Potato spindle tuber viroid (PSTVd) is a circular non-coding RNA of 359 nucleotides that replicates and spreads systemically in host plants, thus all functions required to establish an infection are mediated by sequence and structural elements in the genome. The PSTVd secondary structure contains 26 Watson-Crick base-paired stems and 27 loops. Most of the loops are believed to form three-dimensional (3D) structural motifs through non-Watson-Crick base pairing, base stacking, and other local interactions. Homology-based prediction using the JAR3D online program revealed that loop 27 (nucleotides 177–182) most likely forms a 3D structure similar to the loop of a conserved hairpin located in the 3' untranslated region of histone mRNAs in animal cells. This stem-loop, which is involved in 3'-end maturation, is not found in polyadenylated plant histone mRNAs. Mutagenesis showed that PSTVd genomes containing base substitutions in loop 27 predicted by JAR3D to disrupt the 3D structure were unable to replicate in Nicotiana benthamiana leaves following mechanical rub inoculation, with one exception: a U178G/U179G double mutant was replication-competent and able to spread within the upper epidermis of inoculated leaves, but was confined to this cell layer. Remarkably, direct delivery of the U178G/U179G mutant into the vascular system by needle puncture inoculation allowed it to spread systemically and enter mesophyll cells and epidermal cells of upper leaves. These findings highlight the importance of RNA 3D structure for PSTVd replication and intercellular trafficking and indicate that loop 27 is required for epidermal exit, but not epidermal entry or transit between other cell types. Thus, requirements for RNA trafficking between epidermal and underlying palisade mesophyll cells are unique and directional. Our findings further suggest that 3D structure and RNA-protein interactions constrain RNA sequence evolution, and validate JAR3D as a tool to predict RNA 3D structure. Potato spindle tuber viroid (PSTVd) is a small, circular non-coding RNA that systemically infects host plants. All functions required for replication as well as cell-to-cell and systemic spread are mediated by the RNA genome, whose 2D structure consists of multiple base paired stems and loops. However, a wealth of evidence indicates that most loops in RNA molecules are structured by non-canonical base pairs and other interactions. Here, we show that PSTVd loop 27 forms a 3D structure that is essential for replication. Analysis of an exceptional mutant revealed that loop 27 is also required for transit from infected epidermal cells to underlying palisade mesophyll cells, but is not needed for transport in the reverse direction or into other cell types. Thus, loop 27 is necessary for epidermal exit but not entry, and requirements for transit between epidermal cells and palisade mesophyll cells are unique and directional. PSTVd moves between cells through specialized structures known as plasmodesmata. Combined with previous findings, these studies suggest that plasmodesmal gates interconnecting most and perhaps all plant cell types are unique, allowing precise regulation of RNA transport and the establishment of distinct cellular boundaries. The ability of multicellular organisms to grow, develop, and respond to the environment requires efficient, targeted, and regulated cell-to-cell and systemic communication involving the exchange of both small molecules and macromolecules. Cell boundaries play critical roles in balancing information transfer and cell autonomy, in part by acting as barriers that either facilitate or prevent the exchange of specific nucleic acids and proteins. As viruses and sub-viral pathogens must subvert these barriers to achieve successful infection of their hosts, they serve as useful models to study pathways that mediate macromolecular transfer between cells , 2. At pIn plants, developmental, physiological, and defensive processes involve the spread of specific mRNAs and proteins from the cells in which they are produced into neighboring and distant cells . For exaViruses and viroids achieve systemic infection through cell-to-cell spread and long-distance transport through the phloem. Most plant viruses and all viroids have RNA genomes that provide useful tools to investigate RNA trafficking pathways –28. ViruPotato spindle tuber viroid (PSTVd) to investigate the roles of unique structural motifs in RNA trafficking. To date, several motifs that mediate passage across distinct cellular boundaries have been identified . Us. Us35]. N. benthamiana harbors a protein(s) that binds PSTVd loop 27 in a manner that resembles the SLBP/histone stem-loop interaction in at least some respects. However, there are important differences. First, SLBP binding to the histone stem-loop requires additional bases in a conserved, extended stem that are not present in PSTVd (https://solgenomics.net/) using human, rat, and zebrafish SLBP amino acid sequences as queries also yielded no promising candidates. There are also clear functional differences. SLBP promotes association of U7 snRNP with histone pre-mRNAs, resulting in 3' end cleavage and maturation [That mutants retaining the loop 27 model structure and potential binding sites are systemically infectious suggests that in PSTVd  35]. Fu. FuN. beturation . Cleavagturation . In addituration , 53. MutUndoubtedly the atypical U178G/U179G mutant proved the most informative of the fifteen examined in this study. Despite a sequence predicted to disrupt both the 3D structure and potential protein binding sites within loop 27, this mutant is replication-competent and able to spread between cells in the upper epidermis following rub-inoculation. And while reduced progeny accumulation levels were observed with U178G/U179G , this isN. tabacum) leaves -UTP or digoxigenin (DIG) using plasmid pInter(-) linearized with SpeI as template [Plasmid pRZ6-2-Int and mutant derivatives were linearized with Hind III and employed as templates to generate (+)-PSTVd wild type and mutant template . UnincorN. benthamiana plants were grown in the greenhouse of the Biotechnology Support Facility at the Ohio State University. Two weeks after planting, infection was accomplished by rub inoculating the upper surfaces of the first two true leaves that were previously dusted with carborundum powder. PSTVd (+)-strand in vitro transcripts (300 ng/plant) in water treated with diethylpyrocarbonate (DEPC) served as inoculum. Wild type PSTVd transcripts were a positive control, and mock inoculation was performed using DEPC-treated water. In some experiments, circularized wild type PSTVd and U178G/U179G transcripts (300 ng/plant) were inoculated by multiple needle puncture injections into N. benthamiana stems and petioles using a 10 μl syringe . RNA circularization was performed as described by Beaudry and Perrault (1995) [t (1995) .N. benthamiana protoplasts were prepared and transfected in the presence of polyethylene glycol as described [escribed . Protoplet al. (2006) [RNAzol reagent  was used to extract total RNA from inoculated leaves and systemic leaves collected at 10 days and 28 days after inoculation, respectively. RNA blot hybridization was performed essentially as described by Zhong . (2006) . RNA bloInfected systemic leaves (above the inoculated leaves) were pooled for total RNA extraction using RNAzol reagent (Sigma-Aldrich). In some experiments, RNA was obtained from inoculated leaves. cDNA was prepared by reverse transcription PCR (RT-PCR) essentially as described by Qi and Ding (2002) . The maiN. benthamiana leaves was performed using RNAzol (Sigma-Aldrich). Reverse transcription was performed using Superscript III (ThermoFisher Scientific) with 500 ng of pooled RNA, followed by PCR amplification using Taq polymerase (ThermoFisher Scientific). Actin mRNA was an endogenous control. Sequences of PSTVd PCR primers were: 5'-CGGAACTAAACTCGTGGTTCCT-3' (forward) and 5'-AGGAACCAACTGCGGTTCCA-3' (reverse). Primers used for actin were: 5'-CACCATGGCAGATGGAGAGGATATTCAGCC-3' (forward) and 5'-AGGAACCAACTGCGGTTCCA-3' (reverse). PCR products were subjected to 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining.Total RNA extraction from petioles of N. benthamiana leaf extracts were prepared by grinding young leaf tissue (100 mg) in 1 ml buffer , followed by centrifugation to collect the supernatant. RNA degradation assays were performed by mixing in vitro PSTVd wild type and U178G/U179G transcripts (10 ng) with the supernatant (90 μL), or buffer only as a control, at 28°C. Samples were taken at 0, 15, 30, 60, 120, and 180 minutes and immediately frozen with liquid nitrogen after collection. RNA blot analysis was performed and signals were quantified using Quantity One software . Degradation curves were prepared using the quantified data.in situ hybridization was performed following the protocol described by Traas (2008) [Leaf samples were processed as previously described , 31, 80,s (2008) .S1 FigPresence of the stem-loop in the genomes of 32 viroid species was determined by analysis of sequences and established RNA secondary structures and/or by RNA folding using Mfold. Numbers in red indicate that the stem-loop is present in the majority of variants. NA indicates complete sequence is not available.(TIF)Click here for additional data file.S2 FigSHAPE was performed using BzCN as described Materials and Methods. Normalized original data for wild type loop 27 (A) and selected mutants, including U177A (B), U178G/U179G (C), and A182G (D) are shown. For the mutants, unit-length linear PSTVd transcripts (starting point nucleotide 88) were gel purified and incubated with BzCN or DMSO (negative control). Peaks indicate strong stops following reverse transcription using primer PSTVd-321. In parallel, ddCTP was added to reverse transcription reactions containing wild type PSTVd RNA to generate a sequence ladder. Aliquots of completed ddCTP reactions were added to completed BzCN and DMSO reactions, indicated ddCTP (+) and ddCTP (-), respectively, followed by capillary sequencing.(TIF)Click here for additional data file.S3 Figet al., 2015 [PSTVd structure has been probed using three chemicals in four separate studies. The region shown includes terminal loop 27 and the closing U177-A182 pair, the adjacent base-paired stem, and two bases of loop 26. (A) SHAPE reactivity using BzCN from this study see . (B) SHAl., 2015 . (C) SHAl., 2015 . (E) SHAl., 2015 . SHAPE r(TIF)Click here for additional data file.S4 Figet al., 1997 [in vitro transcription four PSTVd containing products are possible, depending on whether both sites are cleaved, none are cleaved, or cleavage occurs on one side or the other with respect to the PSTVd insertion. Residual inoculum refers to these products. The invariably high infection rate (10/10 plants) of wild type PSTVd in both local (shown here) and systemic leaf assays served as a positive control in all experiments.Total RNA was collected from inoculated  leaves of 10 plants infected with wild type PSTVd. An RNA blot assay is shown. Mock inoculation (M) was a negative control. Control lanes contained linear inoculum (I) and circular form (C) PSTVd RNAs (1 ng each). Ribosomal RNA (rRNA) stained with ethidium bromide was a loading control. The position of circular progeny genomes in infected plants is noted, as is the linear residual inoculum. Plasmid pRZ6-2 used to prepare inoculum contains a cDNA copy of PSTVd flanked by ribozyme cleavage modules adjacent to a T7 promoter . Follow(TIF)Click here for additional data file.S5 FigN. benthamiana protoplasts. Inocula consisted of 6 μg of (+)-PSTVd transcripts together with 20 μg of a GFP-encoding plasmid, which served as an indicator of PEG-mediated transformation efficiency. Wild type (WT) PSTVd was a positive control, and GFP plasmid alone was a negative control. (A) Protoplasts were photographed ~18 hours post-transfection in a fluorescence microscope using a filter to block red chlorophyll autofluorescence and image only GFP-expressing cells (top panel). Green, blue, and red channels were used to visualize all cells (bottom panel). Transfection efficiencies were similar in all cases (~30%). (B) PSTVd RNAs in transfected protoplasts were detected by RNA blot analysis. rRNA, visualized by ethidium bromide staining, was a loading control. All mutants appeared to replicate by this assay. However, progeny sequencing revealed that all had reverted to wild type and acquired new mutations (Five mutants that failed to replicate by the inoculated leaf replication assay were transfected to utations . Images (TIF)Click here for additional data file.S6 FigShown are BzCN-SHAPE probing results for wild type (WT) loop 27 (A), U177A (B), U178G/U179G (C), and A182G (D). DMS modification results for wild type Loop 27 are shown in (E). The region shown includes terminal loop 27 and the closing U-A pair (U177—A182), the adjacent base-paired stem, and two bases of loop 26. SHAPE and DMS reactivity is indicated by color: red = high (>0.85), orange = intermediate (0.40–0.85), black = low (0–0.40). Low reactivity indicates a higher probability of base pairing. In E, gray indicates G and U bases that are not modified by DMS.