Although Qβ replicase has been traditionally used as an enzyme for this purpose, we planned to use norovirus replicase (NV3Dpol) due to its structural simplicity in the scope of in vitro autonomous evolution of the protein. Characteristics of the enzyme NV3Dpolin vitro were re-evaluated in this context.The isothermal amplification of RNA pol, synthesized by using a cell-free translation system, represented the activities which were reported in the previous several studies and the reports were not fully consistent each other. The efficiency of the initiation of replication was dependent on the 3’-terminal structure of single-stranded RNA template, and especially, NV3Dpol preferred a self-priming small stem-loop. In the non-self-priming and primer-independent replication reaction, the presence of -CCC residues at the 3’-terminus increased the initiation efficiency and we demonstrated the one-pot isothermal RNA (even dsRNA) amplification by 16-fold. NV3Dpol also showed a weak activity of elongation-reaction from a long primer. Based on these results, we present a scheme of the primer-independent isothermal amplification of RNA with NV3Dpolin vitro.NV3Dpol can be used as an RNA replicase in in vitro RNA + protein evolution with the RNA of special terminal sequences.NV3D In vitro evolution of RNA has been performed with a given replicase In vitro e method , RNA-Z [e method , etc.E.coli and is a hetero-tetramer, in which only β subunit is encoded in the viral genome and other three subunits  are recruited from the host-cell translation machinery [Qβ RNA replicase is from an RNA phage Qβ of achinery . For theachinery . When thachinery . Yomo anachinery .in vitro virus method or mRNA display [in vitro virus” will evolve autonomously in a flow reactor. Qβ RNA replicase is not adequate for our purpose because it must recruit three proteins from the cell-free replication system and this situation makes population growth dynamics very complex in the test tube containing the cell free translation system. Norovirus RNA replicase  is a candidate for our in vitro virus, because it consists of a single polypeptide chain and its molecular weight is not so large (56 kDa).Yomo’s compartment method is just a cell-type strategy of phenotype-genotype linking and was applied to an experiment of the coupled RNA/replicase evolution . We are  display ,11. Thispolin vitro[There are several preceding reports on the characteristics of various modes of RNA replication catalyzed by the NV3Dlin vitro-20. Theypolin vitro in order to investigate whether we can use this enzyme as an RNA replicase in evolution experiments in vitro, instead of Qβ replicase.Therefore we re-examined the activities of NV3Dpol was a gift from BML Inc., that is, it was same as Ref. [pol-strep, we sequenced it and confirmed ours was triple mutant at amino acid sequence level from the sequence in Ref. [pol-strep protein was observed on SDS-PAGE. From the incorporation of FluoroTect™ GreenLys tRNA into the protein during the cell-free translation, only a sharp single band was detected under a fluorescence detection mode  and Temp(GGG-CCA), RNA templates (50 nts) were amplified as indicated with black arrow in Figure pol made a dsRNA from an ssRNA in a primer-independent manner and amplified it. This result is consistent with the report by Rohayem et al.[et al.[et al.[et al.[Four kinds of single stranded RNA (ssRNA) of which sequences were different in the 3’-terminal three nucleotides  RNA, the RNA template was amplified linearly up to 60 min and reached at plateau near 240 min. On the denaturing PAGE . When 4 pmol of NV3Dpol and 8 pmol of Temp(GGG-CCC) RNA were incubated and quantified on the denaturing PAGE, the amplification curve was shown by closed circle in Figure pol (closed square versus closed circle). Activity of our enzyme varied between different lots. But within the same lot the activity was fairly reproducible as shown in Figure The time-course of the primer-independent amplification reaction of Temp(GGG-CCC) with NV3Det al.[pol initiated the replication on an oligo cytidine. Second, NV3Dpol added some cytidines at the 3’-terminus of the synthesized strand. From these results, they suggested that NV3Dpol prefers a C-stretch for the initiation of replication in primer-independent manner.Rohayem et al. indicatepol at 30°C. The time-course measurements of each case were analyzed on a denaturing PAGE , Temp(GGG-UCC), Temp(GGG-CCC) or Temp(GGG-CCCC) RNA, which have from 1 to 4 cytidine(s) at their 3’-terminus  were noet al. [pol does not have an RNA synthesis activity on poly(A)-tail, Fukushi et al. [pol has this activity. In order to re-examine this activity, we synthesized an RNA bearing an A-stretch of 22 nts at the 3’-terminus (named TD257-735-A22), and incubated with NV3Dpol. Our NV3Dpol showed a slight activity to poly(A)-tail and the amplified products appeared after 30 min , the amplified product, sampled at 240 min of incubation, reaching at plateau, was sequenced. The replicated complementary RNA (RNA(-)) was reverse-transcribed into cDNA using Y-ligation technique shown in Methods. The determined sequences were shown in Table pol at 30°C and analyzed on non-denaturing PAGE. The secondary structures of these two RNA templates at 30°C predicted by Mfold [pol replicates a blunt end dsRNA preferably rather than a sticky end dsRNA.It was reported that the sticky end formed by this TNT activity is favourable for the next round replication of the dsRNA . We re-eby Mfold  were shopol, TD257-735g734c RNA and NV3Dpol were incubated at the ratio of 1 : 20 in a reaction tube. Each aliquot was analyzed on a non-denaturing PAGE followed by the staining with SYBRgreenII and quantified  RNA with NV3Dpol was performed at the same time. The reaction products were analyzed on a non-denaturing PAGE or an 8M urea denaturing PAGE, and imaged , 4 for (iv)).When a small stem-loop structure can be formed at 3’-terminus, the self-priming is more favorable initiation reaction for an ssRNA template than the primer-independent manner as seen in Figure et al.[et al.[ACGCG-3’  and got a double length replication product. They explained it is the result of the back-priming at MluI palindrome. This explanation is not acceptable based on thermodynamic considerations. We propose another explanation that it was the result of self-priming starting from a small stem-loop  made of ---CGUACGCG-3’.In this context we comment the self-priming case by Belliot et al., and Weil.[et al.. They uset al.[Fullerton et al. investigThere were at least two amplification reaction products of the template Temp(GGG-GGG) -tail, we showed that NV3Dpol initiated replication reaction on A-stretch at the 3’-terminus of TD257-735-A22 template -tail. The differences between Rohayem’s system and ours are the amino acids sequence of NV3Dpol, the length of poly(A)-tail, and enzyme concentration used. On the other hand, Fukushi et al. [pol has the initiation activity of primer-independent RNA replication on poly(A)-tail. We supposed that the actual 3’-terminal sequence of RNA template used in Fukushi’s work was ---(A30)UGCGC.Although Rohayem et al.  indicatei et al.  already pol.It is reported that Qβ replicase also favours C-stretch. The initiation efficiency is high when 3’-terminal sequence is ---CCC-3’ or ---CCA-3’. The efficient parasite RQ RNA has also the sequence ---CCC-3’. By the way, we have not yet observed any parasite sequence in amplification with NV3Dpol initiates the replication to dsRNA which has a sticky-end preferably rather than the blunt-end [pol might initiate the replication from the blunt-end cytidines at the 3’-terminus preferably rather than from sticky-end cytidines. We investigated only about the addition of one cytidine. Although the addition of three cytidines was rare  at 3’-terminus in our replicate chain may affect the reduced number of addition. We also observe addition of A and G, which accords with the reports by Rohayem et al.[et al. (Sapovirus 3Dpol) [Fukushi et al. reportedem et al.,18. Theys 3Dpol) .pol has the same template-switching activity as MMLV, the band corresponding to about 100 nts, observed in the replication reaction of Temp(GGG-GGG) RNA  adds some deoxycytidines at the 3’-terminus of reverse transcribed DNA, and switches the template utilizing this overhung dC-stretch . If NV3Dpol, respectively. In the former, the shape of amplification curve showed two phases; linear amplification phase up to 90 min and plateau phase near 240 min, because the enzymatic activity of NV3Dpol is limited by about 120 min, as indicated in previous reports [In the non-self-priming and primer-independent replication shown in Figure  reports . The aut reports .et al.[in vitro RNA amplification for an in vitro evolution. Based on the results of this study together with preceding reports, we propose the following model for the isothermal RNA amplification with NV3Dpolin vitro . Thus, in order to sustainable replication, 5’-terminus of the template must be –GGG. NV3Dpol prefers, however, a small stem-loop structure at the 3’-terminus and begins RNA synthesis in the self-priming manner. Thus, both terminal sequences should not make such a small stem-loop. At the end of template synthesis, NV3Dpol sometimes adds several C using its TNT activity. Thus, 3’-terminus of the replicated strand (RNA(-)) has a C-stretch just as the template (RNA(+)) or sometimes a slightly longer C-stretch, because NV3Dpol recognizes the C-stretch at 3’-terminus of dsRNA, often accessible when it is breathing, and begins synthesis of the next generation strand using the chain dissociation replication activity.Rohayem et al. showed ao Figure  for temppol, prepared with cell-free protein synthesis system, was able to perform the isothermal amplification of RNA (even dsRNA) in vitro. But the initiation efficiency of replication is dependent on the 3’-terminal sequence. And we have to use the 3’-terminal sequence which does not make a small stem-loop, in order to avoid a parasitic self-priming product. Thus, NV3Dpol can be used as an RNA replicase in in vitro RNA evolution with the RNA of special terminal sequences. NV3Dpol, which is a single chain protein, is a candidate of a model protein in in vitro autonomous protein evolution in the form of an in vitro virus [In this study, it was confirmed that NV3Dro virus .pol gene was kindly provided from BML Inc. PCR was performed with KOD-plus- DNA polymerase (TOYOBO) and the PCR products were purified with QIAquick PCR Purification Kit (QIAGEN). NV3Dpol gene was amplified from pVL3Dwt by PCR using prNV3Dstart(+) 5′-ATGGGAGGTGACGACAAGGGC-3′ and prNV3Dstop(-) 5′-TTATTCGACGCCATCTTCATTCACA-3′. NV3Dpol gene was modified with the coding region for Strep-tag II sequence (WSHPQFEK) [TTACTTTTCAAACTGCGGATGGCTCCATTCGACGCCATCTTCATTC-3′  by PCR. At the downstream of the stop-codon, KpnI restriction site was added using prNV3Dstart(+) and prStrep-kpn1(-) 5′-GGGGTACCTTACTTTTCAAACTGCGGATGGCTCC-3′  by PCR. Then the PCR product was digested with KpnI (TaKaRa) and