HnRNP A1 regulates many alternative splicing events by the recognition of splicing silencer elements. Here, we provide the solution structures of its two RNA recognition motifs (RRMs) in complex with short RNA. In addition, we show by NMR that both RRMs of hnRNP A1 can bind simultaneously to a single bipartite motif of the human intronic splicing silencer ISS-N1, which controls survival of motor neuron exon 7 splicing. RRM2 binds to the upstream motif and RRM1 to the downstream motif. Combining the insights from the structure with in cell splicing assays we show that the architecture and organization of the two RRMs is essential to hnRNP A1 function. The disruption of the inter-RRM interaction or the loss of RNA binding capacity of either RRM impairs splicing repression by hnRNP A1. Furthermore, both binding sites within the ISS-N1 are important for splicing repression and their contributions are cumulative rather than synergistic.DOI:http://dx.doi.org/10.7554/eLife.25736.001 Post-transcriptional control of gene expression is a tightly regulated process in higher organisms, which involves a large variety of RNA-binding proteins that associate with newly transcribed messenger RNAs. Among them, hnRNP A1, one of the most abundant and ubiquitously expressed nuclear proteins, participates in a wide range of nucleic acid processing events, and hence constitutes a major regulator of gene expression (reviewed in ). For inSMN) genes. This splicing event is critically associated with the neuromuscular disease spinal muscular atrophy (SMA) and is one of the most studied examples of a splicing regulated exon. SMA originates from a deletion or a mutation within the SMN1 gene, which impairs the production of the SMN protein. The SMN2 gene is nearly identical to SMN1 but fails to rescue the inactive SMN1 because a critical C-to-U change at position 6 of exon 7 strongly weakens exon 7 splicing , which contains two closely related RNA recognition motifs (RRM1 and RRM2) responsible for the specific RNA-binding properties , they are neither redundant nor functionally equivalent. Indeed, chimeric proteins constructed by duplication, swapping or deletion of the RRMs affect hnRNP A1 alternative splicing properties differently . HoweverHnRNP A1 RRMs are interacting with one another and adopt a single defined orientation, as observed by X-ray crystallography and solution NMR . Since RSMN splicing repression and that their binding contributions are cumulative rather than synergistic.To address those questions, we studied each RRM of hnRNP A1 in isolation and showed that both RRMs have similar sequence-specificity and affinity. But while RRM2 specifically recognizes three nucleotides, RRM1 is able to recognize a longer motif. We also investigated how hnRNP A1 RRMs bind the ISS-N1 RNA and showed that both RRMs can bind this RNA simultaneously, RRM2 binding its 5´part. This RNA-binding topology directly depends on the relative orientation of the two RRMs and we showed that disrupting the inter-RRM interface impairs hnRNP A1 function in splicing. We also showed that both RRM binding sites within ISS-N1 are important for In order to better characterize the differences in terms of specificities of each RRM, we decided to study each hnRNP A1 RRM in isolation. We thus performed NMR titrations of the isolated RRMs with several short RNA ranging from 6 to 8 nucleotides, each containing a core AG dinucleotide central for hnRNP A1 binding  Appendi. The RNAWe solved the solution structure of hnRNP A1 RRM1 in complex with 5´-UUAGGUC-3´ using 2560 NOE-derived distance restraints including 117 intermolecular ones. This large number of constraints allowed us to obtain a precise structure with a backbone r.m.s.d. over the entire domain of 0.40 ± 0.11 Å for the 20 conformers ensemble .10.7554/1 to G5), four of which are sequence-specifically recognized through a set of intermolecular hydrogen bonds to the Watson-Crick edges of the bases   , where ten of A3 . This inen of A3 . In 10 ctructure . The fla2 for G5 . Althougensemble , possiblensemble . The nucWe solved the solution structure of hnRNP A1 RRM2 in complex with 5´-UCAGUU-3´ with 2253 NOE-derived distance restraints including 111 intermolecular ones. As for RRM1, we obtained a precise structure with a backbone r.m.s.d. of 0.45 ± 0.08 Å for the 20 conformers ensemble .2 to G4), two of which are sequence-specifically recognized through a set of intermolecular hydrogen-bonds  folds into an α-helix only upon binding to RNA. This folding event explains the large chemical shift variations seen for this entire region upon RNA binding  are absent from both the free and bound spectra, reflecting a conformational dynamic for this loop. As a consequence, intermolecular contacts between this region and U1 cannot be observed in solution, although intermolecular contacts were seen to the equivalent of U1 in the structure of UP1-DNA .Therefore, in order to further confirm or infirm our NMR-derived RNA-binding consensus for each RRM, we measured the affinity of the isolated RRM1 and RRM2 for RNA sequences varying at the two positions flanking the AG core using ITC. In addition, in order to quantitatively estimate the importance of the protein-RNA contacts seen in the structures  and 2, w1 and contribute to the RNA-binding affinity. Removal of the side-chain  has no effect on RNA binding , and could confidently assign the RNA around the two AG dinucleotides present in the ISS-N1 sequence . Overall11A12G13 .11A12G13 motif for RRM2 and the A22A23G24G25 motif for RRM1, and placed a 4-thio-uridine spin-label downstream of G25. The spin-label should not perturb RNA binding at this position, but it should affect the resonances of the RRM binding the 3´-AG motif. We know from our structures of the single RRMs, that the spin-label should be capable of sampling multiple conformations resulting in the perturbation of a large area on one side of the domain. In addition, to exclude that the R140A mutation influences the binding topology, the PRE data were also recorded for the modified ISS-N1 bound by UP1 wild-type  experiments with a nitroxide spin-label attached to the 3’-end of the RNA. For this purpose, we left the two RNA binding motifs unchanged, namely the Cild-type .10.7554/vs. reduced spin label shows that the signals affected by the presence of the paramagnetic probe originated mostly from RRM1 and the interdomain linker matching perfectly our previous experimental data. We observe this local perturbation in RRM1 and the interdomain linker for both UP1 constructs . Our structural model  differs SMN2 splicing , possibly due to a strong topological constraint imposed by the IRL.Our structural investigation of the mode of binding of the ISS-N1 by hnRNP A1 hints at the importance of both RRMs for interacting with the RNA. For RRM1 to bind with high affinity, the IRL must be positioned such as to sandwich the RNA together with the β-sheet surface of RRM1. This proper positioning of the IRL is achieved by RRM2 contacting RRM1 through a network of interactions including two salt bridges (Arg88-Asp157 and Arg75-Asp155). If the nterface , which interface . NeverthSMN1 minigene  and RRM1 (r1R2)   . This efSMN1 exon 7 ) had a very strong effect on exon 7 inclusion  but not to the same level as in the wild-type SMN2-minigene (24%). Removal of either the upstream or downstream AG  also increases exon 7 inclusion to 48% and 46%, respectively. This can be partially suppressed to 32% and 30%, respectively, by hnRNP A1 overexpression for both ISS-N1 mutants (Mutation of both AG core dinucleotides to AA (ISS-N1-13a24a) raises as expected  mutants . To be s mutants , which d mutants .The effect observed for the ISS-N1-13a24a mutant is approximately the sum of the effects of the two single-substitutions, suggesting an additive rather than a synergistic effect of the two motifs for splicing repression. The same holds true when hnRNP A1 is overexpressed, with the RNA double-mutant being less efficient for targeting a splicing repression by hnRNP A1 than the single mutants. Altogether, this shows that both RNA-binding sites of the ISS-N1 are equally important for splicing repression of exon 7.As stated before, our structural model suggests that a minimal spacer of at least 4 nucleotides is necessary to allow binding of both RRMs of a single hnRNP A1 molecule to ISS-N1. Therefore, we tested the effect of shortening the 9-nucleotide spacer by steps of 2 nt (ISS-N1 −2 to ISS-N1 −8 mutants) .SMN2 minigene containing the 2-nt deletions showed a significant increase in exon 7 inclusion for ISS-N1 −6 and ISS-N1 −8. In contrast the ISS-N1 −2 and ISS-N1 −4 SMN2 minigene mutants showed no aberration of the splicing pattern  in a context where both RRMs interact intramolecularly as depicted in SMN pre-mRNAs. We could show that both RRMs can bind ISS-N1 in vitro, with RRM2 binding the upstream AG motif. Structure-based mutations and splicing assays in cells are consistent with this mode of binding. Altogether, our findings have important implications for understanding the RNA-binding mode of hnRNP A1 and its mechanism of action in splicing regulation as discussed below.In this report, we investigated the RNA-binding properties of hnRNP A1 RRMs, and particularly, the RNA-binding mode of hnRNP A1 to the ISS-N1 motif that silences splicing of the CAGG-3´ and 5´-U/CAGN-3´, respectively. The same preference was found by Jain and co-workers using HTS-EQ on the HIV ESS3 hnRNP A1 binding site -HSQC or (1H)−1D NMR spectra. Short RNA oligonucleotides were purchased from Dharmacon, deprotected according to the manufacturer’s instructions, lyophilized and resuspended in the NMR buffer . NMR samples of protein–RNA complexes were prepared at 0.8–1.0 mM at a ratio of 1:1 in a sample volume of 250 µL. Long RNA oligonucleotides, that is, ISS-N1 wild-type and mutants, were prepared by in vitro transcription with T7 polymerase . RNA was purified by anion-exchange high-pressure liquid chromatography