When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures  RNA structure is a jack-of-all-trades. Besides being crucial to the function of structural non-coding RNAs (ncRNAs), RNA structural elements are now widely recognized as key players in almost any essential biological process, ranging from transcriptional and post-transcriptional control of gene expression, to catalysis and sensing of environmental stimuli such as metabolite and temperature changes .n nucleotides-long RNA can theoretically fold in up to 1.8n distinct conformations  (A widely used approach to define the putative structure of an RNA based on its sequence relies on predicting its minimum free energy (MFE) structure, the theoretically most stable structure, based on a set of experimentally-determined thermodynamic parameters known as r rules) . Unfortur rules) . This isr rules) . The accr rules) .in vivo, while the majority of them are only suitable for in vitro and ex vivo RNA structure probing experiments. Two classes of compounds are mainly used to date. On the one hand, nucleobase-specific probes, such as dimethyl sulfate (DMS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), can be respectively used to probe adenine (A) and cytosine (C), or guanine (G) and uracil (U) bases  bases ,14. On tU) bases , as theyU) bases ,17, withU) bases ,19.Typical readout of RNA probing experiments is the detection of reverse transcription (RT) drop-off events induced by the probing reagent-modified RNA bases . More rein vivo RNA probing by synthesizing six new SHAPE reagents and by assaying their ability to probe in vivo Gram-positives, Gram-negatives and mammalian cells. One of these compounds, 2-aminopyridine-3-carboxylic acid imidazolide (2A3), showed both increased reactivity with RNA and higher permeability to biological membranes, resulting in a significantly higher signal-to-noise ratio when compared to NAI. Importantly, when used to perform experimentally-informed prediction of in vivo RNA structures, 2A3 produced markedly more accurate predictions than NAI.While the use of SHAPE compounds over nucleobase-specific probes has in theory a considerable advantage, as they enable the unbiased probing of all four RNA bases, reagents such as DMS still exhibit a higher signal-to-noise ratio in MaP experiments. To this end, we here sought to optimize Ka close to (or slightly above) 7. The pKa values in water were obtained (where possible) using the DataWarrior tool  and added to D2O to achieve quantifiable concentrations. Samples were incubated at 25°C or 37°C and kinetic 1H NMR scanning was performed. The disappearance of product resonances and reappearance of starting materials (correlating to hydrolysis) was monitored as previously described . 2 ml aliquots were collected by centrifugation at 1000 × g (4°C) for 5 min. Cell pellets were resuspended in 1 ml of Resuspension Buffer , and lysozyme was added to a final concentration of 100 μg/ml. After incubation at 22°C for 5 min and on ice for 10 min, protoplasts were collected by centrifugation at 5000 × g (4°C) for 5 min. Pellets were resuspended in 120 μl Protoplast Lysis Buffer , supplemented with 0.2 μg/μl Proteinase K. Samples were incubated for 5 min at 22°C and for 5 min on ice. Sample was then extracted 2 times with phenol:chloroform:isoamyl alcohol , and once with chloroform. 20 U SUPERase•In™ RNase Inhibitor  were then added and RNA was equilibrated at 37°C for 20 min prior to probing.Deproteinized escribed , with miE. coli rRNAs in RNA folding buffer, pre-equilibrated at 37°C for 20 min, were mixed with 20 μl of each SHAPE compound . RNA was then allowed to react at 37°C for 15 min, with moderate shaking, after which 200 μl of 1 M DTT were added, to quench the reaction. Samples were then vortexed briefly and 1 ml of ice-cold QIAzol Lysis Reagent  was added to each sample, followed by extensive vortexing.180 μl of deproteinized E. coli K-12 DH10B was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000 × g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 100 μl of Buffer A , supplemented with 5 U SUPERase•In™ RNase Inhibitor, by vigorously vortexing for 30 s. 25 μl of Buffer B , supplemented with 100 μg/ml final lysozyme, were then added, followed by 30 s vortexing. Samples were incubated 1 min at room temperature, after which 125 μl of Buffer C  were added to lyse protoplasts. Samples were then incubated at room temperature for 5 min, after which 100 μl of lysate were transferred to 1 ml ice-cold QIAzol and thoroughly mixed by vortexing.A single colony of Bacillus subtilis 168 was picked and inoculated in LB medium without antibiotics, then grown to exponential phase (OD600 ∼ 0.5). 2 ml aliquots were collected by centrifugation at 1000× g for 5 min. Cell pellets were resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by brief vortexing. Cells were allowed to react at 37°C for 20 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 17 000 × g for 2 min (4°C) and supernatant discarded. Cell pellets were then resuspended in 200 μl TE Buffer , supplemented with 1.1% final SDS. 0.25 g glass beads were then added and samples were beated in a Mini-Beadbeater-24 (Glen Mills) for 1 min. Samples were then incubated on ice for 1 min, after which beating was repeated once. 1 ml ice-cold QIAzol was then added, followed by vigorous vortexing. Samples were then incubated at 70°C for 20 min, to allow complete cell lysis.A single colony of d-glucose), supplemented with 10% heat-inactivated FBS, 0.1 mM NEAA, 1 mM sodium pyruvate, 25 U/ml penicillin and 25 μg/ml streptomycin, at 37°C (5% CO2), to ∼75% confluence. Cells were then covered with a thin layer of trypsin–EDTA (0.25%) solution, incubated at room temperature for 1 min, then dissociated by pipetting up and down with complete medium. Cells were centrifuged at 180 × g for 1 min and medium discarded. Cells were then resuspended in 450 μl 1× PBS (pH 7.4) and 50 μl of each SHAPE compound were added, followed by gentle mixing by tapping the tube. Cells were allowed to react at 37°C for 15 min, with moderate shaking, after which 500 μl of 1 M DTT were added, to quench the reaction. Cells were pelleted by centrifugation at 10 000 × g for 1 min (4°C) and supernatant discarded. Cells pellets were lysed by direct addition of 1 ml ice-cold QIAzol, followed by a brief incubation at 56°C for 5 min to completely dissolve cell aggregates.HEK293 cells were grown in DMEM (4.5 g/l g for 15 min (4°C). After centrifugation, the upper aqueous phase was transferred to a clean 2 ml tube, and mixed with 2 volumes of 100% ethanol by vigorous vortexing. The entire volume was then transferred to an RNA Clean & Concentrator™-5 column  and RNA was purified as per manufacturer instructions. RNA integrity was ensured by gel electrophoresis on a denaturing 2% agarose gel. Besides samples treated with I6, all other samples appeared to be perfectly intact. Before proceeding to library preparation, traces of genomic DNA were removed by treatment with 2 U TURBO™ DNase  at 37°C for 20 min.After samples have been collected in 1 ml QIAzol, 200 μl of chloroform was added, followed by vigorous vortexing for 15 s. Samples were then incubated at room temperature for 2 min, after which they were centrifuged at 12 500 × 2], then purified with NucleoMag NGS Clean-up and Size Select beads , supplemented with 10 U SUPERase•In™ RNase Inhibitor, and eluted in 2 μl (for SSII and HIV RT) or 2.5 μl (for TGIRT-III) NF H2O. Eluted RNA was supplemented with 0.5 μl 20 μM random hexamers and either 0.25 μl (for SSII) or 0.5 μl (for HIV RT and TGIRT-III) dNTPs (10 mM each), then incubated at 70°C for 5 min and immediately transferred to ice for 1 min. Reverse transcription reactions were conducted in a final volume of 5 μl. For HIV RT, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 5 U HIV RT . For TGIRT-III, reaction was supplemented with 1 μl 5× RT Buffer , 0.25 μl DTT 0.1 M, 5 U SUPERase•In™ RNase Inhibitor and 50 U TGIRT-III RT . For SSII, reaction was supplemented with 1 μl 5× RT Buffer , 0.5 μl DTT 0.1 M, 0.25 μl MnCl2 120 mM, 5 U SUPERase•In™ RNase Inhibitor and 50 U SuperScript II RT . Reactions were incubated at 25°C for 10 min to allow partial primer extension, followed by 2 h at 37°C (for HIV RT), 2 h at 57°C (for TGIRT-III) or 3 h at 42°C (for SSII). SSII and HIV RT were heat-inactivated by incubating at 75°C for 20 min. As TGIRT-III tightly binds the cDNA-RNA complex, 0.5 μg of Proteinase K were added and reaction was incubated at 37°C for 20 min, after which 0.5 μl of a 1:2 dilution of protease inhibitor cocktail  in water was added to stop the reaction. For SSII, the buffer exchange step was omitted. Instead, 6 mM final EDTA was added to chelate Mn2+ ions, followed by 5 min incubation at room temperature and addition of 6 mM final MgCl2. Reverse transcription reactions were then used as input for the NEBNext® Ultra II Non-Directional RNA Second Strand Synthesis Module . Second strand synthesis was performed by incubating 1 h at 16°C, as per manufacturer instructions. DsDNA was purified using NucleoMag NGS Clean-up and Size Select beads, and used as input for the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® , following manufacturer instructions.Total RNA was first fragmented to a median size of 150 nt by incubation at 94°C for 8 min in RNA Fragmentation Buffer {3})’. For each hairpin-loop, we then calculated the median reactivity on the first and last two bases of the loop and the median reactivity on the three bases of the stem preceding and following the loop. The signal-to-noise value was then calculated as the ratio between the median reactivity across all the loops and the median reactivity across all the stems.E. coli cells, under conditions previously shown to preserve the native RNA folding  and HIV RT (N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT) adducts (HIV RT). Analysis of the distribution of per-base mutation frequencies across 16S and 23S rRNAs showed that, while TGIRT-III has lower background mutation frequencies compared to SSII (median DMSO control: 0.75 × 10−3 for TGIRT-III versus 2.19 × 10−3 for SSII) as previously reported , we sought to find the best reverse transcription conditions. To this end, we extracted deproteinized total RNA from  folding ,23,33 and HIV RT ,24,34. Treported , SSII shreported . Accordie models  and inspe models . Altogetin vivo probing experiments); (ii) high signal over background ; (iii) easy and safe to synthesize. The two best-characterized in vivo SHAPE reagents to date are NAI and FAI. These compounds can be easily synthesized via the carbonyldiimidazole (CDI)-mediated activation of carboxylic acids, resulting in the corresponding carboxylic acid imidazolide, an activated carbonyl, that can acylate nucleophiles present in the system, especially 2′ hydroxyl groups of RNA. We decided to adopt the same strategy, trying to optimize the acylating group. To this end, we selected seven commercially-available carboxylic acids, preferring those containing nitrogen groups or for which the putative adduct-forming group had a pKa in water close to (or slightly above) 7  permeable to biological membranes  as it was too reactive, hence