integrated into the pTD1 expression vector (SHIMADZU Biotech) [pol-strep. NV3Dpol-strep expression construct DNA was amplified using prTD161-179 (5′-GCAGATTGTACTGAGAGTG-3′) and prTD845-827 (5′-GGAAACAGCTATGACCATG-3′), and transcribed in vitro with RiboMAXTM Large Scale RNA production system-T7 (Promega). The transcript, named NV3Dpol-strep mRNA was purified with NICK column . NV3Dpol-strep mRNA was translated with Transdirect insect cell cell-free protein synthesis kit (SHIMADZU Biotech) [Plasmid pVL3Dwt (GenBank: AB039782 ) harbourSHPQFEK)  using prBiotech)  accordinBiotech)  accordinpol was bound onto Strep-tactin Superflow plus resin, pre-equilibrated with binding buffer . The bound protein was washed with the binding buffer and eluted with elution buffer ). The eluted protein was then enriched and buffer-exchanged with buffer A  with Microcon YM-50 column (Millipore), and stored at -80°C. In the enrichment step using Microcon YM-50 column, bovine serum albumin (BSA) (TaKaRa) was used as a carrier protein to avoid the non-specific adsorption of NV3Dpol to the column. The amount of NV3Dpol-strep was quantified on sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) referring a calibration curve with BSA.The translated product was purified with Strep-tactin Superflow plus (QIAGEN). The strep-tag II modified NV3DTAATACGACTCACTATAGGGAATAAGATTTCACAGTTCAGAGAGACATTAAGTTGTTGTTGTTGCCC-3′) (underline indicates T7 Φ6.5 promoter sequence), prT7tempGGG + (5′-GCCAGTCGCCTGCAGTAATACGACTCACTA-3′) and prTempCCC- (5′-GGGCAACAACAACAACTTAATGTC-3′). Other DNA templates, Temp(GGG-GGG), Temp(GGG-CCA) Temp(GGG-UAC), Temp(GGG-UUC), Temp(GGG-UCC), Temp(GGG-GCCCC), Temp(GGG-UCCCC) and Temp(GGG-UCCC) DNA, were generated as same as Temp(GGG-CCC) DNA with the primer listed in Additional file in vitro transcribed with RiboMAX™ Large Scale RNA production system-T7. TD257-735 RNA were also in vitro transcribed from the PCR amplified DNA  in PCR. TD257-735-A22 was made from TD257-735 by adding 22 nts of adenine at the 3’-terminus using prTD757-733 (5′-TTTTTTTTTTTTTTTTTTTTTTGCG-3′) in PCR. All RNA templates were purified on denaturing PAGE and quantified by measuring absorbance at 260 nm.Sequences of RNA template used in this study were summarized in Additional file RNA primer (5′-GGGCAACAACAACAAC-3′) modified with FITC at the 5’-terminus was purchased from Japan Bio Service Inc.pol-strep and in vitro-transcribed RNA template in a reaction buffer ) at 30°C. Reaction products were subjected to denaturing or non-denaturing PAGE followed by SYBRgreenII (Lonza) staining and visualized on Pharos Fx imager (Bio-Rad). Here, the non-denaturing PAGE was performed with 5 or 10% acrylamide, 0.625% bisacrylamide and TBE buffer at 20°C. The denaturing PAGE was performed with 8 M urea plus the same constituent of non-denaturing PAGE at 65°C.RNA replication or amplification was performed with purified NV3Dpol, we could not use a normal sequencing method using a primer which hybridizes 3’-terminal region of the sample RNA. Thus we sequenced the full length of RNA(-) applying the Y-ligation method [pol-strep, the reaction solution was desalted with Micro bio-spin column 30 (Bio-Rad), and ligated with 5’-phosphorylated Y-adapter (5′-CAAAGGGAATAAGATTTCACAGTTCAGAGCTTAGATAATACGACTCACTATAGGGTTAAC-3′) by Y-ligation method. The ligated product was hybridized with prT7g10SD-NV3D(-) (5′-CCTTGTCGTCACCTCCCATGGATATATCTCCTTCTTAAAGTTAACCCTATAGTGAGTCGTATTA-3′) and reverse transcribed with Avian myelobastosis virus (AMV) reverse transcriptase (Promega). The reverse transcribed product was purified on denaturing PAGE, and amplified by PCR using prTempCCC- and prSeq (5′-CCTTGTCGTCACCTCCCA-3′) as primers. TA cloning was performed with pGEM-T easy vector system (Promega). Sequencing was performed by Operon Inc. (Tokyo).To detect the addition of nucleotides at the 3’-terminus of replicated RNA (RNA(-)) by TNT activity of NV3Dn method , as follThe authors declare that they have no competing interests.HA and YH designed the study, analyzed the data and wrote the paper. HA performed the experiments. NN, MS and KN analyzed the data. All authors contributed to the manuscript and have read and approved the final manuscript.pol proteins used in Rohayem’s report (A), in Fukushi’s report (B) and in this report (C). The Amino acid sequence homologies between (A) and (B), (B) and (C), (A) and (C) were 90.4, 99.2, 89.6%, respectively. Amino acid substitutions were indicated in bold letters. Amino acid sequence comparison of NV3DClick here for filepol under the excess BSA condition (lane 3 and 4). Temp(GGG-CCC) RNA (5 pmol) was incubated with NV3Dpol (the amount was not detected) and sampled at 0 and 120 min. In the case of excess BSA +, 500 ng of BSA was added to the standard reaction mixture (BSA -). M1; 10 bp DNA step ladder (Promega). M2; DynaMarker dsRNA ladder (BioDynamics Laboratory Inc.). Ref.; Temp(GGG-CCC) RNA.Primer-independent RNA amplification reaction of Temp(GGG-CCC) RNA with NV3DClick here for filepol (5 pmol) was incubated with (i) Temp (GGG-CCC), (ii) Temp (GGG-GGG), (iii) Temp (GGG-CCA) or (iv) Temp (GGG-UAC) (5 pmol each), and sampled at 0, 60, 120, 180 min respectively (reaction volume = 20 μL), which is the same reactions shown in Figure Supplements to Figure Click here for filepol (the amount was not detected) were incubated at 30°C for 120 min. The reaction was stopped with adding EDTA, followed by PCI  and CIA  extraction, and ethanol precipitation. Then the pellet was resuspended with 1 × S1 nuclease buffer (TaKaRa) and incubated with 13.5 U/μL of S1 nuclease (TaKaRa) at 37°C for 15 min. Each aliquot was added with EDTA to stop the reaction, and analyzed on an 8M urea denaturing 10% PAGE (A) or a non-denaturing 10% PAGE (B), visualized by SYBRgreenII staining. M1; 10 bp step DNA ladder (Promega). M2; DynaMarker dsRNA ladder (BioDynamics Laboratory Inc.). White headarrows indicated in lane 2 were the nucleic acids from cell-free protein synthesis system (e.g. tRNAs).S1 nuclease treatment of the amplification product of Temp(GGG-CCC) . Temp(GGG-CCC) RNA (5 pmol) and NV3DClick here for filePotential small stem-loop structures of 3’-terminus of RNA templates. (A) 3’-terminal sequence and potential small stem-loop structure of Temp(GGG-GGG) (ii), Temp(GGG-CCA) (iii) and Temp(GGG-UAC) (iv). Asterisks indicate the hybridization points between 3’-terminus. In (ii), the potential small stem-loop in the case of addition of 1 - 3 cytidine(s) on 3’-terminus were shown. (B) Potential small stem-loop structures of 3’-terminal sequence used in the previous reports ((a); ,22, (b);Click here for file22 RNA (0.4 pmol) were incubated with NV3Dpol (4 pmol) (reaction volume = 20 μL) and analyzed on a non-denaturing 5% PAGE. M; 100 bp DNA ladder marker (Promega). In our PAGE condition, dsDNA 400 bp marker (Promega) corresponds to dsRNA 430bp marker (BioDynamics Laboratory Inc.).Effect of poly(A)-tail for the initiation efficiency of RNA replication. TD257-735-AClick here for filepol, RNA template, RNA primer and template-primer hybrid, respectively. +/- corresponds to the presence or absence of substrates. M; 10 bp DNA step ladder (Promega). Supplement to Figure Click here for file(a), DNA oligomers (b) used in this work. DNA sequence of 161-757 region in pTD1 (c). Underline indicates T7 ϕ 6.5 promoter sequence.Sequences of RNA templates Click here for fileSchematic illustration of sample preparation for the 3’-terminus sequencing of RNA. As described in Materials and Methods, the reaction procedure is as follows; (1) the replicated RNA (RNA (-), shown in red) was ligated with an adapter DNA (shown in blue) hybridized at the 3’-terminal region of RNA(-) by the Y-ligation method. (2) The ligation product was reverse-transcribed with RT primer (green arrow) to cDNA, (3) and the cDNA was amplified by PCR, cloned and sequenced. Click here for file


Escherichia coli 23S rRNA. The homolog RluF is specific to the adjacent nucleotide in the stem, U2604. The 1.3 Å resolution crystal structure of the complex between the catalytic domain of RluB and the isolated substrate stem-loop, in which the target uridine is substituted by 5-fluorouridine (5-FU), reveals a covalent bond between the isomerized target base and tyrosine 140. The structure is compared with the catalytic domain alone determined at 2.5 Å resolution. The RluB-bound stem-loop has essentially the same secondary structure as in the ribosome, with a bulge at A2602, but with 5-FU2605 flipped into the active site. We showed earlier that RluF induced a frame-shift of the RNA, moving A2602 into the stem and translating its target, U2604, into the active site. A hydrogen-bonding network stabilizes the bulge in the RluB–RNA but is not conserved in RluF and so RluF cannot stabilize the bulge. On the basis of the covalent bond between enzyme and isomerized 5-FU we propose a Michael addition mechanism for pseudouridine formation that is consistent with all experimental data.RluB catalyses the modification of U2605 to pseudouridine (Ψ) in a stem-loop at the peptidyl transferase center of  Escherichia coli (E. coli) is devoted to RNA modifying enzymes, underscoring the biological importance of RNA modification , is the most prevalent RNA modification  has been implicated in catalysis .Sch.SchC-glΨ synthases use two strategies for selecting a target for modification. In eukaryotes and archaea, pseudouridylations of rRNA and snRNAs are accomplished with versatile ribonucleoprotein particles (RNPs) comprising the Ψ synthase Cbf5 (dyskerin in humans), three structural proteins and a small guide RNA, which is responsible for target recognition by base pairing to either side of the target site .All kingdoms of life also have stand-alone Ψ synthases that modify sites that may be buried in folded RNA, thus cannot be recognized by direct sequence readout . Some ofE. coli Ψ synthases, RluB and RluF, selectively modify adjacent bases on a stem-loop of E. coli 23S rRNA. RluB is specific for U2605 whereas RluF is selective for U2604, but also modifies U2605 to a small extent  to block a late step in catalysis  was cloned into a modified pET47 vector using the restriction sites BamHI/XhoI to yield a fusion protein with an N-terminal hexahistidine tag and an HRV 3C protease site. RluB (1–251) was expressed in E. coli BL21(DE3) cells at 20°C for 16 h after induction with 0.3 mM IPTG. Cells were harvested, washed in Tris-buffered saline and resuspended in lysis buffer . The cells were lysed using an Emulsiflex-C5 homogenizer (Avestin) and the lysate was cleared by centrifugation at 32 000 g for 25 min. The supernatant was incubated with nickel-nitrilotriacetic acid (Ni-NTA) at 4°C in the presence of 20 mM imidazole. The resin was washed with 15 column volumes of lysis buffer containing 20 mM imidazole and the fusion protein was eluted with lysis buffer containing 250 mM imidazole. Proteolytic removal of the hexahistidine tag using HRV 3C protease was performed during dialysis of the protein over night at 4°C in 50 mM Hepes pH 