under denaturing conditions, as previously described  and RRM2 (95–196), were expressed and purified as previously described . Mutagenescribed .www.cgl.ucsf.edu/home/sparky/).All NMR measurements were conducted in the same buffer  at a temperature of 303 K. NMR spectra were measured on Bruker AVIII-500 MHz, AVIII-600 MHz, Avance-600 MHz, AVIII-700 MHz, AVIII-750 MHz and Avance-900 MHz spectrometers. Except for the 750 MHz, all spectrometers were equipped with cryoprobes. The data were processed using TOPSPIN 3.x (Bruker) and analyzed with Sparky -HSQC, 2D -HSQC, 3D HNCA, 3D HNCACB, 3D CBCA(CO)NH, 3D HNCO, 3D HN(CA)CO, 3D  HCC(CO)NH-TOCSY, 3D  HCC(CO)NH-TOCSY, 3D NOESY--HSQC and two 3D NOESY--HSQC optimized for the observation of protons attached to aliphatic carbons or to aromatic carbons. In addition, the assignment of aromatic protons was conducted using 2D -TOCSY and 2D -NOESY measured in D2O.Protein resonances were assigned with 2D -TOCSY, 2D -NOESY, and 2D 13C F1-filtered F2-filtered -NOESY -NOESY, 2D 13C F2-filtered -NOESY -NOESY . ProteinH)-NOESY  and 3D 1spectrum , all rec methods  as previ methods .2O; their hydrogen-bond acceptors were identified from preliminary structures as well as from analysis of the characteristic NOE pattern found in α-helices and β-sheets. For protein-RNA intermolecular hydrogen-bonds, donors and acceptors were identified from preliminary structures and confirmed by large NH or CO chemical shift displacement upon RNA binding. In addition, dihedral δ angles of sugars were restrained to the C2´-endo conformation for nucleotides with a strong H1´-H2´ cross-peak in the TOCSY spectrum. We calculated 100 independent structures that we refined in a water shell with the program CNS 1.3 (http://www.pymol.org).Automated NOE cross-peak assignments  and stru CNS 1.3  includin CNS 1.3 . Twenty  CNS 1.3  and the  CNS 1.3 . Overall4U-modified RNA and 10 mM IA-proxyl spin label -HSQC spectra were measured before (paramagnetic sample) and after reduction of the spin-label with the addition of 2 mM ascorbic acid (diamagnetic sample). Backbone chemical shift assignment of the complexes was inferred from the assignment of the UP1-R140A:ISS-N1-u25g complex. Residues with a ratio of the intensity in the oxidized or paramagnetic state over the intensity in the reduced or diamagnetic state (Ipara/Idia) lower than 0.7 were considered as affected by the presence of the spin label.A modified RNA (5´-ACCAGCAUUAUGAAAGG(4-thio-U)A-3´) was purchased from Dharmacon and deprotected according to the manufacturer’s instructions. The 3-(2-iodoacetamido)proxyl (IA-proxyl) spin-label was dissolved in 100% methanol to obtain a 100 mM concentration. The modified RNA was resuspended into a reaction buffer containing 100 mM potassium phosphate pH 8.0, 100 μM sin label . The reat 320 nm . The spi13C/15N-labeled protein in complex with an unlabeled RNA, we could determine the unambiguous intermolecular NOE for the assigned nucleotides . Next, using a filtered NOESY experiments measured on a Model structures of the UP1-R140A:ISS-N1-u25g complex  were obtIsothermal titration calorimetry (ITC) experiments were performed on a VP-ITC instrument . Optical density absorbance at 280 and 260 nm was used to determine the concentrations of proteins and RNAs, respectively. Samples were prepared in NMR buffer (replacing 1 mM DTT with 10 mM 2-mercaptoethanol). The measurements were performed using the protein as titrant and the RNA as titrate. All measurements were taken at a cell temperature of 30°C over (40-)44 injections, each 6 µL, with a spacing time of 300 s and a filter period of 2 s. The reference power was set to 10 µCal/s and the initial delay to 60 s. The data was analyzed using the software Origin version 7.0  and a 1:1 binding model using nonlinear least-squares fitting. For measurements with isolated RRM1 or RRM2 (WT and mutants) the baseline was set manually for all experiments prior to integration, and the data was corrected for nonspecific heats. For measurements with the UP1 mutants no baseline correction was applied and the data was corrected for dilution effects by subtracting the integrated heats of a control experiment of protein titration into buffer prior to fitting.The output of the ITC measurements can be found in SMN1 or SMN2 minigene, respectively, were previously described (HEK293T (human embryonic kidney) cells were obtained from the European Collection of Cell Cultures (ECACC No. 85120602) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine albumin (FBS) and 1% streptomycin/penicillin. The cells were checked for mycoplasma contamination with GATC’s MYCOPLASMACHECK and found to be clean. The pCI-SMN1 and pCI-SMN2 plasmids containing the escribed . For hnRescribed  but withescribed .15 primer and M-MLV Reverse Transcriptase RNase (H-) (Promega). 10% of the resulting cDNA was then used for semiquantitative PCR using a vector specific forward primer (pCI-fwd 5´-GGTGTCCACTCCCAGTTCAA-3´) and a SMN1 specific reverse primer (SMN1rev 5´-AGCCTTATGCAGTTGCTCTC-3´) or a SMN2 specific reverse primer (SMN2rev 5´-GCCTCACCACCGTGCTGG-3), respectively. For SMN2 samples, the PCR was run with 32P 5-labeled SMN2rev primer. The bands corresponding to the product of the splicing reaction were quantified using AlphaView  from radioautography of a 4% polyacrylamide gel for SMN2 samples and by staining the gel with GelRed (Biotium) for SMN1 samples. The ratio of each isoform was normalized to the sum of isoforms. Experiments were repeated three times independently allowing for the calculation of the mean and standard error of the mean for each assay.One microgram of pCI-SMN1 and SMN2 (wild-type (WT) or mutant) was co-transfected with 0.5 µg or 1.0 µg or 2.0 µg of pCGFLAG hnRNP A1 (WT or mutant), respectively, in HEK293T cells counted and seeded 24 hr in advance at 400’000 cells/well, plated in six-well plates using the Lipofectamine 2000 Reagent  according to the manufacturers protocol. After 48 hr total RNA was extracted and 1 µg was used for reverse transcription using Oligo(dT)Fractionation of nuclear and cytoplasmic fractions was done with the NE-PER kit (Thermo Scientific). Protein samples were separated by 15% SDS-PAGE and then electroblotted onto nitrocellulose membranes , blocked with 5% non-fat dry milk in Tris-buffered saline +0.1% Tween (TBS-T). Probing of the blots was done with monoclonal anti-FLAG  in TBS-T and 5% non-fat dry milk, or anti-DDX5  in TBS-T, washed three times in TBS-T and 5% non-fat dry milk, followed by incubation with horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Sigma) for 2 hr in TBS-T and 5% non-fat dry milk. Protein signals were detected with chemiluminescence imaging (Amersham Imager 600RGB).The chemical shifts of hnRNP A1 RRMs in complex with RNA have been deposited in the Biological Magnetic Resonance Bank under accession numbers 34079 and 34080 for RRM1 and RRM2, respectively. The coordinates of the structures have been deposited in the Protein Data Bank under accession codes 5MPG and 5MPL for RRM1 and RRM2, respectively. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by James Manley (Senior Editor) and three reviewers, one of whom, Juan Valcárcel (Reviewer #1), is a member of our Board of Reviewing Editors.Thank you for submitting your article "Tandem hnRNP A1 RRMs act in concert to repress the splicing of survival motor neuron exon 7" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Joint Report:Beusch et al. report a detailed structure/function analysis of the interaction between the two RRMs of hnRNP A1 and an intronic silencer present in the SMN genes, involved in splicing regulation. This addresses a long-standing question in protein-RNA recognition and splicing regulation. The authors work out the arrangement of each of the RRM motifs on the bipartite silencer sequence and come to the conclusion that both RRMs contribute additively to RNA binding and that interaction between the two RRM motifs bound to the adjacent half-sites is important for RNA binding and splicing repression.These results are significant, and may help to identify new binding sites and enhance our understanding of RNA binding and splicing regulation by a paradigmatic RNA binding protein. The results go beyond successive previous structures by crystallography and NMR describing interactions of hnRNP A1 with nucleic acids and establish the architecture of an RNP complex that is the target of an antisense oligonucleotide that has revolutionized the treatment of Spinal Muscular Atrophy. The structural data are of high quality and there is overall good correlation with functional data.Major issues:1) The authors characterize HNRNPA1 RRM1 and RRM2 binding to RNA independently by NMR, and identify binding motifs that differ slightly from previous SELEX motifs. The optimal binding sequence for RRM2 was identified as UCAGUU  Further experimental evidence to rule out that multiple HNRNPA1 molecules bind to the same RNA strand could be provided by co-transfecting cells with HNRNPA1 R1r2 and r1R2. If each ISS-N1 binding site can only interact with a single hnRNPA1 molecule, there should not be an additive or synergistic effect.3) – The protein analysis of 4) Based on the result in 5) A manuscript just appeared in PNAS by Tolbert describing the specificity of hnRNPA1. The authors should compare the two sets of results and evaluate them critically.6) It is a bit unclear if the model of the 2 RRM-RNA structure is a model or a formal structure. This could be clarified, perhaps by showing a table with proper statistics to show what the data actually establish.Reviewer #1:Beusch et al. report a detailed structure/function analysis of the interaction between the two RRMs of hnRNP A1 and an intronic silencer present in the SMN genes, involved in splicing regulation. The authors work out the arrangement of each of the RRM motifs on the bipartite silencer sequence and come to the conclusion that both RRMs contribute additively to RNA binding