(TIF)Click here for additional data file.S1 TableRelative replication/accumulation levels of PSTVd Loop 27 mutants in individual plants (10 plants inoculated for each experiment) were determined by RNA blot signal density. Signals were normalized relative to averaged wild type PSTVd signals from 10 plants, which served as a control in each experiment. PSTVd levels in inoculated leaves  and systemically infected leaves  are shown. For systemically infected leaves, only genetically stable mutants (i.e. mutations were retained without introduction of new mutations) are included. Considerable plant-to-plant variation is common, even with wild type PSTVd. However, averaged values reveal clear trends, suggesting three general groups: progeny accumulate to wild type or near wild type levels (0.86–1.42), moderately reduced levels (0.60–0.68), or substantially reduced levels (0.26–0.36). The atypical U178G/U179G mutant is highlighted in red. Low level accumulation of this mutant is likely due to its restriction to a single cell layer.(DOCX)Click here for additional data file.S2 TableIn two of three experiments performed, progeny clones were recovered from transfected protoplasts and full-length sequences were obtained. In all cases, introduced mutations had reverted to wild type, and all genomes had acquired new mutations, as indicated.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Sequencing of newly synthesised RNA can monitor transcriptional dynamics with great sensitivity and high temporal resolution, but is currently restricted to populations of cells. Here, we develop new transcriptome alkylation-dependent single-cell RNA sequencing (NASC-seq), to monitor newly synthesised and pre-existing RNA simultaneously in single cells. We validate the method on pre-labelled RNA, and by demonstrating that more newly synthesised RNA was detected for genes with known high mRNA turnover. Monitoring RNA synthesis during Jurkat T-cell activation with NASC-seq reveals both rapidly up- and down-regulated genes, and that induced genes are almost exclusively detected as newly transcribed. Moreover, the newly synthesised and pre-existing transcriptomes after T-cell activation are distinct, confirming that NASC-seq simultaneously measures gene expression corresponding to two time points in single cells. Altogether, NASC-seq enables precise temporal monitoring of RNA synthesis at single-cell resolution during homoeostasis, perturbation responses and cellular differentiation. Sequencing of newly synthesised RNA can reveal the transcriptional dynamics in a population of cells. Here the authors develop NASC-seq to bring this sensitivity and temporal resolution to single-cell analysis. In most cases, these approaches have relied on the incorporation of 4-thiouridine (4sU) into newly synthesised RNA during gene transcription and subsequent biochemical separation of 4sU-labelled and unlabelled RNA. As these methods require large amounts of total RNA they have been limited to studies of average cellular responses across cell populations. Recent advances in single-cell RNA sequencing and data analysis have however revealed that responses to stimuli are not uniform across cells7. This highlights the need to monitor transcriptional dynamics in single cells to capture biological variability of processes and cellular phenotypes. Although 4sU-labelled RNA cannot be isolated from single cells in quantities that allow for its sequencing, recent cell population experiments showed that chemical modification of 4sU residues incorporated in total cellular RNA leads to T–C conversions during reverse transcription that can later be read out by sequencing11. Thus, newly synthesised RNA is separated from pre-existing RNA in silico during the computational analysis of the sequencing data.The ability to sequence newly synthesised RNA has led to important insights into the kinetics of RNA transcription, processing, and degradation, as well as the detection of rapid transcriptional responses to cellular stimuli or perturbations8 coupled with Smart-seq2 single-cell RNA-sequencing library construction12. We validate the method on K562 and Jurkat cells and demonstrate that NASC-seq can identify rapidly up-regulated or down-regulated genes during transcriptional responses. We foresee a large variety of applications that can benefit from the ability to monitor the dynamics of RNA synthesis in single cells.Here, we introduce a robust single-cell method for the sequencing of newly synthesised and pre-existing RNA in single cells, that we termed NASC-seq. The method is based on 4sU labelling and alkylation of the 4sU residues13 and then alkylated8. Single-cell RNA-seq libraries were constructed using a modified version of Smart-seq212 (see “Methods” section). Reverse transcription over alkylated 4sU residues triggers the misincorporation of guanines instead of adenosines, leading to T–C conversions that identify newly synthesised RNA in the sequenced libraries8. Indeed, T–C conversions were the dominating type of base conversions that we observed in 4sU-labelled single cells compared to unlabelled cells , intermediate (PDLIM5), or low (GAPDH) RNA turnover revealed that the detected RNAs had high, intermediary, and low numbers of T–C conversions, respectively . We estimated the true conversion probability (pc) taking in consideration the background error probability (pe) that we estimated from the observed amount of natural base pair mismatches in C–T and G–A conversions. Importantly, the binomial mixture model estimated a signal-to-noise ratio (pc/pe) of ~10 in this experiment  and high turnover (top 20%). As expected, genes with high mRNA turnover had significantly higher levels of newly synthesised RNA reads than genes with lower turnover . To this end we performed a control experiment where cells were incubated with 4sU on ice for 1 h followed by FACS sorting and NASC-seq library construction. Importantly, cells only exposed to 4sU on ice for 1 h had no significant increase in T–C conversions compared to unlabelled cells  in contrast to lower levels of newly synthesised RNAs for housekeeping genes (e.g. GAPDH and ACTB)  and ionomycin for 30 min as described15 Fig. . StimulaCTB Fig. . Based oTB) Fig. . OverexpTB) Fig. . To furtTB) Fig. . AltogetEGR1 and FOS . PCA could indeed separate newly synthesised RNAs in stimulated cells from those in non-stimulated cells Fig. . As expepc estimates and higher numbers of poor πg estimates; see “Methods” section), they nevertheless contained a significant  median signal-to-noise ratio of 1.5  . As expected, raw conversion rates and signal-to-noise levels increased with labelling time  Fig. .Fig. 3NAMoreover, several mitochondrial genes were only and reproducibly detected as significantly downregulated at the resolution of newly synthesised transcripts Fig. . The inaFinally, we investigated the level of transcriptome-wide RNA turnover in Jurkat cells using NASC-seq data. On average, 6.5% of the RNA in cells exposed to 4sU-labelling for 30 min were newly synthesised, whereas 10.8% of the RNA in cellular transcriptomes were transcribed during 60-min labelling periods Fig. . On the 8 and single-cell RNA-seq library construction using Smart-seq212. We validate NASC-seq by comparison with RNA-labelling data in cell populations. We show that NASC-seq can separate newly synthesised from pre-existing RNA in single human cells, and that it can also monitor up-regulation and down-regulation of transcription during a rapid cellular response. We show that NASC-seq with as short as 15 min of 4sU labelling can detect rapidly changing gene expression, emphasising the sensitivity and robustness of NASC-seq.In this study, we developed a method (NASC-seq) for the simultaneous quantification of newly synthesised and pre-existing RNA in single cells. NASC-seq is based on RNA labelling with 4sU, RNA modification by alkylation13, scATAC-seq17, and scNMT-seq18. We speculate that future combinations of these methods could expand the available toolset to allow for simultaneous measurements of transcription dynamics combined with DNA accessibility and CpG methylation status in single cells. Moreover, the NASC-seq protocol does not require any dedicated liquid handling or automation hardware and can therefore be performed in any molecular biology laboratory. A detailed step-by-step protocol for NASC-seq is provided through the protocols.io open access protocol repository19.Since we utilised biotinylated oligo-dT primers to immobilise and wash the RNA after alkylation, it is plausible that NASC-seq is directly compatible with simultaneous single-cell DNA-sequencingπg), which can cause problems for downstream analyses. It is possible that using increased 4sU concentrations could benefit short labelling times, even though increased 4sU concentrations seemed to lower RNA yields from the cells. Despite obtaining more unreliable estimates in our 15-min labelling experiment, noise-resistant downstream analyses can still provide valuable insights .The current implementation of NASC-seq leaves room for further improvements. The incorporation of unique molecular identifiers (UMIs) in the single-cell RNA-sequencing library construction would enable the direct counting of newly synthesised and pre-existing RNA, that could mitigate current biases. Short labelling times make it difficult to reliably estimate the fraction of newly transcribed RNA per gene , correlations between genes in general are not expected. However, NASC-seq could inform on any dependencies among closely related genes  although that would likely require NASC-seq data with allelic resolution from thousands of homogeneous cells.The bursting nature of gene expression results in relatively heterogeneous gene expression in a population of seemingly homogeneous cells25, which can further inform on future cell fate24. NASC-seq expands on these possibilities by directly measuring newly synthesised RNA from periods of 4sU exposure without being restricted to genes for which intronic RNA reads are detected or limited in time by the kinetics of