7.5, 300 mM NaCl and 0.5 mM TCEP. The cleaved hexahistidine tag was removed by immobilized-metal affinity chromatography using Talon resin (Clonetech). The protein was concentrated using Amicon centrifugal filter units, loaded on a Superdex S200 (10/30) size exclusion column  equilibrated in 50 mM Hepes pH 7.5, 300 mM NaCl and 0.5 mM TCEP and eluted with the same buffer. Peak fractions were analysed by SDS-PAGE, concentrated to 10 mg/ml and flash-frozen in liquid nitrogen. RluB mutants were expressed and purified as described for the wild-type protein.A C-terminal truncation of 2PtCl4  for 30–45 min, washed two to three times in reservoir solution and cryoprotected in reservoir solution containing 20% (v/v) ethylene glycol.We were unable to crystallize full-length RluB (291 residues) either alone or with small RNA substrates; therefore we used a construct (residues 1–251) lacking the C-terminal 40 residues, which are predicted to be disordered, for crystallization. Crystals of RluB (1–251) were grown at a concentration of 10 mg/ml in 0.1 M tri-sodium citrate pH 5.6, 20% (v/v) isopropanol and 15% (w/v) PEG4000 using hanging drop vapor diffusion by mixing 1 µl protein with 1 µl reservoir solution (500 µl reservoir solution). To make a heavy atom derivative for determining X-ray diffraction phases RluB crystals were soaked with KE. coli 23S rRNA, but with 5-FU substituted for U2605. RNA was purchased from Dharmacon (Thermo Scientific) and deprotected according to the manufacturer’s protocol. The RluB-RNA complex was formed by incubating RluB at a concentration of 5 mg/ml with a 1.2-fold molar excess of 21-mer RNA for 1 h at room temperature. Crystals were obtained in 16% (v/v) polypropylene glycol 400 and 12% (v/v) 1-propanol using hanging drop vapor diffusion. Diffraction data were collected on beam line 8.3.1 of the Advanced Light Source .The 21-mer stem-loop used for crystallization was identical in sequence with nucleotides 2587–2607 of the 2PtCl4-soaked crystals of apo-RluB were not. The structure of the Pt derivative was therefore solved by molecular replacement using the RluF structure (PDB ID: 2GML) edited with Sculptor as a search model, using Phaser  and an oligodeoxynucleotide template in the presence of 0.3 mM cold UTP, 0.1 mM [5-3H]-UTP  and 3.75 mM ATP, GTP and CTP for 3 h at 37°C. The reaction was treated with DNAse I, RNA was extracted with phenol/chloroform and EtOH precipitated. The RNA was further purified by DEAE Sepharose chromatography  using a NaCl gradient. RNA containing fractions were collected at 0.4 M and 0.6 M NaCl. RNA was EtOH precipitated and resuspended in water. Activity assays were carried out at room temperature in 50 mM Hepes pH 7.5, 50 mM NaCl and 1 mM TCEP in a reaction containing 50 nM RluB and 0.5 µM RNA. After 1 h the reaction was quenched with 5% (w/v) activated charcoal (Norit A) in 0.1 N HCl, the sample was centrifuged  and the supernatant was again treated with Norit A, followed by centrifugation. The supernatant was filtered through Ultrafree-MC centrifugal filters (Millipore) to remove residual Norit A. The filtrate was mixed with Aquasol-2 (Perkin Elmer) and released 3H was counted. RluB Y140F had 2.8% ± 0.7% activity (mean of five independent measurements) and RluB R108A had 0.6% ± 1.4% activity of wild-type RluB (mean of three independent measurements).The 21-mer RluB stem-loop was Escherichia coli RluB (1–251) was co-crystallized with a 21-mer RNA stem-loop identical in sequence to the target stem-loop in 23S rRNA except that U2605 was modified to 5-FU to block the last step in catalysis  is comprised of an N-terminal S4 domain  residue,28. The The 21-mer RNA stem-loop binds with the target base 5-FU2605 flipped out into the active site cleft and the loop region abutting the S4 domain. The stem-loop has the same secondary structure as it has in the context of the large ribosomal subunit, with A2602 forming a bulge . In contThe target nucleotide, U2605, is flipped into the active site of the enzyme and RluB has turned over 5-FU2605 to give rise to the C1′-C5 glycoside bond typical for Ψ; RluB (1–251) is catalytically active against the 21-mer A. The 5-8 in In crystal structures of other Ψ synthase complexes with 5-FU substituted substrates, the 5-FU has both isomerized and been hydrated at C6 to give  5-fluoro-6-hydroxy-Ψ 8 in   , though RluB–RNA interactions generally parallel those seen in the RluF–stem-loop complex . The sidArg108 is conserved in the RluA, RsuA and TruA families and using MD simulation we demonstrated how this residue might assist in guiding the target base into the active site of the enzyme TruA . VariantMany of the hydrogen bond interactions between the RNA and RluB are mediated by water. We identified 30 waters in the RNA–RluB interface of complex A (the better ordered of the two complexes in the asymmetric unit), 18 of which were also seen in complex B. The water-mediated nature of much of the interface makes it plastic, allowing RluB and RluF to bind the same stem-loop substrate in native and rearranged forms, respectively, using many analogous interactions. Three of the interface waters are at the active site and these may have important roles in orienting the target base during catalysis or for catalysis itself.Substrate binding by Ψ synthases involves conformational changes to both protein and RNA thus the structures of the apo enzyme and any intermediates along the binding trajectory are important for substrate specificity . The crySupplementary Figure S1).Apo-RluB is similar to apo-RluF  with an 10 helix and bind to the RNA near the bulge , however individually the subdomains align closely. Alignment of both subdomains, allowing for flexibility in their relative orientations , resulthe bulge C.10 helix following α1 ; however, since this stretch is probably flexible and not part of the catalytic core it is unlikely to drive stem-loop rearrangement.RluB-bound to the isolated ribosomal stem-loop, nt 2587–2607, with 5-FU2605 substituted for the target, shows that Arg108 displaced 5-FU2605 from the stem into the active site (just as the homologous Arg in RluF displaced 5-FU2604), where it went through the initial steps of isomerization to pseudouridine. Why does the stem-loop rearrange upon binding to RluF but not to RluB? While the RNA-binding cores of the two enzymes are highly conserved, and their binding interactions with one strand of the RNA stem (A2590- U2596) are equivalent, in RluF the bulge at A2602 is pushed back into the stem and the base pairing is frame-shifted, allowing U2604 instead of U2605 to flip into the active site A. SeekinMore likely, stabilizing interactions between the bulge and residues 131–138 connecting α1 and β4 (RluB nomenclature) prevent stem-loop rearrangement in RluB, while absence of such interactions allow rearrangement in RluF. Residues 131–138 are different between RluBs and RluFs but highly conserved among different species of each enzyme. Specifically, Glu135 (conserved in RluBs) makes a hydrogen bond to the bulge base A2602, while the equivalent Asp, (conserved in RluFs) is too short to make such a contact.in vivo  that directly overlaps the bulge-binding site. The apo-conformation of residues 131–138 might be part of the basis for antagonizing the bulge in RluF. While the frame-shifted state of RNA in the RluF–RNA complex leads to catalysis at U2604, the energy difference favoring frame-shifted over unrearranged stem-loop in the RluF–RNA complex must be rather small since RluF does modify the RluB target, U2605, to a small extent in vivo , and is in vivo .Our RluB-stem-loop structure is the first crystal structure of a Ψ synthase–FU RNA complex in which the enzyme is clearly covalently attached to the 6-position of the rearranged FU, however, the covalently bound amino acid is the highly conserved Tyr140. This result, in the context of previously reported evidence, provides new insights into the Ψ synthase reaction mechanism.et al. -5-fluoro-6-hydroxy-Ψ in the substrate/product site  were performed in H2O18. They showed that the O18 was not incorporated in the expected conserved Asp of the proteins but rather in the 6-hydroxy group of the rearranged FU hydrate  for no relevant reason, conformational changes of the Asp and covalent bond formation to C6. The latter reaction would occur after reaction completion, and represent an ‘accidental’ event. Therefore the acylal mechanism seems overly complicated and unnecessary.The Michael addition to C6 has precedence in numerous mechanistically related enzymes, which adds credence to the analogous mechanism favored here: these enzymes include thymidylate synthase , HmdCMP 5 which would undergo a 1,2-elimination of the leaving group Asp to give highly reactive 7; hydration to give the observed FΨ 6-hydrate, 8, which as described above for the models should be significantly more stable than 5. It can be seen that this mechanism would provide 8 without O-acyl ester cleavage, and hence explain how heat disruption of the covalent complex 5 would give 8 with O18 incorporation at the 6-hydroxyl rather than Asp. With substrate uridine a simple β-elimination on 5, R = H would provide the product Ψ, 6, R = H.7 shortly after release of Asp60 facilitated by the N-1 electrons to form a planar sp2 hybridized C6. Although the phenolic hydroxyl is not a potent nucleophile (pKa ∼10), it is certainly more nucleophilic than water, which does form a FU hydrate in other Ψ synthases. Interestingly, the covalently bound Tyr is on the opposite side of the plane of the FΨ ring as the catalytic Asp and the configuration of the bound Tyr covalent adduct is 5S, 6R—the same configuration as the 6-OH-5-FΨ hydrates found in other Ψ synthase 5-FU-RNA structures; thus, water or Tyr attacks 7 from the same side of the ring.The RluB–FΨ covalent complex described in the present work is readily accommodated by Tyr addition to Supplementary Data (Scheme S1). The reaction invokes three acid-base groups, which are needed to accept protons from O2 and O4 and from C5. These acceptors/donors are as yet undefined; waters or backbone amide groups are possible candidates. The active site of the RluB–RNA complex contains three waters that could serve this role. We point out that in all structures of Ψ synthase–RNA complexes thus far reported, the O4 of the substrate pyrimidine is hydrogen bonded to the backbone amide of Asp110, and O2 accepts a hydrogen bond from the main chain N-Hs of residues 196 and/or 197 (RluB numbering).An accounting of all the proton transfers during pseudouridine formation is shown in the The fortuitous addition of Tyr140 instead of water to C6 shows that these additions at C6 occur after, and independent of the elimination of the Asp from the covalent RNA adduct. The mechanism proposed here is the most parsimonious explanation of this and all reported mechanistic and structural data on Ψ synthases.Coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 4LAB (apo-RluB) and 4LGT (RluB–RNA complex).Supplementary Data are available at NAR Online.