and that interaction between the two RRM motifs bound to the adjacent half-sites is important for RNA binding and splicing repression.These results are important because they differ substantially from previous data describing interactions of hnRNP A1 with nucleic acids and establish the architecture of an RNP complex that is the target of an antisense oligonucleotide that has revolutionized the treatment of Spinal Muscular Atrophy. The structural data are of high quality and there is overall good correlation with functional data.In my opinion the following revision would improve the manuscript:The protein analysis of Reviewer #2:Allain and coworkers address a long-standing question in protein-RNA recognition and splicing regulation. The hnRNPA1 protein regulates alternative splicing and microRNA processing and maturation, and has long been studied as a model to understand RNA recognition as well, However, despite successive structures by crystallography and NMR, a structure of the two RRM protein bound to RNA has been missing and, which would come with it, a thorough knowledge of how this protein binds.Here the authors make several important advances. First, they fully characterize the structure and specificity of each RRM. Then they use this information to optimize the sequence the didomain construct bind to and use this to determine a model of the complete two RRM-RNA structure. Then, they go on to test the biological role of the features discovered in their investigation by using a reporter minigene alternative splicing construct.The advances are significant on a paradigmatic protein. I have however a few suggestions1) A manuscript just appeared in PNAS by Tolbert describing the specificity of hnRNPA1. The authors should compare the two sets of results and evaluate them critically.2) It is a bit unclear if the model of the 2 RRM-RNA structure is a model or a formal structure. This could be clarified, perhaps by showing a table with proper statistics to show what the data actually establish.3) There is too much detail about NMR throughout the manuscript, which would detract from the attention of many readers. Much could be moved to supplementary material.Reviewer #3:HNRNPA1 is an abundant RNA-binding protein with important roles in splicing regulation, which involves binding of its two-RRM UP1 domain to RNA splicing silencer elements. Previous results by the Allain lab revealed a different relative orientation of the RRMs in solution, compared to earlier x-ray crystal structures of UP1 bound to telomeric ssDNA. This manuscript characterizes HNRNPA1 RNA binding by solution NMR, and shows that RRM1 and RRM2 can bind to two nearby motifs on the same RNA strand. Cell-based splicing assays with an SMN minigene suggest that disrupting the interaction between RRM1 and RRM2 inhibits HNRNPA1 repressive activity, and both RRMs are necessary for optimal activity. The authors propose a new model for HNRNPA1 binding to RNA, which may help to identify new binding sites.I do not have the expertise to judge the technical quality of the NMR results, but overall, the data are described clearly and the figures are easy to understand. The results enhance the understanding of RNA binding and splicing regulation by HNRNPA1. Several aspects of the data require clarification:The authors characterize HNRNPA1 RRM1 and RRM2 binding to RNA independently by NMR, and identify binding motifs that differ slightly from previous SELEX motifs. The optimal binding sequence for RRM2 was identified as UCAGUU  The authors characterize HNRNPA1 RRM1 and RRM2 binding to RNA independently by NMR, and identify binding motifs that differ slightly from previous SELEX motifs. The optimal binding sequence for RRM2 was identified as UCAGUU , the authors used a known HNRNPA1 binding site in SMN2 exon 7, with mutations to enhance binding . It is uAs a general principle, we would like to emphasize that exchange regime in NMR complicate the study of complexes. As a result of this, it is not necessarily the complex with the highest affinity that will give the best NMR signals. Similarly, as for the previous point, the choice of the point mutation introduced here (UP1-R140A) was not meant to increase the affinity, but to solely improve the NMR behavior of the complex. In the wild-type complex, some signals at the binding interface disappeared upon RNA addition , which i“Since it is not necessarily the complex with the best affinity that will give the best NMR signals, we then decided to take the opposite approach of weakening the overall affinity of the complex with the aim of reaching the fast exchange regime with improved NMR signals.”In addition, in the main text, this point is now briefly explained as follows:“Since it is not necessarily the complex with the highest affinity that will give the best NMR signals, we then took the opposite approach of weakening the overall affinity of the complex with the protein mutant UP1-R140A .”The authors explain the ~5-fold increase in RNA-binding affinity of UP1 R1r2, compared to RRM1, by the lack of the inter-RRM linker. Although the mutations in UP1 R1r2 are similar to mutations in UP1 r1R2 that appear not to contribute to RNA binding, the increase in binding could also be explained by residual RNA binding to RRM2 of U1 R1r2, which should be discussed or tested by experiments similar to those listed in The reviewers are concerned that the increase in affinity observed for UP1 R1r2 compared to RRM1 could also be explained by residual RNA-binding to RRM2 of UP1 R1r2 despite the mutations in RRM2. In the fitting of ITC data, one of the parameters is the N-value indicative of the stoichiometry of the measured interaction (mentioned on page 17). N is one of the most precisely determined parameters and should be a warning sign for experimental problems, such as a wrong fitting model, when deviations from the expected value do occur . Our data robustly fit with a 1:1 stoichiometry (N = 1)  Further experimental evidence to rule out that multiple HNRNPA1 molecules bind to the same RNA strand could be provided by co-transfecting cells with HNRNPA1 R1r2 and r1R2. If each ISS-N1 binding site can only interact with a single hnRNPA1 molecule, there should not be an additive or synergistic effect.We thank the reviewers for suggesting this experiment. We have performed it and the results can now be found as We comment on these results as follows:“Our structural data proposes the simultaneous binding of both RRMs to the ISS-N1. […] In support of our model, we could not observe any rescue of hnRNP A1 function when co-transfecting with the two RRM mutants .”3) The protein analysis of We acknowledge that the original protein analysis in SMN splicing response remains the same under increased protein amounts.The only mutant that appears to be expressed at a different level under these conditions is hnRNP A1 R1r2. We have tested for dose effects by increasing the protein level by transfection of increased amounts of hnRNP A1 variants . We can In addition, our Western Blots were recorded by CCD-based detection , which offers a dynamic range of 4.8 orders of magnitude allowing for detection across a wide dynamic range for precise quantitation. This allows for a linear detection over a broad range of signals and should cover our range according to the manufacturer. The data obtained during the revisions are overall very similar to those previously obtained. Differences are precisely described in response to point #4.c) of UP1 WT and UP1 INT1-3 mutants. INT3 has, out of the three mutants, a correlation time in the closest range to the wild-type protein. This suggests, that in INT3 the interaction between RRM1 and RRM2 is less disrupted than in INT1 and INT2. This could explain why the INT3 mutant has a behavior closer to the WT protein, explaining its slightly higher activity in the SMN1 splicing assay. However, as the same cannot be seen for the INT2 mutant, which is also more active in the splicing assay, we refrained from discussing this potential correlation in the paper to avoid over interpretation of our data.Finally, the reviewers have raised the concern that INT3 appears most active, despite the apparently lower levels of protein expression. While with the new transfection protocol INT3 amounts do not differ from INT1 and INT2, the reviewers have raised an interesting question. In 4) Based on the result in SMN1 and SMN2 minigene system, hnRNP A1 r1R2 overexpression consistently had an effect and led to a minor repression of exon 7 inclusion compared to the control transfection with an empty plasmid. With the new protocol in place no such effect can be seen in comparison to the control transfection. Taking the new results into account we had to modify our interpretation of the results and their discussion and have now removed any mention of RRM2 being more important than RRM1. In addition, for the set of experiments where the ISS-N1 spacer length was varied, with the new protocol we no longer observe the increase in exon 7 inclusion for SMN2 ISS-N1 -2 which we could not explain earlier.Our new sets of experiments with new plasmid preparation and transfection protocols, lead to a different conclusion regarding the relative importance of the single RRMs. Previously, both in the SMN splicing.In addition, the reviewers mention the ASO tiling experiment performed by Hua et al. in their 2008 publication where steric blocking of the 3’AG had a stronger effect on SMN2 exon 7 inclusion than blocking of the 5’ AG. We believe that steric blocking will work with a different principle and cannot necessarily be compared to a mutation experiment. In the same publication, a mutational experiment was also performed, where individual positions of the ISS-N1 were mutated. In this case, and like in our experiment, in the absence of hnRNP A1 overexpression, targeting either AG had similar effects on SMN2 exon 7 splicing could be a result of unequal amounts of overexpressed protein. We