pre-mRNA splicing. We note that by measuring new and pre-existing RNA separately, we effectively measure the transcriptomes in a single cell at two distinct timepoints. Although the measure of new RNA is a direct readout of recent transcription, the measure of pre-existing RNAs contain a subset of the cellular transcriptome available before labelling, as these RNAs would be continuously turned over during the labelling time. Still, we believe the simultaneous measure of two cellular transcriptomes per cell will add a temporal dimension to studies of transcriptional kinetics21. Thus NASC-seq is ideally suited to monitor changes in transcription activity with high sensitivity and temporal resolution during cell differentiation, tissue engineering, and organism development.Recent studies showed that information on RNA synthesis can be obtained from the detection of intronic sequences in single cells1. Briefly, three 4-thiouridin (4sU)-labelled and three unlabelled RNA spike-ins were in vitro transcribed using the MEGAscript T7 Transcription Kit (Invitrogen) according to the manufacturer’s instructions. Except, in vitro transcription of 4sU-labelled spike-ins was carried out substituting 10% of UTP with 4-thio-UTP (Jena Bioscience). Spike-ins were purified individually using RNAClean XP beads (Beckman Coulter) according to the manufacturer’s instructions and quantified using the Qubit Fluorometer (Invitrogen). Quality was assessed using the Bioanalyzer 2100 (Agilent) and a final spike-in pool was generated containing equal amounts of all six synthetic spike-in RNAs.Synthetic RNA spike-ins  were generated as in ref. 2. Jurkat cells (E6.1 clone) were acquired from ATCC (TIB-152), K562 cells from DSMZ (ACC-10). K562 cells were authenticated at the DSMZ Identification Service according to standards for STR profiling (ASN-0002). Jurkat cells were purchased from ATCC shortly before use and grown at low passage numbers. Cells were routinely tested for mycoplasma contamination . On the day of the experiment, Jurkat cells were seeded to a density of 1*106 cells/ml and stimulated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μM ionomycin (Sigma) for 30 min at 37 °C under 5% CO2.Cells were grown in RPMI 1640 medium (Gibco) supplemented with 10% foetal bovine serum (Sigma) and 1% Penicillin/Streptomycin (HyClone) at 37 °C under 5% CO2 for 3 h. Total RNA was isolated using TRIzol (Life Technologies) according to the manufacturer’s instructions. RNA spike-in mix was added during RNA isolation. For bulk alkylation, 5 μg of RNA was alkylated as described in Herzog et al.8. Briefly, total RNA resuspended in 1mM DTT was treated with iodoacetamide (IAA) at 50 °C for 15 min in a final alkylation reaction of 50 mM sodium phosphate buffer pH 8.0, 10 mM IAA, and 50% DMSO. The reaction was quenched by adding DTT  and RNA was purified using isopropanol precipitation. RNA was analysed using a Bioanalyzer 2100 system (Agilent). Low-input alkylation was performed as described below (NASC-seq).K562 cells were labelled with 500 μM 4sU (Sigma) at 37 °C under 5% CO1. Briefly, Jurkat cells were treated for 15 or 30 min with solvent control  or with PMA and ionomycin. During the last 5 min of each time point cells were labelled in media with 500 μM 4sU (Sigma) at 37 °C under 5% CO2. Cells were harvested by centrifugation at 1400×g for 2 min. Total RNA was extracted using TRIzol (Life Technologies) according to the manufacturer’s instructions under the addition of spike-ins. RNAs were sonicated using in a Bioruptor Plus instrument (Diagenode). 4sU-labelled RNA was purified from 300 μg total fragmented RNA. Separation of labelled RNA was achieved with streptavidin beads (Miltenyi Biotec). Prior to library preparation, 4sU-labelled RNA treated with DNase (Qiagen), purified , and quantified. Strand-specific libraries were prepared with the Ovation Universal RNA-Seq System (NuGEN). The size-selected and pre-amplified fragments were analysed on a Bioanalyzer 2100 (Agilent). Samples were sequenced on an Illumina NextSeq 500 instrument. Data analysis was performed essentially as in Michel et al. 15. Briefly, paired-end 75 bp reads were mapped with STAR26 (version 2.6.0c) to the hg38 (GRCh38) genome assembly (Human Genome Reference Consortium). Gene expression fold-changes upon T-cell stimulation for each time point were calculated using the R/Bioconductor implementation of DESeq227 setting lfcThreshold = 1. Differentially expressed genes were identified applying a P-value cutoff of 0.05 comparing sample to solvent control measurements. For comparison of NASC-seq data with TT-seq data, we calculated the fraction of new RNA .Two TT-seq experiments were performed in biological duplicates, as described previouslyNA Figs.  and S2c 12. The modifications included the removal of the inactivation step from the RT thermocycling programme, performing the RT on a thermoshaker (Eppendorf ThermoMixer C) and the use of custom barcoded primers for the indexing and amplification after Nextera tagmentation. The resulting libraries were sequenced on a NextSeq500 instrument (Illumina) using either single-end (75-cycle) or paired-end (2 × 150-cycle) sequencing strategies.Cells were labelled in medium with 4sU (Sigma), washed with cold PBS and sorted into lysis buffer  in wells of PCR plates. Plates were frozen at −80 °C until use. Streptavidin beads (MyOne Dynabeads Strepavidin C1) were washed twice with buffer 1 , twice with buffer 2 (0.1 M NaOH), and once with 2x B&W buffer  before the binding reaction . Beads were incubated with agitation at room temperature for 15 min and washed twice with 1x B&W buffer. Cells were lysed at 80 °C for 3 min and beads were added to the cells in a volume of 2 μl. RNA was bound during 20 min of incubation at room temperature on a thermoshaker (Eppendorf ThermoMixer C). 5 μl of alkylation mix (20 mM IAA in DMSO) was added for a final alkylation reaction of 50 mM sodium phosphate pH 8.0, 10 mM IAA, 50% DMSO. After 15 min at 50 °C, the alkylation reaction was stopped by adding STOP solution , incubating for 5 min on a magnet, and removing the supernatant from the beads containing the alkylated RNA. RT and the remaining library preparation were performed according to a modified version of Smart-seq2bcl2fastq (Illumina). Nextera adapters were trimmed with TrimGalore28 (v 0.4.5). All paired-end (2 × 150-cycle) data was then aligned to the hg38 human genome using STAR26 (v 2.5). Early NASC-seq data was sequenced using a single-end (75-cycle) sequencing strategy and was aligned to hg19. For all paired-end data we removed duplicates using the MarkDuplicates command in Picard (v 2.17.6). We then annotated the gene each reads maps to using FeatureCounts29 (v 1.6.2). We further annotated all mismatches to the reference genome within each read with the location of the mismatch for the T–C or A–G mismatches depending on the strand of the annotated gene . We identified mismatches in positions which appear in a high frequency over many cells and marked those positions as single nucleotide variants (SNV) to be ignored. We re-annotated the mismatches for each read with the SNV positions ignored and avoided double counting in overlapping paired end reads. Cells with low numbers of mapping reads were removed; for Jurkat cell experiments, each cell was required to have 600,000 reads mapping to features, while for K562 experiments, cells were required to have 300,000 reads mapping to features.After sequencing, the resulting bcl files were demultiplexed to fastq files with pc) based on the background probability of a mismatch due to error (pe) for each cell. We estimated pe by calculating the mean fraction of C–T and G–A mismatches in the given cell, since we concluded that these mismatches agree with the T–C and A–G mismatches in unlabelled cells. To estimate pc we implemented the Expectation-Maximisation algorithm described in Jürges et al. 14. For each gene in each cell, we estimated the proportion of new reads, πg using the binomial mixture model described in GRAND-SLAM14 (see below). We only ran the estimation if the gene had a minimum of 16 reads mapped to ensure reliable estimates. If the gene had more than 1000 mapped reads, we subsampled down to 1000 reads to shorten runtime.We then estimated the probability of a given position being converted in a new read (https://github.com/sandberg-lab/NASC-seq). To facilitate adoption of our computational approach we have prepared an Amazon machine image that is preloaded with our pipeline as well as example data (see GitHub for more details).All code for the processing of NASC-seq data and the implementation of the binomial mixture model is available on GitHub (πg, we implemented the binomial mixture model described in Jürges et al. 14. In the mixture model, each mismatch is either due to a conversion with probability pc or an error with probability pe. The probability of y positions having mismatches in a read containing n positions which may be converted isTo estimate the proportion of new reads for each gene, πg by building a generative model in the STAN modelling language. We use a beta prior for πg with hyperparameters α and βπg by maximising the log-likelihoodi indicates a read for that gene. The hyperparameters were log-transformed and both initialised at 0, while πg was initialised at 0.5. We will then estimate the mean of the beta distribution, which cannot be 0 or 1 by definition. The mode is therefore more appropriate, which we can calculate byα > 1, β < 1 and 0 if α < 1, β > 1. The other possible cases do not occur in our estimation procedure.We estimated Further information on research design is available in the Supplementary InformationReporting SummaryPeer Review FileSource Data


The majority of reward learning neuroimaging studies have not focused on the motivational aspects of behavior, such as the inherent value placed on choice itself. The experience and affective value of personal control may have particular relevance for psychiatric disorders, including depression.We adapted a functional magnetic resonance imaging reward task that probed the value placed on exerting control over one’s decisions, termed choice value, in 122 healthy participants. We examined activation associated with choice value; personally chosen versus passively received rewards; and reinforcement learning metrics, such as prediction error. Relationships were tested between measures of motivational orientation  and subclinical depressive symptoms.pfamilywise error–corrected < .05). Ventral striatal activations to choice were diminished in participants with subclinical depressive symptoms. Personally chosen rewards were associated with greater activation of the insula and inferior frontal gyrus, cingulate cortex, hippocampus, thalamus, and substantia nigra compared with rewards that were passively received. In participants who felt they had little control over their own behavior , prediction error signals in nucleus accumbens were stronger during passive trials.Anticipating personal choice activated left insula, cingulate, right inferior frontal cortex, and ventral striatum (Previous findings regarding personal choice have been verified and advanced through the use of both reinforcement learning models and correlations with psychopathology. Personal choice has an impact on the extended reward network, potentially allowing these clinically important areas to be addressed in ways more relevant to personality styles, self-esteem, and symptoms such as motivational anhedonia. SEE COMMENTARY ON PAGE 105Disruptions in motivation and reward processing are key elements of many psychiatric disorders, including anhedonia