Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing  Many RNAs become functional before their synthesis completes. Here the authors employ a single-molecule vectorial folding assay mimicking RNA transcription and show that the ZTP riboswitch is kinetically controlled and activated by slower unwinding and strategic pausing. These two domains share an overlapping sequence making their formation mutually exclusive. Ligand binding stabilizes the aptamer and prevents the shared segment from adopting its alternative conformation in which it is part of the expression platform3. The F. ulcerans ZTP riboswitch regulates gene expression by binding to 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate and triphosphate , which are elevated during folate stress6. In the absence of ZMP, a transcription termination hairpin or terminator  forms and terminates transcription before the gene coding region  to the synthesis of enough of the terminator to interfere with binding12. More recently, chemical probing approaches were combined with high-throughput sequencing and used to examine riboswitch structure during roadblocked transcription to obtain more complete maps of RNA folding for all transcript lengths14. However, these approaches cannot measure real-time folding dynamics because RNA conformations are measured long after transcription is stalled by a blockade.Investigations of the kinetic control model are often preceded by the observation of a discrepancy between equilibrium ligand-binding measurements and cotranscriptional riboswitch activation measurements, suggestive of a non-equilibrium  process. This discrepancy is then further examined through monitoring ligand binding and RNA folding kinetics. For example, bulk approaches have made use of structural probing techniques, NMR spectroscopy, and fluorescently labeled ligands to measure the RNA folding and ligand binding kinetics for different lengths of riboswitch RNAs representing different points in transcription15. Supporting the kinetic control model, the adenine aptamer was shown to fold and bind adenine no more than once before the transcription decision, which is not at equilibrium. However, due to technical limitations, the roles of several important parameters, such as elongation speed and pausing, were not tested. In addition, both a drawback and an advantage of the optical tweezer assay are that tension is applied to either the nucleic acid substrates or RNAP, thus examining force-dependent perturbation of the system.Using an elegant optical tweezer assay, Frieda and Block observed the folding process of an adenine riboswitch during transcriptionEscherichia coli RNAP holoenzyme). Assembly of RNAP on labeled nucleotide substrates enables the study of RNAP by single-molecule FRET17 but restricts dye placement to the 5' end of the RNA and requires partial transcription of the RNA in a halted transcription elongation complex, which could alter riboswitch folding through an alternative pathway. As RNAP pausing is an established modulator of riboswitch activation in many systems21, halting transcription could similarly disrupt or facilitate folding. Another issue that arises when using RNAP to examine cotranscriptional RNA folding is that mutations to the DNA template, hence changing the RNA sequence, can affect both RNA folding and interactions between RNAP and nucleic acid substrates. For example, a mutation to the terminator hairpin can result in changes in both its folding and its interactions with RNAP such that a single readout of transcription termination efficiency cannot determine if the mutation acts through changes in RNA folding or RNAP interaction.To date, experiments studying the kinetic control model have primarily used RNAP  RNA folding without additional polymerase characteristics or limitations in dye placement  of ~0.5 μM25. However, ZMP binding to the ZTP riboswitch aptamer is sensitive to 3′-end extension after nt 75, and variants containing as few as five base pairs of the terminator bind poorly to ZMP  and acceptor (Cy5) fluorophores27 and tested for a ZMP-responsive conformational change using single-molecule FRET29 , Δterm adopted a low-FRET efficiency conformation (EFRET ~0.25) in the absence of ZMP, and shifted to a mid-EFRET conformation (~0.6) upon ZMP binding , ~30% of Δterm persisted in the low-EFRET conformation  terminated conformation was observed  aptamer-containing conformation. At 0.1 mM and 1 mM ZMP, 17 and 38%, respectively, of all released RNA showed the aptamer-containing conformation  also decreased as the apparent rate of transcription decreased at lower NTP concentrations  if NTP concentration is sub-μM, far below physiological levels. Overall, less ZMP is required for riboswitch activation when more time is allowed for ligand binding, consistent with a kinetic control model for ZTP riboswitch function under these conditions. This observation was consistent between bulk and single-molecule measurements despite the substantial fraction of readthrough leakiness  evident in the bulk assays, especially at faster transcription speeds  is slow, a pause at nt 54 after these sequences would promote aptamer formation. In cotranscriptional studies using RNAP17, though it is feasible to modulate the overall tendency of RNAP to pause via addition of Nus proteins or RNAP mutations20, it is difficult to introduce site-specific pauses at arbitrary positions. In VF experiments, we mimic a specific pause at nt 54 by using a DNA that hybridizes only to nt 55–94 of the WT riboswitch, allowing nt 1–54 to pre-fold  reduced the ZMP concentration required for riboswitch activation and led to substantially higher readthrough compared to WT in the absence of ZMP . The time difference (Δt) measured between the Cy3 intensity decrease and PIFE peak center corresponds to the time for Rep-X to unwind 32 base pairs of the heteroduplex, from which we estimated the unwinding speed to be 65.0 ± 0.39 nt s−1  although the terminated conformation (EFRET ~0.2) is also observed  terminated conformation within 1.5 s; however, 8% (2/24) of the WT trajectories showed high-EFRET values (≥0.7) for ≥3 s  for ≥3 s in the initial phase of folding  was severely delayed during VF , which could functionally promote transcription readthrough if the conformations persisted long enough for RNAP to pass the terminator, provide a plausible mechanism for leaky readthrough . Taken together, although the 81–83 A mutation only mildly destabilizes the terminator under equilibrium refolding conditions . As in the unwinding speed measurements above, we observed an intensity peak in the Cy3 signal, which we attributed to PIFEU32 Fig. . By fittved Fig. . In the 3 s Fig. , longer 3 s Fig.  vs. time3 s Fig. . Althoug3 s Fig. , 60% , or addition of proteins that modulate transcription rates , which also affect other transcription dynamics. Therefore, it is challenging to probe the effect of specific pause sites in RNAP-based cotranscriptional folding experiments. Using VF assays, we found that using the pause mimics, i.e., initiating unwinding after a slow folding element, promotes riboswitch activation at limiting ZMP concentrations, similar to the effect of slower unwinding speeds. However, using complexes similar to the pause mimics here Fig. , pauses 13 in which strand displacement occurs in the direction of transcription. That is, rather than initiating at G81, which is the most 5′ nucleotide of the terminator, terminator folding via strand displacement cannot begin until U84. As the terminated conformation is still the predominant state in equilibrium measurements of the 81–83 A variant, the kinetic delay, rather than the stability perturbation, is a more important factor for increasing the readthrough observed in the bulk transcription assays. The findings here highlight the advantages of single-molecule approaches, in which folding heterogeneity can be directly observed in real time. VF assays circumvented the challenge of attaching fluorophore on nascent RNA chains encountered in RNAP-based single-molecule FRET (smFRET) cotranscriptional riboswitch folding studies, and thus making it possible to observe a much wider range of folding dynamics in real time. Future in vivo structural mapping studies can further validate the existence and roles of these intermediates.From these experiments, an individual riboswitch is observed to fold through one of many distinct channels or pathways, and the entire folding landscape is made evident from many observations Fig. . This in23 suggest that such an effect is not significant at least in the systems we have examined so far. These interactions between ssDNA and RNA chains can also occur with RNAP, such as those in R-loop structures37. Thirdly, the current unwinding yield of VF assays is ~30%, which is primarily due to the fact that not every construct has a helicase bound at the helicase concentrations we used, and some helicase dissociates before it begins unwinding23. Although 30% should be sufficient for many studies, we anticipate that future developments will improve the yield.A potential drawback of VF assays is that replacing RNAP with a helicase cannot recapitulate some RNAP-specific interactions with nascent chains, though it helps to more clearly dissect the contributions of other key kinetic parameters. In addition, we cannot rule out the effect of potential contacts between the unwound ssDNA and RNA chains, though comparisons made with bulk kinetics and steady-state single-molecule kinetics here and elsewhere13, targeting them with small molecules is a strategy that would, for example, promote riboswitch termination through stabilizing an off-pathway intermediate that competes with aptamer domain folding. This approach is analogous to targeting short-lived excited states rarely accessed by a dynamic RNA39, although riboswitch transcription intermediates are non-equilibrium states that can temporarily dominate a folding pathway. In addition to lower conservation, a drawback in targeting riboswitch transcription intermediates with synthetic small molecules is that the new ligand cannot be developed from the canonical riboswitch ligand and thus the target binding site in the intermediate may have lower information content11 than the canonical binding site.In both bulk transcription and single-molecule unwinding assays, only ~40% of this riboswitch is modulated by ligand binding, suggesting that a large fraction of RNA falls into leaky terminated or leaky readthrough states that do not respond to ligand. As shown here, folding intermediates that prevent proper folding of the aptamer domain promote leaky termination Fig. , and del16. One implication is that cellular RNA helicases or even ribosomes can recommit the fate of these translational riboswitches long after transcription has completed. As many nucleic acid folding processes are kinetically controlled, including those involving ribonucleoprotein complexes41, we believe this method is broadly applicable to many types of RNA and even DNA structures, as it has been demonstrated in ribozymes , RNA aptamers  and even telomeric DNA sequences in an experimental format that mimics co-reverse transcriptional telomeric DNA folding36, and to the studies of nucleic acid-protein complex assemblies.As kinetically controlled switching behavior is observed for this riboswitch with two different motile enzymes (Rep-X and RNAP), the RNA sequence itself contains the kinetic control information, which is interpreted or manifested by the motile enzyme. Consequently, a conserved riboswitch sequence could still function kinetically in the context of different cellular processes , as long as a motile enzyme is present to impose sequential movement. Interestingly, certain translational riboswitches exhibit kinetically controlled folding behaviors27. Briefly, all steps involving fluorescently labeled oligomers were performed in the dark. Unlabeled DNA oligomers, primers, and plasmids , phenol-chloroform extracted, and ethanol precipitated. The Cy3-labeled nt 1–33 RNA and Δ1–33 RNAs were annealed to a DNA splint identical to the cDNA-dT20 used in single-molecule measurements and ligated using T4 RNA ligase 2 (New England Biolabs). Ligated RNAs were purified by 8% denaturing urea-PAGE, recovered via elution in Whatman Elutrap electroelution systems, concentrated, washed with DEPC-treater water, and filtered in 0.22 μm spin filters. Concentrations were calculated by the absorbance of Cy3 at 552 nm (ε552 = 0.15 μM−1 cm−1) and the absorbance of the RNAs at 260 nm (ε260 = 1.16 μM−1 cm−1 for the terminator-containing RNAs and ε260 = 0.982 μM−1 cm−1 for the aptamer-only RNA) after correcting for the contribution of Cy3 absorbance at 260 nm (~5% correction). Ligated RNAs were aliquoted and stored at −20 °C in the dark