resulting in very fast RNA degradation (data not shown). When looking at overall distributions of mutation rates, indoline-5-carboxylic acid imidazolide (I5) and 1-methylimidazole-4-carboxylic acid imidazolide (1M4) showed a lower reactivity towards RNA compared to NAI . Oppositely, the other four compounds, nicotinic acid imidazolide (NIC), benzotriazole-5-carboxylic acid imidazolide (B5), 6-aminopyridine-3-carboxylic acid imidazolide (6A3) and 2-aminopyridine-3-carboxylic acid imidazolide (2A3), all showed increased reactivity compared to NAI , with 2A3 having ∼2.4X higher mutation frequencies than NAI , independently of DMSO background signal subtraction , independently of the probed cell type, partially followed by B5 in B. subtilis and NIC in HEK293 cells.At first, we looked again into the distribution of mutation frequencies for the different reagents. Unexpectedly, while the observed distributions for HEK293 cells approximately mirrored those observed on ied RNAs , we obsee Figure . On the ex vivo-probed RNAs, ROC curves built with respect to unpaired versus paired residues from E. coli, B. subtilis and H. sapiens rRNAs  revealed a marked difference in sensitivity/specificity of NAI compared to 2A3, with 2A3 showing up to 17–20% increased discrimination of unpaired versus paired residues, independently of DMSO background signal subtraction  with respect to NAI , independently of the examined cell type  and the intercept (b). As these parameters depend on the experimental setup and on the employed reagent, we first used the ViennaRNA Package 2.0  and positive predictive value  for both 16S and 23S rRNAs. Notably, grid search already revealed that the accuracy of 2A3-driven predictions was largely independent of the employed slope/intercept pair and that, even the most accurate NAI-driven prediction  was ∼10% less accurate than most 2A3-driven predictions, independently of the used m/b pair and ∼18% less accurate that the top-scoring 2A3-driven prediction . As an example, although incorporation of NAI data slightly increased the accuracy with respect to the unconstrained prediction , the NAI-guided prediction resulted in the misfolding of the 3′ domain of the 16S rRNA, while the 2A3-guided prediction correctly recapitulated most of the known base-pairs from the accepted reference structure . This unwanted side product increased over time, eventually reaching a maximum of ∼90% as compared to 2A3 in solution during an overnight reaction in a sealed vessel. The formation of 2A3 proceeds rapidly as CDI is added to the 2-aminonicotinic acid. The conversion of 2A3 to 3AIA then proceeds at room temperature in the crude reaction mixture. The unwanted side reaction proceeds slowly and can be minimized with vigorous stirring under a constant flow of inert gas.in vivo SHAPE probe, we obtained commercially-available pure 3AIA  and used it to probe living E. coli cells. Notably, 3AIA showed modification frequencies indistinguishable from those of the DMSO control in vivo  showed partial improvements with respect to NAI. Surprisingly, when tested in vivo, 2A3 significantly outperformed NAI and all the other tested reagents under all the analyzed conditions, with the most striking differences being observed in bacterial cells. Indeed, for both E. coli and B. subtilis, several regions of the ribosome resulted to be blind to probing by NAI and most of the other tested reagents, while they were readily probed by 2A3. In this context, 2A3 better captured local nucleotide dynamics, as confirmed by in depth analysis of available ribosome 3D structures. This reveals that, besides showing a higher ability to react with RNA, 2A3 has also a greater capacity to permeate biological membranes. It is conceivable that the peptidoglycan of the bacterial cell wall might represent the main limitation when it comes to in vivo SHAPE probing. Indeed, although 2A3 is able to probe both gram-positive and gram-negative bacteria, we observed higher efficiencies in gram-negatives, suggesting that the thicker peptidoglycan of gram-positives limits the permeability to the SHAPE compound. The presence of an amine rather than a methyl group at position C2, that differentiates 2A3 from NAI, might explain this increased permeability, in agreement with a recent study showing that small molecules containing amine groups are more likely to permeate and accumulate into bacteria  even if providing information on roughly only a half of RNA bases.Furthermore, we showed that the higher signal-to-noise ratio achieved by 2A3 results in a significantly higher RNA structure prediction accuracy when performing 2A3-informed RNA folding. It is worth noticing that 2A3, as opposed to NAI, outperforms probing with dimethyl sulfate (DMS) as well. Indeed, probing with DMS usually shows a higher signal-to-noise ratio with respect to NAI, hence resulting in more accurate experimentally-informed RNA structure predictions . It may be that 2A3 falls in a ‘sweet’ spot for reactivity compared to the other tested electrophiles both in vivo RNA structurome.In summary, 2A3 represents a concrete and significant advance in our ability to interrogate RNA structures in living cells. We can anticipate that, being able to readily probe even regions of ribonucleoprotein complexes that are normally hidden to other compounds, 2A3 will rapidly overtake conventional SHAPE reagents, revealing new and previously unobserved features of the ex vivo probing of E. coli RNA with DMSO and NAI has been previously deposited under the accession GSE151327, as part of another study. Additional processed files are available at: http://www.incarnatolab.com/datasets/2A3_Marinus_2020.php.SHAPE-MaP data has been deposited to the Gene Expression Omnibus (GEO) database, under the accession GSE154563. Data for gkaa1255_Supplemental_FileClick here for additional data file.


The SNM1 nucleases which help maintain genome integrity are members of the metallo-β-lactamase (MBL) structural superfamily. Their conserved MBL-β-CASP-fold SNM1 core provides a molecular scaffold forming an active site which coordinates the metal ions required for catalysis. The features that determine SNM1 endo- versus exonuclease activity, and which control substrate selectivity and binding are poorly understood. We describe a structure of SNM1B/Apollo with two nucleotides bound to its active site, resembling the product state of its exonuclease reaction. The structure enables definition of key SNM1B residues that form contacts with DNA and identifies a 5′ phosphate binding pocket, which we demonstrate is important in catalysis and which has a key role in determining endo- versus exonucleolytic activity across the SNM1 family. We probed the capacity of SNM1B to digest past sites of common endogenous DNA lesions and find that base modifications planar to the nucleobase can be accommodated due to the open architecture of the active site, but lesions axial to the plane of the nucleobase are not well tolerated due to constriction around the altered base. We propose that SNM1B/Apollo might employ its activity to help remove common oxidative lesions from telomeres. The maintenance of genomic integrity is vital for normal cellular processing. Unrepaired, or mis-repaired, DNA lesions act as impediments to DNA and RNA polymerases preventing the faithful replication and expression of genetic information. Accumulation of DNA lesions is implicated in the aetiology of numerous pathologies and states of cellular degeneration; including cancer , neurodePSO2  is a 5′-to-3′ exonuclease, which was first identified as one of three vertebrate orthologues of the yeast DNA repair nuclease, PSO2 . SNM1B iethality . Second,ethality .via its interaction with TRF2 (telomeric repeat-binding factor 2)  ,12,13. Iormation ,7,14–16.ormation ,15,17. Tormation ,19.SNM1B is implicated in DNA damage repair pathways, primarily ICL repair ,21, but CPSF, Artemis, SNM1, PSO2) nucleic acid processing nucleases are a subfamily. All MBL type enzymes possess a characteristic α/β/β/α MBL core fold that provides a scaffold that supports the active site, which coordinates the metal ions essential for catalysis. The β-CASP domain is inserted within the MBL domain and contains three conserved motifs, which contribute to the active site architecture and catalysis  contains the catalytic (MBL and β-CASP) domains, and the C-terminal region the protein-protein interaction regions, including the well-characterised TRF2 binding domain (amino acids 498–509) . SNM1B iatalysis ,25. Few atalysis ; SNM1A aatalysis ; and SNMatalysis  and 28)CPSF, ArtHere, we present a 1.8 Å resolution structure of SNM1B with two 5′-deoxy-adenosine monophosphate (AMP) molecules in its active site. The two nucleotides are proposed to bind in a similar manner to single-stranded DNA, so enabling us to build a model for substrate binding and experimentally probe the molecular basis of SNM1B’s interaction with DNA, its nuclease activity, and substrate selectivity. We have also examined the structure and function of the breast cancer associated SNM1B variant, H61Y, which is suggested to affect cancer risk based on genome wide association studies (GWAS) . The H61via ligation independent cloning (LIC) into baculoviral expression vectors pFB-CT10HF-LIC (GenBank EF199842), except for the D35A/H36A SNM1B mutant, which was cloned in pFB-LIC-Bse (Sf9 insect cells (Sf9 cells at a density of 2 × 106 cells/ml infected with 6 ml of P2 virus stock and cultures were grown for 72 h at 27°C before being harvested by centrifugation in a JLA8.1 rotor (Beckmann-Coulter) at 900 x g for 30 min. Cell pellets were resuspended in 50 ml cold lysis buffer , 1 mM TCEP, supplemented with protease inhibitors ).DNA encoding for the core MBL-β-CASP domains of SNM1B aa 1/2–335) were cloned for expression 5 were cl-LIC-Bse . The tract cells . SNM1B pThawed cell aliquots were lysed by sonication on ice, followed by centrifugation . The supernatant was mixed with Ni-Sepharose slurry  and incubated, on a rotating mixer at 4°C for 1–2 h. Samples were centrifuged 700 g, 5 min), the supernatant decanted, and the Ni-Sepharose beads were washed twice with 50 ml lysis buffer, resuspended in 30 ml wash buffer , 1 mM TCEP), and added to a 25 ml gravity flow column. The column was washed with 10 column volumes (CV) wash buffer, and eluted in 10 CV elution buffer , 1 mM TCEP). SNM1B containing fractions were pooled, and the respective tags cleaved by incubation with TEV protease , whilst being dialysed using a 3.5 kDa molecular weight cut-off SnakeSkin membrane (ThermoFisher Scientific) into a buffer containing 50 mM HEPES, 500 mM NaCl, 5% glycerol, 1 mM TCEP. Following dialysis, the protein solution was added to a 5 ml Ni-Sepharose column. The column was washed iteratively with buffers  containing increasing amounts of imidazole . SNM1B proteins were further purified by size exclusion chromatography using a 16/60 Superdex S200 column  in a buffer containing 50 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol (v/v), 1 mM TCEP. The presence and purity of each SNM1B protein was validated by SDS-PAGE, and intact mass analysis (ESI-TOF-MS) . A ‘nucl min, theCrystallisation of the