in major depressive disorder (MDD), negative symptoms in schizophrenia, and mania in bipolar disorder Derived from self-determination theory is the concept of the locus of causality, describing the source from which a person perceives his or her behaviors to be motivated Factor analysis suggests that the causality orientations have partial overlap with personality concepts described by the NEO Five-Factor Inventory: Control shares variance with agreeableness, and impersonal shares variance with neuroticism, whereas autonomy stands as a separate entity Neurobiologically, the feeling of personal control Specifically, our aims were to 1) verify previous findings concerning choice-anticipatory activation; 2) determine if responses to rewards differ according to whether or not they were personally won or passively received; 3) establish that, with appropriate modification of the original paradigm, computational models of reinforcement learning can explain observed brain activity; and 4) determine whether elicited activation covaries with subclinical depressive symptoms and personality factors relevant to depression, namely, neuroticism and measures of causality orientation. We anticipated that high neuroticism and impersonal scores would be associated with diminished activation to the inherent value of choice because depression has been linked to other types of blunted reward value Individuals were selected from a wider ongoing study [Stratifying Resilience and Depression Longitudinally Neuropsychological data collected included the General Causality Orientations Scale Data were acquired using a 3T magnetic resonance imaging scanner (repetition time = 1.56 seconds) see .The task was adapted from Leotti and Delgado In the original task, the yellow and blue cards shared equal reward contingencies. In our adaptation, they had different contingencies to permit modeling of reinforcement learning: the yellow card was associated with an 80% chance of a 100-point reward, and the blue card was associated with a 20% chance. The alternative outcome was 0 points. We also introduced 1500 to 4000 ms of jitter between selection and outcome phases of each trial, allowing for disambiguation of all three phases.Participants completed 66 trials, 33 choice and 33 no-choice. Trial order and the side of the screen on which the yellow and blue cards appeared were randomized, preventing final action planning. Decisions made by the participant during choice trials were mirrored by the computer with a three-trial lag during no-choice trials in an effort to match the overall rewards received across conditions of interest. Total task length was 14 minutes 59 seconds.Participants were told their objective was to learn by trial and error which color card was more likely to give them points. They were informed that for some trials they would get to choose, but for others the computer would choose for them. During the latter trials, they had to follow the computer’s selection. Participants were also told that the reward contingencies remained consistent regardless of whether they or the computer were doing the choosing. A questionnaire administered after scanning asked participants to rate their desire to win points on a scale of 1 to 10 and their preference for choice or no-choice trials.Two analytic approaches were adopted:1.The basic model was used to (a) verify that appropriate reward responses were seen for the outcome phase contrast of reward 100 > 0, regardless of choice/no-choice; (b) verify the results previously reported by Leotti and Delgado 2.The Pavlovian reward learning model attempted use a computational framework to estimate how much each participant valued being able to choose by fitting a temporal difference learning model to the data. This considered the cue phase choice and no-choice indicators as though they were stimuli to be conditioned on subsequently obtained rewards. We proposed that the degree to which the model’s value estimate accounted for choice anticipatory activation would be dependent on causality orientation, neuroticism, and depressive symptoms.This was modeled at the first level as a series of delta functions convolved with a canonical hemodynamic response function, the onsets of which were denoted by experimental conditions of interest. These were the onsets of the choice and no-choice cues and the onsets of trial outcome, with choice/no-choice and 0/100 points being modeled separately, giving six experimental vectors of interest. Nuisance regressors included the onsets of yellow/blue selection, trials where an incorrect response or no response was received, and motion parameters.Q = .05 across all comparisons, and standardized β values were reported.At the second level, cue phase contrasts of choice > baseline and no-choice > baseline were entered into a random-effects flexible factorial analysis, modeling the factors of participant and choice/no-choice. The outcome phase was considered in a separate 2 × 2 flexible factorial analysis incorporating the contrasts of choice 100 > baseline, choice 0 > baseline, no-choice 100 > baseline, and no-choice 0 > baseline, modeling the factors of participant, choice/no-choice, and reward amount see . For botThe task was also modeled as an instance of classical conditioning, using a temporal difference learning model pfamilywise error–corrected < .05. Given a priori interest in the striatum and dopaminergic midbrain, we also conducted region-of-interest analyses within a structurally defined mask comprising bilateral caudate, putamen, and dopaminergic midbrain . Of participants, 93% preferred making their own choices. Learning continued throughout the task, with the most rewarding card being chosen 79% of the time during the final quarter of the session (p = .003) and impersonal scores  were positively correlated with neuroticism.Median age of participants was 62 years, and 46% were men . There w session . Both QISee Cue phase choice > no-choice revealed strong activation in the cerebellum, left insula, left cingulate/supplementary motor area, and right inferior frontal gyrus (IFG), corrected for whole-brain volume . BilaterNext we examined whether responses to personally earned outcomes differed from those passively received. The outcome phase choice > no-choice contrast showed significant activation in the bilateral insula, anterior cingulate, right IFG, left hippocampus, and left thalamus . Within pfamilywise error–corrected = .038) but not that of 0 > 100 (The contrast of choice (0 > 100) > no-choice (0 > 100) showed activation in right IFG pars opercularis . Conjunc 0 > 100 . Contras 0 > 100 .Figure 4p < .001) (p = .045), whereas impersonal demonstrated the inverse relationship . Within precuneus, control had a positive relationship during outcome: no-choice > choice activation . Conversely, impersonal showed a negative association in the same region  (p = .039). QIDS depressive symptoms were negatively related to left putamen anticipation: choice > no-choice  . During  = .012) . Here QIp = .008) and greater choice > no-choice value estimates in anterior caudate (p = .019). Again during the cue phase, control orientation demonstrated a positive relationship with learning rate in the right superior temporal sulcus (p = .017).The final analytical thread considered whether the ability to choose was intrinsically rewarding in itself, within a reinforcement learning context. During the cue phase of each trial, there were no main effects of choice/no-choice or learning rate. However, as anticipated, there was significant covariation with several metrics of interest . Increasp < .001). Conversely, there was an effect of increasing α in the right anterior insula and supplementary motor area (p < .006). Learning in ventral striatum therefore appears to operate over a longer timescale than in insula and supplementary motor area. Finally, impersonal showed a stronger PE representation for no-choice > choice in bilateral nucleus accumbens (p < .004)  in ventral striatum, with a lower α being associated with greater PE representation ( < .004) . For intIn this study, we modified Leotti and Delgado’s 2011 inherent reward of choice task We verified the striatal anticipatory response to choice as seen in Leotti and Delgado Responses to personally chosen outcomes were enhanced compared with those that were passively received: insula/IFG and cingulate cortex were apparent, as were hippocampus, thalamus, and substantia nigra. Right IFG pars opercularis demonstrated a choice × reward interaction, whereby there was an enhanced response when participants personally failed to win. Right IFG pars opercularis plays a specific role et al.Right IFG and insula showed a choice > no-choice response across the sample during the outcome phase, which was enhanced by high autonomy but diminished by high impersonal scores. The concept of locus of causality is not far removed from that of learned helplessness, which inspired an animal model of MDD and gave ventral prefrontal cortex (IFG) particular prominence in a recent update by its architects Maier and Seligman The precuneus showed a no-choice > choice response during the outcome phase, especially so in participants with high control, with the opposite being seen with high impersonal scores. Precuneus is part of the default mode network and generally deactivates during goal-directed tasks The final analysis phase attempted to capture the learning process underlying how the choice/no-choice cues developed their inherently rewarding character and how this related to participants’ characteristics. The use of a computational model potentially allows for a more mechanistic understanding of the activation observed and highlighted relationships with personality metrics that were not detected during the basic analysis. Highly autonomous people encoded value during presentation of the no-choice cue within right amygdala, suggesting that either no-choice cues A number of study participants were unable to perform the task correctly, suggesting that it was subjectively hard to understand or that a potentially important section of the population was excluded. We have not examined trial-by-trial assessments of choice preference or changes in stay/switch behavior, which are also believed to covary with depressive symptoms Our findings suggest that the modified inherent value of choice task could provide useful insights into the neurobiology of MDD. Within this large sample of healthy control subjects, we have shown how personal choice modulates activation within areas known to be disrupted in MDD. This covaries with how inclined participants are to see themselves as drivers of their actions, to look to the outside world for their cues, or even to feel at a loss as to why they act at all. Being able to tease apart how particular manifestations of personality impact one’s vulnerability to MDD is likely to be important to stratification. Characteristics such as causality orientation arguably build on more stable and heritable measures such as neuroticism, as they are more responsive to environmental events and so may provide more timely information regarding the risk of transition to illness as well as offering targets for psychotherapeutic interventions. The hope is that by examining the reward system in a manner that ties self-perception to behavior, more clinically applicable insights can be drawn. For example, a particularly effective therapeutic strategy for individuals having a high impersonal/low autonomy style might be to both enhance dopaminergic transmission and challenge self-orientation beliefs during cognitive behavioral therapy.