The goal of this work was to study mutational patterns in the evolution of RNA secondary structure. We analyzed bacterial tmRNA, RNaseP and eukaryotic telomerase RNA secondary structures, mapping structural variability onto phylogenetic trees constructed primarily from rRNA sequences. We found that secondary structures evolve both by whole stem insertion/deletion, and by mutations that create or disrupt stem base pairing. We analyzed the evolution of stem lengths and constructed substitution matrices describing the changes responsible for the variation in the RNA stem length. In addition, we used principal component analysis of the stem length data to determine the most variable stems in different families of RNA. This data provides new insights into the evolution of RNA secondary structures and patterns of variation in the lengths of double helical regions of RNA molecules. Our findings will facilitate design of improved mutational models for RNA structure evolution. Molecules of RNA perform biological functions which require that they fold into specific secondary and tertiary structures. Conservation of these structures may be as important as, or more important than, sequence conservation during the course of RNA evolution Although many structural elements  are conserved within a given RNA family, there is also variation in the presence or absence of certain stem-loops and pseudoknots across evolution, and there is variation in the length of corresponding double-helical regions RSEARCHThe patterns of RNA base and base pair changes have been both studied and modeled. One of the earliest models was developed by Knudsen et al. Recently, evolutionary models that address structural variation have been proposed. Holmes Our primary goal in this study was to determine the evolutionary and mutational patterns in double helical regions of RNA secondary structures that are responsible for variability in stem length, focusing on those that lead to stem-insertion and deletion. We chose to work with tmRNA (found in bacteria and organelles), RNaseP A , RNaseP B  and eukaryotic telomerase RNA sequences. This selection was motivated by the availability of large, well annotated databases for these RNA sequences and structures Saccharomyces, Kluyveromyces telomerase RNA structural alignments were obtained from Rfam Saccharomyces and the Kluyveromyces telomerase RNAs, respectively. We chose K. lactis structure as a consensus for all 6 species of Kluyveromyces, as the telomerase database contains the annotation for the conserved segments only and Rfam has the alignment only for Saccharomyces species. Therefore, we used the K. lactis structure as a consensus and predicted additional helices in the segments which are unique to other Kluyveromyces species using RNAfold at default parameters (ViennaRNA-1.8.4) We obtained structural alignments for tmRNAs and RNasePs from the tmRNA database RNApastaExcept for RNaseP A and RNaseP B, the numbers of sequences used in our study are greater than or equal to the number of sequences present in the seed alignment of the Rfam database. We excluded RNaseP A and RNaseP B sequences that did not have corresponding rRNA sequences in the ribosomal database project Kluyveromyces telomerase RNAs, corresponding rRNA sequences were obtained from the comparative rRNA website Saccharomyces, the reference tree was obtained from the Saccharomyces phylogeny website (http://www.genetics.wustl.edu/saccharomycesgenomes/yeast_phylogeny.html).We obtained rRNA sequences for the same species whose sequences were in the tmRNA and RNaseP datasets from the Ribosomal Database Project MrBayes3.1.2 program MrBayes parameters is given in supplementary The reference phylogenetic trees were built by Mesquite (version 2.74 (build 550)) program Mesquite with maximum parsimony. Given the tree and observed character distribution, this method finds the ancestral states that minimize the number of steps of character change. The cost of change for the continuous data from state x to state y is (x–y) which can be linear or squared; we used the default squared method as it can handle the trees with polytomies. Dnapars(version 3.5c)Phylip suite, was used to construct the hypothetical ancestral sequence at each node of the tree. This program counts the number of changes of bases needed on a given tree. We generated the hypothetical ancestral sequences by turning on the user tree and printing the sequences at the node of the tree options.We used the We manually determined which stems were variable across the phylogenetic tree; if one of the branches at the nodes containing closely related species were variable with respect to stem-loops then all the RNA sequences belonging to that particular node were used in the further analysis. We collected the underlying sequences for those stems from our alignment file. Afterwards, we created two types of base pair substitution matrices for each type of RNA under study. The base pair substitution matrices summarize information about the mutations that affect the pairing ability of the RNA molecules. The first matrix was created by counting the base changes that occur in the stem regions of extant sequences (those at the leaves of the trees). The second matrix was created by comparing the changes that occurred with respect to reconstructed ancestral sequences present at the corresponding node in the tree. Similarly, we also created two single nucleotide substitution matrices.ijklA = log2 ((ijklf)/(ijfklf)) where ijklA is the value in any cell of the matrix, ijklf is the frequency of base pair change for that cell, ijf and klf are frequency of individual base pair involved in that change. Similarly, in the single nucleotide substitution matrix, observed/expected values were calculated by the formula: ijA = log2 ((ijf)/(i fjf)) where ijA is the value in any cell of the matrix, ijf is the frequency of single nucleotide change for that cell, if and jf are frequency of single nucleotide involved in that change. The expected values were calculated by obtaining the frequencies of nucleotides/base pairs within the matrices.We transformed the counts in each cell of the matrices into observed/expected values using the formula: RNApasta program. Prior to performing PCA, we clustered data by k-means clustering k) of clusters; we grouped the RNA molecules from different species based on similarity in their stem lengths. Both of the above analyses were performed using the R (R 2.9.1) statistical programming language.We also performed principal components analysis (PCA) on the stem length data obtained from the RNApastamesquite, we showed the ancestral state of each stem in terms of the presence or absence of it at the root node using these arc diagrams  chloroplasts have this stem. M. viride represents the earliest diverging green plant lineage Prochlorococcus and Synechococcus sequence during structure reconstruction) of RNA molecules of all these species. The alignment  in cyanobacteria and chloroplasts' tmRNA. In cyanobacteria, this pseudoknot divides into two small pseudoknots PK4A and PK4B lignment  clearly B. thetaiotaomicron, P. gingivalis, F. yabuuchiae and completely absent in C. limicola and C. tepidum. These species belong to Bacteroidetes/Chlorobi group. A reconstruction of the ancestral sequence . For prokaryotic tmRNA and RNaseP, we investigated possible relationships of the first two principal components with biological properties of the organisms, including oxygen requirements, temperature, energy source and motility. However, we did not find any significant relationship between the biological properties and principal components. Detailed results of the clustering and symbols representing the species are presented in the Supplementary We displayed the clusters on a PCA biplot to investigate further variance in stem lengths. The first 2 principal components explain 45% of the overall variance in stem lengths for tmRNA. The biplot of the first 2 principal components for tmRNA  shows thOur analysis of RNA secondary structures centers on documenting the mutational patterns responsible for the variation in the double helical regions, including insertion and deletions of whole stems as well as changes in the stem lengths. Our approach differs from previous studies of tmRNA 2 = 16.8019, df = 2, p-value = 0.0002247).Based upon the variability obtained by mapping the structure characters onto the tree, we were able to determine the level of variability shown by every stem of each RNA family under study . We deteWe constructed matrices to summarize the changes in bases and base pairs that occurred in stems that were variable across the phylogenetic tree. Since we also reconstructed the ancestral sequences, we were able to compare ancestral sequences with extant sequences as well as extant sequences with each other. All the methods available for ancestral sequence reconstruction have their limitations From the reconstructed ancestral state of each stem, obtained using mesquite, we found that in vertebrate telomerase RNA, stem D  is absenWe used principal components analysis to identify co-variable stems among the RNA molecules under study. The observation that stem U1 (involved in the formation of RNA pseudoknot PK4) is variable among the tmRNAs  is consiTetrahymena paravorax separates from all other species. A comparison among the ciliate telomerase RNA sequences indicates that this is due to the absence of stem B in the T. paravorax telomerase RNA. In the previous studies of ciliate telomerase RNA, this helix has been suggested to be a primitive telomerase RNA structural feature and deletion of this stem in T. paravorax and in other hypotrich telomerase RNAs is considered to be example of convergent evolution In the Ciliate PCA biplot , we founIn summary, we implemented a new approach to analyzing RNA structure from an evolutionary prospective. From this analysis, we conclude that different types of mutations are responsible for the variation in the lengths of double helical regions of RNA. We documented the associated substitution patterns in log-odds matrices. We also demonstrate the usefulness of PCA in the analysis of the RNA structure alignment. PCA in combination with clustering can easily determine the outliers from the large structure alignment of RNA which can then be subjected to further analysis. Further studies like these of the evolutionary variability of RNA structure and the associated mutational patterns will be essential for improving computational programs that model RNA structures.Figure S1Reference tree for all RNA families under study. Reference tree for sequences of A) tmRNA B) RNaseP A C) RNaseP B D) Vertebrate E) Ciliate F) Saccharomyces G) Kluyveromyces telomerase RNA; node numbers are indicated in the circle on each tree.(TIF)Click here for additional data file.Figure S2RNA secondary structure displaying stem variability drawn by RNApasta. RNA secondary structure diagram labeled with RNApasta annotation for A) tmRNA B) RNaseP A C) RNaseP B D) Vertebrate telomerase RNA E) Ciliate telomerase RNA F) Saccharomyces telomerase RNA and G) Kluyveromyces telomerase RNA; the black, brown and red color of stems indicates that single length distribution is present in 71–100%, 41–70 and 1–40% of the species, respectively. The intersecting lines connecting two loop region indicates a pseudoknot. Each alphabet in the figure represents a RNA stem (RNApasta notation).(TIF)Click here for additional data file.Figure S3RNApasta arc diagram showing ancestral state of each stem. RNA secondary structure diagram labeled with RNApasta annotation showing the ancestral state of each stem in terms of presence/absence of it, for A) tmRNA B) RNaseP A C) RNaseP B D) Saccharomyces telomerase RNA and E) Kluyveromyces telomerase RNA; the black, red and brown color of the each stem indicates the presence, absence and ancestral state not resolved, respectively. A crossing pattern of arcs indicates a pseudoknot. Each alphabet in the figure represents a RNA stem (RNApasta notation).(TIF)Click here for additional data file.Figure S4PCA Biplot for RNaseP A, RNaseP B and vertebrate telomerase RNA. Biplot of principal components for A) RNaseP A B) RNaseP B and C) Vertebrate telomerase RNA; arrows followed by alphabet indicates RNA stems; points in different shape represents clusters of species; partial RNaseP A sequences were excluded from the analysis.(TIF)Click here for additional data file.Table S1Accession numbers of cytochrome B sequences used in vertebrate reference tree creation.(DOC)Click here for additional data file.Table S2Detailed mcmc parameters used in MrBayes for reference tree creation.(DOC)Click here for additional data file.Table S3The process responsible for variation in the stem length.(DOC)Click here for additional data file.Table S4Observed/expected base pair substitution matrices combining the mutations among all three RNA families for the extant/extant sequence comparison.(DOC)Click here for additional data file.Table S5Observed/expected base pair substitution matrices combining the mutations among all three RNA families for the ancestral/extant sequence comparison.(DOC)Click here for additional data file.Table S6The species present in the each cluster in all studied RNA families in PCA.(DOC)Click here for additional data file.Text S1Alignment of tmRNA used in the study.(TXT)Click here for additional data file.Text S2Alignment of RNaseP A.(TXT)Click here for additional data file.Text S3Alignment of RNaseP B.(TXT)Click here for additional data file.Text S4Vertebrate telomerase RNA alignment.(TXT)Click here for additional data file.Text S5Ciliate telomerase RNA alignment.(TXT)Click here for additional data file.Text S6Saccharomyces telomerase RNA alignment.(TXT)Click here for additional data file.Text S7Kluyveromyces telomerase RNA alignment.(TXT)Click here for additional data file.