apo forms of WT and H61Y SNM1B was achieved by sitting drop vapour diffusion, at 4°C, in conditions containing 6% PEG 20 K, 0.1 M bicine, pH 7.3 for WT SNM1B; and 6% PEG 3350 K, 0.1 M HEPES, pH 7.0, 0.2 M sodium formate for H61Y SNM1B. Protein concentrations were 15 and 12 mg/ml for WT and H61Y SNM1B, respectively. For co-crystallisation of WT SNM1B with 2′-deoxy-5′-AMP, WT SNM1B (at 15 mg/ml) was incubated with 50 mM 2′-deoxy-5′-AMP  for 30 min on ice. Crystallisation was achieved by sitting drop vapour diffusion at 20°C, in conditions containing: 20% PEG 3350 K, 0.2 M ammonium chloride. Crystals were cryoprotected in the reservoir solution supplemented with 20% ethylene glycol, and plunged into liquid nitrogen.Data were collected at Diamond Light Source utilising beamlines I02 (H61Y and WT apo form), and I04 (nucleotide form). Diffraction data were processed using DIALS. The structures of SNM1B (apo form) and H61Y SNM1B were solved by molecular replacement using PHASER with PDB: 5AHO used as a search model. The structure of SNM1B (nucleotide form) was also solved using molecular replacement, using SNM1B (apo form) as a search model. Model building was performed using COOT, and subsequent refinement of the native dataset using REFMAC. Data collection and refinement statistics are in Table 32P-dATP (Perkin Elmer) by terminal deoxynucleotidyl transferase , incubated together at 37°C for 1 h. This solution was passed through a P6 Micro Bio-Spin chromatography column (BioRad), and the radiolabelled DNA was annealed with the appropriate unlabelled oligonucleotides (1:1.5 molar ratio of labelled to unlabelled oligonucleotide)  were labelled with 3.3 pmol of α-leotide) .2, 0.05% Triton X-100, 5% glycerol and 1 nM of SNM1B. Reactions were started by the addition of DNA substrate (10 or 100 nM), incubated at 37°C for the indicated time period, and quenched by the addition of 10 μl stop solution  and incubating at 95°C for 3 min.Standard exonuclease assays were carried out in 10 μl final volume reactions containing 20 mM HEPES-KOH, pH 7.5, 50 mM KCl, 10 mM MgClReactions were analysed by 20% denaturing polyacrylamide gel electrophoresis  and 7 M urea (Sigma Aldrich)) in 1× TBE (Tris-borate EDTA) buffer. Electrophoresis was carried out at 700 V for 75 min; gels were subsequently fixed for 40 min in a 50% methanol, 10% acetic acid solution, and dried at 80°C for 2 h under a vacuum. Dried gels were exposed to a Kodak phosphorimager screen and scanned using a Typhoon 9500 instrument (GE).Real-time fluorescence nuclease assays were carried out using a modified version of that previously described  24,35).,35.24,3510 SNM1B concentration, and binding curves were fitted using a non-linear regression (curve fit) in GraphPad Prism .Reactions (volume 20 μl) were carried out in black 384 well plates . SNM1B protein was serially diluted from 5 μM to 0.288 nM and incubated with DNA substrate  at 25 nM1–355 comprising the catalytic MBL and β-CASP domains, as production of the full-length protein is not well tolerated in bacterial or insect cell expression systems. Small amounts of the full-length SNM1B protein have been previously purified by other research groups  with two 2′-deoxy-5′-adenosine monophosphate (AMP) molecules at the active site (denoted SNM1B (nucleotide form)). All biochemical and crystallographic experiments were carried out with truncated SNM1Bh groups ,16, and Similar to the previously reported structure of truncated SNM1B , our strThe two 2′-deoxy-5′-AMP (AMP_1 and AMP_2) molecules are bound in the active site and are arranged in a position consistent with that predicted for the terminal nucleotide products from hydrolysis of a ssDNA substrate. The P–P distance is 6.1 Å, commensurate with the distance between two phosphate groups of adjacent nucleotides in ssDNA (∼6.6 Å). The electron density map Figure  reveals via the main chain amides of S258 and D275, the sidechain hydroxyls of Y182, T257, S258 and S274, and a single positively charged sidechain of K186  is bound in a well-defined pocket formed in the cleft between the MBL and β-CASP domains Figure . The 5′-6 Figure , D. Whil6 Figure , involvi1–355, we performed an alanine substitution analysis of residues implicated in phosphate binding . We havkcat and, (more unexpectedly) KM values compared with WT SNM1B. The D275A point substitution completely abrogated nuclease activity  in the low micromolar range 1.67 ± 0.17 μM. Most of the phosphate binding pocket variants showed similar or increased apparent dissociation constants (KDapp) with K186A and T257A being moderately affected (KDapp 2.73 ± 0.3 and 2.12 ± 0.18 μM respectively); KDapp for Y182A 1.1 ± 0.09 μM, being slightly lower than the wildtype for unclear reasons; and D275A exhibiting the greatest increase in KDapp (3.28 ± 0.4 μM), consistent with this substitution also showing the greatest decrease in nuclease activity. It is important to note, however, that the conditions of the turnover and FP binding assays are different and they use different reporter molecules.The effects of these substitutions on DNA binding were investigated, using a 3′ overhang DNA substrate and a fluorescence polarization (FP) assay Figure . WT SNM11–355 MBL fold supported active site  indicates both metal ion-binding sites are fully occupied. X-ray fluorescence analysis (XRF) of the crystals revealed that a mixture of nickel and iron were present in roughly equal proportions , coordinated by H31 and H33 , H99 , D120 , and a water molecule. Octahedral coordination is completed by the 5′-phosphate oxygen (O3) of AMP_2. The second metal ion (M2 site) was modelled as an Fe; it is coordinated in an octahedral arrangement by D35A and H36A , D120 , an oxygen (O1) of the 5′-phosphate group of AMP_2, and two water molecules  we solved a structure of unliganded SNM1B1–355 to 2.8 Å resolution, hereafter denoted SNM1B1–355 (apo form)  Table . This mem) Table , with a 2+, Fe2+ . D35 whi2+, Fe2+ .1–355 (apo form) and SNM1B1–355 (nucleotide form)  Figure  reveal t) Figure , with si) Figure ) is very) Figure . There a1–355 was confirmed by inductively-coupled-plasma mass-spectrometry (ICP-MS)  . However(ICP-MS) ,35), alte Figure ), it is 1–355 (nucleotide form), and the geometry of the active site, support the outlined mechanism proposed for SNM1B ..1–355   . The oveolution) .1–355 (nucleotide form) structure as a guide for modelling SNM1B1–355 complexed with a relevant substrate. Across the front surface of SNM1B, and adjacent to the active site, is a prominent cluster of positively charged residues, with appropriate dimensions for a DNA binding groove. Utilising the orientation of the two active site bound AMP molecules, it was possible to model a B-form duplex DNA substrate. This substrate contained a short 3′-overhang, to reflect the predominant physiologically relevant (telomeric) SNM1B substrate , whilst R300A and R259A exhibited an increased KDapp, and S183A exhibited the largest increase of around 3-fold , and completely inhibited by the addition of 10 μM unlabelled substrate (a 100-fold molar excess) (One observation made from the WT (nucleotide form) structure and from the model of duplex DNA with SNM1B excess) .in vitro   acted as a complete barrier to digestion. SNM1B was able to digest up to the site of the lesion (i.e. four exonucleolytic events from the 5′ end), but was unable to digest past the thymine glycol leaving a 22 nt substrate with the 5′ end being the thymine glycol modified base  , and SNMo SNM1A) . We exame Figure . Thyminee Figure .We assume that in order to digest fully past a lesion modified bases must be accommodated in both the hydrolysis and 5′-phosphate position, and we have used our SNM1B DNA binding model to try and understand the structural basis of this specificity. The general exonucleolytic promiscuity appears to be the result of the open nature of the active site, with modifications which are planar to the nucleobase (such as the common 8-oxo-G), being well-tolerated in both the hydrolysis and 5′ phosphate position Figure . In cont1–355 complexed with two AMP molecules in the active site (1.8 Å resolution) provides insight into its mechanism, and substrate recognition. The position of AMP_1 in the extended active site of SNM1B1–355 enables definition of a phosphate binding pocket, which likely confers the strict 5′-phosphate requirement for the exonucleolytic activity of SNM1B (and SNM1A). The identification of this phosphate-binding pocket, whilst previously hypothesised . Our structure of SNM1Bthesised , had notthesised  PDB: 6TT1–355’s phosphate binding revealed varying detrimental effects on activity. The effects on DNA binding within this region were similarly varied, but less pronounced, with mutations having, at most, moderately negative effects. These results are consistent with the observation that SNM1B1–355 will bind  a 5′-hydroxyl-containing DNA substrate, but shows a complete inability to exonucleolytically digest it, compared to a substrate bearing a 5′-phosphate. Nine hydrogen bonds are made within the context of the phosphate-binding pocket and the pocket likely provides an important contribution to DNA binding , 1–355 bound model enabling us to probe the contribution of residues within the putative DNA binding cleft to DNA binding and nuclease activity through site-directed mutagenesis. Individual substitutions within this region had varying effects, likely representing the importance of the entirety of this region, and not the sole importance of single residues. Consistent with their importance, however; R19, S183 and R259 are conserved across chicken, fish, mouse, human, chimpanzee, and pig SNM1B. R20 and K260 exhibit conservation of charge  across the aforementioned species.From this nucleotide bound structure, we generated a duplex DNA/SNM1B1–355 to digest past DNA damage lesions and modelled the corresponding lesions in the active site of SNM1B1–355, using our DNA bound model. Intriguingly, we observed that SNM1B1–355 can accommodate  planar nucleobase lesions; by contrast, in the case of thymine glycol, where the C5 methyl group protrudes axially from the ring of the pyrimidine, SNM1B1–355’s exonuclease activity is ablated. Due to their sequence and structure, telomeres are particularly sensitive to reactive oxygen species (ROS)-mediated strand hydrolysis and base oxidation  is associated with cancer risk due to its correlation with alternative splicing of the SNM1B and PHTF1 transcripts, each also associated with increased cancer risk  has previously been considered a two metal-ion-dependent nuclease , and the1–355 in complex with two deoxyribonucleoside (product mimicking) molecules provides insight as to how it recognises 5′-phosphate residues, hydrolyses phosphodiester bonds, and interacts with DNA substrates and is able to digest past a subset of key oxidative (and other DNA damage) lesions, a role that might be important for the maintenance of genome integrity, particularly at telomeres.In summary, the structure of WT SNM1BAtomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data bank under accession numbers: 7B9B, 7B2X and 7A1F.gkab692_Supplemental_FileClick here for additional data file.


