We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a ‘gate’ that prevents cellular RNAs from generating productive complexes that can signal.RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5′ triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5′-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high  RIG-I like receptors, found in most cell types, are cytosolic sensors of viral RNAs. These receptors include RIG-I, MDA5 and LGP2, which are superfamily 2 RNA helicases/ATPases with the ability to recognize specific RNA features and trigger an immune response against a wide variety of commonly found RNA viruses –5. RIG-IThe current model of RIG-I activation is based on three key crystal structures ,24,25. TKd) and the ATPase turnover rate (katpase) of RIG-I complexes with blunt-ended and non-blunt ended dsRNAs. We correlate these parameters to the ability of the RNA molecule to stimulate cell-based signaling activity and find a good correlation between signaling and katpase/Kd ratio and Kd, but not with the katpase. Non-blunt-ended dsRNAs without 5′ppp and with a 5′-overhang have the lowest RNA affinity and ATPase turnover rate and signal poorly whereas 3′-overhang is not as deleterious. By carrying out studies with full-length RIG-I (referred to as RIG-I from here on), RIG-I domains and interface mutant, we show that CARD2-Hel2i interface plays a key role in RNA selection by acting as a ‘gate’ to allosterically regulate both RNA affinity and ATPase activity. Thus, the molecular switch used in autoinhibition also governs RNA selection.In this study, we use fluorescence and ATPase-based RNA binding assays to quantify the RNA equilibrium dissociation constant  were deprotected and resuspended in 20 mM potassium phosphate buffer pH 7.0. The RNA concentration was determined in 7M guanidine HCl using the NanoDrop spectrophotometer at And CARD-Helicase-RD (97–925), CARDs (1–228) were overexpressed in E. coli strain Rosetta (DE3) (Novagen) as soluble proteins. The isolation of pure proteins involved three chromatographic steps: affinity column , hydroxyapatite column  and heparin sepharose column . An additional gel-filtration chromatography step  was added to purify RIG-I. Purified helicase-RD was further dialyzed overnight at 4°C into 50 mM HEPES pH 7.5, 50 mM NaCl, 5mM DTT, 10% glycerol, snap frozen in liquid nitrogen and stored at –80°C as reported previously (E. coli BL21 Star (DE3) cells and the soluble fraction was purified to homogeneity using a Ni2+affinity column, cation exchange  and gel filtration chromatography.All the RIG-I constructs were sub-cloned into a modified pET28b vector with N-terminal SUMO fusion. Human RIG-I (1–925), Helicase-RD (232–925), and 2eviously . The RD The double mutation in RIG-I gene was introduced using the QuikChange II XL site-directed mutagenesis kit from Agilent Technologies. The mutagenic primers used were:5′-GGAGTATAGATTACTTTTAAAAGCTGCACAACCAGAATTTAAAACC-3′ (Forward)5′-GGTTTTAAATTCTGGTTGTGCAGCTTTTAAAAGTAATCTATACTCC-3′ (Reverse)Purification of the mutant was carried out using the same protocol as RIG-I.2, 5 mM DTT, 0.01% Tween 20 (γ-32P]ATP and RNA substrates (1 μM) in Buffer A at 15°C or 37°C. The reactions were stopped at desired time points using 4N HCOOH and analyzed by PEI-Cellulose-F TLC (Merck) developed in 0.4 M potassium phosphate buffer (pH 3.4). The TLC plates were exposed to a phosphorimager plate, which was imaged on a Typhoon phosphor-imager and quantified using ImageQuant software. The ATPase turnover rate was determined from the plots of [Pi] produced versus time and dividing the ATPase turnover rate by the respective enzyme concentration.The ATP hydrolysis assays were performed in 1X Buffer-A at 15°C unless otherwise mentioned. 1X Buffer A: 50 mM MOPS-Na (pH 7.4), 5 mM MgClTween 20 . The ATProbs) was plotted as a function of protein concentration (Pt) and fit to Equations .Fluorescence anisotropy measurements were carried out using FluoroMax-4 spectrofluorimeter (Horiba JobinYvon) in Buffer A as previously described . Fluoresquations  and 2)  robs) waKd values were consistently observed in titrations repeated 2 to 3 times.The reported RNA γ-32P] ATP). A time course (0–60 min) of the ATPase reactions was performed in Buffer A at 15°C or 37°C and the initial velocities were plotted as a function of time to obtain the ATPase rate. The error bars were obtained from the fit of the time-course of the ATPase reactions. The quenched reactions were analyzed as indicated above to obtain the ATPase turnover rates that were plotted as a function of RNA concentration and fitted to hyperbolic equation (The ATPase turnover rate was measured at constant protein 5 nM or 25 nM) and increasing RNA (0.1 nM–2 μM) in the presence of 1 mM ATP (spiked with γ-2P ATP). 5 nM and 4 cells/well density and transfected with each of the RNA ligands  or Poly I:C (700 ng/well) using Lipofectamine transfection reagent (Life Technologies). After 20 h the activities of firefly and Renilla luciferases were measured sequentially with the Dual-Luciferase reporter assay kit (Promega). Data were collected in quadruplicate sets and the relative luciferase activities were calculated. The error bars represent the standard error of the mean (SEM).HEK293T cells were grown in 6-well plates to 60% confluence and co-transfected with firefly luciferase reporter plasmid (2.5 μg), Renilla luciferase reporter plasmid (500 ng) and a plasmid carrying the wild type (wt) RIG-I gene under CMV promoter or an empty plasmid (2 μg). The firefly luciferase gene is under the interferon β promoter and the Renilla luciferase plasmid is under the constitutively active TK promoter. The plasmid transfections were carried out with X-tremeGENE HP DNA Transfection Reagent (Roche). Cells were re-plated in 96-well plates the next day at 2 × 10in vitro transcribed RNAs with questionable RNA-ends , the C-terminal repressor domain (RD) and the CARD2-Hel2i interface mutant , which was estimated by fluorescence anisotropy/intensity based titrations that are non-disruptive, providing accurate Kd values, especially of weak complexes. The presence of fluorescein does not perturb binding to RIG-I . The 5′-end fluorescein labeled dsRNA (with 5′ppp or 5′OH at the other end) was titrated with increasing concentration of RIG-I . Replacing 5′ppp with 5′OH results in a ≈15-fold lower affinity (Kd = 6 ± 0.4 nM) . The KdIn vivo, RNA binding occurs in the presence of ATP; hence, we measured the RNA binding under ATPase cycling conditions by measuring the ATPase turnover rate as a function of increasing RNA concentration . The ATPase turnover rate versus RNA concentration curves fit well to the 1:1 (RIG-I:RNA) binding stoichiometry  to the on-rate (6 × 108 M−1s−1) yielded a Kd of 2.5 pM for 5′ppp RNA . Thus, deletion of CARDs increases the affinity of RIG-I for blunt-ended dsRNAs by 150–250-fold, indicating that the CARDs regulate RNA affinity. We found that adding CARDs in trans to the Helicase-RD weakened RNA affinity by about 2-fold .The CARDs are responsible for interacting with the downstream adapter proteins to relay the signal, however their role in RNA discrimination is not known. Deleting the CARDs (Helicase-RD) increases the binding affinity for both 5′ppp and 5′OH blunt-ended dsRNAs. Data fitting to the quadratic equation provided A Figure , but theKd,app values of non-blunt ended complexes range from ≈0.1 nM for 5′ppp and 3′- and 5′-ovg RNA complexes and ≈1.5 nM for 5′OH and 3′- or 5′-ovg RNA . Similarly, ssRNAs binds to the Helicase-RD with Kd,app of ≈1600 ± 500 nM for 5′OH ssRNA and ≈500 ± 100 nM for the 5′ppp ssRNA but does not bind to RIG-I . These results indicate that the CARDs are preventing RIG-I from interacting with the non-blunt ended dsRNAs and ssRNAs. Interestingly, the ratio of the Kd values of RIG-I and Helicase-RD shows that the presence of CARDs is critical for discriminating against binding of dsRNAs with 5′ovg and stem RNAs .Surprisingly, the Helicase-RD construct of RIG-I binds to all non-blunt ended dsRNAs with a high affinity in the sub-nanomolar range Figure . The Kd,The duck RIG-I structure shows that CARDs are interacting with Hel2i when RIG-I is not bound to RNA and ATP . To inveKd,app of complexes of CARD2-Hel2i interface mutant R109A/L110A with blunt-ended and non-blunt ended dsRNA using the ATPase based assays , indicating that a disruption the CARD2-Hel2i interface results in a loss of discrimination against 5′OH RNA. The CARD2 interface mutant also binds to non-blunt-ended dsRNAs with 1.5- to 4.6-fold higher affinities relative to wild type RIG-I . Interestingly, the ATP Km values do not show a clear trend and remain essentially constant (Supplementary Table S3). The lower ATPase turnover rates of RIG-I with non-blunt ended RNAs is not due to weak RNA binding, because these reactions were carried out at 1 μM dsRNA concentration, which is well above the Kd values of these complexes.Although the exact role of RIG-I's ATPase activity is not known, its importance in mediating RIG-I's signaling function is well documented. Mutations that abrogate or compromise RIG-I's ATPase function lead to defects in their signaling activity –32. We fkcat, and the kcat/Km values of the Helicase-RD are almost the same with blunt-ended and non-blunt-ended dsRNAs, differing only by about 2-fold . The 10-bp dsRNAs did not induce signaling possibly because of the instability of short dsRNAs at 37°C conditions of the cell-based assays; therefore, we prepared 23-bp dsRNAs of the same sequence and containing the same end-modifications as the short dsRNAs. To prevent a second RIG-I molecule from binding to the dsRNA at the other end, we introduced three base pairs of dsDNA at one end, since RIG-I is not capable of binding DNA. We used ATPase measurements at 37°C to measure the The blunt-ended 5′ppp RNA showed the highest signaling activity, followed by the 5′OH RNA and 5′ppp 3′ovg RNA whose signaling activity is only ≈2-fold lower relative to the 5′ppp RNA Figure . The reskatpase, Kd,app and katpase/Kd,app of the 23-bp dsRNA against the signaling activity. We find no correlation between signaling and ATPase turnover rate . This is interesting, because it has been suggested that the role of ATPase activity is to facilitate active dissociation of RIG-I from non-PAMP RNAs, thus preventing RIG-I from forming signaling competent complexes with such RNAs   . Under our cell-based assays, only RNAs between 1 and 10 nM Kd,app and 1–20 × 109 M−1 s−1katpase/Kd,app show significant signaling activity  Figure  as well ty Table . Thus, RActivation of RIG-I is an essential step in establishing an antiviral response in the cell. Equally important is assuring that RIG-I is activated selectively by non-self viral RNAs and not by self RNAs present in the cytoplasm. Insights into the molecular mechanism for how RIG-I selects non-self dsRNA ends from self were obtained by studying the binding affinity and ATPase activity of dsRNA with various end-modifications using the full-length RIG-I and different domain constructs of RIG-I. Our studies show that all the domains of RIG-I play a role in RNA selection. The C-terminal RD has high affinity for blunt-ended dsRNAs with and without 5′ppp, compared to non-blunt ended RNAs, and aids in RNA selection. The helicase domain provides additional binding sites for dsRNA and is critical for high affinity binding of 5′ppp dsRNAs carrying 3′-overhang. Most interestingly, CARDs regulate RNA affinity and ATPase turnover activity; even though, there is no evidence that CARDs interact with RNA.versus non-self RNA selection.The regulation by CARDs involves an allosteric mechanism whereby interactions between CARDs and Hel2i subdomain stabilizes the autoinhibited state of RIG-I, which is not efficient in RNA binding and ATPase activation. Deletion of CARDs increases both the RNA binding affinity and the ATPase activity, such that all types of RNAs including non-blunt-ended dsRNAs bind tightly and stimulate the ATPase rates of the Helicase-RD. This indicates that in the autoinhibited state, CARDs are selectively preventing binding of ssRNAs, dsRNA stem structures and dsRNA with 5′-overhang, but not hindering binding of blunt-ended dsRNA and dsRNA with 3′-overhang. The data also imply that once CARDs are released from its interactions with the helicase domain, RIG-I binds with a high affinity to all types of RNAs. Deleting CARDs drastically reduces RNA selectivity for blunt-ended 5′ppp dsRNA over non-blunt ended dsRNAs from ≈3000-fold to ≈150-fold. Similarly, RNA selectivity is reduced from ≈3000-fold to ≈750-fold when the CARD2-Hel2i interface is mutated. Thus, the CARD2-Hel2i interface known for its role in autoinhibiting RIG-I, is also involved in self Kd, but this could be tight enough to form an initial complex to aid in the search for the correct RNA end by translocation . The helicase-RD contains a pore with basic amino acids at the interface between the RD and Hel1 domains, where the 3′ovg was accommodated with only minor additional protein rearrangements . This explains why siRNAs containing 2-nt 3′-ovg and 5′ppp are able to activate RIG-I  and why location . This fllocation ,43. Similocation .versus non-self RNA selection by RIG-I , which initiates the intrinsic apoptotic signaling pathway ,51. The Supplementary Data are available at NAR Online.