have responded to this concern as a part of reviewers’ comment #3.Finally, the reviewers have raised the concern that a difference in We understand that each of our experiments only allows us to look at a single part of the UP1:ISS-N1 interaction decoupled from the others. Taking our results together as well as the new experiment added in response to reviewers’ comment #2, we are nevertheless very confident in our interpretation of the experiments.5) A manuscript just appeared in PNAS by Tolbert describing the specificity of hnRNPA1. The authors should compare the two sets of results and evaluate them critically.CAGG-3’for a wider description of the preference.Indeed, a manuscript by Tolbert and co-workers appeared in PNAS at the point of submission of our work. Using HTS-EQ and cross-validation with ITC they have found a consensus motif of 5’-YAG-3’ for hnRNP A1. Furthermore, it was found that an additional guanine immediately 3’ to the motif is favored too. Together this matches our findings for a strong preference of 5’-UAG-3’, which can be extended to 5’-U/We have added this analysis of the manuscript by Tolbert and co-workers to the Discussion.6) It is a bit unclear if the model of the 2 RRM-RNA structure is a model or a formal structure. This could be clarified, perhaps by showing a table with proper statistics to show what the data actually establish.The two RRM-RNA structure is not a formal NMR structure, and we refer to it as a “structural model” throughout the manuscript. Although we did use similar protocols as for regular NMR structure calculations, the ensemble that we have obtained cannot be seen as a formal NMR structure. The main reason for this is that the RNA-spacer separating the two RRM-bound motifs of the ISS-N1 was not restricted in our modelling procedure, since we did not assign intermolecular NOEs to this region. For this reason we do not consider it as an actual structure.In order to make this point clearer for the reader, we have expended the statement in the figure legend of In addition, in the main text, we have added the following sentence: “Combining all the structural information obtained on this complex, we could build a structural model showing how hnRNP A1 RRMs can assemble on the same pre-mRNA stretch (See Appendix 2 for details).”And in the Appendix 2 we have added the following paragraph:“Important remark: It is important to note that this model only illustrates one possible path of the ISS-RNA from RRM2 towards RRM1. […] Nevertheless, this model illustrates that the RNA-spacer is long enough for hnRNP A1 RRMs to loop out RNA and bind simultaneously to the 5´- and 3´-motifs.”


Orthomyxoviridae family and their genome consists of eight (-) sense viral RNA (vRNA) segments of different lengths coding for at least 11 viral proteins. A heterotrimeric polymerase complex is bound to the promoter consisting of the 13 5′-terminal and 12 3′-terminal nucleotides of each vRNA, while internal parts of the vRNAs are associated with multiple copies of the viral nucleoprotein (NP), thus forming ribonucleoproteins (vRNP). Transcription and replication of vRNAs result in viral mRNAs (vmRNAs) and complementary RNAs (cRNAs), respectively. Complementary RNAs are the exact positive copies of vRNAs; they also form ribonucleoproteins (cRNPs) and are intermediate templates in the vRNA amplification process. On the contrary, vmRNAs have a 5′ cap snatched from cellular mRNAs and a 3′ polyA tail, both gained by the viral polymerase complex. Hence, unlike vRNAs and cRNAs, vmRNAs do not have a terminal promoter able to recruit the viral polymerase. Furthermore, synthesis of at least two viral proteins requires vmRNA splicing. Except for extensive analysis of the viral promoter structure and function and a few, mostly bioinformatics, studies addressing the vRNA and vmRNA structure, structural studies of the influenza A vRNAs, cRNAs, and vmRNAs are still in their infancy. The recent crystal structures of the influenza polymerase heterotrimeric complex drastically improved our understanding of the replication and transcription processes. The vRNA structure has been mainly studied in vitro using RNA probing, but its structure has been very recently studied within native vRNPs using crosslinking and RNA probing coupled to next generation RNA sequencing. Concerning vmRNAs, most studies focused on the segment M and NS splice sites and several structures initially predicted by bioinformatics analysis have now been validated experimentally and their role in the viral life cycle demonstrated. This review aims to compile the structural motifs found in the different RNA classes  of influenza viruses and their function in the viral replication cycle.Influenza A viruses (IAV) are responsible for recurrent influenza epidemics and occasional devastating pandemics in humans and animals. They belong to the  Orthomyxoviridae family that comprises seven genera: Alphainfluenzavirus, Betainfluenzavirus, Gammainfluenzavirus, and Deltainfluenzavirus, as well as Isavirus , Thogotovirus (transmitted by arthropods), and Quaranjavirus (which predominantly infects insects and birds). Among this family, the major threat for public health is the Alphainfluenzavirus genera. Influenza B and C share common ancestry with Influenza A virus (IAV). However, Influenza B (IBV) and C (ICV) viruses mainly infect human and are genetically less diverse. IBV, like IAV, has 8 genome segments coding for at least 11 proteins , whereas Influenza C has only 7 genome segments coding for at least 9 proteins. The segments are either numbered from the longest to the shortest or named after the main protein they code for, e.g., in influenza A viruses, segments 1–8 are also named PB2 (Polymerase Basic 2), PB1 (Polymerase Basic 1), PA (Polymerase Acid), HA (HemAgglutinin), NP (nucleoprotein), NA (NeurAminidase), M (Matrix), and NS , respectively.Influenza viruses belong to the Each segment constitutes an individual replication unit coding for at least one essential protein and form viral ribonucleoproteins (vRNPs). They are composed of a viral RNA (vRNA) of negative polarity, a heterotrimeric polymerase complex bound at the paired 5′ and 3′ termini of the vRNA and multiple copies of NP .de novo and are used as template for producing new vRNA molecules that will be packaged.After viral entry in the host cell, vRNPs are imported in the nucleus, where transcription and replication take place. During these steps, two different classes of RNA of positive polarity are synthetized: complementary RNA (cRNA), which is the exact copy of vRNA, and viral messenger RNA (vmRNA) . The latThe RNA secondary structure is biologically important for many RNA viruses, governing key processes, including viral replication, RNA packaging, RNA editing, and mRNA splicing regulation. Influenza viruses are no exception and several mechanisms during their life cycle are controlled by RNA structure. Steps such as splicing, the switch between transcription and replication, vRNA packaging and recognition by the host immune system have been proposed to be under the control of RNA structure. Only few things are known about the structure of the vRNAs and even less about the structure–function relationships, but things are starting to change dramatically thanks in part to new methodologies exploiting the potential of next generation RNA sequencing.This review aimed to compile the structural motifs found in the different RNA classes of influenza viruses and their function in the viral replication cycle. A previous review was published many years ago and focused on the differences and similarities between the RNA structure in IAV and the genome structure of other RNA viruses .For convenience, in this review, the numeration of nucleotides forming the promoter will be as follows: from the 5′ terminus, nucleotides will be numbered as X′, while from the 3′ terminus, they will be numbered as X.Figure 1B). They all share some features such as base pairing between certain parts of the 3′ and 5′ ends but are quite different when it comes to the structure of the first nucleotides at each end. Figure 1B-1) arising from sequence complementarity between the 3′ and 5′ vRNA ends. They proposed that this panhandle structure would be found only within vRNPs, and that no interaction between the two ends would take place in ‘naked’ vRNAs. The same year, Figure 1B-2). Such a pairing would occur in naked vRNAs but the addition of NP would melt the structure. The same year, Figure 1B-3). In this model, two short stem-loops are formed within the 3′ (nucleotides 1–9) and 5′ ends (nucleotides 1′ to 9′) and a five base-pair ‘duplex region’ is formed between both ends from nucleotides 11′ and 10 to nucleotides 13′ and 12 . The 5′ stem-loop (or ‘hook’) is formed by two canonical base-pairs flanked at both sides by non-canonical A-A base pairs, and this compact structure binds in a pocket at the interface of the PA and PB1 polymerase subunits  only in the absence of the polymerase. Accordingly, Figure 1B-1) is unclear. When the polymerase complex is bound to the promoter, a structural rearrangement occurs with the formation of a hook in the 5′ end proximal region, leaving the 3′ end single stranded .The vRNA promoter of IAV is used for two main mechanisms, transcription (which leads to the synthesis of capped and polyadenylated vmRNA) and replication (synthesis of cRNA which will be used as template for vRNA synthesis). The fork model described earlier was proposed in order to explain the initiation of the transcription, where the polymerase complex first binds the single-stranded 5′ end of the vRNA and then the single-stranded 3′ end . Howeversubunits . These rsubunits , as it sstranded . The disAs mentioned earlier, the duplex region of the promoter can be extended in a segment-specific way. In the case of the HA segment, this duplex can be extended by forming a base pair between nucleotides 13 and 14′. Figure 1B-3)   . Of notere 1B-4) . Using