After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA–RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA–RBP pairs from natural components remains challenging while such synthetic RNA–RBP pairs could significantly expand the RNA–RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 10  RNA and RNA-binding protein (RBP) pairs, natural or engineered, are used as adaptors to link an RNA of interest with a protein of interest for various applications. For example, the specific interaction between the bacteriophage MS2 coat protein (MS2CP) and MS2 RNA stem–loop  has beenA reasonable strategy to generate novel RNA–RBP pairs is to re-engineer the binding characteristic of an existing RNA–RBP pair. This has typically been done in two steps. (i) Mutate either the parental RNA or RBP to abolish binding ,14. (ii)While these in vitro selection technologies usually involve the selection of an RNA or protein library for binding to a single target, an alternative approach is library-vs-library selection (or screening) which has been employed for identification of protein–protein , proteinEscherichia coli  to develop a method to coevolve RNAs and RBPs through library-vs-library in vitro selection Figure . First, hia coli , and in hia coli ,32,37–45E. coli Rosetta-gami B 5615 strain is not commercially available, because the manufacturer has discontinued production. Accordingly, we generated the strain in the laboratory as follows: pAR5615 plasmid DNA encoding isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible wild-type T7 phage capsid gene was purified from BLT5615 strain (Merck-Millipore) using Zyppy Plasmid Miniprep Kit (Zymo Research). The plasmid DNA was transformed into Rosetta-gami B competent cells (Merck-Millipore), and transformants were selected on an LB plate supplemented with antibiotics (50 μg/ml carbenicillin and 34 μg/ml chloramphenicol). MagStrep ‘type3’ XT beads (2-4090-002) was purchased from IBA Lifesciences. FG HM-NeutrAvidin beads (TAB8848N3171) and magnet stand (TAB4899N12) were purchased from Tamagawa SEIKI.Unmodified oligo DNAs were purchased from Eurofins Genomics, FASMAC, or Integrated DNA Technologies (IDT) Japan. Randomized oligo DNAs containing NNK  codons were purchased from Ajinomoto Bio-Pharma or IDT Japan. DNA oligo pool was purchased from Twist Bioscience. Synthetic genes were purchased from Eurofins Genomics, IDT Japan, or Twist Bioscience. Biotinylated DNA and 2′-OMe RNA were purchased from FASMAC. Q5 High-Fidelity 2× Master Mix and HiScribe T7 High Yield RNA Synthesis Kit were purchased from New England Biolabs (NEB) for polymerase chain reaction (PCR) and in vitro transcription, respectively. SuperScript IV reverse transcriptase was purchased from Thermo Fisher Scientific for reverse transcription (RT). All chemicals used for buffer preparation were purchased from Nacalai Tesque or FUJIFILM Wako Pure Chemical Corporation. Difco LB broth Miller was purchased from BD for bacterial culture. E. coli BL21 (DE3) strain. Transformants were pre-cultured overnight at 37°C in 2 ml of LB medium supplemented with 100 μg/ml carbenicillin and transferred to 100 ml fresh LB medium supplemented with 50 μg/ml carbenicillin. After 1–2 h culture at 30°C, IPTG was added at a final concentration of 0.4 mM, and the cells were further cultured for 20 h at 30°C. The cells were harvested by centrifugation at 6000 × g for 10 min at 4°C and suspended in 20 ml ice-cold His-tag purification buffer A . After cell disruption by ultrasonication, lysozyme , Triton X-100 (0.1% v/v), MgCl2 , DNase I , and RNase A  were added to the crude cell extracts and incubated at 37°C for 30 min. The cell extracts were centrifuged at 8000 × g for 20 min at 4°C to remove cell debris, and the supernatants were filtered. Soluble proteins were loaded onto TALON metal affinity resin (TaKaRa) in a PD-10 (Cytiva) column and washed with 10 ml His-tag purification buffer A. His-tagged proteins were eluted by His-tag elution buffer , and the eluents were incubated with Strep-Tactin XT Superflow high capacity resin (IBA Lifesciences). After 5 min incubation, the resin was washed twice with 0.5 ml His-tag purification buffer A, and Twin-Strep-tagged proteins were eluted twice with 0.5 ml 1× Buffer BXT Strep-Tactin XT Elution Buffer (IBA Lifesciences). The eluents were desalted and concentrated using an ultrafiltration device  and buffer-exchanged into protein storage buffer . Quality of the purified proteins was confirmed by SDS-PAGE  followed by Coomassie blue staining   were subcloned into a pET vector backbone. The bacterial expression vectors  were tra TaKaRa) . The conin vitro transcription. RNA aptamers were synthesized by in vitro transcription at 42°C for 4 h using HiScribe T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer's manual for in vitro transcription of short templates  was injected into all flow cells at a flow rate of 25 μl/min for 7 min to activate carboxylic acid groups. After injection of ultrapure water for 1 min, 0.3 mg/ml NeutrAvidin in 10 mM sodium acetate (pH 5.2) was injected by setting the immobilization level to 5000 RU. Finally, 1 M ethanolamine–HCl (pH 8.5) was injected for 7 min to quench the activated carboxylic acid. To remove the non-covalently bound NeutrAvidin on the chip surface, 10 mM NaOH was injected three times at a flow rate of 20 μl/min for 20 s. Setting the immobilization level to 400 RU, biotinylated 2′-OMe RNAs (5′-/bio/cguucugugucuuucgucgau-3′) (1 μM) in high salt buffer  was injected and the remaining biotin-binding sites on the chip were quenched by injection of 1 μM free biotin.Binding kinetics were determined using Biacore T200 (Cytiva) at 25°C. CM5 chip surface was washed by three injections of 10 mM NaOH at a flow rate of 20 μl/min for 30 s. Next, a mixture of 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mM 2, 0.25 mM TCEP-Na, 0.1 mg/ml BSA, 25 μg/ml yeast tRNA).RNA aptamer solutions (500 nM) were prepared in water and denatured by heating at 80°C for 3 min. After cooling at room temperature for few minutes, the RNA solutions were diluted to 10–50 nM with the high salt buffer. Various concentrations of protein solutions were prepared in SPR running buffer  was determined from the ratio of the association and the dissociation rate constants (KD= koff / kon). The figures were generated using GraphPad Prism software 6.0h (GraphPad Software). The measurements were repeated at least twice using different flow cells (or different SPR chips) in different days to ensure reproducibility.RNAs were injected at a flow rate of 10 μl/min for 30 s, and then the running buffer was injected for 1 min. Proteins were injected at a flow rate of 80 μl/min for 100 s (or 200 s) to monitor association, and then the dissociation kinetics was monitored for 420 s (or 1500 s) in SPR running buffer. The sensor surface was regenerated with 2 μl injection of 10 mM NaOH followed by 6 M urea at a flow rate of 20 μl/min and the running buffer was injected for 1 min. The raw data were analyzed by Biacore T200 Evaluation Software 1.0 (Cytiva) using 1:1 Langmuir interaction model. Background signal from the reference flow cell was subtracted from that of the sample flow cell, and no-protein sample (SPR running buffer only) was injected in each experiment (double referencing). Dissociation constant  before use.MagStrep ‘type3’ XT beads were rinsed three times with ultrapure water and stored in blocking buffer  at 4°C. FG HM-NeutrAvidin beads (100 μl) were rinsed once with immobilization buffer , and mixed with 1.64 nmol of biotinylated capture oligo DNA (5′-/bio/CGTTCTGTGTCTTTCGTCGAT-3′) in the same buffer and incubated at room temperature for 5 min. After washing with the same buffer three times, FG beads were stored in blocking buffer at 4°C. The immobilized oligo DNA was estimated to be ∼6.3 pmol per μl suspended beads. The beads were pre-equilibrated with selection buffer , and the sequence was verified by Sanger sequencing. The partially randomized DNA library encoding L7Ae mutants was prepared by overlap extension PCR (8). The PD library was amplified with logarithmic phase of Rosetta-gami B 5615 strain in 200 ml scale (multiplicity of infection < 0.01). Expression of Gp10A was induced in advance by addition of 1 mM IPTG. Upon lysis , 5 g of sodium chloride was added to the lysate and the E. coli debris was removed by centrifugation at 8000 × g for 20 min at 4°C. After collection of the supernatant, 21 g of polyethylene glycol (PEG) 8000 (Promega) was added and incubated at 4°C overnight. Lysate-PEG mixture was centrifuged at 8000 × g for 40 min at 4°C and the supernatant was discarded. The precipitated phage particles were suspended in T7 phage stock buffer , and subjected to ultrasonication for few minutes. The phage solution was filtered through a 0.45 μm membrane filter , and stored at 4°C until use. Titer of the amplified phage was quantified by plaque assay. Bacteriophage T7-displayed L7Ae or dL7Ae was confirmed by Western blotting  of the purified DNA was used as a template in an in vitro transcription reaction at 37°C for 4 h in 200 μl volume . The transcription product was treated with 2 U TURBO DNase (Thermo Fisher Scientific) at 37°C for 60 min and purified by phenol-chloroform extraction followed by ethanol precipitation. Purified RNAs were dissolved in 1× RNA loading dye (NEB), heat-denatured at 80°C for 3 min, and separated by 8% PAGE. The separated sample was visualized by UV shadowing to excise the gel fragment and the RNA was eluted overnight in TE buffer. The eluents were filtered through a 0.22 μm membrane filter , and concentrated using an ultrafiltration device . RNA concentration was determined from absorbance at 260 nm using NanoDrop One (Thermo Scientific).The template DNA was prepared by primer extension as described previously with modifications . Two olig for 10 min at 4°C. The phage particles were dissolved in 500 μl selection buffer and was incubated with 10 μl pre-equilibrated MagStrep ‘type3’ XT beads at room temperature for 30 min. The phage-immobilized beads were washed once with 500 μl selection buffer and re-suspended in 295 μl of the same buffer. RNA solution of an appropriate concentration