Monoamine transporters control neurotransmission by removing monoamines from the extracellular space. They also display channel properties, but their regulation by PIP2 has not been reported. The psychostimulant amphetamine acts on monoamine transporters to stimulate transportermediated currents and efflux and thereby increases the levels of extracellular monoamines.Neuronal functions, such as excitability or endo- and exocytosis, require phosphatidylinositol-4,5-bisphosphate  reduced membrane PIP2 dependence of reverse transport and current in monoamine transporters.These data demonstrate for the first time PIP


GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed.The RNA guanylyltransferase (GTase) is involved in the synthesis of the  During mRNA synthesis, eukaryotic mRNAs undergo a series of essential modifications before being exported to the cytoplasm where they are translated into proteins A number of different microbial pathogens code for their own enzymes involved in the synthesis of a cap structure During the past few years, both the RNA triphosphatase and the RNA (guanine-N7) methyltransferase (N7-MTase) components of the RNA capping machinery have been major targets for the development of drugs directed against RNA cap synthesis in vitro studies have shown that foscarnet is a potent inhibitor of the GTase reaction Recent In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. We demonstrate that mycophenolic acid, a compound which is currently used both in cancer and immunosuppressive chemotherapy, is an inhibitor of the GTase reaction. The biological implications of these findings for the MPA-mediated inhibition of RNA capping are discussed.Chlorella virus: 1CKN and 1CKO; Candida albicans: 1P16; S. cerevisiae: 3KYH). The chemical databases used in our virtual screening included the Sigma-Aldrich, Ambinter, ASINEX, IS Chemical technology, MolPort, and Vitas M Laboratory catalogs. Collectively, these 6 databases offered a collection of 13,800,000 small-molecule compounds.To identify potential candidate compounds that can bind to GTases, the crystal structures of various GTases were retrieved from the Protein Data Bank (http://pubchem.ncbi.nlm.nih.gov/). Based on the binding models of these compounds predicted by DOCK, the X-SCORE program (Version 1.1) was applied to obtain an estimate of the binding affinities of these compounds The molecular docking program DOCK (Version 4.0) was used to perform the virtual screening S. cerevisiae (Ceg1), vaccinia virus (D1R), Chlorella virus (A103R), and human (HCE) were expressed and purified as described before Chlorella virus (ChVLig) was also expressed and purified as described previously The RNA guanylyltransferases from 32P]GTP in a buffer containing 50 mM Tris-HCl, pH 8, 5 mM DTT, and 5 mM MgCl2 for 5 min at 30°C. The reactions were stopped by the addition of EDTA to 10 mM and SDS to 1%. The reactions were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS. The radiolabeled proteins were visualized by autoradiography of the gel. The extent of covalent complex formation was quantitated by scanning the gel with a PhosphorImager (Amersham Biosciences).The assay was performed by incubating the enzyme (0.1 µM) with 10 µM [α-32P]ATP or [α-32P]GTP to the transcription reaction.An RNA substrate of 81 nucleotides was synthesized with the MAXIscript kit (Ambion) using T7 RNA polymerase. The RNA transcript was synthesized from the pBS-KSII+ plasmid (Stratagene) that had been linearized with HindIII. The RNA substrate was purified on a denaturing 20% polyacrylamide gel and visualized by ultraviolet shadowing. The corresponding band was excised and then eluted from the gel by an overnight incubation in 0.1% SDS and 0.5 M ammonium acetate. The RNA was then precipitated with ethanol and quantitated by spectrophotometry. Alternatively, radiolabeled RNA substrates were also synthesized by adding GTP and a 5′-diphosphate acceptor RNA. The reaction products were then analyzed on a denaturing polyacrylamide gel. As can be seen in The ability of MPA to inhibit the GTase of the model organism 32P]GTP. The radiolabeled covalent enzyme-GMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Our results indicate that very high concentrations of MPA are required to inhibit the formation of the enzyme-GMP covalent complex  and 27 internally labelled guanosine residues. The RNA was then incubated with the GTase in the presence of GTP or MPA. The products of the reaction were extracted with phenol/chloroform and the RNA acceptor molecules were recovered by ethanol precipitation. Aliquots of the RNA samples were then digested with nuclease P1 and alkaline phosphatase and analyzed by polyethyleneimine-cellulose thin layer chromatography . However, the binding of RNA to the enzyme-GMP/MPA complex was severely decreased in the presence of MPA  C. albicans GTase OB domain (aa 244–390) aligns to the Chlorella virus GTase OB domain with 1.8 Å rmsd over 72 amino acids (28% identity) Chlorella virus, as well as the corresponding human enzyme. Our results demonstrate that MPA inhibits all these GTases to the same extent than the S. cerevisiae homolog  and second step (∼620 µM) of the GTase reaction were observed when MPA was added to the reactions catalyzed by the vaccinia, Chlorella, and human enzymes. Overall, these results indicate that MPA inhibits the activity of different GTases through a similar mechanism that mainly prevents the catalytic transfer of the GMP moiety onto an acceptor RNA.GTases are members of the RNA/DNA nucleotidyltransferase superfamily that share six conserved sequence motifs  and a si homolog . IC50 vaChlorella virus.GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases Chlorella virus DNA ligase. MPA was shown to inhibit the strand-joining activity by 50% at 700 µM. We next set out to investigate which of the first two catalytic steps of the ligation reaction is inhibited by MPA. The first step of the reaction, i.e. the formation of the enzyme-AMP covalent intermediate, was monitored by incubating the purified enzyme in the presence of [alpha-32P]ATP. The radiolabeled covalent enzyme-AMP complex was then visualized by autoradiography following electrophoresis on a denaturant polyacrylamide gel. Similarly to what was observed in the case of the GTase activity, our results indicate that very high concentrations of MPA are required to inhibit the first step of the reaction, i.e. the formation of the enzyme-AMP covalent complex , as recently suggested through proteomic analysis de novo synthesis of GTP through IMPDH inhibition, and both molecules can inhibit RNA capping, albeit through different mechanisms of inhibition.What is the biological relevance of the present finding? MPA has been shown to cause a reduction of the cellular GTP pools through the inhibition of IMPDH which is required for the


The specific interaction between hepatitis B virus (HBV) polymerase (P protein) and the ε RNA stem-loop on pregenomic (pg) RNA is crucial for viral replication. It triggers both pgRNA packaging and reverse transcription and thus represents an attractive antiviral target. RNA decoys mimicking ε in P protein binding but not supporting replication might represent novel HBV inhibitors. However, because generation of recombinant enzymatically active HBV polymerase is notoriously difficult, such decoys have as yet not been identified.in vitro reconstitution system exploiting a recombinant truncated HBV P protein (miniP), to identify potential ε decoys in two large ε RNA pools with randomized upper stem. Selection of strongly P protein binding RNAs correlated with an unexpected strong enrichment of A residues. Two aptamers, S6 and S9, displayed particularly high affinity and specificity for miniP in vitro, yet did not support viral replication when part of a complete HBV genome. Introducing S9 RNA into transiently HBV producing HepG2 cells strongly suppressed pgRNA packaging and DNA synthesis, indicating the S9 RNA can indeed act as an ε decoy that competitively inhibits P protein binding to the authentic ε signal on pgRNA.Here we used a SELEX approach, based on a new in vitro SELEX approach. Effective suppression of HBV replication by the S9 aptamer provides proof-of-principle for the ability of ε decoy RNAs to interfere with viral P-ε complex formation and suggests that S9-like RNAs may further be developed into useful therapeutics against chronic hepatitis B.This study demonstrates the first successful identification of human HBV ε aptamers by an  Hepadnaviridae, is the causative agent of B-type hepatitis Hepatitis B virus (HBV), the prototypic member of the A unique characteristic of HBV replication is the protein-primed reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA), which takes place within viral capsids (core particles) Aptamers are the high affinity ligands derived from libraries of randomized molecules through ″SELEX″ , a high-flux screening technique involving repeated rounds of partitioning and amplification in vitro selection has been demonstrated for the related duck HBV (DHBV) after recombinant DHBV P protein had successfully been reconstituted into priming-active RNPs in vivo effects of aptamer sequences replacing the authentic ε-sequence in the DHBV genome have recently been reported in vitro SELEX-based screening for such aptamers was not possible until very recently, when Hu and coworkers succeeded in reconstituting RNP formation with HBV P protein in vitroin vitro SELEX procedure by which we successfully isolated high-affinity RNA aptamers against recombinant truncated HBV P protein (miniP) from two large RNA pools. In one pool (termed AS), the upper ε stem was completely randomized, in the other (termed S), the naturally conserved apical loop sequence was maintained. Among various strongly binding aptamers, the one with the highest affinity and specificity for P protein, S9, inhibited HBV replication strongly in transiently cotransfected HepG2 cells, and still substantially in the stably HBV producing HepG2.2.15 line. As shown below, this inhibition occurs most likely by competition of the aptamer with the authentic ε signal on pgRNA. The in vitro SELEX-based aptamer selection thus represents a powerful strategy to identify decoys that might become therapeutically applicable to reduce viral loads in chronic HBV infection.Previously, the feasibility of identifying aptamers specifically binding a hepadnaviral P protein by E. coli strain BL21-CodonPlus(DE3) and purified using immobilized metal affinity chromatography (IMAC) performed as previously described for the DHBV P protein  and the C terminal 231 aa including the RNase H domain were deleted. Analogous DHBV miniP constructs display authentic, ε-dependent priming activity  protein  [33],  fraThe miniP protein was expressed from pET-MBP-TEV-HP1-199/292-601 in BL21-CodonPlus(DE3) as His-tagged fusion protein with the maltose-binding protein  and purified as previously described N)29GAACAGTAGGACATGAACAGCCCTATAGTGAGTCGTATTAattc-3′) and DepsNuppS(−) (5′-GGTACCTGTCCATGCCCCA(N)12GCACAG(N)11GAACAGTAGGACATGAACAGCCCTATAGTGAGTCGTATTAattc-3′), respectively with a (+)-polarity T7 promoter oligo; randomized positions are indicated by underlined N. The