This requires a scalable methodology for sensing, transmission, and actuation, which could be assembled into larger signaling networks. Here, we present a biochemical strategy to design RNA-mediated signal transduction cascades able to sense small molecules and small RNAs. We design switchable functional RNA domains by using strand-displacement techniques. We experimentally characterize the molecular mechanism underlying our synthetic RNA signaling cascades, show the ability to regulate gene expression with transduced RNA signals, and describe the signal processing response of our systems to periodic forcing in single live cells. The engineered systems integrate RNA–RNA interaction with available ribozyme and aptamer elements, providing new ways to engineer arbitrary complex gene circuits.Organisms have different circuitries that allow converting signal molecule levels to changes in gene expression. An important challenge in synthetic biology involves the  Natural signal transduction systems allow organisms to adapt to fluctuating environments, often by exploiting subcellular localization, molecular cascades and protein allostericity ,2. A majtrans-acting riboregulator. This corresponds to a four-molecule system, where the first one is the signal molecule, either a small molecule or sRNA, and the last one is a cis-regulated mRNA as system's readout. The other two molecules (two components) correspond to the sensor and mediator, which can be switched ON/OFF in presence/absence of the signal molecule, respectively.In this direction, pioneering work in synthetic biology inserted known aptamer domains into 5′ untranslated regions (UTRs) of messenger RNAs (mRNAs) to sense small molecules , and alsThe devised system shares properties with natural signaling systems . On the de novo design without automation. We have previously demonstrated that an automated design methodology is able to generate de novo riboregulation in live cells  with gene expression. We will first describe the development of a methodology for nucleotide sequence design, and then we will present a mechanistic characterization to assess the self-cleavage activity of the regazyme. Subsequently, we will show results assessing signal transduction with time-dependent induction in bacterial cells, allowing the characterization of the dynamic regulatory properties at both population and single cell levels.We developed a Monte Carlo Simulated Annealing  optimizassrA degradation tag  and then subcloned into plasmids pSTC1 or pSTC2. These two plasmids contain a pSC101m replication origin (a mutated pSC101 ori giving a high copy number) and a kanamycin resistance marker . The pSTC2 vector is based on our previously reported vector pSTC1  by removtion tag . Dysfunc32P]-labeled RNA probe after separating the different RNA samples by denaturing polyacrylamide gel electrophoresis (PAGE). RNA preparations were mixed with formamide loading buffer for denaturation, followed by PAGE separation in 5% polyacrylamide gels including 8 M urea and TBE buffer. Gels were stained with ethidium bromide. Membranes were hybridized overnight, imaged by autoradiography, and then hybridization signals quantified by phosphorimetry (Fujifilm FLA-5100). See more details in Supplementary Materials and Methods.Processing extent of regazyme at different time points  was analyzed by northern blot hybridization using a complementary [Cells were grown overnight in LB medium, and were refreshed in culture tubes with LB medium in order to reach stationary phase. Cells were then diluted 1:200 in 200 μl of M9 minimal medium in each well of the plate (Custom Corning Costar). The plate was incubated in an Infinite F500 multi-well fluorometer (TECAN) at 37°C with shaking. It was assayed with an automatic repeating protocol of absorbance measurements (600 nm absorbance filter) and fluorescence measurements (480/20 nm excitation filter – 530/25 nm emission filter for sfGFP) every 15 min. All samples were present in triplicate on the plate .et al.  , which wet al. . All imaOur modular strategy consists in designing switchable functional RNA domains, which is implemented by exploiting strand-displacement principles together with the engineering of allosteric conformational states. This way, we can design chains of several domains that are activated in cascade. Without loss of generality, we considered a system composed of two transcriptional units: regazyme and mRNA of a reporter gene (e.g. a gene coding for a green fluorescent protein − GFP), but our methodology could be generalized to an arbitrary number of transcriptional units containing switchable functional elements. To engineer such a synthetic RNA system implementing the transduction of molecular signals into changes in gene expression, we took advantage of a standard physicochemical model (based on Watson-Crick and wobble pairing) predicting RNA secondary structure and free energy  to be uscis-repressed ribosome-binding site − RBS), and actuator (mRNA with cis-repressed RBS) , mediator  Figure . To crea) Figure . The sen) Figure . To be nWe here propose a new methodology to engineer one-to-two-component signal transduction, which combines the advantages of subcellular independence of one-component systems and of modularity of two-component systems. We constructed a combinatorial optimization problem to explore the sequence space of the transducer module Figure , where aAs shown by a random sampling of 1000 sequences Figure , an optiet al. (RAJ11 and RAJ12)    and one . (RR12)   of the solution space for a particular couple of aptazyme and riboregulator. We expect, nevertheless, a higher designability when sequences are allowed to vary in length, as it is the case for our algorithm. It is instructive to align multiple solutions to reveal conserved nucleotide positions. Figure To analyze the mechanism of the signaling pathway, we first carried out a kinetic and dose-dependent study of the catalytic activity. The predicted secondary structure of the small-molecule-sensing regazyme theoHHAzRAJ12 in the uncleaved state Figure  shows, a−1 for theoHHAzRAJ12 with 4 mM Theo, although the model does not capture finely the experimental trend . According to previous experimental results in vitro without RNA production and degradation . The maximal cleavage fraction reported here is almost 25% , which shows a big discrepancy with those previous in vitro results (∼75%). One possible explanation is that the expression of the sRNA with 100 ng/ml aTc does not saturate the system, because the regazyme is expressed from a strong constitutive promoter, and also because of a high effective dissociation constant. Of relevance, this regazyme has much lower leakage , which could be important in case of sensitive systems. Moreover, we observed higher heterogeneity in the dynamic response (from cell to cell) for this sRNA-sensing regazyme, which could be a result of a heterogeneous expression of the sRNA or even of a certain heterogeneous sRNA–regazyme interaction. As a result, by predicting RNA structures and quantifying cellular RNA extracts, we have shown the precise signal sensing and subsequent cleavage to release a functional riboregulator.To monitor the dynamic RNA processing of the system, we performed a gel assay from cellular RNA extracts. Cells expressing regazyme theoHHAzRAJ12 or breakHHRzRAJ12 were induced with different concentrations of Theo or aTc and lysed at several time points. The gel assays in both cases showed fast dynamic RNA processing, reaching steady states in almost 16 min. The observed cleavage rate  is 0.15 minradation , the obsin vitro  revealedin vitro transcription of systems theoHHAzRAJ12 and breakHHRzRAJ12. The experiments showed similar cleavage fractions (with respect to the in vivo assays) after 30 min of reaction . For theoHHAzRAJ12, 70% of the molecules were cleaved in vitro, whereas 80% were in vivo. For breakHHRzRAJ12, 25% of the molecules were cleaved both in vitro and in vivo. We also observed that theoHHAzRAJ12 was cleaved in higher extent in absence of ligand. Because in vitro we can neglect degradation, the cleavage fraction is expected to increase with time, in presence of ligand and also in absence of it due to the basal activity of the ribozyme. We further performed a time-course assay to study the cleavage of the regazymes. As shown in our experimental results, the fraction of cleaved products of theoHHAzRAJ12 in test tubes was only ∼10% larger when Theo was present . However, when the same RNA was monitored in bacterial cells, the apparent level of induction of cleavage activity by theophylline was >2.5-fold. The difference in cleavage in vitro in case of breakHHRzRAJ12 with respect to the presence or not of Break1 (introduced as DNA oligo) was more remarkable . There is certain number of reasons why an RNA might exhibit different behaviors in vitro than in vivo. For example, in vitro the regazyme might exist in thermodynamic equilibrium with its ligand, resulting in a different effective dissociation rate, or differ slightly in length from the strands in vivo. In addition, in vitro we used T3 polymerase for transcription (without terminators) instead of Escherichia coli polymerase and a higher Mg2+ concentration than in vivo, which might result in differences in folding and cleavage kinetics of the regazyme. In the following, we present the net effect of regazyme cleavage and riboregulator release on GFP expression in vivo (both at the population and single cell levels) with and without the ligand, showing a regulatory behavior as designed.To further confirm that our devices behave as expected, we performed d-thiogalactopyranoside (IPTG) and aTc in E. coli cells expressing constitutively the repressors LacI and TetR. Thus, our systems implement multi-input AND logic circuits  by using a dysfunctional mutant removing the regazyme element P different implementations of circuits with P regazymes, including cascades and feedback loops .All together, these results demonstrate the modularity of our designs: (i) the same sensor module can transduce the signal to different mediators (riboregulators), and (ii) the same riboregulator (RAJ12 in this case) can be coupled with different sensor modules. We also analyzed experimentally the orthogonality between regazymes. To this end, we constructed new genetic systems based on non-cognate pairs (between riboregulators and 5′ UTRs), in order to test pression . Computapression . Howeverpression  and thenTo characterize the dynamic response of the designed regazymes at the single cell level, we constructed microfluidics devices according to previous work ,37. TherBy collecting all single cell measurements, we can analyze the response of the population and its heterogeneity (quantified as the coefficient of variation − CV − in the state ON). The fluorescence is significantly shifted in all cases after addition of the inducers Figure . Then, wcis-repressed gene, which may allow the sensing and regulation in different subcellular localizations.We have developed a new kind of synthetic RNA molecules (here termed regazymes) able to transduce signals in a modular way in live cells, introducing the one-to-two-component signal transduction paradigm Figure . The desThe use of an optimization algorithm, relying on intra- and intermolecular structures and their corresponding free energies as model variables, has allowed us to solve the design problem of the fusion between RNA-based sensors and regulators. However, the computational methods used here to predict free energies and conformational states do not consider three-dimensional contacts neither intermolecular contacts arising in cellular environments , which partly limits the predictability of the performance of our designs. By exploring the sequence space, we have identified ideal regions to design functional regazymes without affecting the stability and specificity of the aptamers and riboregulators. The three energetic terms for deriving the objective function have proved effective to capture the functionality of the system Figure . AlthougNucleotide-level energy models, such as the one presented in this work, can provide the underlying explanations for molecular interactions, which can then be used in the construction of higher-level biological systems . Our stuin vitro with the same ribozyme core . One example would consist in exploiting as mediator module a sRNA able to guide the CRISPR-associated catalytically inactive dCas9 protein to block transcription initiation or elongation . ThiSupplementary Data are available at NAR Online.


Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus–infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, -1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP’s RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus. Influenza is an infectious disease of birds and mammals caused by the influenza viruses belonging to the family Orthomyxoviridae1456Influenza A virus nucleoprotein (NP) is a major virion structural protein that has been predicted to interact with negative-strand viral RNA during viral nucleocapsid formation11et al. have been reported that the tail loop binding pocket as a potential target for antiviral developmentet al. reported that several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficientlyA substantial quantity of RNA is wrapped around each NP monomer, with a stoichiometric ratio of 20 nucleotides of RNA per 1 NP1416Accordingly, we proposed that the surface of the groove, which contains numerous conserved residues  is able to interact with the RNA residue . In this4-3′, was used as a probe in the SPR experiments. The dissociation constants, KD (kd/ka), for the various NP and RNA complexes were obtained from kinetic analyses of SPR experiments , indicating the side chain of Y148 increase RNA residence times in NP  of the purified WT and mutant NPs using CD in which the absorbance at 220 nm was analyzed as a function of temperature (Tm of approximately 0.7 to 6.2 °C (from 67.3–72.8 °C), indicating that Y148 contributes significantly to the structural stability of the NP. Compared to WT and other mutant NPs, Y148F had a lowest Tm value. The results indicate that deletion of the tyrosine hydroxyl would significantly reduce the stability of NP, suggesting tyrosine hydroxyl is required for proper NP folding.To examine the thermodynamic stabilities of H1N1 WT and mutant NPs including Y148F, Y148S, Y148R, and Y148A, we measured the melting temperatures  than other mutant NP-RNA complexes  and dissociation constants (kd), respectively, and the dissociation rate constant (KD) to determine the effects of these four compounds on the RNA-binding affinity toward NP. In the presence of compounds under saturation conditions, the affinity of NP bound to RNA (KD = 19.3 nM) was higher than those for the other compounds. The NP exhibited weaker RNA binding in the presence of H7, with a 2-fold increase in the dissociation constant. The KD values for H1, H3, and H5 were 10.4, 9.7, and 11.7 nM, respectively, which are at the same level as the KD without drug  on the RNA-binding capacity of NP by SPR experiments. Only H7 decreased the RNA-binding capacity of NP by more than 10% . We alsod to RNA  for H7  of 10.9 μM.To determine if H7 interacts with NP of the influenza A virus, we assessed the effects of H7 after an early step involved in virus replication. NP associates with viral RNA to form a helical nucleocapsid after the first 2 h. We performed a time-of-addition experiment to detect the viral RNA and viral protein synthesis in virus-infected A549 cells. Different concentrations of H7 were added to A549 cells after virus infection (T2: 2 h after infection), and culture supernatants were collected to determine the viral RNA after one viral replication cycle. The matrix protein 1 (M1) viral RNA expression levels during replication were normalized based on the vehicle control (same volume of DMSO added at T2). As shown in 50) value was >27.48 μM. Overall, the time-of-addition experiment indicated that H7 exerts its antiviral effects through RNA-binding capacity with NP after the viral absorption stage.We next investigated whether H7 inhibited viral RNA synthesis in virus-infected A549 cells, which would also reduce viral protein synthesis. The expression level of NP in the influenza virus–infected cells as compared to the viral control was reduced by 20% upon H7 treatment in the later steps of the viral cycle. As shown in NP is the most abundant RNA-binding viral protein in influenza virus–infected cells and is responsible for recognizing RNA and forming a filamentous nucleocapsidKd values of WT, R184A, R214A, and R236A were similar, a finding that is consistent with previous studies showing that the single amino acid substitutions at R184A, R214A, or R236A had no effect on viral-genome replication and transcriptionka) for RNA binding to most of the mutants was decreased significantly, except that the Y148R mutant is similar to that for WT, suggesting that Y148R may participate in RNA-binding of NPs via electrostatic interactions.In this study, we investigated the mechanism of influenza virus NP bound to single-stranded RNA. Using SPR, we explored the effect of single amino acid substitution at 13 hot spots on the surface of the groove between the head and body domains on the RNA-associated properties of NP, including the kinetic behavior of NP bound to RNA, by evaluating the SPR association and dissociation phases. The association phase mainly reflects the entry of RNA into the NP groove, whereas the dissociation phase measures the hydrogen bonding environment within the groove that accommodates the RNA molecules. The structure of the full-length N protein should be taken into consideration in our studies. Moreover, in gel filtration we obtained and checked the uniformity of the purified construct NP. In addition, in the SPR experiment we designed the 28nt in length RNA oligomer, and the length of RNA oligomer can be bound by one protein molecule. During data analysis, we fit the SPR sensorgram of the wild and mutant NP bound to RNA by using 1:1 Langmuir model. We showed that the dissociation rate of NP from the single-stranded RNA increased in all 13 mutants, while the presence of Y148A, R150A, R152A, R156A, R174A, R175A, R195A, R199A, R213A, and R221A mutants significantly decreased the rates of association between NP and RNA, suggesting these 10 residues play a crucial role in RNA binding. Interestingly, the No structural data were available regarding influenza virus NP binding to single-stranded RNA. Thus, to understand the structural interactions responsible for the RNA recognition by influenza virus NP, we modeled the structure of influenza virus NP in an RNA-bound state . This moet al. identified a small-molecule compound called nucleozin that can trigger oligomeric NP aggregates and inhibit their nuclear entryet al. identified several nucleozin analogues that can inhibit the replication of the influenza virus in MDCK cells by inducing the severe aggregation of NP molecules and by preventing RNP formation during the production of viral particleset al. discovered a series of influenza replication inhibitors that interfere with NP-dependent processes via formation of higher-order NP oligomerset al. reported that some small molecules targeting E339…R416 from virtual screening were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozin-resistant strains21et al. discovered naproxen, which interferes with the RNA-binding activity of the influenza virus NP and inhibits viral titersCurrent antiviral drugs developed to treat influenza virus infections primarily target the viral M2 channel and neuraminidase. However, the use of M2 inhibitors, such as amantadine and rimantadine, has been limited by the propensity of these drugs to cause central nervous system side effects and the rapidly increased number of drug-resistant viral strains27According to our mutational analyses of the influenza virus NP, the tyrosine residue on NP (Tyr148) appears to interact with RNA bases via stacking and hydrogen-bonding interactions and to play a crucial role in protein stability. Docking results suggest that several hits can bind to Y148 of the NP-NTD’s groove using virtual screening. Using fluorescence titration and an SPR assay of the NPs, we identified one potential natural compound, curcumin (H7), that targets Y148 of influenza virus NP and potently interferes with its RNA-binding activity. We also found that H7 could inhibit influenza virus replication and the efficacy of H7 is relatively diverse. Previous studies demonstrated that curcumin exist no or low cytotoxicity in normal cellsNdeI and XhoI, and the DNA fragments were cloned into pET21b (Novagen) using T4 ligase (New England Biolabs). DH5α competent cells that were transformed with the resultant plasmid were grown in culture. Protein expression was induced by supplementing the culture media with 1 mM IPTG, followed by incubation at 10 °C for 24 h. After the bacteria were harvested by centrifugation (6000 × g for 15 min at 4 °C), the bacterial pellets were lysed with lysis buffer in the presence of RNase . The soluble proteins were isolated from the supernatant following centrifugation  to remove the precipitate. The recombinant NPs carrying an N-terminal 6×His-tag were purified using a Ni-NTA column (Novagen) with an elution gradient that ranged from 15 to 300 mM imidazole. The pure fractions were collected and dialyzed against the buffer that lacked imidazole. Purified NPs (with greater than 95% purity) were analyzed by gel filtration with Superdex 200  and Coomassie blue staining (To generate full-length wild-type (WT) and mutant NPs encoded by the A/TW/12/2001 (H1N1), NP genes were amplified with the polymerase chain reaction (PCR) using various primers. The PCR products were digested with staining . The pro4-3′, was used as a probe in our SPR experiments. Experiments were conducted by injecting NP at different concentrations in 50 mM Tris (pH 7.5) with 150 mM NaCl (Sm) between NP and RNA, based on the equation,The affinity, association, and dissociation between the NPs and RNA were measured using a BIAcore 3000A surface plasmon resonance (SPR) instrument  equipped with a SA5 sensor chip from Pharmacia. The repeated sequence, 5′-bio(UCCAAAC) mM NaCl . We calcL [RU] = Immobilization level, Rmax [RU] = Maximum binding response,RA [Da] = Molecular weight of analyte, and MWL [Da] = Molecular weight of ligand.MWm values of various NPs were calculated to be 0.8 ~ 1.3. Thus, we can ensure that the binding stoichiometries of RNA to various NPs are 1 to 1 and compare the effect of NP mutations on the RNA-binding activity of the protein. The sensorgrams were fit to the 1:1 Langmuir model using the BIAevaluation software (version 3) to determine the association and dissociation rate constants .The SThe circular dichroism (CD) spectra were obtained using a JASCO-815 CD spectropolarimeter. The temperature was controlled by circulating water at the desired temperature in the cell jacket. Each protein was dissolved in 50 mM Tris-HC (pH 7.3) and 150 mM NaCl. The CD spectra were collected between 250 and 190 nm with a 1-nm bandwidth at 1-nm intervals. All of the spectra were obtained from an average of five scans. The photomultiplier absorbance did not exceed 600 V during the analysis. The CD spectra were normalized by subtraction of a background scan with buffer alone or RNA alone. The mean residue ellipticity, [θ], was calculated based on the equationMRW is the mean residue weight, θλ is the measured ellipticity in millidegrees at wavelength λ, l is the cuvette path length (0.1 cm), and c is