until further use.The following method was used to prepare fluorescently labeled RNA and RNA-DNA complexesFluorescently labeled RNAs for double PIFE helicase rate measurements Fig.  were con20  were prepared in the buffer containing 50 mM HEPES-KOH (pH 7.4) and 150 mM KCl. The reaction aliquots were slowly cooled from 95 °C to 20 °C in a thermocycler (30 s per 5 °C). Annealing success was evaluated on 8% nondenaturing PAGE (in 1X TBE) by comparison to reactions lacking either biotin-DNA-Cy5, cDNA-dT20, or both. Reactions lacking cDNA-dT20 were also saved for single-molecule measurements. Gels were scanned with a Typhoon Trio Variable Mode Imager  to identify bands containing fluorescent labels.To form annealed heteroduplexes for single-molecule measurements, 20 μl reaction aliquots containing a 1:0.8:10 ratio of Cy3-RNA:biotin-DNA-Cy5:cDNA-dT20 (cDNA-speedmer-dT20) was used for annealing reactions in the presence of an 18-nt RNA oligo labeled at the 3´ end with Cy3 (18-speedmer), the WT Cy3-labeled RNA, and biotin-DNA-Cy5. Reactions contained a 1:0.8:9:10 ratio of Cy3-RNA:biotin-DNA-Cy5:cDNA-speedmer-dT20:18-speedmer to saturate cDNA-speedmer-dT20 with 18-speedmer. Annealing success was evaluated via nondenaturing PAGE as above.For dropoff-PIFE experiments Fig. , a longe20:Cy3-oligo:Cy5-oligo  were prepared in the buffer containing 50 mM HEPES-KOH (pH 7.4) and 150 mM KCl. The reaction aliquots were slowly cooled from 95 °C to 20 °C in a thermocycler (30 s per 5 °C). Selecting doubly labeled surface-tethered molecules ensured that the molecules being imaged contained all four oligos.For dropoff experiments , 150 mM KCl and 10 mM MgCl2. Free Rep-X was washed out and unwinding was initiated by adding the unwinding buffer. The unwinding buffer contained 50 mM HEPES (pH 7.4), 150 mM KCl, 10 mM MgCl2, 1 mM ATP, and different concentrations of ZMP. To stabilize the remaining aptamer conformation and distinguish it from the terminator conformation, 1 mM ZMP was supplied into the imaging channel after 1 min of vectorial folding. To observe the heteroduplex unwinding and riboswitch folding in real-time, imaging was started ~5 s before the addition of the unwinding buffer. The loading and unwinding buffers used during imaging contained additional 4 mM Trolox, 0.8 % wt vol−1 glucose, 165 U ml−1 glucose oxidase, and 2170 U ml−1 catalase to reduce the photobleaching rate43.Vectorial folding (VF) was performed with the following methodFor VF multiple turnover experiments with Rep-X Fig. , 50 nM R2 and different concentrations of ZMP. The reaction aliquots were slowly cooled from 95 °C to 20 °C in a thermocycler (30 s per 5 °C). When refolded at 1 mM ZMP, RNAs were imaged within 5 min after refolding to minimize the loss of ZMP-bound aptamer due to terminator hairpin formation.To refold RNAs for single-molecule measurements, 20 μl reaction aliquots containing a 1:0.8 ratio of Cy3-RNA:biotin-DNA-Cy5 (160 nM Cy3-RNA and 128 nM biotin-DNA-Cy5) were prepared in the buffer containing 50 mM HEPES-KOH (pH 7.4), 150 mM KCl, 10 mM MgCl44. Briefly, fluorescently labeled samples were immobilized on DT20 or PEG surfaces through a biotin-NeutrAvidin linkage46. Samples were imaged with a prism-based total internal reflection fluorescence (TIRF) imaging microscope47. A 532-nm laser (Coherent Compass 315 M) and a 633-nm laser (Research Electro-Optics) were used for Cy3 and Cy5 excitation, respectively. A water immersion objective  and an EMCCD camera (Andor Technology IXon 897) were used to collect and record signals with 0.075 s time resolution. The fluorescence emission was filtered by a long pass filter (Semrock BLP02-561R-25) and a notch filter (Chroma ZET635NF) to block the excitation lasers. Spatially separated single molecules were picked by a custom IDL code and their intensities were extracted for further analysis. To plot the trajectory overlay, we synchronized trajectories according to the PIFE peak center. The percentage of aptamer-like fold is calculated as the ratio of the aptamer population (magenta) over the sum of the terminator (green) and aptamer populations.Single-molecule data acquisition and analysis were performed with the following methodn being the number of individual molecules.For traces containing multiple PIFE peaks Fig. , peak ce−1) with the known distance of 34 nt separating the two events. Traces containing more than one PIFE peak were excluded from analysis (9 traces excluded). The average rate is reported as the mean ± s.e.m. with n being the number of individual molecules .For dropoff-PIFE traces Fig. , the droFor dropoff experiments , and 10 μM of a 26 nt oligonucleotide complementary to the C-less region. Transcriptions were restarted by addition of various concentrations of NTPs and ZMP prior to incubation at 37 °C for the duration of the time course, removing aliquots as needed and quenching reactions by addition of loading dye containing 8 M Urea, 20% sucrose, 0.1% SDS, 0.01% bromophenol blue, 0.01% xylene cyanol and placing them on ice or storing them at −20 °C. Reactions were then separated via 8% denaturing urea-PAGE and analyzed49. To determine apparent reaction rates for appearance of terminated and readthrough transcription products, time courses were fit by single exponential fits using Origin. For single time point titrations, reactions containing different concentrations of ZMP were incubated for 20 min at 37 °C, and results were fit to a 1:1 binding isotherm to determine T50 values.Transcription reactions were performed by halting transcription by omission of CTP and restarting transcription by adding NTPs and ZMP50 with different lengths of ZTP riboswitch transcripts as inputs.Sequential folding in silico was performed by using Mfold51, using a MicroCal iTC200 . Briefly, RNAs were refolded by heating at 95 °C for 2 min and immediately placed on ice. MgCl2 was added to 10 mM, the sample was brought up to volume, and the RNA was incubated at 37 °C prior to performing titrations at 37 °C in a buffer containing 50 mM Hepes-KOH, pH 7.4, 150 mM KCl, and 10 mM MgCl2. Typically the cell containing 20 μM RNA was titrated with 200 μM ZMP, but higher concentrations were used for weaker binders.All ITC measurements were performed and analyzed with the following methodFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5′-untranslated region (5′UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597–599 