resulting partial duplex DNAs were used as templates for in vitro transcription using the T7 MEGAshortscript kit (Ambion). The mutant RNA, which has been previously proved to be an in vitro P binding-deficient RNA cagaTAGGACCCCTATAGTGAGTCGTATTAattc-3′) as transcription template; the mutated positions are indicated by underlined lowercase. The wt HBV ε RNA was obtained by in vitro transcription of the corresponding plasmid (pBS-A1) after linearization with Eco RI of the above described sequences. The products were analyzed by electrophoresis in 12% denaturing polyacrylamide gels, followed by silver staining; RNA concentrations were determined by measuring the absorbance at 260 nm. Subsequent RNA pools were produced analogously, but using the duplex RT–PCR products as template.T7 RNA polymerase mediated run-off transcription was performed as described previously in vitro binding reaction for selection of strong miniP-binders, approximately 100 ng purified miniP proteins were incubated with Hsp90β , Hsp70 , Hdj1 , Hop , and p23 , together with the randomized RNAs at a final concentration of 12286M. For a 102286l in vitro binding setup for EMSAs-based detection, the amount of individual proteins was exactly added as described recently In a 30 µl 2+NTA agarose beads (Qiagen) were added to the in vitro reconstitution reactions 2, 40 mM KCl, 100 mg/ml yeast tRNA). Finally the beads were suspended in 100 µl TMK buffer and the bound RNAs were purified by phenol extraction. The extracted RNAs were precipitated, and dissolved in 15 µl TE buffer.Isolation of miniP-binders was performed as previously described 5′-CAATCTGCAGTCTAGATAAGGTACCTGTCCATGCCCCA-3′) and M-MLV reverse transcriptase (Promega) as per the manufacturer's instructions. Subsequently, the RNA template was degraded by alkaline hydrolysis and an aliquot of this solution was amplified using Taq DNA polymerase (Promega). RT–PCR products were directly sequenced using the Thermo Sequenase Cycle Sequencing Kit  as recommended by the supplier. The RT-PCR products from the third selection round were cloned into pUC19 vector (Invitrogen) via the terminal Xba I and Eco RI sites and plasmid DNAs from 45 individual colonies were sequenced.An aliquot of 2 µl of the isolated RNA solution was reverse transcribed using the reverse primer (32P ATP (3000 Ci/mmol) as described 32P ATP was removed using Quick Spin columns (Roche). For direct EMSA (protein-binding affinity), the 32P-labelled RNAs were used at 50 nM final concentration (specific activity ∼2×105 cpm/pmol). For competitive EMSA (protein-binding specificity), a mixture of 32P-labeled wt ε RNA  plus 1 µM unlabelled selected RNAs was used. Following incubation, the samples were analysed on 5% (w/v) polyacrylamide (37.5∶1 acylamide:bis acrylamide) gels containing 0.5× TBE. Labeled RNAs and RNP complexes were detected by autoradiography of the dried gels. Signal intensities were determined by phosphorimaging using OptiQuant 5.0 software (Perkin Elmer).RNAs were 5′ terminally labeled by dephosphorylation and rephosphorylation with γ-Relative quantitative analysis was performed to calculate intensities of the shifted signals using OptiQuant 5.0 (PerkinElmer).TM 2000 (Invitrogen) according to the manufacturer's instructions, using 24 µg DNA per 10 cm diameter plate. pSUPER constructs were analogously transfected using 8 µg DNA per 6 cm diameter. For co-transfections, mixtures of 12 µg pCH-9/3091 and 12 µg pSUPER plasmid per 10 cm diameter dish were used. Cytoplasmic core particles were isolated from transfected-cells as previously described 2, and incubated with 20 U DNase I (Fermentas) plus 15U RNase A (Fermentas) at 37°C for at least 6 h. Cytoplasmic core particles were then precipitated with 6.5% polyethylene glycol.HepG2 32P-labelled random-primed probe specific for the HBV sequence.To analyze HBV DNAs by Southern blotting, the isolated core particles were incubated with 50 U micrococcal Nuclease S7 (Fermentas) to remove the nonencapsidated DNA completely. Then core DNA was extracted, separated by 1% agarose gel electrophoresis, and hybridized to a 4Cl, 1 mM EDTA, 25 mM MgCl2, 0.1% h-mercaptoethanol, 0.5% Nonidet P-40) supplemented with 0.5 mM each of dCTP, dGTP, and dTTP, and 10 µCi α-32P-dATP (3000 Ci/mmol). The resulting 32P-labeled reaction mixtures were directly electrophoresed on a 1% native agarose gel and then subjected to dry gel autoradiography.EPA was performed as previously described in For native western blotting, isolated core particles were electrophoresed on 1% TAE agarose gels and transferred to PVDF membrane. To normalize the transfection efficiencies, aliquots corresponding to 20 µl of 1 ml cytoplasmic lysate from a 10 cm diameter plate were subjected to electrophoresis in 15% SDS polyacrylamide gel and transferred to PVDF membrane. Immunoblotting was performed using antibodies against native and denatured HBc (both from DAKO). Horseradish peroxidase-conjugated anti-rabbit secondary antibody and enhanced chemical luminescence (ECL) were employed to visualize either assembled HBV core particles or translated core proteins. For normalization of the HBV signals to the number of cells, the housekeeping protein β-actin present on the same blots was detected using an anti-β actin antibody (Abcam).32P DNA probes specific for HBV.Viral pgRNA from intracellular core particles was prepared as described in th transfected cells from a 10 cm-diameter plate were suspended in 1 ml DMEM medium, of which 100 µL were seeded into the wells of a 96-well plate. MTT assays were performed using the Vybrant® MTT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer's instructions.24 h post-transfection, aliquots corresponding to 1/10Table S1Classification of individual isolated RNA aptamers.(DOC)Click here for additional data file.Table S2Transfection of pSUPER vector encoding the S9 aptamer is not detectably cytotoxic.(DOC)Click here for additional data file.


RNA Helicase associated with AU-rich element (RHAU) (DHX36) is a DEAH -box RNA helicase that can bind and unwind G4-quadruplexes in DNA and RNA. To detect novel RNA targets of RHAU, we performed an RNA co-immunoprecipitation screen and identified the PITX1 messenger RNA (mRNA) as specifically and highly enriched. PITX1 is a homeobox transcription factor with roles in both development and cancer. Primary sequence analysis identified three probable quadruplexes within the 3′-untranslated region of the PITX1 mRNA. Each of these sequences, when isolated, forms stable quadruplex structures that interact with RHAU. We provide evidence that these quadruplexes exist in the endogenous mRNA; however, we discovered that RHAU is tethered to the mRNA via an alternative non–quadruplex-forming region. RHAU knockdown by small interfering RNA results in significant increases in PITX1 protein levels with only marginal changes in mRNA, suggesting a role for RHAU in translational regulation. Involvement of components of the microRNA machinery is supported by similar and non-additive increases in PITX1 protein expression on Dicer and combined RHAU/Dicer knockdown. We also demonstrate a requirement of argonaute-2, a key RNA-induced silencing complex component, to mediate RHAU-dependent changes in PITX1 protein levels. These results demonstrate a novel role for RHAU in microRNA-mediated translational regulation at a quadruplex-containing 3′-untranslated region. Guanine-rich nucleic acids are prone to fold into unique structures known as a G4-quadruplexes. These quadruplexes typically consist of four tracts of guanines arranged in parallel or anti-parallel strands that align in stacked tetrad planes. These tetrad guanine planes are stabilized by Hoogsteen hydrogen bonds and a monovalent cation . QuadrupRNA Helicase associated with AU-rich element (RHAU), also known as DHX36, is a member of the DEAH-box family of RNA helicases that has emerged as a key enzyme capable of binding and unwinding both inter- and intramolecular quadruplexes in both DNA and RNA (53–105) is sufficient for specific quadruplex interaction; however, the full-length protein containing the helicase domain is necessary for the quadruplex unfolding activity of RHAU  studies of Dicer that demonstrate similar and non-additive increases in PITX1 protein on Dicer and RHAU knockdown. Additionally, argonaute-2 (Ago2) knockdown is found to prevent RHAU siRNA-dependent upregulation of PITX1. This study, for the first time, identifies PITX1 as a quadruplex-containing RNA that is bound by RHAU at the 3′ UTR to regulate protein expression.N-methyl-mesoporphyrin IX was purchased from Frontier Scientific . All standard laboratory chemicals and reagents were purchased from Thermo-Fisher Scientific . The recombinant full-length RHAU protein and RHAU53–105 truncation were purified as described previously  cell lines were a gift from Dr Spencer Gibson. The monoclonal murine anti-RHAU antibody was a gift from Dr Yoshikuni Nagamine. Cell culture and monoclonal antibody purification was performed as described previously . The foleviously .g for 5 min at 4°C. The cell pellet was resuspended in 5 ml of cytoplasmic lysis buffer . The cells were lysed on ice for 5 min and centrifuged at 5000 rpm in a bench-top microfuge for 5 min at 4°C. The supernatant was set aside on ice, and the cell pellet was resuspended in 5 ml of nuclear lysis buffer  sucrose, 350 mM NaCl, 0.01% (v/v) NP-40 supplemented with protease/phosphatase/RNase inhibitors). To lyse the nuclei, the suspension was vortexed for 30 s followed by passage through a 20-gauge needle. The nuclear and cytoplasmic fractions were combined, and insoluble material was removed by centrifugation at 14 000rpm in a bench-top microfuge at 4°C for 10 min. A total of 150 µg of monoclonal anti-RHAU antibody was added to the cell lysate and incubated for 30 min at room temperature with end-over-end mixing. After 30 min, 500 µl of Pierce Protein A/G magnetic beads (Thermo-Fisher Scientific) pre-equilibrated in lysis buffer was added to the sample, and mixing was continued for 30 min at room temperature. The magnetic beads were subsequently washed 3-fold with cytoplasmic lysis buffer, followed by three washes with nuclear lysis buffer and finally three washes with a 1:1 mixture of cytoplasmic and nuclear lysis buffer.To screen for novel RHAU binding RNAs, cells from 20 confluent 150-mm dishes were scraped into 10 ml of cold phosphate-buffered saline per plate and pelleted by centrifugation at 1500Escherichia coli DNA Polymerase , 2 U RNase H (New England Biolabs), 300 µM dNTP mix (New England Biolabs) and 10 µl NE Buffer 2 (New England Biolabs). The reaction was brought to a final volume of 100 µl and incubated at 15°C for 2 h. After 2 h, 10 U of T4 DNA polymerase was added to the reaction and incubation continued for an additional 5 min. The double-stranded cDNA was then purified using the GeneJet PCR purification kit (Thermo-Fisher Scientific). Purified cDNA was blunt-end cloned into the pJet1.2 vector using the CloneJET PCR cloning kit (Thermo-Fisher Scientific) and transformed into chemically competent E. coli. Plasmid DNA was isolated from transformants using the GeneJet plasmid DNA mini-prep kit (Thermo-Fisher Scientific) and sequenced .To isolate