the protein concentration in g/mL. In addition, the Tm was determined from the polynomial fitting of the observed curve and taken as the temperature corresponding to half denaturation of the NP. The first derivative of the absorption with respect to temperature, dA/dT, of the melting curve was computer generated and used to determine the Tm.where The small molecules tested were primarily obtained from an in-house collection of compounds and were included for testing of NP inhibitors. In the drug-induced fluorescence change experiment, a final concentration of 4 μM NP was added to a buffer that contained various concentrations of compound, and the samples were incubated at 25 °C for various durations. The buffer consisted of 50 mM Tris (pH 7.5) and 100 mM NaCl. The tryptophan fluorescence was measured using a Hitachi F-4500 fluorescence spectrophotometer that was equipped with a cuvette with a 1-cm light path. The excitation wavelength was 288 nm, and the emission data were collected between 300 and 400 nm. All static measurements were recorded in triplicate. The relative fluorescence titration intensity was determined using the following equation:FLNP is the NP fluorescence in the absence of a test compound and FLNP-drug is the fluorescence of the NP-compound complex.where F) from three separate experiments at 3 h after addition of a test compound were averaged and fit with the Hill equation using the GraphPad Prism software program  as follows:To determine the binding constants between a test compound and the various NPs, the compound-induced fluorescence changes , were grown in RPMI1640 medium (Gibco). Madin-Darby canine kidney (MDCK) cells (ATCC accession no. NBL-2) were grown in Dulbecco’s modified Eagle medium (Gibco). All media were supplemented with 10% fetal bovine serum (Gibco), 100 U/mL of penicillin and streptomycin, and 2 mM L-glutamine. WSN viruses were provided by the Clinical Virology Laboratory of Chang Gung Memorial Hospital . Viruses were amplified by using MDCK cells. Virus titer was determined by a plaque assay using MDCK cells.In the time-of-addition experiment, A549 cells were infected with WSN (MOI 1) and incubated in E0 medium; at T2 time points after infection, H7 was added at different concentrations. The cells were rinsed with PBS to remove the drug and culture medium, and the cell lysates were applied to detect the viral RNA and viral protein at 8 h post-infection.5 A549 cells were seeded into six-well plates, allowed to reach confluence, and then challenged with virus (MOI 1). Total RNA was extracted from cells using the TRIzol reagent . Following phenol-chloroform extraction and isopropanol precipitation, the RNA pellet was washed, dried, and dissolved in 20 mL of RNase-free water. The protocol of RT-PCR amplifications and Q-PCR were as described by Hsu et alA total of 5 × 10The cell lysates were collected and lysed with lysis buffer  for detecting the target proteins. Proteins with sample buffer were subjected to SDS-PAGE and electroblotted onto Hybond ECL membranes (Amersham). Blocking and incubation with antibodies were done using 0.05% Tween 20 and in Tris-buffered. Western blot membranes were incubated with 1:10000 dilution of anti-NP rabbit polyclonal Ab .Cell cytotoxicity of inhibitors was determined by MTS assay. A549 cells were grown (5000 cells/well) in 96-well plate for 24 h. The medium was replaced with that containing serial diluted compound after virus infection (T2: 2 h after infection) and the cells were further incubated for 8 h. The culture medium were removed and added 100 μL including MTS and PMS mixture solution, the plate was incubated for 30 min. MTS and PMS are purchased from sigma and prepared in PBS . To identify a 96-well microtiter plate, 2 ml reagent containing both MTS and PMS at the ratio of 20:1 was mixed immediately with 8ml serum-free DMEM. Each drug concentration was performed with four repeats. The optical density was measured at OD490nm in multi-well spectrophotometer (ELISA reader).et al. were used41The crystal structure of the influenza A virus NP (Protein Data Bank code 2IQH) lacks a defined tertiary structure in the regions between amino acid residues 73–91, 397–401, and 429–437. The SWISS-MODEL program was used to model the complete structure of the NP. We used the complete structure of the NP as a template to construct a plausible NP-ssRNA complex using the molecular modelling programs Discovery Studio 2.5 and HADDOCKHow to cite this article: Liu, C.-L. et al. Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development. Sci. Rep.6, 21662; doi: 10.1038/srep21662 (2016).


The chromatin-remodelling complex B-WICH, comprised of William syndrome transcription factor, the ATPase SNF2h and nuclear myosin, specifically activates RNA polymerase III transcription of the 5S rRNA and 7SL genes. However, the underlying mechanism is unknown. Using high-resolution MN walking we demonstrate here that B-WICH changes the chromatin structure in the vicinity of the 5S rRNA and 7SL RNA genes during RNA polymerase III transcription. The action of B-WICH is required for the binding of the RNA polymerase machinery and the regulatory factors c-Myc at the 5S rRNA and 7SL RNA genes. In addition to the c-Myc binding site at the 5S genes, we have revealed a novel c-Myc and Max binding site in the intergenic spacer of the 5S rDNA. This region also contains a region remodelled by B-WICH. We demonstrate that c-Myc binds to both sites in a Max-dependent way, and thereby activate transcription by acetylating histone H3. The novel binding patterns of c-Myc and Max link transcription of 5S rRNA to the Myc/Max/Mxd network. Since B-WICH acts prior to c-Myc and other factors, we propose a model in which the B-WICH complex is required to maintain an open chromatin structure at these RNA polymerase III genes. This is a prerequisite for the binding of additional regulatory factors. Transcription by RNA polymerase III (RNA pol III) in eukaryotic cells is closely associated with cell growth and proliferation. The highly expressed transcripts are short non-coding fragments of RNA that are directly involved in translation, transcriptional elongation and splicing. RNA pol III genes are regulated by nutrients and cellular stress, as well as by proliferative signals generated throughout the cell cycle and differentiation ,2. SeverRecent genome-wide profiling has shown that the chromatin structure in the vicinity of active RNA pol III genes resembles that of active chromatin at RNA pol II genes –20. The To study the role of chromatin in RNA pol III transcription we have investigated the role of chromatin-remodelling complexes in RNA pol III transcriptionr. We report here investigations into the mechanism by which the B-WICH complex, comprised of WSTF (William Syndrome Transcription Factor), SNF2h and nuclear myosin 1 (NM1), activates RNA pol III transcription. We demonstrate that B-WICH remodels the chromatin locally in the vicinity of the 5S rRNA and 7SL genes, and that this affects transcription. Within the 5S rRDA loci, a region in the intergenic spacer (IGS) is remodelled by B-WICH, and this site is required for the acetylation of histone H3. The remodelled region exposes a DNA-binding site, an E-box, for c-Myc-Max dimers. Binding of Myc-Max is part of a wide gene regulatory network, in which the action of c-Myc is repressed by the alternative interaction of Max with Mxd1 at promoters. The finding that B-WICH is required for the binding of c-Myc-Max to the 5SrDNA locus connects RNA pol III transcription to the Myc/Max/Mxd network that operates on RNA pol II genes . SurprisHeLa cells were grown in DMEM high-glucose supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin and 50 μg/ml streptomycin. Cells were treated with 75 μM 10058-F4 (Sigma), dissolved in DMSO, for 16 h.HeLa cells were transfected at 50% confluence with WSTF siRNA, c-Myc siRNA, Max siRNA, Brf1 siRNA or scrambled siRNA . SiRNA was delivered using lipofectamin RNAi Max (Invitrogen) and the cells were harvested 30–40 h after transfection start, as stated in the text. c-Myc expression vector was delivered using Lipofectamin 2000 (Invitrogen).ChIP was performed as previously described . Immunop280, and the amount of MNase was titrated in each experiment. The digested DNA was evaluated by qPCR, and analysed by calculating ΔCt between the reaction with no MNase and the digested reaction at each concentration. The primer pairs used are given in the Supplementary Information (Supplementary Tables S2 (5S rDNA) and S3 (7SL RNA gene 1).Cells were cross-linked with 1% formaldehyde for 20 min, and chromatin was prepared as for ChIP, but washed with a buffer containing 25% glycerol, 5 mM magnesium acetate, 50 mM Tris at pH 8.0, 0.1 mM EDTA and 5 mM DTT, as described in Petesch and Lis  and modiCell extracts were prepared from nuclei in an extraction buffer containing 0.7 M KCl, 0.05% NP40, 1 mM EDTA and 10 mM Hepes at pH 7.6 . Four huImmunoblotting was performed using a PVDF membrane (Millipore).RNA was prepared using Qiazol (Qiagen) from cells following the manufacturer's manual. The RNA preparations were subjected to DNAse (1 U for 30 min), and converted to cDNA  using random primers according to the manufacturer's instructions.Protein concentrations were determined using the Bradford reagent (Bio-Rad).To investigate the underlying mechanism by which the B-WICH complex regulates RNA pol III genes, we first investigated its effect on the binding of components of the RNA pol III transcription machinery. The binding of RNA pol III and its factors, TFIIIA, TFIIIB (Brf1) and TFIIIC (TFIIIC220), to 5S rRNA and the 7SL RNA genes was severely compromised in cells in which the WSTF protein had been knocked down (WSTF KD cells) Figure . For genMost regulatory factors of RNA pol III transcription bind directly to the RNA pol III machinery. This led us to investigate whether the WSTF protein interacts directly with RNA pol III and the general RNA pol III transcription factors. However no clear interactions were detected; WSTF antibodies did not precipitate RNA pol III, TFIIIA, TFIIIB-Brf1 or TFIIIC220 Figure , whereasp = 0.0006, Student's t-test). At the 7SL RNA gene, two positions were less accessible on either side of the gene, both upstream (p = 0.025) and downstream (p = 0.008), in WSTF KD cells  on cross-linked chromatin isolated from cells transfected with scrambled siRNA or WSTF siRNA, and analysed the regions around the genes with primer pairs that produced adjoining 100-bp products. The positions of the amplicons are shown in the diagrams of the 5S rDNA and 7SL RNA locus Figure  and B. Ws Figure . The appAs the effects of B-WICH were seen outside of the 5S rRNA and at the edges of the 7SL RNA genes, we investigated the association pattern of WSTF and SNF2h in the loci. In contrast to RNA pol III, which associated mainly within the 5S rRNA and 7SL RNA genes, the WSTF and SNF2h signals from ChIPs peaked outside of the genes Figure  and F. TIt is possible that the closed chromatin configuration at the 5S rDNA and 7SL RNA locus upon WSTF knocks down was due to a