and 603–605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344–596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome–HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5′UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF. Chronic hepatitis C virus (HCV) infection is a major cause of liver fibrosis, cirrhosis, and hepatocellular carcinoma ,2,3. AboMoreover, the HCV genome possesses an alternative open reading frame (ORF) overlapping the core coding region in the +1 frame (known as core+1 ORF or ARF). This core+1 ORF encodes additional proteins ,12, inclConcerning the biological relevance of core+1 protein, specific antibodies ,29,30,31Interestingly, phylogenetic analysis and RNase structure mapping have identified three highly conserved RNA secondary structures within the core coding region of HCV that flank the core+1/S initiator codons, which are known as SL47, SL87, and SL248 ,39. We hHere, we provide evidence that SL47 and SL87 also function as a novel cis-acting element that shares main properties of an IRES element and directs core+1/S internal translation initiation at the site of core+1 frame methionine codons 85/87.To examine whether the HCV core sequence comprising the SL47, SL87, and SL248 elements A can dirSpecifically, the C1 plasmid series contains the core nucleotide sequence nt 344-665 of the HCV-1 (genotype 1a) inserted into the intercistronic region of Rph, in the 5′-3′ (sense) or 3′-5′  orientation and the F-Luc gene fused in the frames core−1 (C1_−1) or core+1 (C1_+1) B. This sThe C1_+1 construct supported efficient F-Luc activity levels that were about 20-fold those of the negative control Rph empty vector C. HoweveTo verify the internal translation initiation site(s) that drive the F-Luc expression, the core+1/S initiation codon 87 (nt 603–605) was converted to a stop codon  or mutated SL47 and SL87 sequences from JFH1 isolate (genotype 2a), which have been described previously . The mulp < 0.001). The negative control Rph empty vector-derived RNA was exclusively accumulated in free RNPs. These data are consistent with the assumption that SL47 and SL87 are important RNA elements for the translation of the HCV core+1 frame.To confirm the capacity of SL47 and SL87 structures to mediate the core+1 frame translation, we analyzed the association of wild-type and mutated structures with polysomes, which represents an alternative approach to assess actively translating RNA population. Specifically, we compared the distribution of C1_+1 RNA with that of the respective SL47/SL87 deletion mutant (C3_+1), in the ribosomal fractions of transfected Huh-7 cells, to examine differences in their translation efficiency. 5′UTR IRES and Rph (empty vector) derived bicistronic RNAs were used as positive and negative control, respectively. In this series of experiments, we used uncapped in vitro transcripts to completely abrogate the minimal levels of cap-mediated R-Luc translation, thus restricting the binding of ribosomes exclusively to the second cistron (F-Luc). Since the Rph-derived RNAs are non-replicative, electroporation was used to optimize the transfection efficiency and subsequent ribosome association levels. Prior to the biochemical assay, the ability of the uncapped RNAs to drive F-Luc translation was tested by luciferase assay A. As expIn total, all data from the bicistronic constructs suggest the presence of a cis-acting element embedded in SL47 and SL87 of the HCV core region that drives internal translation initiation from the core+1 frame AUG codons 85 (nt 597–599) and 87 (nt 603–605).Recently, we reported that core+1 ORF is expressed in the context of a bicistronic JFH1-based replicon when fused with the N-terminus of the F-Luc gene and demonstrated the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics . The corFirst, to confirm that C+1‒Luc translation from AUG codons 85/87 is efficient at 6 h post transfection, we examined the luciferase activity of replicon C+1‒Luc, in comparison to C-Luc and to the mutant C+1‒Luc/Val79stop. The latter carries a stop codon at position 79 (nt 579–581), converting Val79 to a stop codon  and 87 (nt 603–605) are active as initiators, both AUGs were converted to GGG  having encapsidated the subgenomic JFH1 replicons C‒Luc, C+1‒Luc wild-type, or mutated variants thereof, or the negative control C−1‒Luc were produced in Huh7.5[CoreE1][E2p7NS2] helper cells after electroporation  constitute a novel cis-acting element that directs the internal translation initiation at the AUG codons 85/87 (nt 597–599 and 603–605) of the alternative core ORF (core+1), which resembles the functional properties of an IRES.TCP) carrying JFH1 replicon constructs, a system simulating native HCV entry. These experiments were combined with an extensive site directed mutagenesis approach and an analysis of ribosome-HCV RNA association.We have earlier shown that the AUG codons 85/87 of the core+1 frame function as a translation initiation site of the short core+1 protein (core+1/S) in transfected cells ,16. FurtWe showed that nt 344–665 of the core-encoding region supported efficient F-luc expression when inserted only in the sense orientation in the Rph intercistronic region, suggesting for the presence of a cis-acting translation element with 30–40% efficiency as compared to the 5′UTR IRES activity. Notably, this alternative element directed translation initiation at the core+1 frame native AUG codons 85/87, as well as at the initiator AUG of the F-Luc gene fused at nt 665, which is 60 nt downstream of the proposed core+1 initiator codons. These findings suggest that the ribosome possibly enters the core region and scans downstream until an initiation codon in an appropriate context is encountered; thus, other AUG codons located close to codons 85/87 may be functional. The presence of such an AUG in the core frame has been reported in clinical isolates derived from cancer patients with high viral load of genotype 1b HCV and resistance to IFN/ribavirin  and has Interestingly, mapping of the genetic element responsible for core+1/S translation initiation at codons 85/87, in both the conventional bicistronic Rph constructs and the viral subgenomic replicon system, supported the critical role of the SL47 and SL87 stem-loops. Their potency to direct translation at the position that corresponds to the internal core+1/S initiation codon 85 (nt 596) was observed when this initiator