the copurified RNA, beads were resuspended in 300 µl of resuspension buffer from the Thermo-Scientific GeneJet RNA cleanup and concentration micro kit, and the RNA was purified according to the manufacturer's instructions. Ribosomal RNA was removed from the preparation using the Ribo-Zero rRNA removal kit . RNA was then reverse-transcribed to complementary DNA (cDNA) using the Revertaid H minus first strand cDNA synthesis kit according to the manufacturer’s instructions (Thermo-Fisher Scientific). Second strand synthesis was performed by combining 25 µl of the first strand synthesis mixture with 40 U 53–105 competition assays, 5 µM RHAU53–105 was added to the binding reaction for 30 min before addition of antibody. For the 6×His-RHAU53–105 immunoprecipitations, the binding reaction was supplemented with 300 nM 6×His-RHAU53–105 for 30 min before addition of the anti-6×His Tag antibody. Cycling conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 s and 55°C for 30 s. The PCR products were analyzed by agarose gel electrophoresis following amplification to ensure reaction specificity. The primer sequences used are as follows: PITX1 coding sequence (CDS) forward—TCTCCACCAAGAGCTTCACCTTCT, PITX1 CDS reverse—GCCGGTGAGGTTGTTGATGTTGTT, PITX1 UTR forward—TTTACAGCGTCCCTGTGTATG, PITX1 UTR reverse—CACGCTCGGACTATGGTTT, hTR-forward—TCTAACCCTAACTGAGAAGGGCGT, hTR-reverse—TGCTCTAGAATGAACGGTGGAAGG, BCYRN1 forward—TAATCCCAG CTCTCAGGGAGGCTAA, BCYRN1 reverse—GGTTGTTGCTTTGAGGGAAGTTACGC, GAPDH-forward—ACCCACTCCTCCACCTTTG and GAPDH-reverse—CTCTTGTGCTCTTGCTGGG.For real-time PCR analysis, RNA was isolated and purified as aforementioned using a single confluent 150-mm dish. A total of 2 µl (∼20 ng) of the purified RNA was used directly with the iScript one-step reverse transcriptase–polymerase chain reaction (RT–PCR) kit  according to the manufacturer's protocol with an annealing temperature of 55°C. Fold enrichment values were calculated relative to 25 ng total RNA. The cDNA was amplified by quantitative PCR with primers specific to the RNA transcripts of PITX1, TERC, BCYRN1 and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). For RHAU53–105 were resolved on 10% (w/v) polyacrylamide gels.To identify probable quadruplex-forming regions within the PITX1 mRNA, the sequence (NCBI Accession # NM_002653.4) was analyzed using the QGRS Mapper software ,35. Thre6 cells per condition as described earlier in the text. A binding reaction was prepared that contained 500 µl of cell extract, 50 ng/µl Poly I-C, 250 µl of binding buffer  glycerol) and 250 nM biotin-labelled probe. Binding reactions were incubated for 30 min at room temperature, and 40 µl of Pierce streptavidin magnetic beads were subsequently added . Following a 30-min incubation at room temperature, beads were washed three times in a 1:1 mixture of cytoplasmic and nuclear lysis buffers, followed by three washes each in cytoplasmic and nuclear lysis buffers and finally three washes in RIPA buffer . After washing, the beads were resuspended in 50 µl 1× SDS loading dye , boiled for 5 min and the isolated proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and assayed for binding by western blotting.To assess RHAU interactions with the predicted quadruplex regions within the PITX1 mRNA, a streptavidin pull-down assay was performed with 5′-biotin-labelled PITX1 RNAs. Cell extraction was performed on 20 × 10—GAUCCUAUGUUCAAUCUAA (Cat. # s23754) and AGO2—GGUCUAAAGGUGGAGAUAA (Cat. # s25931). As a control, all experiments were performed using an equal amount of the Medium GC StealthRNAi Universal Negative Control duplex (12 935-300) from Invitrogen Corporation (control siRNA). An siRNA-resistant silent mutant of the DHX36 cDNA was developed by site-directed mutagenesis of each codon within nucleotides 1713–1728 of the DHX36 mRNA (accession # NM_020865) from TCGGAAAATAGTAATT to GAGAAAGATTGTTATA in the pCDNA3 vector (Invitrogen). An ATPase-dead mutant of RHAU (RHAU E335A) was generated as previously published (ΔRSM) was generated by deleting nucleotides 241–270 (NM_020865) corresponding to amino acids 54–63 (NP_065916) in a synthetic gene that comprised the 5′ region of the RHAU coding sequence through until an internal EcoRI restriction digest site. Plasmid transfections were performed using Turbofect (Thermo-Fisher Scientific) according to the manufacturer’s protocol. Western blotting was performed as described previously (The siRNA transfections were carried out using lipofectamine RNAiMAX (Invitrogen) using the reverse transfection protocol supplied by the manufacturer. For long-term siRNA knockdowns, cells were split and retransfected at 48 h post-transfection. Prevalidated siRNA duplexes were obtained from Invitrogen. The sequences of the siRNAs are as follows: DHX36—GGUGUUCGGAAAAUAGUAA (Cat. # s46823), PITX1—GCAACGUACGCACUUCACA (Cat. # s194685), DICERCat. # s254 and AGl duplex  935-300 A 1071 nt sequence corresponding to the PITX1 3′ UTR was synthesized by Genscript Corporation . The PITX1 3′ UTR was cloned into a version of the PGL3-promoter vector  in which the luciferase cDNA was replaced with cDNA encoding the β-galactosidase gene. The 3′ UTR was amplified by PCR using the following primers: B-gal-PITX1 UTR forward: GGCGGCGAATTCCAGCTGAGCGCCGGTCGCTACCATTACCAGTTGGT CTGGTGTCAAAAATAATAATAACCGCCCCGCCGCACCACGCGGGCCGGCGG and B-gal-PITX UTR reverse: ACGACGTCTAGATGCTTAGCACGCTCGGACTATGGTTTTAATAGACGTACATGGA and cloned using EcoRI and XbaI restriction enzyme sites. The 3′ UTR was cloned into the PGL3-promoter vector using an EcoRI site within the 3′ end of the β-galactosidase cDNA, allowing for direct fusion of the 3′ UTR following the β-galactosidase stop codon.Quadruplex disrupting mutations were introduced into the reporter vector by site-directed mutagenesis with primers in which an internal run of guanines was mutated to cytosines. The following primers were used along with the appropriate corresponding antisense oligonucleotide: PITX1 Q1MUT sense: GGCCGGAGC GGGGAACCCCGCGGGCG, PITX1 Q2MUT sense: GATCCGTGTTGGGGCCCCCGTTGGGTTTGGGGG and PITX1 Q3MUT sense: AGCGGGCAGTGCCCCCCTGGCGGGAGG. Site-directed mutagenesis was performed using a previously published two single-primer reaction method . PITX1 1To performed reporter assays, cells were either left untransfected, or transfected with control or RHAU siRNA. After 48 h, cells were split 1:3 and retransfected. At 72 h cells were transfected with the β-galactosidase reporter vectors containing the PITX1 3′ UTR and various mutants. Lipofectamine RNAiMAX and lipofectamine LTX were used for the siRNA and cDNA transfections, respectively (Invitrogen). After an additional 24 h, cells were harvested and levels of β-galactosidase were assessed using the Promega β-galactosidase enzyme assay system (Promega Corporation). Absorbance measurements were made on a Biotech Epoch 96-well spectrophotometer (Fisher Scientific).21–50) of the mature mRNA (accession # NM_002653.4). The following DNA oligonucleotide probes were used to digest at the 3′-end: Set 1—PITX11311–1340, Set 2—PITX11941–1970, Set 3—PITX12081–2110, Set 4—PITX12283–2303 and Set 5—PITX12358–2376.To identify the RHAU binding site on the PITX1 mRNA, DNA oligonucleotide-directed RNase H digestion of the protein-bound mRNA was performed. RNA immunoprecipitation was performed as described earlier in the text with a single confluent 150-mm dish of cells per RNase H digestion condition. Following binding of the protein–RNA complexes to the Protein A/G beads, the beads were washed once with each of the lysis buffer conditions described earlier in the text. Following washing, digestion reactions were prepared in a 100 µl volume that contained 10 µl of RNase H reaction buffer, 5 µM of each DNA oligonucleotide probe and 25 U of RNase H (New England Biolabs). Beads were resuspended in the digestion reaction and incubated for 45 min at 37°C with mixing every 10–15 min. Following digestion, the beads were washed once more with each buffer condition, and the remaining bound RNA was purified as described earlier in the text. A total of 5 µl of the purified RNA was amplified by quantitative PCR and compared with the undigested (RNase H without DNA oligonucleotide) sample. The RNA fragments that remained bound to the magnetic beads via RHAU interaction were detected by using primer sets within the PITX1 coding sequence (nucleotides 974–1143) and near the end of the 3′-UTR (nucleotides 2283–2376). All digestion reactions contained the same 5′ oligonucleotide cutting probe  containing the RSM is sufficient to bind quadruplexes with both high specificity and affinity (N-methyl mesoporphyrin IX (N-methyl mesoporphyrin IX (Mut) did not stain with the fluorescent dye. Following fluorescent visualization, the gel was subsequently stained with the total RNA stain toluidine blue to visualize the negative control RNAs.Previous studies have identified RHAU as a quadruplex-specific helicase with high affinity for both DNA and RNA inter- and intramolecular quadruplexes ,13,39,40affinity ,14. To iaffinity ,35. Threaffinity A. Synthehyrin IX B. As poshyrin IX . The puthyrin IX B. In con53–105). Both the full-length protein and RHAU53–105 demonstrated a specific interaction with the quadruplex RNAs in vitro, indicating a direct interaction  to eliminate circularization of the mRNA. RNase H digestion with PITX121–50 alone demonstrated a marginal reduction in the amplification with both the coding sequence (CDS) and UTR primer sets (21–50 and a DNA oligonucleotide immediately at the 3′ end of the PITX1 coding sequence (PITX11311–1340) resulted in a loss of the CDS template, but the UTR template was retained via RHAU interaction on the beads. This result strongly suggests that the RHAU interaction exists within the 3′-UTR, as the 3′-UTR remains bound to RHAU when the 5′ UTR and coding sequence are dissociated by targeted digestion. Similar results were obtained using oligonucleotide sets that progressively truncated the mRNA from the 5′ end and removed all three putative quadruplex-forming regions of the PITX1 mRNA . Cutting further 5′ to this region with PITX12283–2303 and PITX12358–2376 resulted in ablation of the UTR primer binding sites and restoration of the amplification with the CDS primer set (2081–2110 and PITX12283–2303). These data narrow the binding site of RHAU on the PITX1 mRNA to a ∼170 nt region that does not contain any of the predicted quadruplex-forming regions discussed previously; however, the results do not preclude the possibility that RHAU acts enzymatically on the identified quadruplexes while being retained at the mRNA via an as yet uncharacterized interaction.To detect the RHAU-bound RNA, primer sets were used within the PITX1 coding sequence (CDS Primers 974–1143) and near the end of the 3′-UTR (UTR Primers 2283–2376). All digestions shared a common 5′ targeting oligonucleotide complementary to nucleotides 21–50 of the PITX1 mRNA (PITX1mer sets B. In conimer set B. Retent53–105) and the endogenous RHAU protein. HEK293T cell lysates were supplemented with 5 µM RHAU53–105 for 30 min before RHAU immunoprecipitation. Co-precipitating RNAs were detected by quantitative real-time PCR. Primer sets were used for PITX1 as well as the telomerase RNA. The interaction