changed nucleosome density. However, no significant difference in H3 occupancy was observed at the 5S rRNA genes or the 7SL RNA genes in WSTF KD cells compared to that found in cells transfected with scrambled siRNA Figure . Next, wTo determine the effect of the B-WICH complex on the association of HATS with the 5S rDNA and the 7S RNA locus, we first tested whether binding pattern of different HATs. We found that the p300 and GCN5, but not the PCAF, associate with the 5S RNA genes Figure , while aSince p300 has been reported not to function as a HAT at the 5S rRNA genes , we focuSeveral factors such as c-Myc and RNA pol II have been suggested to regulate the chromatin structure at RNA pol III genes –20,37–39Genome-wide studies have revealed that c-Myc binds in a broad peak over RNA pol III genes . We inveThe protein level of c-Myc was lower in WSTF knock-down cells, a finding that was also reflected in the binding of c-Myc to the chromatin factor . In order to ensure that the reduced binding upon WSTF knock-down at the 5S rRNA and 7SL RNA genes was not a result of the lower c-Myc level, we overexpressed c-Myc in cells in which WSTF had been knocked down by siRNA. Overexpression of c-Myc did not result in large increases of the 5S rRNA, 7SL RNA and tRNA, while a large increase was observed in the snU6 RNA level . ChIP experiments investigating the binding of c-Myc in WSTF KD cells overexpressing c-Myc showed that the binding at the genes and in the IGS was reduced at the 5S rDNA Figure  and the The peak of c-Myc observed in the IGS of the 5S rDNA and the 7SL RNA locus prompted us to examine the region for E-boxes (CACGTG), to which c-Myc binds as a dimer with Max . SeveralInterestingly, we detected Max associated with the 5S rRNA gene Figure , suggestTo study the function of the association of c-Myc at the 5S rRNA and 7SL genes, we investigated factor binding in c-Myc knock-down cells. Transfection with c-Myc siRNAs for 30 h reduced the level of c-Myc mRNA and the levels of 5S rRNA, 7SL RNA, tRNA, snU6 RNA and the mRNA of cyclin D, but not that of the actin mRNA Figure ,26,40, bTo investigate whether c-Myc and Max bind directly to DNA in the IGS, we treated cells with the compound 10058-F4, which specifically inhibits the binding between c-Myc and Max, and their binding to E-boxes in DNA . TreatmeNext, we knocked down Brf1 to study the role of the c-Myc site at the gene. The knock-down of Brf1 reduced the levels of 5S rRNA, 7SL RNA and tRNA, but not that of snU6 RNA Figure  in line WSTF knock-down had a greater effect on the factor binding at the 5S rDNA locus than c-Myc or Max knock-down. This led us to investigate the order of events between WSTF and c-Myc in transcriptional activation. To determine whether c-Myc knock-down and the subsequent reduction of histone H3 acetylation lead to a similar remodelling of the chromatin structure as that observed in WSTF KD cells, we performed high-resolution MNase walking. In contrast to WSTF knock-down Figure , c-Myc kThis study demonstrates that chromatin is an important regulator of RNA pol III transcription and that chromatin-remodelling complexes are required to alter the chromatin structure in the vicinity of the genes for transcriptional activation. We have studied the order of events upon transcriptional activation of 5S rRNA and 7SL RNA genes and demonstrated that chromatin remodelling is required early in the process. We demonstrate that the B-WICH complex is necessary for the remodelling of chromatin in the vicinity of the 5S and 7SL to allow the binding of other factors that induce RNA pol III transcription Figure . The sitIt has previously been proposed that transcription factors affect the chromatin structure and the profile of histone modifications in the vicinity of RNA pol III genes. These factors mainly prevent nucleosomes from forming at the promoters of the genes. One example is TFIIIC, which remains bound at low levels in the absence of transcription ,19,28,43We have identified c-Myc as one factor that requires B-WICH to remodel chromatin at the 5S rDNA. c-Myc binding was also B-WICH-dependent at the 7SL RNA genes, but RNA pol II is also a factor that depends on B-WICH for binding. This suggests that B-WICH is not specific to any one factor, but is used to activate genes by remodelling chromatin at specific sites. Previous studies have shown that c-Myc binds to RNA pol III genes via Brf1 in the TFIIIB complex in a Max-independent manner –16. We sWe suggest that the association of c-Myc to both a DNA binding site, probably in the IGS, and the binding site in the promoter is required for transcription. These sites were both required for the recruitment of HATs and the subsequent histone acetylation of the locus. Since interfering with binding of c-Myc at the promoter affected the binding of c-Myc-Max at the IGS, we suggest that the two sites in the repeat co-operate. It has been reported that c-Myc acts as a linker forming functional chromatin loops at the nucleolar rDNA loci . Thus, sThe identification of a novel c-Myc-Max site links for the first time 5S rRNA transcription to the Myc/Max/Mxd gene regulatory network, and this may explain the deregulation of RNA pol III transcription often seen in oncogenic transformation . HoweverSupplementary Data are available at NAR Online.


We have previously shown that the T362I substitution leads to a lower fidelity RdRp, and viruses encoding this variant are attenuated in a mouse model of poliovirus. Given these results, it was surprising that the nucleotide incorporation rate and nucleobase fidelity of the Sabin I RdRp is similar to that of wild-type enzyme, although the Sabin I RdRp is less selective against nucleotides with modified sugar groups. We suggest that the other Sabin amino acid changes  help to re-establish nucleotide incorporation rates and nucleotide discrimination near wild-type levels, which may be a requirement for the propagation of the virus and its efficacy as a vaccine strain. These results also suggest that the nucleobase fidelity of the Sabin I RdRp likely does not contribute to viral attenuation.The Sabin I poliovirus live, attenuated vaccine strain encodes for four amino acid changes ( Th. Th24]. Other amino acid substitutions in the Sabin I RdRp may also change RdRp function. The Y73H substitution has been shown to interfere with the initiation of RNA synthesis , which mIn this article, we show that although the T362I substitution by itself lowers RdRp fidelity, the Sabin I RdRp, encoding all four substitutions, discriminates against nucleotides with incorrect nucleobases at the same level as wild-type (WT) enzyme. These results may suggest that there was evolutionary pressure during the selection of the Sabin I virus to maintain an optimal level of RdRp fidelity. In contrast, the Sabin I RdRp is less faithful when selecting against 2′-modified nucleotides, which would not be under the same selection pressure.i.e., D53N/Y73H/K250E/T362I). To ensure that the Sabin I RdRp was amenable to the single nucleotide incorporation assays, we first examined its ability to interact with RNA. We have previously shown that the T362I substitution does not significantly weaken interactions between enzyme and RNA compared to WT RdRp ATP and [α-32P]UTP (>7000Ci/mmol) was from VWR-MP Biomedical ; nucleoside 5′-triphosphates and 2′-deoxynucleoside 5′-triphosphates  were from GE Healthcare Bio-Sciences . 3′-Deoxyadenosine 5′-triphosphate (cordycepin) was from Trilink Biotechnologies . All RNA oligonucleotides were from Dharmacon Research, Inc. . T4 polynucleotide kinase was from New England Biolabs, Inc . [Methyl-13C] methionine was from Cambridge Isotope Laboratories . HisPur Ni-NTA resin was from ThermoFisher Scientific . Q-Sepharose fast flow resin was from GE Healthcare Bio-Sciences. Polyethylenimine-cellulose thin layer chromatography (TLC) plates were from EM Science . QuickChange site-directed mutagenesis kit was from Stratagene . The plasmid DNA isolation miniprep kit was from Qiagen . All other reagents were of the highest grade available from Sigma-Aldrich  or ThermoFisher. Met-labeled PV RdRp [1H, 13C HSQC (heteronuclear single quantum coherence) NMR spectra were collected on a Bruker Avance III 600 MHz spectrometer equipped with a 5-mm “inverse detection” triple-resonance  single axis gradient TCI probe at 293 K. NMR samples generally contained 250 μM RdRp, 1000 μM s/s RNA, and 4 mM UTP, 8 mM 2′-dUTP or 16 mM ATP.NMR sample preparation followed procedures described previously using [ PV RdRp ,30. 1H, pol-coding sequence of viral cDNA, pMovRA, overlap PCR was performed with oligonucleotides PV-3D-D53N-for (5′-GAT CCC AGG CTT AAG ACA AAT TTT GAG GAG GCA ATT TTC-3′), PV-3D-D53N-rev (5′-AGA AAA TTG CCT CCT CAA AAT TTG TCT TAA GCC TGG GAT C-3′), PV-3D-Y73H-for (5′-ATT ACT GAA GTG GAT GAG CAT ATG AAA GAG GCA GTA GAC-3′), PV-3D-Y73H-rev (5′-GTC TAC TGC CTC TTT CAT ATG CTC ATC CAC TTC AGT AAT-3′), PV-3D-K250E-for (5′-GCT TGG TTC GAG GCA CTA CAA ATG GTG CTT GAG AAA ATC GGA-3′), PV-3D-K250E-rev (5′-TCC GAT TTT CTC AAG CAC CAT TTC TAG TGC CTC GAA CCA AGC-3′), PV-3D-BglII-for (5′- GGC AAA GAA GTG GAG ATC TTG GAT GCC AAA GC-3′), PV-3D-EcoRI-ApaI-polyA-rev (5′-CGC TCA TCG ATG AAT TCG GGC CCT TTT TTT TTT TTT TTT TTT TCT CC-3′) and the pMoV-3D-BPKN-I92T-T362I plasmid as template [. PCR products were purified and digested with Bgl II and Eco RI and the digested PCR product was ligated into pMovRA vector.To introduce the four mutations, D53N, Y73H, K250E and T362I, into the RdRp/3Dtemplate . PCR proApaI and purified with Qiaex II suspension (Qiagen) by following the manufacturer’s protocol. RNA was then transcribed from the linearized plasmid DNAs in a 20-μL reaction mixture containing 350 mM HEPES, pH 7.5, 32 mM magnesium acetate, 40 mM dithiothreitol (DTT), 2 mM spermidine, 28 mM nucleoside triphosphates, 0.025 μg/μL linearized DNA, and 0.025 μg/μL T7 RNA polymerase. The reaction mixture was incubated for 4 h at 37 °C, and magnesium pyrophosphate was removed by centrifugation for 2 min. The supernatant was transferred to a new tube, and RQ1 DNase  was used to remove the template. The RNA concentration was determined by measuring absorbance at 260 nm, assuming that an A260 of one was equivalent to 40 μg/mL, and the RNA quality was verified by 0.8% agarose gel electrophoresis.The pMo-3D-D53N-Y73H-K250E-T362I plasmid was linearized with HeLa cells were transfected by electroporation with 5 µg of viral RNA transcript and these cells were serially diluted and plated onto HeLa cell monolayers. Cells were allowed to adhere to the plate for 1 h at 37 °C and then the medium/PBS was aspirated. Cells were covered with 1X DMEM/F12 plus 10% fetal bovine serum and 1% agarose. After 2–4 days of incubation, the agarose overlay was removed and the cells were stained with crystal violet.BglII-for and PV-3D-EcoRI-ApaI-polyA-rev as primers. The presence of all four mutations was determined by sequencing of the nucleic acid obtained in second PCR step with oligonucleotides PV-3D-seq100-for (5′-GAA GGG GTG AAG GAA CCA G-3′) and PV-3D-seq500-for (5’-AGG TTG AGC AGG GGA AA-3′).HeLa cells were transfected by electroporation with 5 µg viral RNA transcript, added to HeLa cell monolayers and incubated at 37 °C. Upon cytopathic effect (CPE), viruses were harvested by three repeated freeze-thaw cycles, cell debris removed by centrifugation. Viral RNA was isolated with QiaAmp viral RNA purification kit (Qiagen), as recommended by the manufacturer. The 3Dpol cDNA was prepared from purified viral RNA by reverse transcription with MMuLV-RT (New England Biolabs) with Random Hexamer Primers (manufacturer). The resulting DNA product was then PCR amplified using SuperTaq DNA polymerase  and oligonucleotides PV-3D-