was prepared in 200 μl volume, and the RNAs were folded by heating at 80°C for 3 min followed by incubation at room temperature. The folded RNAs were mixed with 4 μl yeast tRNA , 40 U Murine RNase Inhibitor (NEB), and 395 μl of the phage-immobilized magnetic beads. After washing with selection buffer, phage particles were eluted with 50 μl 1× Buffer BXT Strep-Tactin XT Elution Buffer  (IBA Lifesciences).A detailed summary of the selection parameters is shown in 2, 5 mM dithiothreitol) were added to another aliquot of the beads and incubated at 30°C for 15 min. The beads were separated by a magnet and the supernatant containing phage particles was collected.In the 2nd purification, the eluent from the 1st purification was mixed with 10 μl of capture oligo DNA-immobilized NeutrAvidin beads and 440 μl selection buffer and incubated. After washing with the same buffer, the beads were re-suspended in 20 μl of TE buffer. The bead suspension was split into two aliquots for separate RNA and phage recovery. To recover RNAs, 20 pmol of reverse primer N20L_Rv,  was addeThe recovered RNAs were reverse transcribed at 60°C for 15 min in 20 μl volume using SuperScript IV, and the reaction was quenched by incubation at 85°C for 5 min. The cDNAs were amplified by PCR  with appropriate primers  or two-step PCR (for PD library). The RNAs were mixed with a barcoded reverse transcription primer  according to manufacturer's instructions. PhiX control v3 DNA  was spiked into the libraries as an internal control, and sequencing was performed on Illumina MiSeq sequencer using MiSeq Reagent Kit v3 (150 cycle single read or 300 cycle paired-end read). Raw sequence data (fastq files) were analyzed and visualized by custom Python scripts.The oligo pool (426 fmol) containing 2000 distinct sequences was amplified by PCR  using N20L_Fw and N20L_Rev  as primeE. coli BL21 (DE3) strain. The transformants were cultured overnight at 30°C in 2 ml of MagicMedia Medium (Thermo Fisher Scientific) supplemented with 50 μg/ml carbenicillin. The cells were harvested and suspended in ice-cold His-tag purification buffer B . After cell disruption by ultrasonication, Triton X-100 (0.05% v/v) was added to the crude cell extract. The extract was centrifuged at 18 000 × g for 10 min at 4°C to remove cell debris. The supernatant was incubated with TALON metal affinity resin  for 10 min at 4°C. After washing several times with the same buffer, His-tagged protein was eluted with His-tag elution buffer. His-tag purified proteins were confirmed by Western blotting with Strep-Tactin-HRP conjugate   .MagStrep ‘type3’ XT beads  were rinsed three times with ultrapure water and then incubated with blocking buffer  at 4°C overnight. His-tag purified proteins were incubated with 10 μl of MagStrep ‘type3’ XT beads at 4°C for 30 min. After washing with 500 μl each of high salt buffer  and tTBS  , the beads were stored in blocking buffer at 4°C.RNA solution (0.2 μM) was prepared in 500 μl selection buffer supplemented with 0.5 M urea, and the RNAs were folded by heating to 80°C for 3 min followed by incubation at room temperature. The folded RNAs were mixed with 1 μl yeast tRNA  and 5 μl recombinant protein-immobilized beads, and incubated at 37°C for 30 min. After washing eight times with 500 μl sample buffer, RNAs were eluted with 50 μl 1× Buffer BXT Strep-Tactin XT Elution Buffer (IBA Lifesciences). The eluents were purified and concentrated by ethanol precipitation and then dissolved in 10 μl TE.We designed the following strategy to enrich complexes of RNA and phage-displayed protein from unbound species via two-step affinity purification Figure . First  which isE. coli to be amplified. The amplified RNA and phage pools are then used for the subsequent round of selection. After several rounds of selection, the enriched RNA and phage-displayed RBP sequences are analyzed by next-generation sequencing (NGS).Through this two-step affinity purification protocol, complexes of RNA and phage-displayed protein are enriched. The beads are split into two aliquots to recover the enriched RNAs and phage particles separately. RNAs and phage particles are recovered by heating and ribonuclease H (RNase H) treatment, respectively. As RNase H cleaves the RNA strand in DNA–RNA hybrid, it helps to reduce phage particles that non-specifically bind to the capture DNA, NeutrAvidin, or magnetic beads. The recovered RNAs are amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR) and in vitro transcription (IVT). The recovered phage particles are used to infect A. fulgidus L7Ae that lacks affinity for box C/D Kt as a negative control protein  between L7Ae and box C/D Kt was 2.13 × 10–11 M in physiological salt and magnesium concentrations at pH 7.4  measured by stopped-flow kinetic analysis  of a mock PD library was immobilized on StrepTactin magnetic beads. Next, the beads were incubated with 0.2 μM mock RNA library in 500 μl buffer for 10 min, and the beads were washed three times with the same buffer. The phage particles and the RNAs were eluted with D-biotin and then incubated with DNA-immobilized NeutrAvidin magnetic beads in 500 μl of buffer for 10 min to capture RNA-phage complexes. After washing three times, RNAs were eluted, reverse transcribed, and amplified by PCR. Since dKt was designed to be 5-nt longer than box C/D Kt, relative abundance of the two RNA species could be evaluated by gel electrophoresis of the PCR products  and five residues  in the loop connecting α4 helix and β4 sheet were randomized. We did not randomize R41 because it is considered to form a universal hydrogen bond with a phosphate group in Kt RNA (10 ( = 208), diversity of the actual library is limited by the efficiency of in vitro packaging reaction of the phage genomic DNA which was estimated to be approximately 4.0 × 108 based on plaque assay. The PD library size is comparable to those of the previously reported T7 PD libraries , 5 and 6 were analyzed by Illumina sequencing. As expected, almost all sequences in the initial RNA and PD pools (99.8% of RNA and 99.2% of PD) were sequenced only once Figure  and C. RSequencing of the enriched RNA and LS protein sequences does not indicate which RNA interacts with which LS protein. To discover specific RNA–LS protein pairs, we prepared recombinant LS1–LS20 and L7Ae proteins and immobilized them individually on magnetic beads. We also synthesized the 1991 most abundant RNA sequences after the 6th round and 9 control RNAs  ,42 by inA simple inspection of the RNA sequences after PD-SELEX revealed two highly enriched consensus sequences. Consensus sequence 1  was found in 57% of the population, and consensus sequence 2 (UCCAUGACGC) was found in 25% of the population after round 6. The 2000-sequence RNA library also contained 59% and 33% of the consensus sequence 1 and 2, respectively. In contrast, the unselected pool contained 0.0109% (consensus sequence 1) and 0.0011% (consensus sequence 2) of the two sequences. Consequently, we focused on binding of these consensus sequences to the individual LS proteins.Enrichment of the RNAs possessing consensus sequence 1 or 2 was statistically analyzed and visualized by box plots Figure  and C. JFurther inspection revealed that LS4 has low EF values (<0.5) for consensus sequence 2, and LS12 and L7Ae have low EF values (<0.5) for consensus sequence 1 Figure  and C. TKD of 7.36 × 10–11 M, and LS12-1 RNA bound LS12 with a KD of 1.05 × 10–11 M  of CS1 RNA–LS4 and CS2 RNA–LS12 using 0.5 nM Cy5-labeled RNA showed binding profiles characteristic of titration regime  and measured their binding affinities using SPR Figure . RNAs weM Figure  suggesteM Figure , a minimA Figure  which coA Figure . AccordiA Figure  whose KDn regime  which in 10–10 M .–8 M, and that of CS1 RNA and L7Ae was 1.49 × 10–9 M •A (Hoogsteen) type base pairs (KD = 2.21 × 10–9 M) for LS4 but its affinity for LS12 and L7Ae was not measurable (KD > ∼1 μM). This prompted us to characterize additional mutants at these positions . CS1-M2b RNA exhibited respectable binding selectivity for LS4 (1273-fold over LS12) with a lower KD (8.65 × 10–11 M). The possibility of tuning the binding affinity while preserving selectivity by mutating CS1 RNA may be advantageous for adapting the RNA–RBP pair for specific applications.It is known that G•A mismatches in the core of Kt RNAs form se pairs . We founse pairs . To exam–7 M KD which is 15800-fold greater than that for LS12 (7.24 × 10–12 M). On the other hand, the KD value of CS2 RNA and L7Ae was 5.71 × 10–12 M (We next examined the binding affinities of CS2 RNA with LS4 and L7Ae. CS2 RNA bound LS4 at 1.14 × 10 10–12 M  which wa 10–12 M . Therefo 10–12 M .KD except for M3. Impact of the M3 mutation on binding affinity was more pronounced for L7Ae (KD = 2.00 × 10–7 M) than for LS12 (KD = 6.02 × 10–9 M)  in the asymmetric internal loop of CS2 RNA  Figure . LS12 an 10–9 M) . This ob 10–9 M) . It was  10–9 M)  which coKD = 5.99 × 10–7 M and 1.45 × 10–8 M, respectively) compared to the box C/D Kt – L7Ae interaction (KD = 2.13 × 10–11 M). (The affinities of LS4 and LS12 for the box C/D Kt RNA were significantly impaired (0–11 M). . Therefo0–11 M).  and B.KD value of 2.13 × 10–11 M  based on Förster resonance energy transfer (FRET) measurement using a stopped-flow mixer   ,42. We a 2.5 nM) ,63. More 2.5 nM) . The RNA nM) (KD . While t nM)   . Moreoved Lilley , suggestd Lilley . While iE. coli or yeast cell-based in vivo library-vs-library selections have been reported to identify protein-protein  that are represented more frequently in the randomized RNA library (relative to larger and more complex RNA motifs) may have contributed to the successful enrichment the RNA–RBP pairs. In principle, RNA–RBP pairs with lower affinity (e.g. nanomolar KD) should also be selectable by PD-SELEX. In Bowley et al., an antigen-antibody pair with a KD of 50 nM was selected from phage-displayed antibody and yeast-displayed antigen libraries  show low picomolar affinities which are among the strongest RNA-protein interactions observed in natural or synthetic systems . We expeAll data and materials are available upon request.gkab527_Supplemental_FileClick here for additional data file.