In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed  E. coli, this RNA chaperon is involved in many cellular processes controlled by small non-coding RNAs (sRNAs), including modulation of RNA transcription, translation and degradation2. A gene for the Hfq protein is present in half of the sequenced bacterial genomes including many pathogens4, though no Hfq orthologs have been found in Actinobacteria. Hfq was shown to act as a virulence factor in several bacterial pathogens5, but it is not clear whether it can be considered as a general factor required for the virulence of all Hfq-containing pathogens. Hfq also has DNA-binding activity: it is classed as one of the twelve nucleoid-associated E. coli proteins, and, in addition, it induces negative supercoiling into plasmid DNA8.In all organisms, RNA-binding proteins participate in modulating all the steps in the life cycle of RNA, including transcription, folding, translation and turnover. Hfq is a RNA-binding protein playing a pivotal role in the control of gene expression in bacteria. In 9. Base pairing with sRNAs also frequently affects the half-life of mRNA targets, which can either be stabilized or become more prone to degradation. Hfq is one of the proteins interacting with RNase E10, and the recruitment of RNase E by Hfq leads to its targeting of some mRNA-sRNA hybrids and to their rapid degradation by RNase E13. Besides RNase E, Hfq has also been reported to associate with Rho, poly(A)polymerase (PAP), and RNA polymerase16.Hfq binding stabilizes sRNAs and facilitates their pairing with multiple target mRNAs, thereby controlling their translation either positively or negativelyE. coli has revealed that Hfq interacts with numerous proteins including interactions with subunits of RNA polymerase, RNase E and other degradosome components18. The web site http://www.ebi.ac.uk/intact/main.xhtml provides an open source database and analysis tools for protein interactions mainly based on the systematic analyses of protein-protein interactions. First, the ASKA His-tagged ORF clone library was used in large-scale pull-down assays, and proteins co-purifying with His-tagged baits on Ni2+-NTA column were identified by MALDI-TOF MS18. In the second method, Sequential Peptide Affinity (SPA) or Tandem-Affinity-Purification (TAP)-tagged derivatives were used to isolate the interacting protein partners using two rounds of affinity chromatography, which were then identified by MS17. Overall 79 interactants were found to bind Hfq, but somewhat worryingly, only two interactants are common to both sets of data; the ribosomal proteins RpsD and RplB. Moreover, further analysis of these data showed that most interactions originated from spoke-expanded co-complexes (i.e the prey protein is part of a complex which interacts with the Hfq bait protein). After filtering so that the prey protein interactions with all other proteins of the complex were removed from the matrix model, binary complexes were reduced to 2, Hfq with itself and Hfq with Rho. In addition, a recent resource details a global landscape of cell envelope protein complexes in E. coli. Hfq appears in 3 complexes: two large complexes comprising 83 and 48 subunits and a smaller complex of 7 subunits 19. Overall these results strongly imply that Hfq is a “sticky” protein capable of multiple interactions, which may not all be specific.Many sensitive high-throughput techniques have been developed to define protein-protein interactions. Systematic analysis of protein-protein interactions by pull-down assays and/or co-purification in 20. In addition, due to its very high affinity for RNA, Hfq purification requires very stringent conditions to ensure RNA elimination23.Characterizing protein-protein interactions is challenging in the case of Hfq as the previously used global approaches can be prone to artifacts. In particular Hfq is one of the proteins exhibiting a false-positive binding activity during Ni-affinity purificationin vivo. Altogether 246 non-ribosomal proteins were found to interact with Hfq with 55 detected by the two approaches. Since RNase A treatment disrupts the interaction of 42 out of the 141 complexes identified after TAP-tag purification, our results suggest that most of the interactions of proteins with Hfq in E. coli are mediated through RNA or involve RNA. Interestingly we show that RNA may stabilize the Hfq-protein interaction or compete with the other partner to interact with Hfq as shown for Rho or poly(A)polymerase.The main goal of this study was to exploit alternative approaches to confirm previously identified Hfq-interacting proteins, to search for new interacting proteins and to determine whether their interactions with Hfq are mediated by RNA. To this purpose, we have used TAP-tag purification combined with RNase A treatment, and protein-protein crosslinking, which freezes protein-protein complexes formed The strains and plasmids used in this study and their constructions are described in Table hfq was transformed by the plasmids pHfqTT, a derivative of pTX381, expressing Hfq with the TAP-tag (HfqTT) from the hfq promoter and by pCATT as a control. In this latter case the TAP-tag peptide is expressed from an IPTG inducible promoter by addition of 5 10−5 M IPTG (Table hfqpCAhfqTT) to control ptsG expression as efficiently as when it is expressed from its own promoter (IBhfq95Δhfq phfqTT)  and after 16 h (stationary phase). Extracts were prepared essentially as described in24 with the following modifications. Cells, resuspended in lysis buffer, were passed through a French press . The cell extracts were clarified by centrifugation and the supernatants submitted to two rounds of affinity chromatography. Half of each sample was treated with 0.1 µg/µl RNase A and incubated for 30 min at 37 °C, after the IgG Sepharose beads column and TEV cleavage and before the affinity purification on calmodulin beads. The extracts were then analysed by mass spectrometry by the “Plate-forme Protéomique Paris 5 (3P5) Descartes”.Strain IBhfq95ΔTG Table . This coT) Table . Bacterihfq was transformed by the plasmids pHfqH6, a pACYC184 derivative expressing HfqH6, or pACYC184, as a control (Table 650 = 1), pellets were washed twice in PBS and resuspended in 0.25 mM DSP (Dithiobis (DSP). To this end, strain IBhfq95Δhfq mutant, was transformed with low copy number plasmids23 expressing the genes encoding HfqTT (TAP-tagged) or HfqH6 (His6) proteins (both tags at the C-terminus). These proteins were expressed from the native hfq promoter  the Tandem-Affinity-Purification (TAP-tag) which allows the rapid purification of complexes under gentle native conditions, including (or not) an RNase A treatment to improve the specificity and to distinguish protein- and RNA-mediated interactions and (ii) an er Table  to conse31 Table . The carhfq mutant strain was transformed with the pHfqTT plasmid, encoding the TAP-tagged Hfq, or with the control pCATT plasmid, encoding the TAP-tag peptide induced by IPTG. Cultures were harvested in exponential and stationary phase since Hfq is maintained at a level characteristic of the rate of cell growth33. Proteins, which were detected in the TAP-tag-only control experiment with the pCATT plasmid including H-NS, SecA, enolase, NifU and numerous ribosomal proteins, were not further taken into account  with DTT, to reverse the crosslinking, and loaded on SDS-PAGE gel  was added to growing IBhfq95Δgel Fig. . Each lagel Fig.  Sheet 1.gel Fig. .Figure 120 (data not shown), which were not further taken into account. DSP crosslinking revealed 246 Hfq interactants of which 205 were non-ribosomal proteins  revealed a few false positive candidates, able to bind to Ni-NTA Agarose columns in the absence of a H6 tag, such as GlmSns Table . Of the in vitro35 and/or that the complex is stabilised by the RNA.The highest scores of the RNA-dependent partners of Hfq detected by TAP-tag concern essentially proteins belonging to RNA metabolism  were found after TAP-tag and DSP procedures and the TAP-tag complexes were sensitive to RNase treatment. Similarly PNPase was fished by both techniques. This agrees with recent data showing that PNPase, Hfq and sRNA can form in vitro a ternary complex in which the ribonuclease plays a non-destructive structural role. Such ternary complexes were proposed to arise in vivo at least transiently36. In addition van Nues et al. reported that IsrA sRNA (McaS) associated with Hfq in a core complex with other proteins such as RNA polymerase, CsrA, S1 and ProQ37.Hfq was reported to co-purify with RNA polymerase in an S1-dependent mannerRho, which was the only Hfq interactant remaining after filtering out the “spoke-expanded co-complexes” from the previous high-throughput experiments was found here to interact with Hfq by both TAP-tag and DSP crosslinking. The amount of Rho which interacted with Hfq was higher in exponential than in stationary phase, and in both phases was sensitive to the presence of RNase A Table  sheet 3.13. Moreover, in another report the sRNA MicC was clearly required in vitro to mediate the interaction between Hfq and the recognition core (part of the C-terminus) of RNase E38. We provide here further evidence that RNA plays a major role in the formation of RNase E-Hfq containing complexes since an interaction between Hfq and RNase E was detected in vivo by using the two approaches, and it was disrupted upon RNase treatment of the TAP-tag samples. The observation that RhlB and PNPase (Pnp) are also RNA-dependent interactants in the TAP-tag experiment and crosslinked to Hfq by DSP could be due to their proximity inside the degradosome. Enolase (Eno), which is also part of the degradosome, was only found in the DSP experiments , but not with C-terminally truncated RNase E, incapable of forming a degradosome. Neither PNPase nor RhlB (both components of the degradosome) was required for the RNase E-Hfq interactionts Table , sheet 1ts Table  sheet 3.Known RNA binding proteins like ProQ and CsrA were also found interacting with Hfq in a RNA-dependent manner. YhbS, an uncharacterized putative N-acetyl transferase scored highly in the RNase dependent category Table  sheet 3 42.Our criteria for RNA independent interactants in the TAP-tag experiment was a score reduced by no more than 30% after RNase treatment but for many proteins the number of peptides detected after RNase treatment increases considerably reflecting the loss of all the RNA dependent interactions Table . This ob43. Both GyrA and RecA were among the interactants detected by both techniques as was the heat shock factor, protein chaperone, DnaK. The Hfq protein is thermostable, therefore it is possible that its interaction with DnaK is related to its chaperon function to assist the folding of nascent proteins.Another class of interacting proteins are GyrA, HUα and β (DbhA and B), StpA, and RecA which represent DNA binding or histone-like proteins and thus confirms previous reports of Hfq binding the chromosome44. It is worth noting that these RNA-independent candidates are particularly present in stationary phase which may reflect an unsuspected function of Hfq in conditions where it is released from nucleic acids, as in stationary phase.Other proteins found to be directly interacting with Hfq included several enzymes  Table . Two sub7, and via its association with sRNAs, it plays a role in controlling translation initiation by binding at or near the ribosome-binding site. In addition Andrade et al. very recently reported that Hfq could act as a novel auxiliary ribosome biogenesis factor45 making a functional interaction between Hfq and ribosomal proteins possible.Ribosomal proteins were strongly represented amongst the interactants with Hfq after crosslinking and in the TAP-tag experiment, but they were also found in the TAP-tag-only control, suggesting they are false-positives. Moreover, nearly all the ribosomal proteins were found associated with Hfq, not just those near the mRNA binding site. These interactions could be a non-specific consequence of the abundance of ribosomes in the cell. However it should be remembered that Hfq was initially reported as being associated with ribosomesThe interactions of Hfq with PAP, RNA polymerase, EF-Tu, S1 and Rho were further investigated by using far western analysis and Microscale Thermophoresis (MST) in order to clarify the role of RNA in these interactions.The interaction of Hfq with several proteins susceptible to interact with Hfq either directly (EF-Tu) or together with RNA  and also ribosomal protein S1 was tested by a far-western experiment. Purified proteins were separated on three identical SDS gels, two of which were blotted to nitrocellulose membranes. After denaturation and renaturation of the proteins, one membrane was incubated with Hfq (far western