RNA binding motif protein 47 (RBM47) as a suppressor of breast cancer progression and metastasis. RBM47 inhibited breast cancer re-initiation and growth in experimental models. Transcriptome-wide HITS-CLIP analysis revealed widespread RBM47 binding to mRNAs, most prominently in introns and 3′UTRs. RBM47 altered splicing and abundance of a subset of its target mRNAs. Some of the mRNAs stabilized by RBM47, as exemplified by dickkopf WNT signaling pathway inhibitor 1, inhibit tumor progression downstream of RBM47. Our work identifies RBM47 as an RNA-binding protein that can suppress breast cancer progression and demonstrates how the inactivation of a broadly targeted RNA chaperone enables selection of a pro-metastatic state.The mechanisms through which cancer cells lock in altered transcriptional programs in support of metastasis remain largely unknown. Through integrative analysis of clinical breast cancer gene expression datasets, cell line models of breast cancer progression, and mutation data from cancer genome resequencing studies, we identified DOI:http://dx.doi.org/10.7554/eLife.02734.001 Tumors form when mistakes in the genes of a single cell allow it to multiply uncontrollably. Sometimes further mutations in genes allow the cancerous cells to escape from the tumor, enter the bloodstream and start a second cancer elsewhere in the body. However, many of the genetic changes behind this process, which is called metastasis, are poorly understood—especially those changes in genes that occur rarely, but can still help the cancer to spread.RBM47 was often switched off in these cells, and patients with a low level of RBM47 tended to have a poor clinical outcome.Vanharanta, Marney et al. have looked at data on which genes are switched ‘on’ or ‘off’ in metastatic breast cancer cells. A gene called RBM47 in cancer cells that had spread from the breast to either the lungs or the brain, and then injected these cells into mice. Few of these cells were able to invade lung and brain tissues in the mice. However, switching off the RBM47 gene in breast cancer cells had the opposite effect; these cells invaded the lungs of mice more efficiently.To test the function of the gene, Vanharanta, Marney et al. switched on RBM47 encodes a protein that sticks to molecules of messenger RNA: molecules that transport the instructions encoded in DNA to the machinery that builds proteins. Vanharanta, Marney et al. found that the wild-type RBM47 protein increased the levels of 102 different messenger RNA molecules, but decreased the levels of another 92. Further experiments showed that RBM47 also slows the rate at which messenger RNA molecules are broken down inside cells: this results in the accumulation of proteins that slow down the growth of tumors. Without RBM47, tumor growth is unleashed. Further work is needed to test if increasing RBM47 activity could be used as a new treatment for some types of cancer.DOI:http://dx.doi.org/10.7554/eLife.02734.002 TP53 and PIK3CA, most studies also report a long tail of rarely mutated genes. While many of these mutations are likely to be passenger events, some of them are potential mediators of tumor phenotypes. How to identify such low-frequency driver mutations remains a challenge.Cancers arise through an evolutionary process that feeds from stochastic genetic alterations and selection . The ideIn addition to mutations that directly promote tumorigenesis through specific alterations in cell signaling and repair pathways, many aberrations found in cancers do not affect cell signaling pathways directly, but rather, they support the stabilization of altered transcriptomic profiles that facilitate the emergence of pro-tumorigenic and metastatic traits. Mutations in epigenetic regulators fall into this category . The phePrecise regulation of RNA metabolism is fundamental in the generation of biological complexity in both normal and disease states . The conRNA binding motif protein 47 (RBM47) as a suppressor of breast cancer progression. By analyzing the transcriptome-wide RBM47 binding patterns we demonstrate that RBM47, a previously uncharacterized RNA-binding protein, modulates mRNA splicing and stability. Loss of RBM47 function thus provides a specific example of the power of global RNA modulatory events in the selection of pro-metastatic phenotypic traits.We hypothesized that combining cancer genome resequencing data with gene expression information from both clinical data sets and experimental model systems of metastasis would allow the identification of rarely mutated cancer genes with potential functional significance. This approach identified RBM47, a gene encoding a previously uncharacterized putative RNA-binding protein was the only one that fulfilled all these criteria , the association with brain metastasis remained statistically significant  onwards  database  and brain metastatic (BrM2) derivatives of the MDA-MB-231 triple negative breast cancer cells (MDA231 for short), respectively . All clones exhibited dose-dependent RBM47 mRNA upregulation upon doxycycline induction  per replicate. 75% of the tags mapped to regions corresponding to UCSC/Refseq genes, with a high degree of reproducibility of binding observed between replicates  with increasingly stringent filters : tags pe and IL8 .SLK, MDM4, LIMCH1, MBNL1 and SEC31A  exon junctions as described previously for Mbnl2 , and an d SEC31A .10.7554/By mapping normalized RBM47 CLIP tags associated with RBM47-dependent splicing changes on a composite transcript  we generI281fs expression. This revealed a set of 102 mRNAs that were upregulated and 92 that were downregulated, respectively, in cells expressing the wild type RBM47 , when compared to cells expressing the mutant . This repe RBM47 . Importape RBM47 .10.7554/To determine whether the 194 RBM47-responsive genes displayed clinically meaningful expression patterns, we conducted an unsupervised hierarchical clustering analysis of the TCGA cohort of breast cancer specimens. Two main clusters emerged, one of which harbored characteristics of the RBM47-low phenotype . The closest homologs of RBM47 are Apobec1 complementation factor (A1CF) and hnRNP-Q, which regulate RNA editing  and spliOur data demonstrate widespread and reproducible RBM47 binding to target mRNAs predominantly in introns and 3′UTRs, with the most robust binding occurring in 3′UTRs. Recent RNA-compete studies have proposed a binding motif for RBM47 in vitro . Our in We find that RBM47 binds robustly to ∼2500 gene transcripts in human breast cancer cells, with only a subset showing steady state level change or alternative splicing upon RBM47 reintroduction. Given the stringent criteria used to define RBM47-bound and regulated targets and the generally low level of intronic RNA in a cell, it is likely that this subset is an underestimation of the number of RBM47-regulated transcripts, and does not take into account the potential for regulatory events, such as re-localization, that may not alter steady state transcript levels. The complex interplay of RBPs in agonistic and antagonistic modulation of mRNA is becoming increasingly apparent. For example, the RRM-domain containing protein HuR modulates the destabilizing effects of miRNAs  and AUF1DKK1, is stabilized by RBM47 and partially mediates RBM47 tumor suppressive function. Interestingly, rbm47 knockdown in zebra fish embryos leads to a headless phenotype mediated via up-regulation of the wnt8a pathway .For RBM47 restoration, DKK1miRm: TGCTGTTGACAGTGAGCGACGGGTCTTTGTCGCGATGGTATAGTGAAGCCACAGATGTATACCATCGCGACAAAGACCCGGTGCCTACTGCCTCGGADKK1miRn: TGCTGTTGACAGTGAGCGACACCTTGAACTCGGTTCTCAATAGTGAAGCCACAGATGTATTGAGAACCGAGTTCAAGGTGGTGCCTACTGCCTCGGADKK1miRo: TGCTGTTGACAGTGAGCGCCAACTCAATCCTAAGGATATATAGTGAAGCCACAGATGTATATATCCTTAGGATTGAGTTGATGCCTACTGCCTCGGADKK1miRp: TGCTGTTGACAGTGAGCGACAGTAAATTACTGTATTGTAATAGTGAAGCCACAGATGTATTACAATACAGTAATTTACTGCTGCCTACTGCCTCGGADKK1miRq: TGCTGTTGACAGTGAGCGAAACGGAAGTGTGATATGTTTATAGTGAAGCCACAGATGTATAAACATATCACACTTCCGTTCTGCCTACTGCCTCGGAFor RNAi-mediated gene silencing, RBM47 pGIPZ shRNA constructs  were obtained from Open Biosystems . The DKK1 shRNAmiR constructs were designed based on the rules described by The pGIPZ empty vector was used as a control.t test and Wilcoxon rank-sum test, respectively. Brain images were acquired with a Leica SP5 up-right confocal microscope and Zeiss AxioVert 200 M using 20X and 5X objectives. Image analysis was performed with Metamorph and ImageJ softwares.All animal experiments were performed in accordance with a protocol approved by MSKCC Institutional Animal Care and Use Committee. Lung metastasis assays were conducted in 5–7 week old female NOD/SCID mice. Brain metastasis and mammary tumor assays were carried out using 5–7 week old female athymic nude mice. In vivo bioluminescence imaging was performed using the IVIS Spectrum Xenogen machine . For orthotopic mammary tumor assays, cells were mixed with Matrigel. Injections were confirmed and tumor growth was followed by bioluminescent imaging. Statistical significance of tumor and metastasis free survival was assessed by the log-rank test. Differences in raw and normalized bioluminescence signal was assessed by the Student's TBP was used as a housekeeping control gene. mRNA stability was assessed by performing quantitative PCR after actinomycin D treatment. For immunoblotting, antibodies recognizing RBM47 , α-tubulin  and ACTB (Sigma) were utilized. Secondary antibodies were HRP  or fluorescence  conjugated. Immunostaining for RBM47  was performed according to standard protocols in the MSKCC Molecular Cytology Core Facility on paraffin embedded tissue blocks. Secreted protein was detected by ELISA .Total RNA was extracted using PrepEase RNA spin kit . We used Transcriptor First Strand cDNA Synthesis Kit  for cDNA synthesis. Quantitative PCR was performed using predesigned Taqman gene expression assays  and the 7900HT or ViiA 7 real-time PCR systems (Applied Biosystems/Life Technologies). 