was fused either at the core+1 or core−1 frame. Deletion of both RNA stem-loops SL47 and SL87 or disruption of their integrity upon the insertion of previously reported substitutions  was detrTCP) carrying the viral replicons.The core+1/S internal translation initiation at codons 85/87 in the context of the bicistronic subgenomic JFH1‒Luc replicon and the role of SL47 and SL87 in this mechanism was verified also under conditions simulating HCV entry, upon infection of cells with HCV trans-complemented particles  are also predicted to have SL47/87-like RNA structures in their core region (data not shown). The presence of evolutionarily conserved RNA structural patterns in viral coding sequences has been linked to translational regulation , as in tOur results are also analogous to those reported for other RNA viruses that use IRES elements to express overlapping ORFs directing non-canonical translation initiation at internal codons. Dicistroviridae members  use the intergenic region IRES to translate a short ORF (ORFx) that is shifted by +1 nucleotide relative to the structural polyprotein region, analogous to the HCV core+1 ORF ,44. In tNon-canonical translational mechanisms have been reported to regulate the expression level and/or timing of expression of various viral genes under certain conditions . Thus, tSeveral positive sense RNA viruses keep surprising us with their ability to increase the coding capacity of their genomes by the use of alternative translation mechanisms based on the exploitation of secondary and higher-ordered structures as cis-regulatory elements. The new evidence on a role of SL47/87 RNA structural elements on translation of the core+1 ORF and ongoing studies on the biological function of its products would contribute to a better understanding of HCV translation and pathogenesis.v/v) fetal calf serum (referred to as complete DMEM). The HCV packaging cell line Huh7.5[CoreE1][E2p7NS2] , was cultured in complete DMEM supplemented with 5 μg/mL blasticidin. For the replication and infection-permissive Huh7.25-CD81 cells , which , Japan, ), which All nucleotide (nt) numbers refer to the JFH1 sequence . Rph R. The corThe bicistronic subgenomic replicon constructs pFK-I630-core-Luc/EI-NS3-3′-JFH1-dg A that co2, 2 mM spermidine, 40 mM dithiothreitol (DTT), 2.5 mM of each deoxyribonucleoside triphosphate, 5 mM m7G(5′)ppp(5′)G  , 1 U of RNasin  per μL and 0.6 U of T7 RNA polymerase  per μL. After incubation for 1.5 h at 37 °C, 0.3 U of T7 RNA polymerase/μL reaction mixture was added, followed by another 1.5 h of incubation at 37 °C. Poly(A) tailing was performed after increasing the transcription reaction volume by 5 times and adding 1X E-PAP buffer, 2.5 mM MnCl2, 1 mM ATP, and 6 U of E. coli poly(A) polymerase I  for 1 h of incubation at 37 °C. Transcription and poly(A) tailing reactions were terminated by the addition of 1.2 U of RNase-free DNase  per μg of plasmid DNA and 30 min of incubation at 37 °C. For in vitro synthesis of uncapped polyA-tailed Rph RNA molecules, the same protocol was used, but no cap analog was included. The RNA was extracted with acidic phenol and chloroform, precipitated with isopropanol, and dissolved in RNase-free water. Denaturing agarose gel electrophoresis was used to assess the integrity of RNA, and the concentration was determined by measurement of the optical density at 260 nm. HCV subgenomic replicon constructs were linearized with MluI and used for in vitro transcription as described previously [E2p7NS2] packaging cells were performed by electroporation (10 μg of in vitro transcribed RNA was mixed with 400 μL of cell suspension containing 1 × 107 cells/mL in the case of Huh7 and 1.5 × 107 cells/mL in the case of Huh7.5 packaging cells), as described elsewhere [Transfections of Rph-capped polyA-tailed RNAs into Huh7 cells ‒Luc replicons, were harvested, passed through 0.45 μm pore size filters, and spun in Amicon Ultra-15 100 K centrifugal filter devices at 4000× g for 20 min, using a swinging bucket rotor. Huh7.25-CD81 cells were seeded at a density of 4 × 104 cells per well of a 48-well plate and, 24 h later, they were inoculated with 100 μL of viral particles preparation diluted 1:2 (with complete DMEM) for 4 h. Luciferase activity was determined 16 h later, and viral RNA levels were quantified by RT-qPCR 48 h later, as described below.Concentrated stocks of HCVTCP particles, F-Luc activity was measured using Luciferase Assay System. Measurements were taken in a GloMax 20/20 single tube luminometer (Promega) for 10 s. Luciferase activities were normalized to the total protein amount determined using the Bradford assay reagent (Pierce).Cells were lysed with Passive Lysis Buffer , and luciferase activity was quantified. For cells transfected with Rph constructs, F-Luc activity was measured using a Luciferase Assay System (Promega), as recommended by the manufacturer, and R-Luc activity was measured using 12 μM coelenterazine (Promega) in assay buffer . For cells transfected with HCV replicons together with an R-Luc expressing RNA used as normalization control, F-Luc and R-Luc activities were determined using the Dual Luciferase Reporter Assay (Promega), according to the manufacturer’s instructions. Finally, for cells infected with HCVTotal RNA was extracted from cells using TRIzol reagent , according to the manufacturer’s instructions. Rph RNAs and viral replicons were quantified with reverse-transcription (RT) and quantitative real-time polymerase reaction (qPCR). RT was performed using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase , and oligo (dT) for the Rph RNAs, or the JFH1 IRES specific primer 5′-GGATTTGTGCTCATGGTGCA-3′ (reverse) for viral replicons. For qPCR, KAPA SYBR FAST qPCR Master Mix  was used. Rph RNAs were quantified using the F-Luc specific primers 5′-AGGTGGCTCCCGCTGAAT-3′ (forward) and 5′-CATCGTCTTTCCGTGCTCCA-3′ (reverse). Viral replicons were quantified using the JFH1 IRES specific primers 5′-GGCCTTGTGGTACTGCCTGATA-3′ (forward) and 5′-GGATTTGTGCTCATGGTGCA-3′ (reverse). The housekeeping gene YWHAZ was employed as an internal control (primers 5′-GCTGGTGATGACAAGAAAGG-3′ and 5′-GGATGTGTTGGTTGCATTTCCT-3′).Denaturing SDS-polyacrylamide gel electrophoresis and Western blotting was performed as described elsewhere . Dilutio2, 15 mM Tris-HCl, pH 7.4, 100 μg/mL CXH, and 1% Triton-X 100. The cell lysates were collected, and nuclei were pelleted