tre 1B-4) .During viral transcription, polyadenylation of vmRNAs is also regulated by the vRNA promoter structure. Indeed, The results discussed above show that the vRNA promoter structure is essential for the whole transcription process, from the beginning (cap-snatching) to the end (polyA tailing). In addition, the switch between transcription and replication of the vRNA could also be due, at least partly, to the promoter structure. Indeed, even though the IAV promoter is highly conserved across viral strains and genomic segments, it comes in two variants. The promoter of the three segments coding for the polymerase subunits have a C residue at position 4 (C4 promoter), while the promoter of the five remaining segments has a U residue at this position (U4 promoter). Using C4 and U4 promoters, in vitro is sufficient to induce IFN production and more importantly that the degree of complementarity in the proximal step of the promoter is critical for RIG-I activation   (A bioinformatics study of the RNA structure of segment M in both (+) and (-) strands indicated that the first structure found on both strands is similar, consisting of a stem-loop (nucleotides 219-240), named SL3-10, the difference being that on the (+) strand the loop is 8 nts long, while it is only 4 nts long in (-) strand, but with a longer stem (gure 3A) . The samgure 3B) . Mutatiogure 3B) .in vitro in the absence of NP protein and polymerase complex, they most likely do not reflect the actual vRNA structure found within vRNPs in the infected cells or in the viral particles. Nevertheless, the presence of multiple single stranded regions suggests that they could constitute potential interaction sites between the different vRNAs leading to an efficient and specific packaging . To date this remains the only inter-vRNA interaction that has been unambiguously demonstrated to play a role in packaging.Such interactions have been described man H3N2 ,b and anman H3N2 . It was man H3N2 . These iarticles . The inttrans-compensatory mutants the observed defects could also be attributed to alteration of functionally important intramolecular structures. In the same study, SHAPE-MaP experiments  were performed to compare the flexibility profile of each vRNA nucleotide in vitro and ex vitro, after removal of the vRNP-associated proteins . The results suggest that the structure is more flexible in vitro with uneven NP binding along the genome. Interestingly, a recent HITS-CLIP (High Throughput Sequencing of RNA isolated by CrossLinking ImmunoPrecipitation) study showed that NP does not bind evenly along the vRNAs: in each vRNA, there are zones of low NP density  . Most segments could potentially interact with at least two other vRNAs, and NA and NS are the vRNAs that form the fewest interactions. Mutations aiming at disrupting the proposed intermolecular interactions resulted in packaging defects ; however, in the absence of  density , which m density . Moreove density . These rAltogether, these recent studies suggest that both intermolecular vRNA-vRNA structures and intramolecular vRNA structures in NP-devoided regions play a major role in the selective packaging process of influenza viruses.While most of the structural elements that will be described below are specific to cRNA or mRNA, there is a common feature shared by these two molecules. In 2013, a study investigated binding of the NS1 protein to RNA and its specificity using SELEX  experiments . It was Unlike vmRNAs, cRNAs have a terminal promoter that is required for vRNA synthesis and is assumed to be organized in the same way as the vRNA promoter. However, due to the imperfect complementarity between the 5′ and 3′ RNA termini, the vRNA and cRNA promoters must be slightly different. Comparing the NMR structure of short vRNA and cRNA promoters in the absence of polymerase, Figure 1B-3), whereas in the cRNA promoter A10′ can form a base pair with U11, while U10 is unpaired . It was proposed that these differences between the promoters induce a different recognition by the polymerase complex  that plays a key role in the recognition of the polymerase complex. However, the distal stem of the cRNA promoter is one base pair shorter than its vRNA counterpart, its 3′ single-stranded region is one nucleotide longer (due to the unpaired U11), and the single-stranded nucleotides 3, 5, and 8 are different. Interestingly, nucleotides at positions 3 and 8 interact in a base-specific manner with the viral polymerase  in the whole Influenza genome (both strand orientations). GORS revealed ‘excess’ thermodynamic stability of wild-type RNA sequences versus random RNA of the same composition. This study demonstrated that GORS only exist in the (+) strand of segments PB2, NP, and NS  and thatThe vRNA and cRNA promoters are conserved in all segments of all influenza strains and thus their structure attracted a lot of attention. However, influenza A vmRNAs might also contain segment-specific structural motifs that regulate processes such as translation, export, or splicing. The later process, which is required for production of the NS2 and M2 proteins, has been the subject of several studies.Figure 5). These results were supported by an independent bioinformatics analysis , it was suggested that the equilibrium between the two structures could regulate splicing. Indeed, a single mutation predicted to induce misfolding of this RNA region strongly reduced production of the NS2/NEP mRNA and attenuated viral replication, whereas introduction of a second site mutation predicted to restore RNA folding restored splicing and viral replication, demonstrating the importance of the folding of this RNA region for splicing regulation . Disruption of this secondary structure reduced transcription of the NS1 vmRNA. However, this structure was questioned by Figure 6B), again using a bioinformatics approach. Figure 6C). They used RNA chemical probing and isoenergetic microarray techniques to confirm that this hairpin structure exists in the mRNA in vitro    but chemical and enzymatic RNA mapping, mutagenesis and NMR all support the multi-branch structure  displays the same structural feature as the 3′ splice site of segment NS, namely an equilibrium between a pseudoknot and a double hairpin structure that was first proposed by bioinformatics analysis (igure 7)  and suppigure 7) , as welligure 7) . As for igure 7) . A regiogure 8A) . This retructure . Howevertructure .Figure 9A), that they slightly revised based on chemical and enzymatic probing experiments .Within the NP vmRNA, nucleotides 1051–1171 were predicted to be structured . Indeed,2+ and has been proposed to regulate translation of PB1-F2 and N40.Another pseudoknot was predicted on the PB1 vmRNA (nucleotides 65–126); it contains the start codon of two alternative polypeptides encoded by this segment, PB1-F2 and N40. This structure was predicted using bioinformatics  and confThe polymerase complex of Influenza B virus bound to the vRNA promoter was crystalized and its X-ray structure was determined. Indeed, the IBV polymerase binds the vRNA promoter in the same way as the IAV polymerase and the IBV vRNA promoter also adopts the hook conformation when bound to the viral polymerase . RegardiTo determine possible structural constrains acting on Influenza viruses, protein-coding sequences of IBV were used to perform a survey for conserved secondary structure in the (+) and the (-) RNAs. The GORS analysis revealed similarities between IAV and IBV and GORS have been found in both RNA orientations in NP, NS, and PB2 segments . In genePrevious studies on IAV described a role for RNA structural motifs in the splicing of M and NS vmRNAs. The corresponding regions of IBV and ICV genomes have been scanned with the RNAz program and regions of high thermodynamic stability and conserved RNA secondary structure overlapping the 5′ splice sites of IBV segment NS and ICV segments M and NS were detected. In contrast, the structural motifs detected close to the IAV 5′ splice sites of segments M and NS do not overlap with these sites .Segment 8 (NS) of IAV is homologous to segments 8 and 7 of IBV and ICV, respectively, and in all cases NS2/NEP mRNA is produced by splicing. In both IBV and ICV, the 5′ splice site is located into the stem of a conserved hairpin, possibly sequestering the splice site. Additionally, a conserved hairpin is also predicted specifically in segment 6 (M) of ICV, in which the M1 protein is produced by splicing, in contrast to IAV and IBV, in which M1 is translated from the unspliced vmRNA . MoreoveIn this review, we focused on the relation between the RNA structure of influenza viruses and its function in the replication cycle. Most of our knowledge about influenza RNA structure and function deals with the vRNA/cRNA promoters and the splice sites of vmRNAs. Concerning the promoter, the recent X-ray structures of the viral heterotrimeric polymerase alone and bound to the vRNA promoter  shed a nConcerning the splice sites, several different structures have been proposed based on bioinformatics predictions. In several cases, chemical and enzymatic mapping experiments helped identify the actual structure. Interestingly, the IAV 3′ splice site of both segment M and NS mRNAs share the same structural feature, which has been shown to regulate splicing and viral replication. By contrast, the M and NS 5′ splice sites adopt different structures.Until recently, our knowledge of the structure of the internal part of vRNAs within vRNPs was almost inexistent. However, rapid progress in this area has been made within the last months thanks to the coupling of several classical RNA biochemistry tools with next generation sequencing  and obtaDF, MC, AP-S, RPS, J-CP, and RM conceived the review topic. DF, MC, and RM drafted the manuscript and DF generated the figures. AP-S, RPS, and J-CP corrected and edited the manuscript. All authors read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors DF, MC, AP-S, RS, J-CP, RM.