2 on a bed of ice . Samples were processed for HITS-CLIP as previously described . Purified RBM47-bound RNA fragments were polyA tailed , and reverse transcribed  in the presence of Br-dUTP (Sigma). Unique polydT-NV RT-primers were used per replicate, containing Solexa sequences separated by an abasic furan (that serves as an ApeI cut site), a 6 nt degenerate region and a 6 nt index sequence to allow for multiplexing during sequencing.6GATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGAT20VNRT Primer 1pGCACTGTTN6GATCGTCGGACTGTAGAACTCT/idSp/CAAGCAGAAGACGGCATACGAT20VNRT Primer 2pGCGAAACTN231BrM2 tet-on FLAG-RBM47 cells were treated with 1 ng/ml doxycycline for 3 days before 254 nm UV crosslinking at 400 mJ/cmescribed  using anBrdU-cDNA was stringently purified by IP  using ProteinG dynabeads (Invitrogen), eluted from the beads via BrdU competitive elution (Sigma), and re-immunoprecipitated. cDNA was circularized on bead , washed and digested with ApeI  to relinearize. cDNAs were eluted from the beads by heating to 98C in Phusion HF Buffer (NEB), then PCR amplified using Phusion DNA polymerase (NEB) and SYBR Green I (Invitrogen) in an iQ5 real-time PCR machine in order to monitor amplification, with the samples being removed when the RFU signal reached ∼1000.P5—aatgatacggcgaccaccgacaggttcagagttctacagtccgacgP3—caagcagaagacggcataPCR products were purified using MinElute columns  as per manufacturer's instructions and quantified using Quant-It (Invitrogen). cDNA was multiplexed and sequenced using Illumina Hi-Seq . All data analysis was done using the Galaxy platform , as prevn-1, n and n+1 cycles, lane 4 contains an absence of reverse transcriptase control (−RT), with all other lanes corresponding to replicate samples of the indicated cell type amplified to n cycles. IR and ΔI calculated using ImageJ  and WT10 cells  as previously described . In all g ImageJ .affy, gcrma and hs133ahsentrezgcdf (version 10). Unsupervised hierarchical clustering was performed using the function heatmap.2 with Pearson's correlation coefficient as the similarity metric. For survival analysis, a Cox proportional hazards model was utilized as implemented in the coxph function in the R-package survival. For RNA-seq analysis of the cell lines, raw paired-end sequencing data were mapped to human genome (hg19 build) with STAR2.3.0 . Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 2 reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Loss of the multifunctional RNA-binding protein RBM47 as a source of selectable metastatic traits in breast cancer” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.eLife. However, the following issues would need to be addressed before we can consider the work further for publication in eLife.The reviewers felt that the identification of the RBM47 RNA-binding protein as a regulator of metastasis was an important contribution that could be published in Major comments:eLife experiments to demonstrate a specific role of RBM47 in metastasis, as opposed to general cell proliferation, are required.1) A critical issue is whether RBM47 has a specific role in regulating metastasis as opposed to more broadly affecting cell proliferation and growth. More specifically, 2) Given the pleiotropic effects of the RBM47 protein on cellular RNA metabolism, it is possible that multiple target RNAs are involved and stronger evidence that the DKK1 and Wnt signaling pathways are the primary targets would be required. Possible experiments include:a) Examining whether RBM47 expression is inversely correlated with Wnt gene signatures in the human breast tumor datasets described in b) Examining if weaker DKK1 shRNAs have the appropriate effect on tumor growth and metastasis as related to RBM47. This is based on the observation that 3) Most of the experiments were carried out with the metastatic variants of the MDA-MB-231 cell line, and one would like to know how general the effect of manipulating RBM47 levels on breast cancer cell metastasis is. Some experiments were carried out with CN34 cells, a metastatic breast cancer line derived from a pleural effusion at MSKCC, and these data support the conclusion that RBM47 acts to reduce metastasis. In this regard, the authors showed that CN34-BrM2 cells have low levels of RBM47 – did they check if RBM47 expression reduces the level of brain metastases? Have any other breast cancer cell lines been assessed for the effects of RBM47 expression or depletion on metastatic potential (e.g. lines like SKBR3 that have high endogenous RBM47)?Minor comments:4) It would improve the CLIP data to normalize their number of tags to the abundance of that RNA from the RNA-seq data. This might reveal a more relevant binding to intron sequences .5) It is possible that effects on alternative splicing can influence RNA stability due to sequence differences in mature mRNAs. Although not required for publication, it would improve the manuscript if the authors determined whether there is a significant overlap between mRNAs with altered splicing and abundance in order to see if these two sets are related.6) The CLIP experiment was done with over-expressed RBM47 protein and this has the potential to influence the observed binding sites. This caveat should at least be made explicit in the manuscript.7) The key functional data presented in this study  relied o8) It would significantly strengthen the manuscript if the effect of RBM47 on metastasis could be ascribed to a specific step in metastasis .9) Since no biochemical analysis was performed on the RBM47 I281fs mutant, it is unclear whether this mutation completely abolishes RBM47 function. Therefore, in 10) In 11)  We would like to thank the reviewers for their time and insightful comments. We were happy to see that all of you found our work interesting and that the general assessment was positive. The comments highlighted important points, all of which we have now addressed. We feel that this has improved our paper. We have also decided to eliminate the term hnRNP from the paper, as it is an outdated concept, originally meant to refer to abundant nuclear RNA binding proteins that coat hnRNA. However, the term essentially has no current meaning except for historical purposes.1) A critical issue is whether RBM47 has a specific role in regulating metastasis as opposed to more broadly affecting cell proliferation and growth. More specifically,attempt to show that RBM47 suppresses experimental metastases in the brain and the lung. However, without examining whether RBM47 overexpression and knockdown also affect cell proliferation in culture and primary tumor growth in mice, it is unclear whether RMB47 specifically regulates metastasis vs. cell proliferation. Indeed,shows that expression of RBM47 drastically inhibits primary tumor growth, suggesting a possible role of RBM47 in regulating primary tumor growth, with a possible downstream effect on metastasis.aims to demonstrate a critical role of DKK1 downstream of RBM47 in suppressing tumor progression. Sinceshows that DKK1 knockdown resulted in significantly bigger primary tumors, it is again unclear whether there is any specific effect on metastasis. For this work to be of sufficient impact for publication in eLife experiments to demonstrate a specific role of RBM47 in metastasis, as opposed to general cell proliferation, are required.RBM47 expression correlates with poor patient outcome. Moreover, in the breast cancer brain metastasis sequenced by Ding et al. (2010), the RBM47 mutation was highly enriched when compared to the primary tumor. Combined with our functional data, all this evidence suggests that the most aggressive cancer cell populations that are most likely to form metastases benefit from RBM47 loss and hence RBM47 loss is further selected for during metastatic progression.We agree with the assessment that the data presented in the manuscript does not show specific effects of RBM47 as a suppressor of metastatic progression. In fact, the data in We would like to address here the widespread idea that ‘true’ metastasis genes and functions are not involved at all in primary tumor formation. In fact, with the exception of genes that mediate the last stage of the metastatic process – overt colonization of specific organ microenvironments – the majority of genes implicated in metastasis confer advantages to cancer cells already in a primary tumor. Otherwise, clones overexpressing such pro-metastatic genes would not become selected and enriched enough in primary tumors for their expressed genes to score as predictive metastasis markers or signatures. These genes are not tumor initiation or early progression oncogenes. Their functions provide a selective advantage to invasive cancer cells that venture into hostile microenvironments, for example, at the invasive front. The clones expressing these genes are thereby primed for metastatic seeding in distant sites, where their advantageous traits may undergo additional selection and amplification. Extensive clinical and experimental evidence for this concept has been presented in our work (1-5) and the work of others. Therefore, the idea that authentic pro-metastatic genes contribute to tumorigenesis only in distant organs and not at all in primary tumors is incorrect, in our view. We have succinctly annotated the revised manuscript in order to clarify this issue.The evolutionary process that shapes tumor progression at the primary site pre-selects phenotypes that will later on enhance the metastatic fitness of the disseminated cells. As the vast majority of disseminated cancer cells die, this pre-selection is essential for cancer cell survival at the early stages of metastasis initiation. For this reason, the majority of important mediators of metastasis identified to date also have detectable effects on tumor growth at the primary site. This is the case with RBM47 as well.The most important finding of our paper is the demonstration that loss of RBM47, a previously uncharacterized RNA chaperone with widespread binding across the transcriptome, is selected for during metastatic cancer progression. We show that this is partially mediated by enhanced activation the Wnt signaling pathway, but we believe that other pro-tumorigenic signaling pathways could also be involved in the process. Our intention therefore is not to suggest that RBM47 has specific functions suppressing metastasis, but rather that its loss contributes to the establishment of the most aggressive cancer phenotypes that are vital to metastatic dissemination. This is an important concept that highlights the importance of alterations in transcriptomic homeostasis as a source of phenotypic diversity required for cancer progression. In order to clarify this, we have reworded the Abstract and manuscript.2) Given the pleiotropic effects of the RBM47 protein on cellular RNA metabolism, it is possible that multiple target RNAs are involved and stronger evidence that the DKK1 and Wnt signaling pathways are the primary targets would be required. Possible experiments include:a) Examining whether RBM47 expression is inversely correlated with Wnt gene signatures in the human breast tumor datasets described inb) Examining if weaker DKK1 shRNAs have the appropriate effect on tumor growth and metastasis as related to RBM47. This is based on the observation thatshows that RBM47 overexpression resulted in ∼3-fold increase of DKK1 protein level. However, DKK1 shRNAs used in suppression of DKK1 protein to an almost undetectable level; much lower than the level in the control cells without RBM47. Therefore, the observed tumor growth