CYP11B1 also catalyzes 18-monohydroxylation and 11β,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11β-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11β,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11β-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11β-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.Human cytochromes P450  •CYP11B1 and CYP11B2 catalyze steroid hydroxylation for syntheses of cortisol and aldosterone, respectively.•Structural basis for their differences in ability of multiple hydroxylations remains unclear.•A CYP11B1 mutant generated is characterized by monohydroxylase activities.•X-ray crystallography and molecular dynamics simulation reveal spatial restriction in substrate-binding site of the mutant.•Spatial dimension of the substrate-binding sites is crucial for differential production of gluco- and mineralocorticoids. They arCYP11B2) . As theiCYP11B2) . The twoCYP11B2) . The phyCYP11B2) .Previous studies have explored similarities and differences in the catalytic functions of CYP11B1 and CYP11B2 using transfected cells expressing them  or purifKinetic studies on the processivity of aldosterone formation were carried out using purified bovine enzyme. The results revealed that DOC is converted successively by 11β-hydroxylation and then twice by 18-hydroxylation into aldosterone, though most of the intermediate products dissociate . These cS)-enantiomer of fadrozole, the racemic form of which is known as an inhibitor of estrogen-producing aromatase CYP19A1, was reported (R)-fadrozole . Addition of the substrate DOC at 400 μM to both shifted the Soret peak to 392 nm (dashed lines), and addition of DOF also induced similar spectral changes. These spectral data indicate that binding of the substrates displaces heme-bound water, changing six coordinated low-spin state to five coordinated high-spin state. Sodium dithionite partially reduced the substrate-bound enzymes (dotted lines), and formation of ferrous CO complexes (peak at 447.5 nm) was observable (dashed-dotted lines). When a detergent Cymal-5 was added at 5 mM to the substrate-free ferric enzymes, both were found to be converted into high-spin state of the heme iron . In the presence of Cymal-5 the wild type was converted to fully reduced CO form (dashed-dotted line), while the mutant was converted to ferrous CO form at a higher level than in the presence of DOC although formation of P420 was detected as a bump at 420 nm (dashed-dotted line). Partial reduction of heme iron by sodium dithionite may be characteristic of the CYP11B enzymes as described previously  values of the wild type for DOF and DOC were 21.2 μM and 15.1 μM, respectively, and those of the mutant for DOF and DOC were 3.3 μM and 3.8 μM, respectively  are summarized in To compare the catalytic functions between the mutant and the wild type, the three activities, 11β-hydroxylase, 18-hydroxylase, and 11β,18-dihydroxylase, of both enzymes were assayed using DOF and DOC as substrates with a reconstituted electron transfer chain. The products converted from the substrates were analyzed except for 18-hydroxy DOF since its standard chemical was unavailable . Plots okcat 129.4 min−1, Km 28.2 μM; closed circle) and of DOC to corticosterone . Similarly, the mutant catalyzed 11β-hydroxylation of DOF to cortisol  and of DOC to corticosterone .First, the wild type catalyzed primarily 11β-hydroxylation of DOF to cortisol . The mutant also catalyzed 18-hydroxylation of DOC to 18-hydroxy DOC . Thus, the kcat values of the mutant for both 11β- and 18-monohydroxylase activities were slightly lower by 0.7–0.8-fold than those of the wild type. On the other hand, the Km values of the mutant for DOF in 11β-hydroxylation, DOC in 11β-hydroxylation, and DOC in 18-hydroxylation decreased by 2.3–2.7-fold compared to those of the wild type, being parallel with the decreases in the Kd values of the mutant for both substrates in the binding assays  and of DOC to 18-hydroxy corticosterone . By contrast, the mutant did not produce 18-hydroxy cortisol and 18-hydroxy corticosterone from DOF and DOC, respectively  of the monooxygenations at 11β and 18 between the mutant and the wild type, the data obtained above were used for calculation. The mutant was found to have almost two-fold higher catalytic efficiencies of the monooxygenations at both 11β and 18 compared to those of the wild type. This evaluation suggests that the mutation did not alter the structures essential for catalysis of monooxygenation.For comparison of catalytic efficiencies -fadrozole (black arrows). First, the amino acid replacements at the four residues  in helix A′ of the wild type caused a shift of the helix together with a shift of the turn from β1-1 to β1-2. Second, the replacements at the two residues (L244N and W247N) in helix G′ of the wild type caused uncoiling in part and a shift of it. Along with the structural shifts, the substrate access channel has an open entrance . Similar structural shifts as mentioned above were detected when the crystal structure of the (S)-fadrozole-bound wild type CYP11B1 was superposed on that of DOC-bound CYP11B2 (thick arrows). Helices F, G, and H of the wild type of CYP11B1 in comparison to those of CYP11B2 exhibit characteristic shifts . The same set of residues forms the substrate-binding site of the (S)-fadrozole-bound CYP11B1 wild type (gray) and DOC-bound CYP11B2 (light blue). To examine the spatial configuration of their substrate-binding sites, molecular surfaces were calculated with a probe radius of 1.6 Å. The surface representations delineate differences in geometry among them , whereas CYP11B2 has an enlarged space which surrounds the C3 carbonyl side of DOC , creating more space in the innermost area (orange vslight gray), whereas the side chain of W116 in DOC-bound CYP11B2 is located at a higher and right-hand position (light blue). The distance from the C3 carbonyl oxygen atom (O3) of DOC to the Cζ3 atom (CZ3) of the side chain of W116 is 4.1 Å in DOC-bound CYP11B2 (light blue). When DOC is placed into the substrate-binding site of the mutant and the wild type of CYP11B1 at the same position relative to the heme in the CYP11B2 structure, the distances from O3 of DOC to CZ3 of W116 are 2.0 Å and 2.8 Å, respectively. These distance estimations indicate that the innermost area in the substrate-binding site of CYP11B1 could cause steric hindrance more severely in the mutant than in the wild type with DOC which occupied the same position as in the crystal structure of CYP11B2.These spatial differences are attributable mainly to two features. First, the side chains of R120 and E310 form an ionic bond in both CYP11B1  but not ost area . Second,R)-fadrozole-bound CYP11B2 . Similarly, analysis of occupancies of the residue atoms located at distance of ≤4.0 Å from O3 of DOC revealed that the side chain atoms of W116 and F130 and the main chain atoms of A313 and G314 were closer to O3 of DOC in the CYP11B1 mutant than in the wild type but more distant in CYP11B2 . Thus, the space around the C3 side of DOC is more restricted in the mutant than in the wild type of CYP11B1, whereas it is enlarged in CYP11B2 compared to those in both CYP11B1.Subsequently, amino acid residues close to DOC were analyzed by monitoring the residue atoms (except hydrogen) located at distance of ≤4.0 Å from carbon or oxygen atoms of DOC in the MD trajectories. The residues W116, F130, E310, A313, G314, G379, F381, L382, and F478 of the three enzymes were found to meet the conditions with occupancies ≥0.5 , sticks. CYP11B2 , balls. To further explore the positioning of DOC in the substrate-binding site of the simulated complexes, formation of hydrogen bond involving the three oxygen atoms, O3, C20 carbonyl oxygen (O20), and C21 hydroxyl oxygen (O21), of DOC was examined. O3 of DOC hardly formed hydrogen bonds in the mutant of CYP11B1 (average number of 0.03) and rarely in the wild type (average number of 0.16), whereas O3 of DOC in CYP11B2 formed hydrogen bonds to water molecules with average number of 1.1 . In the S)-fadrozole-bound wild type). Regarding the ionic bond between the side chains of R120 and E310, the distance distributions of R120 NH-E310 OE exhibited different patterns in peak shapes between the two enzymes (S)-fadrozole). The results coupled with those from the simulations of the DOC-bound forms provided strong evidence indicating that the spatial restriction in the substrate-binding site of the mutant was caused by the amino acid replacements.In addition to the DOC-bound forms, we performed MD calculations to generate metyrapone-bound forms of the CYP11B1 mutant and the wild type, as another set of the enzymes bound to the same ligand. In comparison of the spatial dimensions of the substrate-binding sites, the distance distributions of W116 CZ3-F130 CG were clearly different between the mutant and the wild type . The ave enzymes . The sho3The present study shows that the functional divergence of the CYP11B enzymes in the ability to catalyze 18-hydroxylation following 11β-hydroxylation depends on spatial dimension in the substrate-binding site. The finding is based on structural analyses of the CYP11B1 mutant deficient in the 11β,18-dihydroxylase activity but retaining 11β- and 18-monohydroxylation activities of the wild type. The correlation between the dihydroxylase activity and the spatial dimension was derived from comparison of the present results with the previous data including the crystal structures of the CYP11B1 wild type and CYP11B2 . MD simuBecause the CYP11B1 wild type is able to catalyze the 11β,18-dihydroxylation, a step necessary for aldosterone synthesis, the enzyme has in part a functional feature of CYP11B2. The deficiency in the dihydroxylase activity of the CYP11B1 mutant generated in the present study means that the mutant is more distantly related to CYP11B2 in terms of catalytic ability. Employing the CYP11B1 mutant, the present study highlights the structural differences in the substrate-binding site between CYP11B1 and CYP11B2. The mutant of CYP11B1 serves as a molecular entity that enlarged the differences in both structure and function between CYP11B1 and CYP11B2.Spatial requirement for successive hydroxylation Assuming that a single enzyme catalyzes hydroxylation of C18 in addition to C11β successively, the substrate-binding site has to position C18 of 11β-hydroxy steroid within the distance of hydroxylation by heme-bound active oxygen. This implies that the space around the C3 side of steroid occupying the substrate-binding site has a key role for the successive catalytic activity. The present study indicates two major structural elements to characterize the innermost area of the