Geobacillus kaustophilus with biologically relevant dissociation constants. Furthermore, of the six sequences we characterize, four show regulatory activity in an Escherichia coli reporter assay. Subsequent footprinting and mutagenesis analysis indicates that protein binding proximal to regulatory features such as the Shine–Dalgarno sequence is sufficient to enable regulation, suggesting that regulation in response to S15 is relatively easily acquired.There are several natural examples of distinct RNA structures that interact with the same ligand to regulate the expression of homologous genes in different organisms. One essential question regarding this phenomenon is whether such RNA regulators are the result of convergent or divergent evolution. Are the RNAs derived from some common ancestor and diverged to the point where we cannot identify the similarity, or have multiple solutions to the same biological problem arisen independently? A key variable in assessing these alternatives is how frequently such regulators arise within sequence space. Ribosomal protein S15 is autogenously regulated via an RNA regulator in many bacterial species; four apparently distinct regulators have been functionally validated in different bacterial phyla. Here, we explore how frequently such regulators arise within a partially randomized sequence population. We find many RNAs that interact specifically with ribosomal protein S15 from  These regulators typically consist of a structured sensor (aptamer) that upon ligand binding alters the conformation of the mRNA to mask or reveal regulatory elements such as ribosome-binding sites, intrinsic transcription terminators or nuclease recognition sites , a metabolic precursor to the tRNA modification queosine (Escherichia coli and one in B. subtilis) in addition to the rRNA binding site, and ribosomal protein S15 interacts with at least four distinct mRNA structures in different bacterial phyla  does not interact with the mRNA regulator originating from E. coli (Ec-mRNA)  ,37, and rpsO open reading frame encoding S15 was polymerase chain reaction (PCR) amplified using whole genomic DNA and cloned into pET-HT overexpression vector similarly to previously described  and purified using isopropanol. The RNA aptamers were reverse transcribed using M-MuLV, and cDNA amplified using mutagenic PCR  at 25°C for 30 min, then RNA-Gk-S15 complexes were isolated by filtering with nitrocellulose. After two washes the bound RNAs were eluted from the filter . Genomic DNA extracted from the species was used as template. For all synthetic sequences the pCR–RNA was used as template to amplify DNA. T7 RNA polymerase was used to transcribe RNA and transcription reactions were purified and eluted as described in SELEX experiment (32P-ATP and purified as previously described (D) and maximum fraction bound (Fmax) were calculated by fitting the fraction of protein bound RNA  at a range of protein concentrations to the equation Fb = (Fmax*[S15])/([S15)+KD) where [S15] is the protein concentration using Excel Solver to minimize the residuals. Mutations to the mRNAs were constructed by site-directed mutagenesis .Binding and Competition Assay: For natural sequences, DNA corresponding to the 5′-UTR of the periment . Purifieescribed . Bindingescribed . Dissoci2CO3 pH 9.0, 1 mM EDTA at 95°C for 7 min. Denaturing T1 reaction (1:10 dilution) was conducted according to manufactures protocol (Ambion). For in-line probing, 5′-labeled RNA was incubated 40 h at 25°C in reaction buffer . The reaction was stopped using Urea loading solution . For lead(II)-probing, protein binding reactions were incubated for 10 min with 15 uM Pb(II)-acetate (300 mM stock prepared directly before use). Reactions were stopped with the addition of EDTA , and the addition of Urea loading solution. Reactions were loaded on 10% denaturing Acrylamide/Bis-acrylamide gel to separate RNA fragments. The gel was dried and visualized using a GE Healthcare STORM 820 phosphorimager and ImageQuant software.Footprinting assays: The RNA–protein binding reaction described above was used for RNAse probing assays. After incubation, 1 μl RNAse A  or VI  was added and the reaction incubated for 15 min at 25°C. The nuclease was inactivated with inactivation/precipitation buffer (Life Sciences) and RNA fragments recovered by ethanol precipitation. Precipitated RNAs were suspended in 10 μl Urea Loading solution (Life Sciences) and incubated 5 min 95°C. Partial hydroxyl cleavage reactions were generated by incubating RNA in 50 mM NalacZ using primers containing EcoRI and SalI restriction sites and template from TOPO PCR 2.1 cloned PCR product . The lacZ sequence requires a start codon from the fused rpsO sequence. All enzymes for molecular biology were purchased from New England Biolabs unless otherwise noted.Plasmid Construction: All synthetic sequences were cloned into the pBS3-RNA plasmid as a translational fusion with E. coli S15 ribosome binding site and an 8 nucleotide linker  preceding the ATG start site and subsequent codons. The reverse primer contained an XbaI site. After digestion, the PCR product was cloned into the pBAD33 vector (ATCC 87402) digested with the same enzymes. All pS15 were sequence verified. S15 mutants were constructed using site-directed mutagenesis.pS15 protein expression plasmids were constructed by amplifying the open reading frame from genomic DNA with a forward primer containing SacI site and a strong ribosome-binding site corresponding to the LacZ regulatory assays: As described previously . A single colony was used to start overnight cultures, grown ± L-arabinose (15 mM) at 37°C, 225 rpm, then diluted the next day to OD600 = 0.15 in fresh media (LB + 100 µg/ml ampicillin + 34 µg/ml chloramphenicol ± 15 mM L-arabinose). At stationary phase (5 h after dilution) 1 mM IPTG was added to induce β-galactosidase expression. After 30 min, 100 µg/ml spectinomycin was used to stop initiation of protein translation, and the cultures assayed immediately according to Miller to determine the levels of reporter expression (P < 0.05) than that observed with an empty pBAD33 vector.eviously , K12: Δrpression . Fold reG. kaustophilus (Gk-S15) with a high affinity . Because there is no regulatory interaction between the mRNA regulator from E. coli (Ec-mRNA) and Gk-S15 . The native E. coli rpsO start codon is retained in the 3′ primer region (bolded), and the native Shine-Dalgarno sequence is not within the randomized region (bolded). Protein-binding RNAs were selected using nitrocellulose filter binding. Mutagenic PCR was used at each round to amplify cDNA as well as increase sequence diversity of the non-primer regions.We carried out 11 rounds of SELEX on a randomized RNA pool to isolate RNAs that bind S15 from d Gk-S15 , and theD of >1 μM in the unselected population to 150 nM at the final round . To assess the affinity of individual sequences in the Round 11 pool, we isolated and sequenced six individuals from this population. Each sequence was unique and the sequences are diverse from one another, containing no common sequence or motif in the randomized region  also varies between 20% and 80%. We identified one sequence, 11–1, which has a binding affinity approximating that of the native mRNA interaction for Gk-S15 . Four of the remaining sequences still strongly bind Gk-S15 (KD = 8.5–20.7 nM), although 11–4 displays a low Fmax of only ∼20%. The final sequence, 11–6, has a relatively weak binding affinity (289 nM). These results suggest that our final sequence pool is likely very diverse and indicate that our SELEX experiment was successful in selecting RNA that interact with Gk-S15.During eleven rounds of selection we decreased the concentration of Gk-S15 (from 1250 nM to 100 nM) and the population binding affinity dramatically increased from a Kn Figure . These sin vivo regulation assay to screen the six cloned sequences. Our regulatory assay consists of a two-plasmid system that assesses whether over-expressed Gk-S15 interacts with an RNA to regulate β-galactosidase expression in the cell. One plasmid contains an RNA-lacZ reporter with a selected RNA sequence cloned upstream and in-frame with lacZ and under the control of an IPTG-inducible pLac promoter. A second plasmid carries the G. kaustophilus rpsO coding sequence under the control of an L-arabinose-inducible promoter (E. coli K12:ΔrpsO strain (600 ≈ 1.5) we performed a 30-min mRNA-lacZ induction and measured the change in gene expression between + and – arabinose conditions. During log-phase growth, over-expressed S15 is rapidly incorporated into the ribosome.To assess whether any of the selected aptamers allowed regulation, we used an promoter . The plaO strain . The regin vitro binding affinity and maximum fraction of RNA bound in vitro (Fmax) do not correlate with regulatory capability. The 11–2 and 11–3 bind Gk-S15 strongly in vitro, yet neither was able to regulate gene expression in vivo. While all of the regulators show weaker regulatory response than the native Gk-S15:mRNA interaction   .G. kaustophilus and Thermus thermophilus both require a mimic of the 3 helix junction (3HJ) in order to enable binding to regulatory mRNA structures, whereas the homolog from E. coli requires a GU/GC motif and a bulged adenosine that is not an obvious mimic of the 3HJ  . This result is surprising because the original RNA pool was based on Ec-mRNA. These results suggest that the selected RNAs likely contain tertiary structures that mimic the 3HJ rather than the more easily identifiable GU/GC motif.Our previous