and brain metastasis formation upon DKK1 knockdown  may not be necessarily related to RBM47. To test this, much weaker DKK1 shRNAs should be used to bring down the DKK1 protein level to the level in the control cells as shown into test its effect on tumor growth and metastasis. Ideally, depletion of the relevant Wnts expressed by these cells would establish the importance of DKK1 as a target for metastasis suppression by RBM47.Our work indicates that RBM47 has multiple target molecules. This is in line with the known principles of nuclear RNA binding protein function; they act as part of large protein complexes to facilitate multiple aspects of mRNA biogenesis. Indeed, we provide evidence that RBM47 regulates both mRNA stability and splicing. As the various target mRNAs of RBM47 encode for proteins that function in several biological processes, the phenotypic output of RBM47 loss will therefore have pleiotropic effects. Our data in In the current study, we focused on DKK1 as one of the downstream mediators of RBM47 action. DKK1 was chosen for two reasons: (i) it was one of the most prominent target transcripts of RBM47 as determined by HITS-CLIP and (ii) it is a known modulator of the Wnt pathway. We show that RBM47 can indeed inhibit Wnt signaling by stabilizing DKK1. We also demonstrate that DKK1 knockdown is able to partially rescue the tumor suppressive effects of RBM47. These results suggest, as pointed out by the referees, that RBM47 expression should correlate negatively with a Wnt responsive signature in clinical data sets. In agreement with the prediction, we have now found that tumors with low RBM47 expression tend to have higher expression of Wnt target genes, as determined by pathway analysis using a curated breast cancer Wnt signature (6). Following the option 2(a) suggested by the referees, these new data have been added into the new net positive contribution to metastasis, much in the same way that dysfunctions in other epigenetic regulators do. In this paper we use DKK1 as an important example, but acknowledge the possible contributions of other RMB47 targets.Despite providing evidence of DKK1 as a mediator of the pro-tumorigenic effects of RBM47 loss, we do not mean to suggest that DKK1 is the only important target transcript of RBM47. DKK1 is but one of many RBM47 targets, the balance of which makes a The upshot of our work is that the loss of a promiscuous RNA-binding protein can, despite its large number of functionally unrelated target mRNAs, lead to the emergence of pro-metastatic cancer cell traits. This finding complements the recent findings of mutations in epigenetic regulators in cancer, and highlights the importance of widespread perturbations in RNA homeostasis as a source of cancer cell phenotypes. In order to clarify this point, the revised manuscript highlights other potential mediators of RBM47 effects for further study..3) Most of the experiments were carried out with the metastatic variants of the MDA-MB-231 cell line, and one would like to know how general the effect of manipulating RBM47 levels on breast cancer cell metastasis is. Some experiments were carried out with CN34 cells, a metastatic breast cancer line derived from a pleural effusion at MSKCC, and these data support the conclusion that RBM47 acts to reduce metastasis. In this regard, the authors showed that CN34-BrM2 cells have low levels of RBM47 – did they check if RBM47 expression reduces the level of brain metastases? Have any other breast cancer cell lines been assessed for the effects of RBM47 expression or depletion on metastatic potential (e.g. lines like SKBR3 that have high endogenous RBM47)?We identified RBM47 as a suppressor of breast cancer progression through an integrative approach that combined gene expression information from metastatic breast cancer models with mutational and gene expression data from clinical datasets. Importantly, the clinical cohorts utilized in this study comprise hundreds of tumor samples, providing critical evidence that the observations made in our experimental model systems have more general clinical relevance. To this end, the new analysis in We agree that increasing the number of model systems increases confidence in the general relevance biological data. Our study therefore contains experimental results from several cell line models  as well as clonal metastatic derivatives, including one derived from the CN34-BrM2 cells (WT6). The results on the effects of RBM47 on the brain metastatic potential of these cells is shown in Minor comments:4) It would improve the CLIP data to normalize their number of tags to the abundance of that RNA from the RNA-seq data. This might reveal a more relevant binding to intron sequences .We agree that normalizing HITS-CLIP data to total RNA reads could reveal more prominent intronic binding patterns. However, our existing RNAseq data corresponding to the HITS-CLIP experiments shown in 5) It is possible that effects on alternative splicing can influence RNA stability due to sequence differences in mature mRNAs. Although not required for publication, it would improve the manuscript if the authors determined whether there is a significant overlap between mRNAs with altered splicing and abundance in order to see if these two sets are related.Alternative splicing can alter mRNA stability, e.g. through the introduction of cryptic NMD exons (8). However, at the cutoff levels used in this study, we did not see any overlap between the transcripts that underwent alternative splicing and genes that showed altered message levels upon RBM47 reintroduction. As discussed in Response 4, deeper intronic RNA sequencing could identify additional RBM47-dependent splicing events, which could also reveal links to mRNA stability, but based on current data, alternative splicing does not seem to be a major determinant of altered mRNA stability in the context of RBM47.6) The CLIP experiment was done with over-expressed RBM47 protein and this has the potential to influence the observed binding sites. This caveat should at least be made explicit in the manuscript.The text has been revised as requested.7) The key functional data presented in this study  relied on overexpressing RBM47 in tumor cells. It is unclear how the level of RBM47 overexpression is compared to the physiological level of RBM47 in normal cells or the parental non-metastatic cells. This information is critical to determine whether the observed tumor growth reduction upon RBM47 overexpression is due to physiological effect of RBM47 or due to toxic effects of overexpressed RBM47.We agree that protein overexpression is a potential source of artifacts. In order to control for this, however, we have taken several measures:a) shRNA constructs were used to validate the metastasis suppressive effects of RBM47 in vivo , as wellb) Single cell-derived metastatic cell lines with doxycycline-inducible RBM47 expression were generated. This allowed us to control RBM47 protein levels, as can be seen in c) In the RNA-seq analysis, stringent conditions were applied to ensure that the RBM47 responsive signature consisted of genes that were changed already at the lowest detectable level of RBM47 protein expression. This signature was able to classify human cancer specimens in a way that correlated with RBM47 expression, as is shown in In addition, DKK1 knockdown was able to partially rescue the effects of RBM47 reintroduction in the brain metastasis assay . Thus, w8) It would significantly strengthen the manuscript if the effect of RBM47 on metastasis could be ascribed to a specific step in metastasis .As discussed in Response 2, RBM47 like all nuclear RNA binding proteins is likely to function in multiprotein complexes to facilitate mRNA biogenesis. In line with this, we find that RBM47 binds to multiple target mRNAs. These targets do not belong to a distinct pathway or molecular class, but rather, represent transcripts that encode for proteins that have functional roles in several biological processes. We focused on DKK1 as one of the many target molecules of RBM47 and show that RBM47 can modulate Wnt signaling via DKK1 stabilization. The Wnt pathway can modulate multiple breast cancer phenotypes including proliferation (9-11), migration (6) and stem cell fitness (12). In line with this, we show in 9) Since no biochemical analysis was performed on the RBM47 I281fs mutant, it is unclear whether this mutation completely abolishes RBM47 function. Therefore, in, LM2 cells expressing a vector control should also have been used to compare the effect on expressing WT and MUT RBM47.RBM47  InshRNAs were used to knock down RBM47 in LM2 cells to determine the effects on lung colonization. Western blotting analysis should be presented to show the RBM47 knockdown levels. Instead, it is confusing whyshows the western blot with CN34 cells.As requested, the Western blot related to 11)should include staining for RBM47 in the parental cells or cells expressing a control vector. Otherwise, it is unclear what is the negative RBM47 staining.states these are RBM47 negative tumors, although many show positive signals. It is important to co-stain for a marker specific for tumor cells, so that expression of RBM47 in tumor cells vs. stromal cells can be separated.The I281fs mutation introduces a premature stop codon. For this reason, the epitope against which the RBM47 antibody was developed is missing from the mutant protein. RBM47 mutant expressing cells can therefore be used as a negative control, as is shown in ReferencesEndogenous human microRNAs that suppress breast cancer metastasis. Nature, 2008. 451(7175): p. 147-52.1) Tavazoie, S.F., et al., Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature, 2007. 446(7137): p. 765-70.2) Gupta, G.P., et al., Genes that mediate breast cancer metastasis to the brain. Nature, 2009. 459(7249): p. 1005-9.3) Bos, P.D., et al., Genes that mediate breast cancer metastasis to lung. Nature, 2005. 436(7050): p. 518-24.4) Minn, A.J., et al., Epigenetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal cancer. Nat Med, 2013. 19(1): p. 50-6.5) Vanharanta, S., et al., WNT signaling enhances breast cancer cell motility and blockade of the WNT pathway by sFRP1 suppresses MDA-MB-231 xenograft growth. Breast Cancer Res, 2009. 11(3): p. R32.6) Matsuda, Y., et al., Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence. RNA, 2009. 15(12): p. 2385-97.7) Reid, D.C., et al., NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure. Elife, 2013. 2: p. e00178.8) Eom, T., et al., An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell, 2004. 6(5): p. 497-506.9) Bafico, A., et al., c-Myc transforms human mammary epithelial cells through repression of the Wnt inhibitors DKK1 and SFRP1. Mol Cell Biol, 2007. 27(14): p. 5135-46.10) Cowling, V.H., et al., Dickkopf-1 mediated tumor suppression in human breast carcinoma cells. Breast Cancer Res Treat, 2008. 112(2): p. 263-73.11) Mikheev, A.M., et al., Breast cancer cells produce tenascin C as a metastatic niche component to colonize the lungs. Nat Med, 2011. 17(7): p. 867-74.12) Oskarsson, T., et al.,