blot experiment) Fig.  and then46. We first determined the binding affinity of HfqH6 for E. coli total RNA, poly(A) and poly(G) by MST  and poly(G) samples also range in size, an average size 1 kb of these molecular species was chosen for further calculation as previously used47. While no significant binding of Hfq to poly(G) was found, the half maximal effective concentration  for RNA and poly(A) were both less than micro-molar   experiments, we verified the thermal stability and the secondary structure content of the Hfq constructs by differential scanning calorimetry (DSC) and circular dichroism (CD) (data not shown). Microscale Thermophoresis estimates the modifications of molecular movement along temperature gradients due to changes in conformation or environment and can be used to measure protein-ligand interactionsMST Fig. . We usedly) Fig. . HoweverWe then tested the binding affinity of HfqH6 for PAP Fig. . A bindi14. Our TAP-tag and DSP crosslinking experiments confirm this interaction and also show an RNA dependence. When we attempted to measure this interaction in vitro by MST, no interaction was detected between Hfq and Rho either in the absence or presence of total RNA, whatever partner was labelled. This result was disappointing considering that our data and previous results strongly imply that RNA is required to mediate or stabilize the Rho-Hfq interaction in vivo. In addition using the purified proteins, we only observed a faint interaction between Hfq and the hexameric form of Rho by far western blotting. These negative results with purified proteins could imply that other components may be required to stabilize the interaction .It has previously been shown that the Rho factor physically interacts with Hfq conferring an anti-termination activity to the tandem complex on mRNA43. Furthermore, many studies have demonstrated the association of Hfq with the various machines of RNA metabolism, for its synthesis, translation and degradation5. Systematic analysis of protein-protein interactions in E. coli had implied that Hfq interacts with a large number of partners18. Our results support and extend these observations. By using TAP-tag and in vivo crosslinking, we identified 96 and 205 partners , respectively, with 55 in common , IciA, StpA and H-NS were all previously shown to copurify with Hfq53. Other high scoring candidates as novel Hfq interaction partners, include YhbS a putative acetylase, and various metabolic enzymes like AceA (isocitrate lyase), or some involved in fatty acid metabolism, FabB and LpxD, or ATP synthase subunits. Their potential to interact with Hfq could reflect a novel role of Hfq and/or have unforeseen consequences on the ribocontrol by Hfq of gene expression in bacteria. It remains to be determined whether the novel interactions identified in this work depict functional consequences on either or both of the interacting partners. RNA binding proteins often exhibit degenerate binding sites allowing a dynamic cycling of ligands which can easily enter and leave complexes or be temporarily sequestrated. In this context the large number of high scoring candidates for a non-RNA dependent interaction with Hfq are a starting point to look for other roles of Hfq. Although it must be admitted that some of these candidates, like EF-Tu, could be false positives just because of their high cellular abundance, others like YhbS, could interact with Hfq in the presence or in the absence of Hfq depending on the growth phase.Our results also reveal several strong candidates, which interact with Hfq in the absence of RNA. First amongst these is EF-Tu, an abundant protein with both tRNA and ribosome binding activities. A weak signal is detected by far-western supporting a direct binding of Hfq to EF-Tu. Any consequences of an interaction of Hfq and EF-Tu on cell metabolism merits investigation. Proteins with important roles in DNA function also interact with Hfq such as RecA and GyrA. As in the case of the ribosome, direct consequences of their interaction with Hfq need to be explored. We speculate that the Hfq-RecA interaction may be one clue to explain the high sensitivity of the Pseudomonas aeruginosa54. It is possible that some of the numerous interactants in E. coli may target Hfq to specific sRNAs or mRNAs, conversely the activity of these interactants may also be modified with significant consequences on transcription in the case of RNA polymerase or DNA-binding histone-like proteins. In agreement with this hypothesis Hfq was shown to affect mRNA levels independently of mRNA degradation2. Despite its wide spectrum of known functions, the capacity of Hfq to interact with many proteins has to be taken into account to reevaluate the global role of Hfq, which may be under appreciated.An important result from our study is that the majority of the interactions of Hfq with the proteins of RNA metabolism, from synthesis to degradation  are essentially dependent upon RNA. The next question will be to determine whether specific sRNA or RNA fragments are part of these ribonucleoparticles. It was recently shown that Crc affects the binding of the sRNA PrrF2 to the proximal side of Hfq in Table S1Table S2Figure S1Table S4Table S3Table S5


Central to the “RNA world” hypothesis of the origin of life is the emergence of an RNA catalyst capable of RNA replication. However, possible replicase ribozymes are quite complex and were likely predated by simpler non-enzymatic replication reactions. The templated polymerisation of phosphorimidazolide (Imp) activated ribonucleotides currently appears as the most tractable route to both generate and replicate short RNA oligomer pools from which a replicase could emerge. Herein we demonstrate the rapid assembly of complex ribozymes from such Imp-activated RNA fragment pools. Specifically, we show assembly of a newly selected minimal RNA polymerase ribozyme variant (150 nt) by RNA templated ligation of 5’-2-methylimidazole-activated RNA oligomers <30 nucleotides long. Our results provide support for the possibility that complex RNA structures could have emerged from pools of activated RNA oligomers and outlines a path for the transition from non-enzymatic/chemical to enzymatic RNA replication. RNA polymerase ribozymes (RPR) generated by in vitro evolution are credible candidates for a modern-day reconstruction of such a RNA replicase. However, how such large and complex ribozymes could have emerged from the pools of short RNA oligomers accessible from prebiotic chemistry (<30 nts) is currently unclear. A plausible approach towards the synthesis of long, more complex RNAs involves their hierarchical assembly from short RNA oligomers by iterative ligation reactions. This approach has previously been exemplified enzymatically for the templated assembly of complex ribozymes from RNA oligomers (for the sunY self-splicing intron), a cross-chiral RNA ligase, a RPR by the hairpin ribozyme, or more recently from RNA oligomer and trinucleotide triphosphates. However, these approaches all suppose the pre-emergence of a ligase ribozyme activity to drive assembly. Here, we explore a non-enzymatic approach based on the widely used phosphorimidazolide (Imp) activation chemistry (de novo (non-templated) and templated RNA polymerisation,9, seeking to connect enzymatic  RNA polymerisation to the simpler non-enzymatic RNA polymerisation reactions that must have preceded it. We first defined optimal reaction conditions that would allow RNA polymerase ribozyme (RPR) activity to proceed in eutectic ice phases, where both de novo as well as templated non-enzymatic polymerisation of RNA could be demonstrated.,11The cornerstone of a hypothetical primodial RNA-dominated biology (an “RNA world”)hemistry , which h2+ concentration ([Mg2+]) dependence (for MgCl2) of RPR activity in ice over a [Mg2+] range of 5–30 mM and a pH range of 8.3–10.5 revealed a trend towards enhanced activity at higher pH values with decreasing [Mg2+]  that resulted in a 20-fold drop in [Mg2+] required for optimal in-ice RPR activity ,12 reducing RNA degradation.We could identify improved reaction conditions for in-ice RPR activity based on a switch of the buffer system and a slight increase of the pH . Investig [Mg2+] . As thisg [Mg2+] , it likeactivity  in compain vitro evolution.,7 Starting from RPR variants Z, W, Y using different randomised linker lengths  connecting the RPR catalytic core to the accessory domain (We further optimized RPR activity (while minimizing RPR size) using in–ice selection by CBT  y domain . After 8y domain . All thr without loss of activity ( resulting in an even shorter RPR variant (F) of only 150 nt, which retained robust polymerase activity but only in ice  chemistry is increasingly able to generate mixed sequence oligomers suggesting that mixed RNA oligomer sequence pools in the ≤30 nt size range may be prebiotically plausible. However as Imp activated monomers are preferentially introduced, ligation of oligos is much less efficient, which hindered polymerisation of 5’-Imp activated RNA oligomers so far.In particular one clone (named K) showed improved RNA polymerase activity on the new primer/template combination in comparison to the best RPR variant (Z) and additionally could be shortened to 180 nt compared to Z (195 nt)activity . The K ry in ice  and in py in ice . After h2+, CHES, pH 9) the spontaneous assembly of the full length catalytic core sequence –activated RNA eicosamers (20 nt) by dividing the class I ligase derived catalytic core of the RPR (100 nt) into five 20-mer segments. Upon addition of the complementary RNA splints (20 nt) and applying the same optimised reaction conditions identified for in-ice activity of the RPR  in the same manner. To this end the whole F RPR sequence was divided into four pieces, each comprising ~38 nt and their 5’ phosphate termini were activated with 2-MeImp. After addition of four complementary RNA splints (24 nt each), we again observed assembly of the whole functional F ribozyme sequence (24 h at 37°C) with a total yield of 1.7% . This shin vitro transcribed version, Finally, we sought to assemble the whole F RNA polymerase ribozyme from RNA oligomers short enough to be accessible from non-enzymatic polymerisation (≤30 nt). This requires the definition of six mutually exclusive  assembly sites in the RPR sequence, with a concomitant steep increase in the potential for mis-assembly. To this end we split the F ribozyme sequence of 150 nt into seven pieceswith arbitrary ligation sites  and activated the 5’-phosphate termini with 2-MeImp. After the addition of the corresponding six complementary RNA splints , we observed the ligation of the full-length ribozyme sequence . Correct1234567F) from up to 7 short (20–30 nt), non-functional 5’-2-methylimidazole-activated RNA oligonucleotides via splint assisted non-enzymatic ligation in a “one pot” reaction. The final yield of the full-length ribozyme from 7 pieces is currently relatively modest (0.5 %), which is likely related to the chemical instability and short half-life of the phosphorimidazole activating groups in aqueous solution, the incomplete formation of ligation junctions due to RNA misfolding and incomplete ligation of assembled junctions. Furthermore, the chemical reactivity of 5’-phosphorimidazole activated RNA oligonucleotides is in general reduced, compared to the equivalent activated RNA monomers, also related to the fact, that the latter is assisted by beneficial interactions of the imidazole leaving groups of the incoming monomer and the next downstream monomer that are absent in the oligonucleotide ligation approach. Reactivation of the 5’-phosphates with imidazole in combination with binding to a solid matrix and/or the introduction of a cycling regime, as demonstrated for both templated RNA monomer polymerisation,9d as well as enzymatic ribozyme assembly, may allow improved reaction yields. While enzymatic ligation strategies often require specific ligation sites that have to be selected (or engineered) prior to ligation, the here described non-enzymatic ligation approach shows a much more general ligation behaviour i. e. with no apparent dependency on the nucleotide identity at the ligation junctions.Our results show how complex RNA structures could have been generated non-enzymatically in a plausible prebiotic setting by the iterative ligation of multiple phosphorimidazole activated RNA oligomers. We provide a proof-of-principle demonstration for the spontaneous assembly of a functional 150 nt RNA polymerase ribozyme , while the substantial decrease in Mg2+ concentrations are beneficial regarding both RNA degradation and dsRNA strand separation.The new minimised RNA polymerase ribozyme (F) (comprising only 150 nt) represents one of the shortest RPR, that shows robust RNA polymerase activity on difficult template sequences in the preferential medium of water-ice at–7°C. This could pave the way for future RPR catalysed RNA self-replication, based on a combination of RNA oligomer synthesis by enzymatic NTP polymerisation and non-enzymatic templated ligation of these oligonucleotides based on phosphorimidazolides at ambient temperatures by applying the same overall reaction conditions (10 mM Mg,15 until more efficient RNA replicase ribozymes emerged at a later stage. Hence, assembly of short RNA oligomer into longer strands would have been a critical transition in boosting pool complexity allowing more efficient enzymatic replicators to emerge. These may have initially similarly utilized oligomer substrates before transition into the more abundant short tetra-, tri- and dinucleotide and eventually mononucleotide substrates, i. e. RNA replicators increased in size and complexity, while their substrates decreased in size. The here explored combination of enzymatic RNA polymerisation and non-enzymatic RNA ligation based on 2-methylimidazolide activated oligonucleotides thus represents a proof-of-principle for a plausible step in a transition scenario from prebiotic oligomer pools to RNA self-replication. Nevertheless, it remains possible and even plausible that different enzymatic and non-enzymatic RNA polymerisation-/ligation-based replication systems might have operated in parallel, coexisting by exploiting orthogonal substrate pools and competing for resources, dependent on substrate availability and environmental conditions.The first primitive genetic systems likely propagated themselves by non-enzymatic RNA replication initiated from pools of short oligomer substrates provided by prebiotic chemistryhttps://doi.org/10.1002/syst.201900004Supporting information for this article is available on the WWW under Supp Info