work indicates that different S15 homologs recognize RNA regulatory sequences using different motifs. The rRNA binding site for S15 is bipartite and consists of both a three-helix junction and a GU/GC motif one helical turn distal from the junction. S15 homologs from  the 3HJ ,46. By arpsO operon interact with a conserved set of amino acids in the S15 protein, all of which fall on the same protein face  and E. coli (Ec-S15). We find that both RNAs are only able to bind Tt-S15, and not Ec-S15  and the other of which is not 11–2). Previous studies have shown that the naturally occurring RNA regulators of the ein face ,36,47. W5 Figure  and B. T. PreviouWe further examined the 11–1 RNA–protein recognition in our regulatory assay using several Gk-S15 mutants . We wereAll of our sequences retain the native Shine–Dalgarno sequence and start codon, yet only some of them regulate gene expression. While RNA-S15 binding must occur to regulate gene expression, it is not necessarily sufficient. To further investigate this, we examined the binding interaction between Gk-S15 and the best performing regulatory RNA, 11–1 to more clearly establish how Gk-S15 recognizes the RNA to enable regulation. RNA footprinting experiments were performed to elucidate the secondary structure features in 11–1 that may be essential for regulation in response to Gk-S15 Figure –E. UsingUsing this structure probing data, we predicted a structure for 11–1 when bound to Gk-S15 Figure . OverallThe footprinting data suggest that the central part of the RNA sequence, G36 through C47, folds into a hairpin. As Gk-S15 concentration is increased, there is increased VI-cleavage product intensity at nucleotides G36, A37, C39, C47, indicating that Gk-S15 does not protect this region, and it is double stranded. Also, in the presence of Gk-S15, the lead(II)-cleavage of the 11–1 RNA increases for nucleotides C41 and U43; there are IL-cleavage products for A40 through U43 Figure  as well To confirm our secondary structure model, a variety of mutations to the 11–1 RNA sequence were designed and the ability for these to bind Gk-S15 was tested using filter-binding assays Table  suggestsTruncations to the 3′ end of the RNA sequence confirm that many of these nucleotides are not required for binding Gk-S15 Table . A 22 nuMutations to the central core of the putative RNA 11–1 structure drastically affect Gk-S15 binding Table . Based oin vivo. To better understand what elements allow this RNA to interact with the protein in vitro, but result in no in vivo functionality, we performed footprinting assays on this RNA. Again, we used RNase VI, RNase A, RNase T1, lead(II)-probing and in-line probing on 5′-end labeled RNA sequence in the presence and absence of Gk-S15 .Protein binding does little to affect the cleavage patterns observed and our data suggest that Gk-S15 does not bind any part of the sequence that may be considered important for regulation. In the lead(II)-footprint we observe reduced cleavage of nucleotides 26 through 29 and of the region 51 through 56 Figure . This stin vitro selected aptamers (Figure Based on the 11–1 and 11–2 examples we hypothesize that protein binding to RNA regions proximal to the native regulatory features is sufficient to allow regulation. To provide further evidence for this hypothesis we examined an additional two selected RNAs, 11–3 and 11–4. One of which regulates, the other of which does not Figure  and C. Rs Figure .in vitro selection to explore how readily regulators responding to G. kaustophilus S15 could be recovered from a randomized sequence population. From our selected pool we examined six individual aptamers. Each of the sequences is unique and all of them interact with Gk-S15. To our surprise two thirds of the in vitro selected aptamers allowed robust regulation in E. coli. Our characterization experiments demonstrate that although the aptamers have no detectable sequence similarities in their variable regions, they all likely interact with the same binding face of Gk-S15, and that they respond to the same set of S15 homologs. This finding suggests that the number of RNAs that may interact with this protein is quite large, and therefore RNA sequences that enable regulation are relatively frequent in sequence space.Our goal was to use One limitation of our study is the fixed sequences at the 5′ and 3′ ends of the sequence and the sequence for nucleotides 48–72 was not completely randomized. Past studies have shown that primers sequences have negligible impact on the results of SELEX ,37,46 suT. thermophilus, S15 uses a ‘displacement’ mechanism where S15 directly competes for binding the mRNA transcript with the ribosome (E. coli, S15 uses an ‘entrapment’ mechanism to regulate expression of the rpsO operon in which both Ec-S15 and the ribosome pre-initiation complex bind the mRNA simultaneously. This interaction ultimately prevents 70S assembly and halts translation (in vitro, but biophysical modeling has shown entrapment may allow lower affinity interactions to still regulate efficiently (The mechanism of action utilized by our selected RNA regulators for regulation is only partially explored here. The natural S15 aptamers use a variety of mechanisms to inhibit translation. In ribosome . In E. cnslation . Entrapmiciently . Our resiciently ,37. BaseThe S15:mRNA regulatory interaction can take different forms, and many naturally-occurring regulatory RNA structures have been described that all perform analogous regulatory functions in different bacterial species ,25,26,35E. coli mRNA, none of our selected regulators interacts with Ec-S15, indicating that selection experiments with different S15 proteins will yield different pools of sequences.One of the observations driving this work is the identification of several natural RNAs interacting with the same small-molecule ligand, or with homologous proteins. Our results suggest that ribosomal protein S15 responsive regulators are likely to be independently derived. We find that solutions to this biological problem are frequent in the sequence space we explored. Furthermore, similar to the natural solutions, our selected regulators display specificity for some of the proteins over others. Despite starting with a sequence population derived from the native


The enzyme Dicer is best known for its role as a riboendonuclease in the small RNA pathway. In this canonical role, Dicer is a critical regulator of the biogenesis of microRNA and small interfering RNA, as well as a growing number of additional small RNAs derived from various sources. Emerging evidence demonstrates that Dicer's endonuclease role extends beyond the generation of small RNAs; it is also involved in processing additional endogenous and exogenous substrates, and is becoming increasingly implicated in regulating a variety of other cellular processes, outside of its endonuclease function. This review will describe the canonical and newly identified functions of Dicer. The Dicer enzyme is a member of the ribonuclease (RNase) III family. It is most well known as the endonuclease that functions in the RNA interference (RNAi) pathway to cleave long double-stranded RNA (dsRNA) molecules into short dsRNA molecules, known as small RNAs, including microRNA (miRNA) and small interfering RNA (siRNA). Indeed, Dicer is considered a key factor in the biogenesis of most small regulatory RNAs, and the majority of Dicer studies have focused on this role. However, increasing evidence shows that Dicer also has functions outside of the small RNA pathway. Dicer's endonuclease function is not only involved in small RNA biogenesis, but also in the processing of other endogenous and exogenous substrates. Furthermore, its function is not limited to cleavage, but may regulate other cellular processes. This review will describe the canonical and newly identified functions of Dicer.Although a full-length mammalian Dicer has not been crystallized, many studies have predicted its structure and the positions of its domains and interacting partners –5. The mSccharomyces cerevisiae .,13.12,13revisiae . The evorevisiae . Both threvisiae ,20. Hower2 genes . In Dros pathway . Consist pathway . This evDrosophila, the helicase domain of Dicer1, which cannot hydrolyze ATP, selectively interacts with the loops of precursor miRNAs (pre-miRNAs) and inhibits cleavage of long dsRNAs [Caenorhabditis elegans, Dicer also needs ATP and the helicase domain to process long dsRNAs with blunt or 5′ overhanging termini; mutation of this domain results in weak production of endogenous siRNAs but maintains the ability to dice pre-miRNAs [Arabidopsis thaliana, DCL1 mainly facilitates the biogenesis of imperfect stem-loop RNAs into 21 nt miRNAs [Drosophila Dicer2 [Selective Dicer-mediated cleavage of various RNAs is driven by the evolutionarily conserved helicase domain  21,24,2,224,25. g dsRNAs . In conte-miRNAs ,30. In At miRNAs  and alsot miRNAs . Howevera Dicer2 ,34, and a Dicer2 ,36.In the canonical pathway, the biogenesis of most small RNA classes, including miRNAs and many siRNAs, occurs through a stepwise process in the cytoplasm. This process involves association with Dicer and specific members of the large family of Argonaute (AGO) proteins, and assembly into various effector complexes, including the RNA-induced silencing complex (RISC). First, RNAs form a pre-RISC with Dicer , aided bIn addition, Dicer controls the fate of many other RNA species . Dicer-dDicer is important for the production of transfer RNA (tRNA)-related fragments (tRFs), a heterogeneous class