all sixteen RNA bases  and the RNA molecule related by the 2-fold ncs axis, (“RNA II”) from G0′ to G8′. The cap moiety occupies the GTP binding pocket while the remaining of the RNA chain makes minimal interactions with the protein and appears stabilized by intra-molecular interactions as described in the next section. Base G0 is sandwiched between Phe25 of the MTase GTP binding site and base A1. In addition, residues Lys14, Leu17, Leu20 and Asn18 together with several water molecules make interactions with the G0pppA1 moiety  Guanine-guanine N7-N1 carbonyl-amino contacts between G2 and G8 with the formation of two hydrogen bonds. The complete list of interactions between both RNA chains (labeled RNA I and RNA II) is listed in 2+ and Mn2+) that are key players in stabilizing RNA structures + and Na+ ions.No direct interactions are formed between the MTase and the rest of the RNA chain: Gg’ loops . Within  solvent . Eight n0pppA1 moiety of the RNA binds to the GTP binding pocket with the 3′-OH of A1 facing the protein, in the “S1 conformation” observed by using a short cap analogue 1 was suggested to be incompatible with a longer RNA chain, due to obstruction by Pro152 and the surrounding residues. Interestingly, our structure shows that despite adopting a similar “S1-like” conformation, the continuation of the RNA chain at the 3′-OH of A1 is not affected by Pro152 and the surrounding residues. Instead, the bases and riboses of A1 and G2 form interactions with Pro152. As seen for short capped RNA analogues0 and A1 moieties. This suggests that the conformation of the RNA octamer adopted here is not compatible with a catalytically productive interaction with the MTase RNA binding groove. Therefore, other -probably longer- RNA chains mimicking the stem-loop architecture that is conserved at the 5′end of the flaviviral RNA genome are required to trap a complex fully relevant to methyl transfer. The atomic distance between the methyl donor in the SAM binding pocket and the N7 acceptor of G0 is about 16 Å in our structure. Therefore, it is conceivable that during the N7 methylation, the extension of the triphosphate linker will bring the G0ppp moiety (more than 14 Å in length) in the immediate vicinity of the methyl group of SAM, allowing methyl transfer  is enzymatically linked with the release of pyrophosphate. Subsequently, conformational rearrangements must occur in order to bring the viral genomic RNA substrate in close proximity to the methyl donor to allow methyl transfer. Results from the PISA server O methylation could trans-complement one another, resulting in double methylated 7meGpppAO-me2′- product O methylation are sequentially executed by separate domains pppA cap analogue  competent cells were transformed with pGEX4T-1 DENV-3 MTase (1-272 amino acids and grown in LB plates containing 100 µg/mL ampicillin and 50 µg/mL chloramphenicol at 37°C overnight. A single colony was picked and inoculated into 5 mL LB medium containing 100 µg/mL ampicillin and 50 µg/mL chloramphenicol and grown at 37°C overnight. The overnight cultures were then transferred to 500 mL fresh LB media and the cultures were incubated at 37°C with shaking (220 rpm) to an OD595 between 0.6 to 0.8. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. The cultures were incubated overnight with shaking (220 rpm) at 16°C. Cells were then harvested by centrifugation at 6000 rpm for 10–15 minutes at 4°C. Cells were then lyzed by sonication for 30 minutes followed by centrifugation at 20 500 rpm for one hour. The protein supernatant was then purified using glutathione S-transferase (GST prep FF16/10) affinity column. The column was washed with buffer A  for five column volumes after sample injection. Proteins were eluted with a linear concentration gradient of reduced glutathione (GSH) from 0 to 10 mM. The collected fractions were dialyzed in buffer A to remove GSH, with the concomitant cleavage of the thioredoxin tag by thrombin digestion at 4°C overnight. The protein was then further purified by gel filtration using HiLoad 16/60 Superdex 75 column that was pre-equilibrated with 20 mM Tris-HCl pH 7.5, 200 mM NaCl, 2 mM DTT and 10% glycerol. Collected fractions containing pure MTase were pooled and concentrated to 16 mg/ml before storing at −80°C.pppAGAACCUG-biotin-TEG-3′ 2SO4 and 0.1 M sodium-citrate pH 5.0, was mixed with an equal volume of MTase-RNA solution. Small, hexagonally shaped crystals were observed after incubation at 18°C for one week. For data collection, crystals were transferred to its mother liquor supplemented with 20% (v/v) glycerol as cryo-protectant before being mounted and froze in liquid nitrogen. Diffraction experiments were performed at the PXII (X10SA) beam line at the Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland. Indexing, integration, scaling and merging of intensities were carried out using MOSFLM To obtain crystals of the binary complex between MTase and 5′-capped RNA, the biotinylated 5′-capped RNA octamer with the sequence 5′-GAll structures were determined by molecular replacement using the MOLREP program from the CCP4i package using DENV-2 MTase (PDB code: 2P3O) as search probe. Structure refinement was carried out using REFMAC5 with TLS refinement The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 2XBM.Table S1Buried surface areas. To assess the stability of the quaternary assembly of MTase molecules "A, B, C, D", we calculated buried interfaces. Monomer A is bound to F and monomer B to E. We note rather small interfaces for protein-protein interactions (by comparison stable antigen-antibody interfaces bury at least 1200 Å2), but a rather large RNA-RNA interface of 405 Å2 (RNA molecules are labeled "E" and "F"). Thus RNA would play a major role in stabilizing such a quaternary assembly in the virus replication complex.(0.05 MB DOC)Click here for additional data file.Figure S1Comparison of the paths taken by the RNA fragments in our structure and in structure 2PXC. One Mtase monomer (yellow) of the crystallographic dimer from 2pxc is superimposed with one bound MTase monomer A of our structure (green). The Gp moieties at the 5′ end are superimposable. . From the β-phosphate onwards, the RNA structure forms a loop and protrudes out of the protein in our structure.(0.46 MB TIF)Click here for additional data file.Figure S2After monomer superposition (in green and yellow), the other monomer (pink) of the crystallographic dimer from structure 2pxc has a ∼60 degrees difference in orientation compared to the nearest neighbour of DENV MTase monomer D (green).(0.48 MB TIF)Click here for additional data file.Figure S3Superposition of the crystallographic dimer of 2pxc with DEN MTase monomers A and B (both bound to RNA).(0.52 MB TIF)Click here for additional data file.


We have constructed cDNA libraries from the poly(A)+ RNA of normal colonic mucosa and a liver metastasis from a colonic adenocarcinoma. Differential screening of these libraries using 32P-labelled cDNAs transcribed from poly(A)+ RNAs isolated from specimens of four normal colonic mucosae, five adenocarcinomas, and three liver metastases by Grunstein-Hogness and dot-blot hybridization has identified a number of recombinant cDNA clones homologous to mRNAs that appear to differ significantly in abundance between normal and neoplastic colon and metastases. These cDNA clones, and others identified in the libraries, may be of considerable importance both as diagnostic tools and in defining the phenotypic changes associated with tumour progression and metastasis.