by centrifugation at 12,000× g for 10 min. The supernatants were layered on a linear 10–50% (w/v) sucrose gradient in polysome gradient buffer  and centrifuged at 27,000 rpm for 4 h at 4 °C in a SW41 rotor . Twenty-nine sucrose density gradient fractions of 400 μL were collected from the top, and the distribution of ribosomal RNA along the fractions was determined by agarose gel electrophoresis and quantified by Quantity One software . The fractions were pooled as follows: samples 1–4 (free ribonucleoprotein complexes), samples 5–11 , samples 12–20 (light polysomes), and samples 21–29 (heavy polysomes). Total RNA was extracted from the sucrose density fractions using acidic phenol/chloroform as described above for in vitro transcribed RNA. The distribution of the free and ribosome associated Rph RNA was determined with RT-qPCR, using F-Luc specific primers as described above.To examine the loading of ribosomes on the Rph bicistronic RNAs, polysome analysis was performed using sucrose density gradient ultracentrifugation. Huh7 cells were electroporated with in vitro transcribed uncapped polyA-tailed RNAs and seeded in 100 mm dishes. After 16 h, electroporated cells were washed twice with ice-cold PBS pH-7.4 containing 100 μg/mL cycloheximide (CXH) and lysed with 500 μL of polysomes lysis buffer containing 300 mM NaCl, 15 mM MgCl® .Results were subjected to statistical analysis using Student’s t-test. Statistical calculations were carried out using Excel Microsoft OfficeThe mfold Web Server  was used


RNA acts as an immunostimulatory molecule in the innate immune system to activate nucleic acid sensors. It functions as an intermediate, conveying genetic information to control inflammatory responses. A key mechanism for RNA sensing is discriminating self from non-self nucleic acids to initiate antiviral responses reliably, including the expression of type I interferon (IFN) and IFN-stimulated genes. Another important aspect of the RNA-mediated inflammatory response is posttranscriptional regulation of gene expression, where RNA-binding proteins (RBPs) have essential roles in various RNA metabolisms, including splicing, nuclear export, modification, and translation and mRNA degradation. Recent evidence suggests that the control of mRNA stability is closely involved in signal transduction and orchestrates immune responses. In this study, we review the current understanding of how RNA is sensed by host RNA sensing machinery and discuss self/non-self-discrimination in innate immunity focusing on mammalian species. Finally, we discuss how posttranscriptional regulation by RBPs shape immune reactions. Innate immunity is a first-line host defense mechanism that initiates inflammatory responses against pathogen invasion. The main characteristic of initial innate immune responses is the production of inflammatory cytokines and chemokines, which leads to inflammatory cell activation and recruitment. Innate immune cells are genetically equipped with pathogen sensors, which are called pattern recognition receptors (PRRs). These PRRs recognize not only pathogen-associated molecular patterns (PAMPs) but also endogenous molecules, which are called damage-associated molecular patterns (DAMPs) ,2,3,4. TRNA and its recognition are critical for regulating inflammatory processes at multiple steps. Host RNA sensors recognize foreign RNAs as well as endogenous RNAs from stressed cells. Furthermore, there are numerous RNA-binding proteins (RBPs) that modulate immune reactions by controlling gene expression . PosttraThe innate immune system has nucleic acid sensors that keep non-self nucleic acids under constant surveillance. Among human TLRs, TLR3, TLR7, and TLR8 are endosomal RNA sensors, whereas TLR3 recognizes double-stranded (ds) RNA. In mice, TLR8 is dispensable for RNA recognition, which completely depends on TLR7. TLR7 and TLR8 sense single-stranded (ss) RNA, whereas TLR13 expressed in mice also recognizes ssRNA derived from bacterial 23S ribosomal RNA ,23. RLRsType I IFNs have multiple functions in the fight against viral infection . Type I According to extensive studies, these sensors not only survey pathogen invasion but also recognize host nucleic acids. Immune responses elicited by endogenous nucleic acids are independent of pathogen infection, a process known as sterile inflammation . SeveralBecause self RNAs are distinguished from foreign RNAs in nature, host cells have evolved multiple molecular mechanisms used in the discrimination of self and non-self RNAs. Long dsRNAs in the cytosol are derived from the genome of RNA viruses or intermediate products that are generated during viral replication; they are undetected in healthy cells . TherefoAmong RLR family members, RIG-I recognizes short dsRNAs. For the optimal activation of RIG-I, dsRNAs require: (1) A triphosphate or diphosphate group on the 5’ end of RNA molecules ,37, (2) TLR7 and TLR8 recognize ssRNA ,50 Figua. These Increasing evidence demonstrates that host RNA-sensing mechanisms are also potent in recognizing endogenous RNAs under pathological conditions. Genetic mutations of RNA sensors and abnormal RNA metabolism cause pathologies, which are induced by aberrant activation of type I IFNs called type I interferonopathies. Examples of these are Singleton–Merten syndrome (SMS) and Aicardi–Goutieres syndrome (AGS). The gain of function mutations of DDX58 and IFIH1, which encode RIG-I and MDA5, respectively, are reported in studies on whole-exome sequencing in patients with Singleton–Merten syndrome (SMS) and Aicardi–Goutieres syndrome (AGS) ,58,59,60Yaa chromosome are used as a male-specific lupus model in which IFN responses are enhanced due to TLR7 duplication [On the other hand, aberrant activations of TLR7 and TLR9 have been implicated in type I IFN-related autoimmune diseases, such as systemic lupus erythematosus (SLE), which is characterized by the presence of autoantibodies against nucleic acids. These two TLRs are expressed particularly in plasmacytoid DCs (pDCs) and B cells. The roles of TLR7 and TLR9 in the pathogenesis of SLE have been shown to be quite different. The severity of the disease depends on the strength of TLR7 signaling. Overexpression of TLR7 in mice accelerates the development of SLE, whereas deficiency of TLR7 in lupus-prone mice ameliorates the phenotype . Mice halication ,71. Howelication . In addilication . The diflication . Once trlication ,76. Thislication . This, ilication . TherefoType I IFN signaling inhibits viral replication by inducing a large array of genes. For instance, IFN-inducible RBPs play critical roles in antiviral defense by binding viral RNAs c. ProteiHost RNases also restrict viral infection by directly degrading viral RNAs. RNase L, which is a cytoplasmic endoribonuclease, degrades viral RNA molecules. The activation of RNase L requires dimerization via 2′-5′-oligoadenylate that is synthesized by 2′-5′-oligoadenylate synthetases (OASs) upon recognition of dsRNAs . RNase LAs previously discussed, nucleic acid recognition by PRRs induces type I IFNs and inflammatory cytokines to combat viral infection. Such inflammatory genes are post-transcriptionally regulated by RBPs to control the magnitude and duration of immune responses. In addition, increasing evidence shows that posttranslational modifications, in response to PRR stimulation, tightly regulate RBP function ,91. The An AU-rich element (ARE) in mRNA 3’ UTR is one of the best-studied cis-elements and acts as a code for mRNA decay, translation control, and nuclear export a. ARE isStem-loop structures in inflammatory mRNAs also act as a cis-element recognized by RBPs such as Regngase-1 and Roquin c. These Posttranscriptional control in the innate immune system promptly induces inflammatory responses and simultaneously produces a timely resolution of inflammation. Dysregulation of posttranscriptional mechanisms, especially those involved in mRNA stability and decay, causes sustained inflammation, and leads to autoimmunity. In particular, the biological roles of ARE-binding proteins have been studied extensively. For example, TTP is a well-characterized RBP that is essential for the maintenance of immune homeostasis. Furthermore, a TTP deficiency in mice causes spontaneous development of arthritis and other inflammatory syndromes, such as dermatitis and cachexia, which are mainly due to the overproduction of TNF ,112. ConZFP36L1 and ZFP36L2 play redundant roles in lymphocyte development. During early lymphopoiesis, ZFP36L1 and ZFP36L2 regulate mRNA-encoding cell cycle regulators ,127. TheAUF1 is another ARE-binding protein involved in mRNA destabilization. Among four isoforms, consisting of p37, p40, p42, and p45, p37 profoundly influences mRNA stability . AUF-defStem-loop-binding proteins are also involved in the maintenance of immune homeostasis. Regnase-1 is expressed ubiquitously, and this protein plays an essential role in the immune system. This protein’s importance has been demonstrated in mice lacking Regnase-1, which shows diverse inflammatory phenotypes, including lymphadenopathy, massive infiltration of lymphocytes in the lungs and liver, and the production of autoantibodies . In macr–/– aged mice show mild lymphadenopathy, which is presumably due to sustained activation of IFNγ signaling in macrophages [Although evidence is very limited, Regnase-1 family proteins are also involved in inflammatory responses. Regnase-3 is highly expressed in myeloid lineages, such as macrophages and DCs, and localizes to endosome/phagosome . At a yorophages . Neverthrophages , which srophages . This gerophages . Regnaserophages ,155. BasRoquin also significantly contributes to the immune system. This molecule’s role was initially verified in mice that harbor a single point mutation present in the putative E3 ligase of Roquin, and these are called sanroque mice . These sRNA is involved in diverse biological processes and has a great impact on immune function, which enables innate-acquired interplay. Recently, there has been a significant advance in utilizing PRR-mediated RNA sensing. Intensive work on viral RNA recognition has provided new information on the molecular and structural basis of how TLRs and RLRs induce antiviral responses. Furthermore, nucleic acids are now considered potential adjuvants for therapeutic approaches. In parallel, greater attention is focused on the detrimental effects of PRR activation triggered by host RNAs, which is implicated in the development of autoimmunity. Recent work suggests that an altered threshold of activation of these RNA sensors or failure to maintain host RNAs, mostly non-coding RNAs, under control are keys to understanding how RNA sensors cause uncontrollable inflammation. Nevertheless, knowledge regarding tolerant mechanisms mediated by host RNA is very limited. Future investigations will be necessary to provide novel insights into non-coding RNAs as potential ligands of RNA sensors.Another aspect of RNA regulation is the posttranscriptional control of gene expression that determines the protein output of inflammatory responses. Because there are as many as 1500 RBPs encoded in the genome , a compl


Remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients. The active form of remdesivir acts as a nucleoside analog and inhibits the RNA-dependent RNA polymerase (RdRp) of coronaviruses including SARS-CoV-2. Remdesivir is incorporated by the RdRp into the growing RNA product and allows for addition of three more nucleotides before RNA synthesis stalls. Here we use synthetic RNA chemistry, biochemistry and cryo-electron microscopy to establish the molecular mechanism of remdesivir-induced RdRp stalling. We show that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. This translocation barrier causes retention of the RNA 3ʹ-nucleotide in the substrate-binding site of the RdRp and interferes with entry of the next nucleoside triphosphate, thereby stalling RdRp. In the structure of the remdesivir-stalled state, the 3ʹ-nucleotide of the RNA product is matched and located with the template base in the active center, and this may impair proofreading by the viral 3ʹ-exonuclease. These mechanistic insights should facilitate the quest for improved antivirals that target coronavirus replication. Remdesivir is a nucleoside analog that inhibits the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) and is used as a drug to treat COVID19 patients. Here, the authors provide insights into the mechanism of remdesivir-induced RdRp stalling by determining the cryo-EM structures of SARS-CoV-2 RdRp with bound RNA molecules that contain remdesivir at defined positions and observe that addition of the fourth nucleotide following remdesivir incorporation into the RNA product is impaired by a barrier to further RNA translocation. The RdRp consists of three