of small RNAs. tRFs are generated from tRNAs, and high-throughput sequencing and analysis show that they map to known tRNA genes –54. The in vitro and in vivo studies show that tRFs derived from human tRNA(Gln) are dependent on Dicer [3′ CCA tRFs are processed in a Dicer-dependent manner . Both inon Dicer ,55. The on Dicer . The miRon Dicer .The lupus autoantigen La is an RNA chaperone associated with the miRNA pathway. La functions as a gatekeeper that ensures correct tRNA maturation and protects the miRNA pathway from tRF. In the absence of La (La knockdown cells), various pre-tRNAs bind to Dicer, and the tRFs derived from the 3′ ends of tRNAs are bound to AGO .Dicer plays a role in processing several types of non-coding RNAs, which has implications for various regulatory mechanisms.Giardia lamblia (protozoa) led to identification of small fragments with miRNA characteristics and showed that Dicer is required for miR2 production from box C/D snoRNA [Processing of non-coding small nucleolar RNA (snoRNA) to shorter, more stable snoRNA-derived RNAs (sdRNAs) is widespread, with sdRNAs identified in vertebrates, including humans –68, planD snoRNA . The ideD snoRNA . ACA45 iD snoRNA .A lot of data support the hypothesis that Dicer has direct roles in post-transcriptional gene-silencing activity of many miRNA-like sdRNAs, but the mechanism underlying their biogenesis remains unknown. It has been shown that processing of box C/D RNAs into sdRNAs can be Dicer-independent, while production of box H/ACA-derived sdRNAs requires Dicer ,62. DiceAnother example of non-coding RNA processing by Dicer involves LINE-1 (long interspersed nuclear element-1), a retrotransposon. The LINE-1 promoter has a bidirectional orientation, containing sense and antisense transcripts that form double-stranded long hairpin RNAs, which are then processed by Dicer into repeat-associated siRNAs (rasiRNAs). rasiRNAs are loaded into AGO proteins to silence LINE-1 by RNA-directed DNA methylation. Inhibition of biogenesis of rasiRNAs has been suggested to increase the processing of LINE-1 transcription via Dicer inhibition . Other eGeographic atrophy (GA), an age-related macular degeneration disease of the retinal pigment epithelium, is associated with reduced expression of Dicer. Inhibition of Dicer leads to accumulation of Alu repeat RNAs of ∼300-nt derived from short interspersed elements (SINEs). This accumulation is cytotoxic and triggers interferon-mediated, caspase 8-dependent apoptosis. Injection of Alu repeat RNA into mouse eyes generates GA . This coIn vitro studies showed that single-stranded CGG-RNA is cleaved by Dicer to generate short CGG-RNAs [Dicer has direct and indirect roles in curtailing expression of toxic triplet repeat RNAs. Triplet repeat expansion disorders include myotonic dystrophy type 1 (DM-1), fragile X-associated tremor ataxia syndrome, Huntington's disease (HD), and spinocerebellar ataxia (SCA), which frequently encode RNAs with long internal triplet repeat structures . In vitrCGG-RNAs . Dicer rCGG-RNAs ,80. The CGG-RNAs . The relCGG-RNAs . ImportaIn addition to its role in processing endogenous RNA, Dicer is also important in processing exogenous RNA, including viral-associated (VA) RNA . Viral iRNA genome viruses do not express small RNA in infected cells; this phenomenon is believed to be a mechanism of host immune evasion ,86. HoweRecent studies show that RNA viruses also process miRNA-like RNAs ,89. A smIn addition to the critical role of Dicer in small RNA biogenesis, it also has important roles in genome regulation and surveillance, unconnected to small RNA production. For example, although its RNAi role occurs in the cytoplasm, Dicer can also localize to the nucleus ,93 and iSeveral lines of evidence suggest that Dicer plays important roles in the nucleus. The C-terminal dsRNA-binding domain of Dicer contains a nuclear localization signal, which allows it to shuttle between the nucleus and the cytoplasm . Human DSchizosaccharomyces pombe [Arabidopsis [Drosophila [Nuclear siRNAs corresponding to heterochromatic loci are associated with silencing; Dicer and AGO1 mutations destroy the production of these endo-siRNAs and the assembly of heterochromatin in the fission yeast es pombe ,108. Thees pombe ,109. Sucbidopsis ,111, Droosophila , and mamosophila . In mammosophila . Anotherosophila , suggestosophila . The nucExosomes are small extracellular vesicles with an average size of 30–100 nm, which were simply considered cell debris for many years. Today, exosomes are known to be rich in lipids, proteins, and RNAs, including protein-coding transcripts (i.e. mRNAs) as well as many types of non-coding RNAs  . ExosomeIn addition to recognition of a novel form of cell–cell communication, the discovery of miRNAs in exosomes has suggested new applications for exosomes, such as potentially easy-access biomarkers, or, given that exosomes are non-immunogenic, possibly as novel therapeutics –124. miRDicer, AGO2, and TRBP proteins are detected in exosomes derived from breast cancer cells, but not from non-tumorigenic breast cells . ExosomeExosomes derived from HIV-1-infected cells contain TAR RNA and Dicer, as well as components of the host miRNA machinery . The incC. elegans, leaving a truncated though catalytically active protein consisting only of the C-terminal RNase III and dsRNA-binding domains. This truncated Dicer inactivates its RNase function, gains DNase function, and initiates breaks on chromosomal DNA. The DNA fragments are metabolized to complete apoptotic DNA degradation.Dicer also plays a role in the recognition and processing of DNA in apoptosis . The casAntiviral RNAi is a powerful mechanism to protect against viral infections. Specific features of viral RNA are sensed as foreign, and Dicer cleaves the double-stranded viral RNA into viral siRNA ,131–134.Although VA RNA is a poor substrate for Dicer cleavage, a few VA RNAs are cleaved to ∼22-nt mivaRNAs. mivaRNA association with the RISC is crucial for adenovirus replication . A recenVaccinia virus (VACV) infection shows that inhibition of Dicer protein is associated with reduction in pre-miRNA processing . FlaviviEBV miRNA can be edited by adenosine deaminase acting on RNA to convert adenosine into inosine. A-to-I-edited miR-BART6-3p decreases the efficiency with which the miRNA encoded on the opposite strand, miR-BART6-5p, is loaded into the RISC in human cells. miR-BART6-5p targets human Dicer though four binding sites in its 3′UTR. Therefore, editing of miR-BART6-3p has a positive effect on Dicer expression. In turn, Dicer levels affect the expression levels of multiple genes that regulate the infectious and lytic states of EBV. Thus it is postulated that editing of miR-BART6-3p could be an indirect way to modulate miRNA biogenesis and thereby the viral life cycle .Viruses encode proteins that suppress RNAi in mammalian cells, e.g. E3L (VACV), B2 (Nodamura virus), NSs (La Crosse virus), Tat (HIV), and VP30, VP35, and VP40 (Ebola virus) ,150–154.Virus-derived small RNAs that direct specific antiviral defenses through biogenesis of a small RNA may be dependent on the cleavage activity of Dicer. However, it is still under debate whether infection by virus induces or suppresses antiviral small RNA.A. thaliana encodes multiple Dicer proteins (DCL 1–4) involved in distinct endogenous RNA-silencing pathways and antiviral defenses, which show functional redundancy and specificity [Drosophila. Dicer1 produces mature miRNAs in an ATP-dependent manner. Dicer2 cleaves cell-derived dsRNA precursors to produce endogenous siRNAs [A. thaliana and D. melanogaster suggests an antiviral strategy, but other Dicer types do not produce antiviral small RNAs from viruses. In contrast, humans have one type of Dicer that processes both miRNAs and siRNAs. These various roles of Dicer led us to hypothesize that Dicer might regulate both antiviral and proviral mechanisms.cificity ,156. Inacificity ,158. As s siRNAs  and exogs siRNAs ,161, ands siRNAs . The speInfection of human cells with a wide range of viruses, including yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, HIV-1, or herpes simplex virus 1, failed to reveal any enhancement in viral replication in Dicer-inhibited human cells (human cells with the absence of Dicer) . miRNAs C. elegans oogenesis; Dicer is a putative ERK substrate [The Dicer protein can command specific interactions with other proteins. For example, Ras/ERK signaling and Dicer regulate ubstrate . Phosphoubstrate . An ERK-In addition to other diseases described throughout this review, many groups have reported a correlation between Dicer expression and cancer. For example, Dicer expression shows significant changes in different stages of lung adenocarcinoma . ReducedAccumulating evidence continues to demonstrate the critical role of the Dicer enzyme in not only the biogenesis of miRNA and siRNA in the canonical RNAi pathway, but also in controlling the fate of many other small RNA species. Furthermore, the list of non-endonuclease roles of Dicer continues to grow. Future directions in Dicer research should include: expanding our understanding of Dicer in the biogenesis and regulation of a growing network of endogenous small RNAs, which have interrelated implications for the regulation of complex physiological processes, including cancer; delineating the regulation of Dicer itself, including the causes and consequences of Dicer subcellular localization; and determining the role of Dicer in response to exogenous RNAs, and how this connects to the immune system.