The mutational tolerance of a conserved tripeptide of 3CD, directly involved in this interaction, was investigated. Randomization of the relevant codons and reverse genetics were used to define the space of viability-compatible sequences. Surprisingly, at least 11 different amino acid substitutions in this tripeptide were not lethal. Several altered viruses exhibited wild-type-like phenotypes, whereas debilitated (but viable) genomes could increase their fitness by the acquisition of reversions or compensatory mutations. Together with our study on the tolerance of oriL , the results demonstrate that at least 42 out of 51 possible nucleotide replacements within the two relevant genomic regions are viability-compatible. These results provide new insights into structural aspects of an important viral function as well as into the general problems of viral mutational robustness and evolution.Replication of RNA viruses is generally markedly error-prone. Nevertheless, these viruses usually retain their identity under more or less constant conditions due to different mechanisms of mutation tolerance. However, there exists only limited information on quantitative aspects of the mutational tolerance of distinct viral functions. To address this problem, we used here as a model the interaction between a replicative  The error rate varies in the range of ~10ying act ,3. Thus,position . Consequposition ,6. Neverposition ,8. To coposition . HoweverEnterovirus genus of the Picornaviridae family of animal viruses. Its positive-strand RNA genome harbors a single open reading frame (ORF) encoding the polyprotein, which is eventually processed into a dozen mature proteins [pol, the viral primer protein VPg , and the protease 3Cpro responsible for the polyprotein processing. The ORF is flanked by the 5′- and 3′-untranslated regions, containing, in particular, two cis-elements required for efficient RNA replication: oriL, also known as the cloverleaf because of its secondary structure [oriR, respectively  and complementary (negative) RNA strands [oriL of several viral and host proteins to generate a ribonucleoprotein (RNP) complex [d of oriL with the viral protein 3CD, the uncleaved precursor of 3Cpro and 3Dpol [d is believed to interact with the TGK motif of the 3C moiety of 3CD, since alterations in the structure of the former may be functionally compensated by the mutational changes in the latter [In particular,  strands ,14,15,16 complex ,17,18. And 3Dpol ,20  of the sequence of its tetraloop and two adjoining base pairs, followed by selection and investigation of the viable progeny (the SELEX procedure). In addition to such in vitro selection experiments, certain altered genomes were engineered and investigated. Unexpectedly, it was found that the overwhelming majority, if not all, of the possible point mutations of this highly conserved octanucleotide were compatible with the viral viability. Some of these mutations did not change the viral phenotype, whereas others inflicted functional defects of variable strength, as evidenced by the impairment of viral reproduction, RNA synthesis, and oriL/3CD binding, these three alterations well correlating with each other. The debilitated mutants demonstrated a good potential for phenotypic recovery by either acquisition of advantageous mutations in the octanucleotide or by changes in the TGK motif of the 3CD protein, and in particular its conversion into IGK or TGR.In a previous study  we assayoriL/3CD interaction, the TGK-encoding nonanucleotide of 3C was now subjected to randomization. After deriving some preliminary conclusions from the selection and sequencing of viable viruses, an additional set of mutant genomes was engineered and investigated. At least 11 amino acid replacements in TGK were found to be compatible with the virus viability. The results markedly enlarged the space of 3CD sequences compatible with the virus viability and in combination with our previous study on mutational tolerance of oriL [d of oriL and the TGK-encoding nonanucleotide, together 17 nt-long), at least 42 out of 51 theoretically possible nucleotide replacements are not lethal. These results not only provide further insights into the mechanistic aspects of an important step of enterovirus reproduction but also give a deeper understanding of the mutational tolerance and evolvability of RNA viruses.To further characterize the mutational tolerance of the poliovirus  of oriL , they de5914C, was purchased from Syntol (Moscow Russia).Plasmids with the randomized nucleotides encoding amino acids 154–156 of the viral 3C protein were prepared as follows . A 68 ntSacII and MluI endonucleases  and purified by electrophoresis in 2.5% agarose. The DNA fragment thus generated was inserted into poliovirus genome-harboring plasmids in a two-step procedure aimed to minimize possible contamination with non-mutated genomes. First, it was inserted at the proper position into the plasmid pT7PV1RibMS, which contained the full-genome copy of the type 1 poliovirus RNA with the artificially created unique cleavage sites for the restriction endonucleases MluI (at position 5861/5862) and SacII (5952/5953). To this end, the large fragment obtained after treatment of pT7PV1RibMS with MluI and SacII was purified by electrophoresis in 1% agarose and ligated with the above-described fragment containing the randomized nonanucleotide. The created plasmid was used for the transformation of E. coli TOP10  and the relevant region of a clone  was sequenced and found to lack G5906, resulting in the frameshifting of the open reading frame downstream of the randomized nonanucleotide. This intermediate plasmid was used as a donor of the large SacII/MluI fragment in the ligation reaction with the small SacII/MluI fragment harboring the randomized codons, as described above. Pools of the randomized plasmids were obtained upon transformation of E. coli TOP10 cells.For its PCR-amplification, the reaction mixture containing 0.6 pmole 3CTGK and 20 pmoles each of 3CMluI and 3CSacII primers  was subjoriL and nonanucleotide of the 3C gene, clone #18 (pT7PV1RibMSa) was treated with ApaI and SplI and the larger ApaI/SplI fragment was ligated with the small ApaI/SplI fragment obtained by PCR with the previously described [oriL. The plasmid thus generated was propagated in E. coli and pooled ~2250 plasmids (pT7PV1RibMSb) were further treated as pT7PV1RibMSa in the protocol for the generation of plasmids with the randomized 9 nt in 3C.To generate plasmids with randomized octanucleotide of escribed  synthetiMluI and SacII and used to generate constructs encoding the full-length viral genome, as described above.Since the viruses generated by the SELEX procedure not infrequently contained mutations outside the randomized nonanucleotide, certain analogous genomes lacking these undesired mutations were constructed. To this end, RNA from the preparations of the relevant viruses generated from the randomized plasmids was reverse transcribed and PCR-amplified using 3CMluI and 3CSacII primers. The product was digested with SacII and MluI, and used to generate plasmids encoding the full-length viral genome as above.To generate plasmids encoding viral genomes with novel mutations, two separate PCR amplifications were performed using pT7PV1RibMS as the template and pairs of primers, mut-s and 3EP4, in one reaction, and mut-a and B5594 in the other, where mut-s and mut-a harbored the desired mutation . The proMluI and SacII of the PCR product of the reaction containing pT7PV1RibMS/mut as the template and 3CMluI and 3CSacII as primers. The gel-purified fragments were ligated to the large fragment obtained upon digestion with the same endonucleases of the expression plasmid pQE60-3CD [40A mutation (inactivating the proteolytic activity of the protein) and the N-terminal His-tag.For the experiments requiring purification of mutated 3CD proteins, the small MluI-SacII fragment with the desired mutation was generated by treatment with QE60-3CD , which hThe presence of introduced mutations in all the engineered plasmid clones was verified by sequencing.E. coli TOP10 cells were transformed by the plasmids and the plasmid DNA was isolated from individual or pooled clones as described previously [eviously .EcoRI and transcribed by T7pol , and the transcription mixtures were used for the transfection of Vero cells (ATCC® CCL-81™) as described [The procedures were performed as described in . Brieflyescribed . The conoriL-containing PCR products were prepared with primers Rib2 and DEN3, whereas primers B5594 and 3EP17 were used for the preparation of DNA fragments encoding the portions of 3C that included the relevant tripeptide. The PCR products were gel-purified and sequenced either manually using afmol® DNA Cycle Sequencing System  or by automatic sequencers Beckman Coulter Seq 8000 or ABI 3130 Genetic Analyzer.The material from a plaque or virus suspension was suspended in 0.3 mL of the nutrient medium. RNA was isolated using either a phenol-chloroform extraction, Trizol reagent (Invitrogen), or Qiagen RNAeasy kit , and was reverse transcribed using random or 3EP4 primers . The ori5 cells/well, were transfected with 50 ng RNA transcripts per well, and the total RNA was extracted with Trizol reagent at 0, 12, 16, 20, and 24 h post-transfection (p.t.). Three wells were used as parallels for each time point. One μg of the purified RNA was used for reverse transcription with a random hexamer primer and the Maxima Reverse Transcriptase . The standard curve was generated by serial dilutions of the wild-type transcript supplemented with 1 μg RNA from mock-transfected Vero cells. Real-time PCR was carried out using a ABI 7500 Real Time PCR System analyzer with primers PVL1 and PVR1 and FAM-tagged oligonucleotide PVP1 as the probe  containing 20 mM glucose and 100 μg/mL ampicillin were grown in a rotary shaker for 4–5 h at 37 °C. The medium was changed to the one lacking glucose and containing 2 mM IPTG and incubated in a rotary shaker overnight at room temperature. The cells were subjected to centrifugation and lysed by sonication, and recombinant 3CD was purified by the Ni-chelating chromatography. The electrophoretic pattern of the purified proteins is shown in The procedures used have been described previously . BrieflyoriL-containing fragments of viral RNA and variants of the 3CD proteins was investigated as described in [Direct interaction between the ribed in . Briefly3C structure  visualization and distance labeling was performed with VMD software . ElectroAs described in Materials and Methods, a set of plasmids was engineered, in which nine nucleotides (positions 5897–5905 of the poliovirus genome) encoding tripeptide TGK  were randomized. The randomization was intentionally incomplete: the triplets in the inserted nonanucleotide contained neither A nor T at the thid positions. Although the total number of potentially encoded codons decreased