substrate-binding site. As described, one is ionic interaction between the side chains of R120 and E310, and the other is arrangement of the side chain of W116. In CYP11B1, formation of the ionic bond between R120 and E310 and a lower position of W116 side chain are likely to induce steric hindrance with the C3 side of 11β-hydroxy steroid when it is placed so that C18 is hydroxylated. In CYP11B2, additional space due to the absence of the ionic bond and higher position of W116 side chain avoids such steric hindrance and allows for successive 18-hydroxylation more efficiently.The putative water channel connecting to the innermost space in the substrate-binding site of CYP11B2  was prevFunctional alteration induced by amino acid substitutions outside of the substrate-binding site Previous studies have examined catalytic activities of the human CYP11B mutants carrying an exchange(s) of the isoform-specific amino acid residues between CYP11B1 and CYP11B2 to identify residues responsible for the catalytic differences (ferences . Based oferences . (i) Theferences . (iii) Cferences . The actferences . Thus, Sferences , exhibitferences . Anotherferences , is likeferences  could noRoles of the isoform-specific residues to form the distinct structures Since the present study reveals that the structural difference in the innermost area of the substrate-binding sites between CYP11B1 and CYP1B2 dictates the functional difference, we suggest possible roles of the isoform-specific amino acid residues at 288 and 320 to form the distinct structures to differentiate their catalytic activities . The three residues at the amino-terminus (GTR: residue numbers 25–27) of the mature enzyme were replaced with MATK, and the original carboxy-terminus at residue number 503 was extended with addition of hexahistidine residues. The mutant carried the replacement at six amino acid residues  carrying pGro12 was transformed with the pET17b vectors carrying the CYP11B1 constructs.Two forms, a wild type and a mutant, of recombinant human CYP11B1 were expressed as 486-amino acid residue polypeptide . They haε393 = 96 mM−1 cm−1 for high-spin state induced by binding of substrate to cytochromes P450 (Recombinant proteins were expressed and purified in similar manners as described previously . For indmes P450  because mes P450 .4.26) was cloned into pET17b utilizing NdeI and HindIII restriction sites. Expression and extraction of the recombinant bovine adrenodoxin was carried out as described above for CYP11B1 except that sodium cholate and Tween 20 were omitted. Homogeneous adrenodoxin (A414/A280 = 0.86–0.90 (ε414 = 11.0 mM−1cm−1 (ε450 = 10.9 mM−1cm−1 (cDNA encoding bovine adrenodoxin was kindly provided by Dr. Yasuhiro Sagara  and used.86–0.90 ) was obt.86–0.90 . ConcentmM−1cm−1  and ε450mM−1cm−1 , respect4.3Kd values. Titration with metyrapone (Sigma) was carried out in the presence of DOC at 320 μM as a competitor in order to evaluate the Kd values smaller than the concentrations of CYP11B1. The dissociation constants were calculated from the apparent values by using the Kd values for DOC as the inhibitory constants. To exclude the possibility that binding of cortisol or corticosterone holds the heme iron in low-spin state, effects of 1 mM cortisol (Sigma) or corticosterone (Sigma) as a competitor on the Kd values for DOC or DOF (Sigma) were tested. The steroids and ligands used for spectroscopic analyses were dissolved in dimethyl sulfoxide.Spectroscopic studies were carried out at room temperature using a Jasco UV–visible spectrophotometer V-530. Purified enzymes were diluted to 1.0 μM for the wild type and 0.8–1.2 μM for the mutant with the buffer consisting of 50 mM potassium phosphate (pH 7.4), 150 mM NaCl, 20% (v/v) glycerol, and 0.05% (v/v) Tween 20. Substrate DOC (Sigma) at 400 μM, sodium dithionite, and CO were added sequentially, and absolute spectra were recorded after each step. Cymal-5, unlike other several detergents we used with the enzyme preparations, was found to induce high-spin state of the heme iron and to give higher levels of reduced CO forms of both wild-type and mutant than DOC. The data obtained with 5 mM Cymal-5 (Anatrace) in place of the substrate were also included in 4.42 0.1 mM EDTA, 20% (v/v) glycerol, 5 mM glucose-6-phosphate, 2 U/ml glucose-6-phosphate dehydrogenase (Roche), 1 mM NADPH (Roche), 0.125 μM bovine adrenodoxin reductase, 5 μM bovine adrenodoxin, 2 nM CYP11B1, 100 μM 1,2-dilauroyl-sn-glycero-3-phosphocholine , and 0–80 μM DOF or DOC. Products converted from the substrates DOF or DOC were analyzed by liquid chromatography-tandem mass spectrometry. Increases in concentrations of adrenodoxin reductase from 0.125 μM or of adrenodoxin from 5 μM did not raise turnover numbers of both CYP11B1 enzymes. Steroids and DLPC used for activity assays were prepared by dissolving in ethanol. Reaction was started with addition of NADPH, incubated at 37 °C for 5 min, and stopped by addition of 1 mL chloroform followed by vortexing. Progesterone (Sigma) at 500 nM was included in the reaction mixture as internal standard. Steroids were extracted from the reaction mixtures once with chloroform and evaporated and were dissolved with 50 μL of 50% (v/v) methanol. Aliquots (5 μL) were analyzed using an Nexera UHPLC system coupled with LCMS-8030plus triple quadrupole mass spectrometer (Shimadzu). A C18 column  was used, and the temperature of the column oven was kept at 40 °C. Running solvents used for the chromatography were: (A) H2O + 0.05% (v/v) formic acid and (B) acetonitrile + 0.05% (v/v) formic acid. The following gradients were applied: 0–2.0 min, 5% B; 2.0–3.0 min, 5–30% B; 3.0–12.0 min, 30–98% B; 12.0–18.0 min, 98% B; 18.0–18.5 min, 98-5% B; 18.5–24.0 min, 5% B at flow rate of 0.2 ml/min. MS/MS was carried out with electrospray ionization at positive ion mode, and multiple reaction monitoring transition are outlined in kcat and Km values.Catalytic activities of the purified CYP111B1 enzymes were examined using a reconstituted system including an NADPH-regenerating system, adrenodoxin reductase, and adrenodoxin as described previously . Assay m4.5For crystallization, imidazole bound to the purified CYP11B1 mutant was replaced with metyrapone using ultrafiltration devices  by three cycles of dilution and concentration with a buffer consisting of 20 mM potassium phosphate (pH 7.4), 20% (v/v) glycerol, 150 mM NaCl, 0.05% (w/v) Fos-choline 12 (Anatrace), 0.1 mM Na EDTA, 0.1 mM DTT, and 100 μM metyrapone. Crystals were grown in drops prepared by mixing of 1 μL of 500 μM enzyme with 1 μL of a precipitant consisting of 100 mM potassium phosphate (pH 7.4), 150 mM NaCl, and 4% (w/v) PEG 3350 (Hampton Research) on sitting drop plates at 4 °C, and the reservoir solution used was 20% v/v glycerol.4.64DVQ) was used as the search model for MR. The protein model of MR solution was manually rebuilt using the program Coot -fadrozole-bound wild type CYP11B1 (PDB ID: 6M7X), and DOC-bound CYP11B2 (PDB ID: 4DVQ). The missing N- and C-terminal residues with free amino and carboxyl groups at the termini were modeled by the LEaP program in AmberTools -fadrozole), and DOC, was built by the antechamber and MCPB.py in AmberTools18 (S)-fadrozole, and DOC were determined by the AM1-BCC charge method with GAFF.The force field for the nonstandard residues, i.e., heme, proximal C450, nitrogen coordinating ligands -fadrozole molecules with DOC and equilibrating the systems, a 60-ns production run at 300 K and 1 atm was performed for the DOC complexes of the CYP11B1 mutant and the wild type, as well as for that of CYP11B2. For the metyrapone complexes of the CYP11B1mutant and the wild type, 10 100-ns runs at 300 K and 1 atom were performed using GROMACS 2019 (The initial geometries of the DOC complexes of the CYP11B1 mutant and the wild type and their metyrapone complexes were constructed from their ligand-bound CYP11B1 structures thermally equilibrated at 300 K and 1 atm by 20-ns MD simulations. After replacing the metyrapone or (ACS 2019 . The resACS 2019 . The BerACS 2019 .The atomic coordinates and the structure factors of the metyrapone-bound mutant of CYP11B1 have been deposited in the Protein Data Bank under the accession code 7E7F.Kuniaki Mukai: Conceptualization, Investigation, Visualization, Writing – original draft, Writing – review & editing. Hiroshi Sugimoto: Investigation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Katsumasa Kamiya: Investigation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Reiko Suzuki: Investigation, Visualization. Tomomi Matsuura: Investigation, Visualization. Takako Hishiki: Investigation, Visualization, Writing – original draft. Hideo Shimada: Conceptualization, Writing – review & editing. Yoshitsugu Shiro: Writing – review & editing. Makoto Suematsu: Writing – review & editing. Norio Kagawa: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Although human/eukaryotic ribosomal protein L14 (RPL14/eL14) is known to be associated with a variety of cancers, its role in nasopharyngeal carcinoma (NPC) remains unclear. The aim of this study was to explore the impact of RPL14(eL14) in NPC. The results of quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemical staining revealed that the expression of RPL14(eL14) significantly reduced in NPC tissues and cells. Furthermore, the protein expression of RPL14(eL14) was linked to NPC-related clinical pathological features, including the T and N classification of Tumor Node Metastasis (TNM) staging . Cell counting kit-8 (CCK-8) assay and colony formation assay revealed that RPL14(eL14) overexpression repressed NPC cell proliferation. In cell cycle assay, RPL14(eL14) overexpression significantly blocked NPC cells in S phase. Overexpression of RPL14(eL14) repressed cell migration and invasion in NPC as shown by transwell assay and cell scratch healing assay. In addition, RPL14(eL14) was closely correlated with the expression of epithelial–mesenchymal transition (EMT) biomarkers, including E-cadherin, N-cadherin, and vimentin as detected by western blot. In conclusion, our results revealed that RPL14(eL14) may be considered as an antioncogene in NPC, which greatly suppresses cancer progression.  