between RHAU and human telomerase RNA is well characterized and consists of binding between the RSM and a quadruplex within the 5′ region of the RNA , we performed a competition assay between the isolated RSM . These data confirm the findings of the competition assay performed in To further study the role of the quadruplex binding motif, an RSM-deficient RHAU cDNA was generated that also contained silent mutations within the siRNA targeting region of the mRNA. HEK293T cells were transfected with either the wild-type RHAU or the RHAUsfection D. After 53–105 was added to HEK293T cell lysates at a concentration of 300 nM for 30 min. After this incubation, an equimolar amount of anti-6×His tag antibody was added to the binding reaction, followed by purification of protein–RNA complexes with Protein A/G magnetic beads. This experiment was performed with cells that were either left untreated or transfected with a control or RHAU-specific siRNA for 72 h. The 6×His-RHAU53–105 immunoprecipitation demonstrated strong enrichment of the PITX1 mRNA (∼150-fold) as well as the hTR mRNA (∼40-fold), whereas the GAPDH mRNA showed no significant enrichment (∼1.5-fold) . Further supporting the hypothesis that RHAU is retained at the PITX1 mRNA in a quadruplex-independent manner, an siRNA-resistant and ATPase-deficient RHAU construct (RHAU E335A) did not demonstrate a significantly enhanced affinity for the PITX1 mRNA as compared with an siRNA-resistant wild-type RHAU cDNA when transfected into HEK293T cells in which endogenous RHAU had been knocked down by siRNA .Although the competition assay A and B a.5-fold) E. RHAU kTX1 mRNA E. RHAU kSupplementary Figure S3A and B). To investigate the functional consequences of the interaction between RHAU and the PITX1 mRNA, an siRNA time course was performed in HEK293T cells. Cells were transfected with siRNA specific to RHAU or a negative control siRNA for 96 h, and PITX1 protein levels were monitored every 24 h after transfection by western blot. Forty-eight hours following knockdown, an increase in PITX1 protein expression is evident that peaks at 72 h and persists through until the 96 h time point A. This iP = 0.0002, by unpaired, one-tailed t-test) increase in PITX1 mRNA levels is observed on RHAU knockdown by siRNA  yet significant . Interestingly combined knockdown of RHAU and Dicer resulted in PITX1 protein levels that were similar to either knockdown alone, consistent with a shared mechanism of action. To investigate the possibility that RHAU associates with the PITX1 mRNA indirectly through interactions with an miRNA-dependent RNA-induced silencing complex (RISC), we assessed RHAU association with the PITX1 3′-UTR under conditions of either control siRNA or Dicer knockdown. Dicer knockdown did not significantly impact the interaction between RHAU and the PITX1 mRNA , and therefore the interaction appears to be independent of the presence of a Dicer processed miRNA.As RHAU binds to the 3′-UTR of the PITX1 mRNA, we sought to determine whether RHAU-dependent changes in PITX1 protein expression were related to miRNA-mediated regulatory mechanisms. To investigate miRNA-dependent regulation of PITX1, we performed siRNA knockdown of a key upstream miRNA processing enzyme, Dicer, a method previously demonstrated to significantly reduce levels of mature functional miRNAs . Dicer kSupplementary Figure S4B). Furthermore, RHAU knockdown did not decrease but increased Ago2 association with the PITX1 mRNA . These data establish a link between RHAU and miRNA-mediated regulation of PITX1; however, further study is necessary to determine the precise interplay of factors that modulate translation of the PITX1 mRNA.Small non-coding RNAs possess a myriad of functions within the cell, particularly within the context of post-transcriptional gene regulation . Mammaliin vitro obscurity to be recognized as multifunctional nucleic acid motifs capable of regulating key biological processes . Despite these negative data, an RISC-mediated interaction at the 3′ UTR warrants further investigation, as several reports have demonstrated an interaction between RHAU and mammalian argonaute proteins (RNA fragments that were retained on the beads (and therefore bound by RHAU) were detected by RT-PCR with primer sets within the coding sequence and 3′-UTR. This approach identified the RHAU binding site as a ∼170 nt region near the 3′-end of the UTR that did not contain any of the putative quadruplexes. This assay could not further define the region, as additional DNA oligonucleotides within the region did not exhibit efficient RNase H-mediated digestion, likely due to secondary structure elements or bound protein that prevented hybridization. This region did, however, contain a predicted hsa-miR-19a binding site that was well conserved, raising the possibility that the RHAU interaction was mediated indirectly through RISC binding. However, preliminary studies of hsa-miR-19a using both an microRNA mimic and inhibitor did not yield significant changes in PITX1 expression  was tested and demonstrated similar affinity for the PITX1 mRNA as wild-type RHAU. These results indicate that the RSM is not solely mediating the interaction between the PITX1 mRNA and endogenous RHAU. Therefore, the interaction between RHAU and PITX1 is mediated via an alternative protein–RNA interaction domain in RHAU or is indirect and mediated via protein–protein interactions between RHAU and an unknown RNA binding protein. The precise mechanism of quadruplex-independent interaction of RHAU in this region is currently the subject of further study.The RSM is a well-defined RNA interaction motif within the RHAU protein that confers quadruplex binding specificity and does not exhibit affinity for single- or double-stranded RNA ,14. To t53–105). The PITX1 mRNA was efficiently immunoprecipitated from HEK293T cell lysates with 6×His-RHAU53–105, as was the hTR RNA. This is in contrast to the negative control mRNA GAPDH that demonstrated no appreciable binding to RHAU53–105. To further examine the putative quadruplex-forming regions within the context of the full-length mRNA, we performed RNA-immunoprecipitations with a 6×His-labelled recombinant RHAU truncation consisting solely of the minimal region that exhibits high and specific affinity for quadruplex RNA  as compared with hTR (∼40-fold), whereas immunoprecipitation of full-length RHAU demonstrates the opposite trend (∼20-fold enrichment of PITX1 and ∼100-fold enrichment of hTR). This manded RNA . In ligh53–105 despite that RHAU53–105 is not capable of displacing endogenous RHAU, it raises the possibility of increased quadruplex stability within the PITX1 mRNA in the absence of the helicase activity of RHAU. This supports a probable role for RHAU at the 3′-UTR as a quadruplex-specific helicase.As RHAU knockdown enhanced enrichment of the PITX1 mRNA by 6×His-RHAUThe results obtained demonstrate that the PITX1 mRNA contains quadruplex-forming sequences that are capable of interacting with the RSM both in cell lysates and in electrophoretic mobility shift assays. As the endogenous RHAU protein can bind to the mRNA in a quadruplex-independent manner, the possibility is raised that RHAU functions to unwind quadruplexes within the 3′-UTR even though they are not the principal mechanism by which the protein is bound. This would prevent dissociation of RHAU on quadruplex unwinding, allowing the mRNA to exist in a dynamic state with the helicase activity of RHAU remodelling the 3′-UTR to toggle between quadruplex and single/double-stranded RNA, thereby regulating binding of factors dependent on the particular fold that the RNA adopts.Although the precise mechanism by which RHAU binds to and functions at the 3′-UTR of PITX1 has not yet been fully elucidated, the functional consequence of RHAU knockdown is an elevation of PITX1 protein levels. This result can be rescued by overexpression of an siRNA-resistant RHAU construct and is consistent across several cell lines tested. The elevation in protein levels in MCF-7 and HEK293T cells peaks at 72 h, whereas in HeLa cells the elevation occurs at 48 h and is returned to baseline by 72 h. This delayed response is likely due to the time between siRNA transfection and efficient decreases in the corresponding protein expression, a factor dependent on both the basal expression and half-life of the RHAU protein. The PITX1 mRNA steady-state level is only marginally increased on RHAU knockdown, suggesting the increases in PITX1 protein expression is primarily a result of enhanced message translation. Although previous studies have demonstrated RHAU-dependent changes in protein expression at the level of gene transcription and by modification of mRNA stability, this is the first study to support the possibility of RHAU-dependent translational regulation of an mRNA ,6,20.As the binding site for RHAU resides in the 3′-UTR of the PITX1 mRNA, and as a recent report demonstrated RHAU involvement in miRNA trafficking , we inveThe interplay between RHAU and miRNA machinery components appears complex and is currently the subject of further study. The data presented in this report establish a probable link between RHAU and RNA interference pathways, but our understanding of the mechanisms underlying this link remains preliminary. A recent report identified RHAU as a specific binding partner of the miR-134 microRNA and demonstrated a role in microRNA trafficking in neuronal cells . This raThis work identified a novel quadruplex-containing mRNA binding partner for the quadruplex-specific helicase RHAU. Unexpectedly, RHAU was discovered to bind independent of the quadruplex-forming regions of the mRNA and the quadruplex interacting motif of the protein. A direct consequence of RHAU knockdown by siRNA is observed in that PITX1 protein levels are elevated through translational regulation, mRNA stability or combination of the two factors. Although significant work lies ahead in defining the particular mechanism involved in both the recognition of the RNA by RHAU and the role RHAU plays in translational regulation, this study establishes a firm basis for future studies of the role of quadruplexes and quadruplex helicases within mRNA 3′-UTRs. Further studies of both PITX1 and other novel RHAU targets will paint a clearer picture of the particular RNA–protein and protein–protein interactions that facilitate the observed biological consequences of quadruplexes and their interacting proteins. Expansion of our knowledge of RHAU and quadruplex-mediated gene regulation will strengthen the platform for the development of quadruplex-targeted therapeutic strategies.Supplementary Data are available at NAR Online.Canadian Insitutes of Health Research (CIHR)/Manitoba Health Research Council (MHRC) regional partnership program. MHRC post-doctoral fellowship (to E.B.). Canada Research Chair program (to J.S.). University of Manitoba undergraduate student research award (to R.H.). University of Manitoba Graduate Fellowship and Manitoba Graduate Scholarship (to S.D.). Faculty of Graduate Studies at the University of Manitoba . Funding for open access charge: Canadian Institutes of Health Research [RPA-118069].Conflict of interest statement. None declared.