from 64 to 32, their capacity to translate into all 20 amino acids was retained (the UAA and UGA stop-codons were not encoded). The decrease in the number of codons made the possible nucleotide sequence space more compact, enhancing the probability of representation of codons for all amino acids in relatively small samples of the genomes investigated.To assess the extent of randomization, the segment corresponding to positions 5800–5960 of the poliovirus genome was sequenced in 24 randomly selected plasmids. Sequencing demonstrated that six plasmids did not harbor non-intended alterations in the codons of interest and adjoining regions, two plasmids exhibited some heterogeneity in the randomized sequence, and one plasmid possessed a synonymous substitution outside the randomized region . These nNineteen pools were assembled from different numbers (31 to ~2250) of randomly selected plasmid clones. DNA preparations isolated from these pools were transcribed in vitro and serial dilutions of these transcripts were used to transfect Vero cells. The cell cultures were observed for at least six days. All these pooled transcripts generated plaque-forming viruses, however, with variable specific infectivity, i.e., the number of plaque-forming units (pfu) per µg RNA, which was several orders of magnitude lower than that of the wild type virus , suggestTo characterize the variety of sequences compatible with the viral viability, a portion of the 3C-encoding region (positions 5800–5960) of the RNA from 37 randomly selected mutant clones was sequenced. In 17 such clones, the primary plaques generated upon transfection contained sufficient material for sequencing, but in other cases the material from primary plaques was subjected to one or more bulk passages. The investigated set of genomes contained 22 unique nucleotide sequences.The prevalence of different codons in the randomized RNA region of these viruses differed markedly from that in the plasmids used for their generation compare . For exa156), which were converted upon passages into more comfortable Thr or Val. Similarly, the heterogeneity at this position in one isolate (#17) was likely due to the conversion of poor but acceptable Met in the virus that initiated infection into Val soon after the transfection. Three unstable primary isolates  harbored changed amino acids at both positions 154 and 156; they increased their fitness by the acquisition of a positively charged residue, Lys or Arg, at the latter position. Somewhat distinctly, a primary isolate (#21) with Ser156 (and Thr154) retained this residue upon passages but acquired Arg at the position just preceding the randomized triplet. Another virus (#22), having uncomfortable Met156, already possessed Arg153 upon isolation from the primary passage and appeared to convert, during further passages, its acceptable RTGM tetrapeptide into an even better (wild-type) CTGK.In the selected viruses, the tripeptide at positions 154–156 of 3C was represented by 13 unique variants of amino acid sequences, including the parental TGK . Althoug153 was replaced by Arg.The above results suggest some preliminary regularity. Various amino acids at position 154 are compatible with viral viability but certain ones  are obviously preferable over some others (Leu and Met), the latter tending to be replaced by the former upon passaging. Position 155 appeared to be invariably occupied by Gly. Position 156 requires either Lys or Arg but tolerated to some extent non-positively charged amino acids , especially if CysTo define more exactly the validity of the above-formulated preliminary regularity and to obtain some information on the phenotypic properties of viruses with different 3C sequences, a number of mutant genomes were engineered and their plaque-forming capacities were compared with those obtained through the in vivo selection of the randomized RNA. This was important because the latter procedure could be accompanied by the acquisition of undetected mutations outside the randomized sequence. The results obtained with the engineered genomes are presented in 154 by Met generated a less-fit, unstable genome, which increased its fitness upon passages by either true reversion, or changing this residue to Val or Leu, or by acquiring a mutation (Pro88Ser) in an upstream region of the 3C sequence. However, the genome with Leu154 also exhibited decreased specific infectivity and plaque sizes as well as genetic instability . The SGK-encoding genome appeared to be quasi-infectious, generating plaques at day 8 p.t. in one out of three attempts. This genome should experience two transversions after transfection to acquire a more favorable C154 residue, explaining the poor reproducibility of the experiments.Firstly, mutants with substitutions at position 154 of 3C were engineered. In line with the results obtained with the randomized plasmids, the variants having at this position Val, Ile, or Cys exhibited phenotypes (specific infectivity as well as the time of appearance and size of plaques) comparable to those of the wild-type genome and were genetically stable. The wild-type-like phenotype was exhibited also by the virus with the TGR-encoding RNA. The replacement of Thr156 by Met or Ser resulted in a marked decrease in both specific infectivity and fitness, as evidenced by the very late (days 7–8 p.t.) appearance of plaque-producing viruses with either the true reversion to TGK (in the case of TGM) or a variety of compensatory mutations in other parts of 3C (in both viruses)  or downstream of it (Asp282 to Asn). The appearance of Arg153 in similar circumstances was observed in the above experiment with randomization as well. This prompted us to construct genomes with RTGM and RTGS sequences. Such viruses appeared to be markedly fitter compared to their Cys153-containing counterparts, as evidenced by earlier appearances of plaques, but still were genetically unstable, accumulating various mutations in other parts of 3C (The replacement of Lysviruses) . The setts of 3C . These c88S or I151L into the RTGS-containing RNA resulted in a significant gain of fitness .It should be noted that the modifications of the TGK in the investigated cases were not accompanied with the acquisition of any alterations in its ligand, the domain d could be, at least partially, compensated by the acquisition of some changes in the TGK motif of 3C, such as T154I and K156R [d, for which such a compensatory effect was not observed earlier. The genomes were constructed with IGK- or TGR-containing 3C in combination with either agCUUGcu- or auGAGAgu-containing tetraloops of oriL. The former and the latter oriLs appeared to confer, to the TGK-harboring genomes, low-infectious and quasi-infectious phenotypes, respectively, and both could increase their fitness by some alterations of the mutated tetraloop [Figure 5, both IGK and TGR variants of the genomes with the non-YNMG oriLs demonstrated a markedly higher fitness compared to their TGK-containing counterparts, with a somewhat lesser effect in the case of the auGAGAgu/TGR combination.As indicated in the introduction, previous studies demonstrated that adverse effects of certain alterations in the tetraloop of domain nd K156R ,21,22. Hetraloop . As showoriL/3CD ligands, plasmids were constructed that encoded full-length poliovirus genomes, in which interacting motifs of both partners  were randomized. Seven transcripts of pooled plasmids (~103–1.5 × 104 clones) were used for the transfection of Vero cells. RNA from one of the three pools with ~103 plasmid clones as well as all transcripts of larger pools proved to be infectious, although with a low specific infectivity. Sequencing 113 5′-terminal nucleotides (including oriL) and positions 5800–5960 (a region of 3C gene) of the genome of 12 viral isolates gave the results shown in d in all but one of the primary isolates possessed sequences not belonging to any consensuses with known stable structures, but they clearly tended to acquire YNMG or GNUA sequences, which have been demonstrated to be optimal for the oriL/3CD interaction [oriL/3CD interaction. It may be noted that the unintended mutations in the oriL of the transcripts in pools 1 and 4  tetraloop in oriL D, congrus genome .oriL/3CD interaction, electrophoretic mobility shift assays (EMSA) were performed. As interacting partners, we used the recombinant 3CD proteins and 5′-terminal 113 nt-long fragment of the poliovirus RNA. The results were visualized by staining with ethidium bromide (EtBr). The VGK-, IGK-, and TGR-containing proteins demonstrated a level of oriL-binding comparable (in the case of IGK somewhat lower than in other cases) to that of the TGK-containing control , which could change the protein conformation under the conditions of the EMSA assay .To ascertain whether phenotypic changes caused by the TGK mutations were linked to the impaired  control  in suffiThe replication of RNA-containing viruses is generally error-prone, and in the case of picornaviruses nearly each act of template copying may be associated with the acquisition of a mutation ,2,3. SucTo prevent or diminish the potential harm of replicative infidelity, viruses should possess a significant degree of mutational tolerance. This tolerance is largely due to the degeneracy of codons, phenotype-neutral character of many amino acid substitutions, the ability of diverse sequences in RNA regulatory elements to maintain analogous mutual orientations, and functional equality of certain nucleotides in these elements . Though oriL and 3CD is an essential step of the poliovirus genome replication [d of oriL and TGK tripeptide of the 3C moiety of 3CD [Enterovirus C species, though Thr, being most abundant, could also be occupied by Val, Ile, and Met [The interaction between lication ,18,20,37y of 3CD ,22,38. Nd of oriL [d and nonanucleotide of the 3C gene) are not lethal. If a second point mutation in the TGK-encoding motif is allowed , then the number of the viability-compatible substitutions in it would reach at least 19  and Tyr6 to Gly155 (5.28 Å) points to possible effects of substitutions in CTGK to the mutual orientation of His89 and Tyr6, which could be compensated by substitution of Pro88 by more conformationally flexible Ser, retaining a His89/Tyr6 interaction in the RNA-recognition.Discussing the phenotypic effects of 3C mutations, additional possibilities to accomplish the ns 82–86 ,44, shou 154–156  may presognition . Pro88, r6 of 3C , the diseraction  and are eraction . The clo156Ala replacement was reported to inhibit the capacity of 3CD to stimulate uridylylation of VPg [oriL/3CD interaction [156-containing (rhinovirus A2) 3C proteins: Lys153 of rhinovirus exposes its positive charge to the same surface area as Lys156 of poliovirus, though this area in the former 3C has a somewhat lower overall positive charge, due to a lesser abundance of basic amino acids [For full functionality, position 156 could be occupied not only by the wild-type Lys but also by positively charged Arg, whereas the negatively charged Glu at this position was lethal, suggesting an electrostatic interaction in the tetraloop/3CD affinity. It may also be noted that the Lysn of VPg , which ieraction . The lacno acids ,46  interaction required for the efficient replication of poliovirus, it was demonstrated that at least 11 amino acid replacements in the involved highly conserved key tripeptide (TGK) of the protein ligand did not kill the virus. Combining the present results with those reported by us previously [oriL and 3C gene, respectively) could be tolerated without the loss of viral viability. A significant proportion of these acceptable mutations did not markedly affect the viral phenotype as studied in vitro. The viruses adversely affected by other viability-compatible mutations exhibited a remarkable level of resilience, i.e., the capacity to regain, fully or partially, the fitness by alterations (reversions or compensatory changes) of either RNA or protein ligands. This resilience is based again on the infidelity of viral RNA replication as well as on selection.This study provides new quantitative and mechanistic insights into how the fundamental conflict between the infidelity of genome replication and the need to retain identity may be solved in RNA viruses. This conflict becomes especially biologically relevant in the case of various bottlenecks, often accompanying viral life history. After such bottlenecks, new viral lineages are often initiated by individual or a small number of mutated genomes haphazardly picked up from always highly heterogeneous (quasispecies) viral populations. By using as the exemplary model an essential viral RNA/protein  to other RNA viruses and thus could contribute to better understanding of their conservation and evolvability.