ThereHuman/eukaryotic ribosomal protein L14 (RPL14/eL14) is a member of the L14E ribosomal protein family. The protein consists of five introns and six exons and is located on chromosome 3p21.3. Previous research has linked RPL14(eL14) with esophagus squamous cell carcinomas (ESCC) , lung anThe epithelial–mesenchymal transition (EMT) refers to the biological process of epithelial cells transforming into mesenchymal cells through specific procedures. The EMT is an important mechanism in tumor cell migration, invasion, and involves downregulation of E-cadherin expression and increased expression of vimentin and N-cadherin . In 2012Although RPL14(eL14) has been found to be associated with a variety of cancers, the role of RPL14(eL14) in NPC remains unclear. Therefore, the aim of this study was to examine the RPL14(eL14) expression in NPC and then examined the role of RPL14(eL14) in NPC cell proliferation, cell cycle, cell migration, invasion, and the EMT process.Our data strongly suggested that RPL14(eL14) contributes to the NPC growth and metastasis. Thus, we suggested that RPL14(eL14) may be considered as an antioncogene in NPC.2.2.1.From July 2017 to August 2018, we collected 16 NPC tissues  and 16 chronic nasopharyngitis tissues  from the First Affiliated Hospital of Guangxi Medical University, Nanning, China for quantitative real-time polymerase chain reaction (qRT-PCR) assay. At the same time, 32 chronic nasopharyngitis tissues and 86 NPC tissues were obtained and used for immunohistochemistry assay. MRI and serum Epstein-Barr virus detections were performed on the patients suspected of NPC. If NPC was indeed suspected, nasopharyngeal biopsy could be performed for pathological diagnosis. All the patients had been recently diagnosed with NPC. The diagnosis had been confirmed pathologically. They had no other tumors and had not undergone chemotherapy or radiotherapy prior to sample collection. Two senior pathologists performed the tumor staging, with staging adhering to the Tumor Node Metastasis (TNM) staging method of the International Union Against Cancer (eighth edition). All surgically isolated fresh tissues were placed in labeled 1.5 ml enzyme-free EP tubes and stored in liquid nitrogen before RNA extraction and tissue section.All the participants signed an informed consent form. The study was approved by the ethics committee of the First Affiliated Hospital of Guangxi Medical University, approval number: 2021(KY-E-092).2.2.2.Cell lines  were obtained from the Otolaryngology Laboratory of Guangxi Medical University, Nanning, China. The cells were cultured in complete cell culture medium, which was mixed with 89% RPMI – 1640 medium , 10% fetal bovine serum , and 1% penicillin-streptomycin mixture  at 37°C with 5% CO2.3.After rehydration, the tissue specimens were incubated in citrate buffer (pH = 6.0) at high pressure for 8 minutes, followed by immersion in 3% hydrogen peroxide at room temperature for 15 minutes. After incubation of the tissues with anti-RPL14(eL14) overnight, the tissue specimens were incubated with biotin-labeled goat anti-rabbit secondary antibody at room temperature for 30 minutes, followed by DAB color development for 5 min and hematoxylin staining for 1 minute. The immunohistochemical staining result was judged by the cell staining intensity and the percentage of positive stained cells .2.4.Control vector (LV-pGV) and lentiviral expression clones for RPL14(eL14) (LV-RPL14) were purchased from Genechem, Shanghai, China. Lentiviral infection solution was added to the cells according to the manufacturer’s instructions, and the cells were cultured for 48 hours. Subsequently, stably transfected cells with puromycin for 10 days were selected. Finally, the successful construction of RPL14(eL14) overexpression cell lines was verified by qRT-PCR and western blot assays.2.5.In brief, total RNA was extracted from the tissues and cells by Trizol reagent . Then, cDNA was synthesized using All-In-One 5X RT MasterMix . To detect RPL14(eL14) expression, a qRT-PCR assay was performed using an EvaGreen 2X qPCR MasterMix kit . Primers were synthesized by Sangon Biotech . The primer sequences for qRT-PCR were as follows: GAPDH-forward: 5ʹ- TGGGTGTGAACCATGAGAAG-3ʹ, GAPDH- reverse: 5-’ GTGTCGCTGTTGAAGTCAGA-3ʹ. RPL14(eL14)-forward: 5-’TTAAGAAGCTTCAAAAGGCAGC-3ʹ, RPL14(eL14)- reverse: 5-’TTTTGACCCTTCTGAGCTTTTG-3ʹ.2.6.For protein extraction, a mixture of 90% RIPA lysis buffer and 10% protease inhibitor  was used. After SDS-PAGE gel electrophoresis at 120 V for 1 hour and transfer of the proteins to a PVDF membrane at 150 mA for 90 minutes, the PVDF membrane was incubated with corresponding anti-RPL14(eL14), anti-GAPDH, anti-E-cadherin, anti-N-cadherin, and anti-vimentin  overnight, then incubated in fluorescent antibody  for 1 hour. GAPDH served as a control. The results were quantitative by image J software .2.7.3 cells/well, HK1: 2 × 103 cells/well) was plated in 96-well plates. The absorbance of CCK-8 (OD 450 nm) 0, 24, 48, and 72 hours after cell attachment was then measured. The absorbance at OD 450 nm reflected the cell proliferation rate [The cells were digested by trypsin, and the cell resuspension solution  were plated in six-well plates and cultured in medium for 14 days. Subsequently, the cells were fixed in 4% paraformaldehyde for 20 minutes and then stained with 1% crystal violet for 20 minutes. More than 50 cells counted as one clone. The cell clone formation rate = the number of clones/number of inoculated cells × 100% .2.9.We used 0.25% no EDTA trypsin to digest the cells, followed by resuspension with 70% cold ethyl alcohol at 4°C for 24 hours. Then, 100 µl of 10 mg/ml RNase were added to the cells, and the cells were incubated at 37°C for 30 minutes, followed by cell staining with propidium iodide at 4°C for 30 minutes. The cell cycle was analyzed by flow cytometry  and visualized by Modfit software (version 3.1).2.10.2) were seeded with FBS-free medium into the upper chamber of an 8-μm pore membrane containing Matrigel (transwell migration had no Matrigel). In the lower chamber, 10% FBS medium was added. After culturing for 48 hours, the membrane was fixed with 4% paraformaldehyde for 20 minutes, and the cells were then stained with 1% crystal violet for 20 minutes. The number of cells that passed through the membrane indicated the migration and invasion ability of tumor cells [In the transwell invasion assay, the cells /D0 × 100%.In the six-well plates overgrown with cells, a sterile 200 μl pipette tip was used to draw a blank area on the confluent monolayer of the cells. The wells were then carefully washed three times with a PBS buffer and photographed using an inverted microscope  to record the distance of the scratched area and marked them as D0. The cells were then incubated at 37°C with 5% CO2.12.All data were analyzed using SPSS software, version 24.0. The difference between the two groups was analyzed using the Student’s t-test. The data are presented as mean ± standard deviation. The Mann–Whitney U-test was also conducted, with the data presented as median and interquartile range. The relation between RPL14(eL14) expression and clinical characteristics was analyzed using the χ2 test. Each assay was repeated at least three times. A p value of <0.05 (two-tailed) was considered a statistically significant difference.3.RPL14(eL14) was down-regulated and may serve as an antioncogene in NPC. Multiple experiments revealed that RPL14(eL14) inhibited NPC cell proliferation, invasion, migration and EMT processes. Moreover, RPL14(eL14) blot NPC cells in S phase. All of the results suggested that RPL14(eL14) may affect the occurrence and development of NPC.3.1.Immunohistochemical staining was utilized to test the protein expression of RPL14(eL14) in 32 chronic nasopharyngitis tissues and 86 NPC tissues. Consistent with the mRNA expression, the protein expression of RPL14(eL14) was weakly detected in the NPC tissues, with a 31.395% positive rate. In the 32 chronic nasopharyngitis tissues, 53.125% exhibited high expression of RPL14(eL14) , Table 1Consistent with the results from this study on NPC tissues, RPL14(eL14) mRNA and protein expression levels in the NPC cells (5–8 F and HK1) were significantly reduced when compared with the normal nasopharyngeal epithelial cells (NP69) . Togethe3.2.To further explore the role of RPL14(eL14) in NPC, the overexpression of RPL14(eL14) in 5–8 F and HK1 cells were stably constructed by lentiviral. As shown by the results of both the western blot and qRT-PCR analyses, RPL14(eL14) expression was successfully overexpressed in 5–8 F and HK1 cells by a lentiviral .Figure 2In the CCK-8 assay, proliferation of 5–8 F and HK1 with LV-RPL14 cells was significantly inhibited when compared with that of vector control groups after 24, 48, and 72 hours  in cell cycle. In the 5–8 F cells overexpressing RPL14(eL14), the percentage of cells in the S phase was significantly higher than the percentage of cells in the S phase in the vector control group (37.10 ± 2.05% vs. 30.78 ± 2 .09%) (p < 0.05). However, there was no difference in the percentage of cells in the G1/G0 phase (15.8% vs. 17.43%) and G2/M phase (47.11% vs. 51.79%) . In terms of HK1 cells, the cells of S phase in LV-RPL14 were 40.16±2.51%, which was significantly higher than 32.45 ± 2.19% cells in LV-pGV  overexpression repressed NPC cell migration and invasion.3.4.The effect of RPL14(eL14) in 5–8 F and HK1 cells on the EMT process was determined by western blot. As displayed in 4.It has been widely recognized that ribosomal proteins (RPs) were not only involved in the composition of ribosomes and biosynthesis of protein but also related to the occurrence and development of tumors . For exaWe found that the loss of RPL14(eL14) was responsible for the development of NPC. Consistent with the findings of our research, a previous study revealed that RPL14(eL14) was down-expression and could serve as an early diagnostic biomarker for esophageal squamous cell carcinomas . ShriverOur proliferation assays revealed that RPL14(eL14) suppressed cell proliferation ability in NPC cells. To further analyze the possible roles of RPL14(eL14) in NPC, we performed cell cycle analysis. The results showed that overexpression of RPL14(eL14) arrested cell cycle in the S phase. Cell cycle arrest is a protective mechanism, which can be explained as when DNA is damaged, cell cycle monitoring points can be regulated to initiate cell cycle arrest for DNA repair. Cell cycle is mainly divided into G0/G1, S, and G2/M phases. Previous researches showed that S phase played an important role in the pathogenesis of a variety of cancers . During Distant metastasis is the major reason for poor overall survival and treatment failure among NPC patients . About t5.This research revealed that RPL14(eL14) both down-regulated in NPC tissues and cells as verified by a variety of experiments. The decrease of RPL14(eL14) was associated with few clinical features of NPC, including T and N classification. Moreover, multiple cell biological function experiments revealed that RPL14(eL14) may have inhibited the occurrence and development of NPC. However, these results need to be validated in future studies.