non-structural protein (nsp) subunits, the catalytic subunit nsp126 and the accessory subunits nsp8 and nsp77. Structures of the RdRp of SARS-CoV-2 were obtained in free form8 and with RNA template-product duplexes11. Together with a prior structure of SARS-CoV RdRp12, these results have elucidated the RdRp mechanism. For RNA-dependent RNA elongation, the 3ʹ-terminal nucleotide of the RNA product chain resides in the –1 site and the incoming nucleoside triphosphate (NTP) substrate binds to the adjacent +1 site. Catalytic nucleotide incorporation then triggers RNA translocation and liberates the +1 site for binding of the next incoming nucleoside triphosphate (NTP).Coronaviruses use an RdRp enzyme to carry out replication and transcription of their RNA genome16. Remdesivir inhibits the RdRp of coronaviruses22 and shows antiviral activity in cell culture and animals25. Remdesivir is a phosphoramidate prodrug that is metabolized in cells to yield an active NTP analog21 that we refer to as remdesivir triphosphate (RTP). Biochemical studies showed that the RdRp can use RTP as a substrate, leading to the incorporation of remdesivir monophosphate (RMP) into the growing RNA product22. After RMP incorporation, the RdRp extends RNA by three more nucleotides before it stalls22. This stalling mechanism is specific to coronaviruses because the RdRp of Ebola virus can add five RNA nucleotides after RMP incorporation before it stalls26.The nucleoside analog remdesivir is the only FDA-approved drug for the treatment of COVID-19 patients9, whereas another structure contained RMP in the –1 site10. In both structures, RMP mimics adenosine monophosphate (AMP) and forms standard Watson–Crick base pairs with uridine monophosphate (UMP) in the RNA template strand. Thus, these studies explained how RMP is incorporated into RNA instead of AMP. However, they do not explain how remdesivir inhibits the RdRp because RdRp stalling occurs only after three more nucleotides have been added to the RNA20.Recent structural studies showed RdRp-RNA complexes after remdesivir addition to the RNA product 3ʹ-end. One structure contained RMP in the +1 site22, we observed that RMP is readily incorporated into RNA and that the RNA is subsequently elongated by three more nucleotides before the RdRp stalls , which we synthesized from 1ʹ-cyano-4-aza-7,9-dideazaadenosine (Rem) in four steps  (Methods). The annealed RNA scaffolds were then bound to purified RdRp and subjected to cryo-EM analysis as described11 did not reveal significant differences. The 1ʹ-cyano group of the RMP ribose moiety was located at position –3 and is accommodated there by an open space in the RNA product-binding site of the RdRp  was resolved at 3.1 Å resolution  was resolved at 3.4 Å resolution  and cloned into pFastBac vector 438C (Addgene #154759). Protein was expressed in Hi5 cells for 60 h. After harvesting by centrifugation, cells were lysed in lysis buffer  glycerol, 30 mM imidazole pH 8.0, 5 mM β-mercaptoethanol, 0.284 μg/ml leupeptin, 1.37 μg/ml pepstatin, 0.17 mg/ml PMSF, 0.33 mg/ml benzamidine and 3 mM MgCl2) and opened by sonication. Lysate was clarified and passed through a HisTrap HP 5 ml  preequilibrated in lysis buffer, and the protein was eluted with nickel elution buffer  glycerol, 500 mM imidazole pH 8.0, 3 mM MgCl2 and 5 mM β-mercaptoethanol) directly onto an XK column 16/20  containing amylose resin. Protein was eluted in amylose elution buffer  glycerol, 116.9 mM maltose, 30 mM imidazole pH 8.0 and 5 mM β-mercaptoethanol). Pure protein fractions were digested with TEV protease overnight. Protein solution was after digestion passed through a HisTrap HP 5 ml column and HiTrap Heparin 5 ml column . The flow-through was concentrated and further purified via HiLoad S200 16/600 pg column equilibrated in size exclusion buffer  glycerol, 1 mM MgCl2, 1 mM TCEP). Peak fractions were pooled, concentrated until 102 μM, aliquoted, flash-frozen and stored at −80 °C until use.Preparation of SARS-CoV-2 RdRp was carried out as describedSynthetic genes for nsp8 and nsp7 were amplified by PCR  and separately cloned into pET-derived 14-B vector (Addgene #154757 for nsp7 and #154758 for nsp8). Both nsp7 and nsp8 were expressed and purified in the same way. Proteins were overexpressed in E. coli BL21 (DE3) RIL cells. Bacterial cells were induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside and shaken for 16 h at 18 °C. After expression, cells were centrifuged and immediately lysed by sonication in lysis buffer  glycerol, 30 mM imidazole pH 8.0, 5 mM β-mercaptoethanol, 0.284 μg/ml leupeptin, 1.37 μg/ml pepstatin, 0.17 mg/ml PMSF and 0.33 mg/ml benzamidine). Clarified lysate was loaded onto a HisTrap HP 5 ml  preequilibrated in lysis buffer, and the protein was eluted in nickel elution buffer  glycerol, 500 mM imidazole pH 8.0 and 5 mM β-mercaptoethanol). Eluted protein was dialyzed against a dialysis buffer  glycerol and 5 mM β-mercaptoethanol) and simultaneously digested with TEV protease overnight. After digestion, protein was passed through a HisTrap HP 5 ml column and a HiTrap Q 5 ml column . Flow-through containing the protein of interest was concentrated and further purified on a HiLoad S200 16/600 pg column equilibrated in size exclusion buffer  glycerol, 1 mM TCEP). Peak fractions were pooled, concentrated, aliquoted, flash-frozen and stored at −80 °C until use. All protein purification steps were done at 4 °C.11, except for the following changes. The final concentrations of nsp12, nsp8, nsp7, and RNA were 3 μM, 9 μM, 9 μM, and 2 μM, respectively. The highest concentration of NTPs was 0.5 mM for each nucleotide , followed by a two-fold serial dilution. RNA products were resolved on a denaturing sequencing gel and visualized by Typhoon 95000 FLA Imager . The RNA sequences used for extending RMP-containing RNA oligonucleotides are: rUrGrA rGrCrC rUrArC rGrC- rA or rR- rGrUrG (product) and rArArA rCrArC rUrGrC rGrUrA/3ddC/(template). The extension assay . RNA products were visualized by SYBR Gold (Thermo Fischer) staining and imaged with Typhoon 9500 FLA Imager .RTP synthesis is described in Supplementary Methods. All unmodified RNA oligonucleotides were purchased from Integrated DNA Technologies. The RNA sequence used for the RNA extension assay Fig.  is /56-Fsay Fig.  was perf1) was converted to the 5ʹ-O-DMT-2ʹ-O-TBDMS-protected 3ʹ-cyanoethyl diisopropyl phosphoramidite  in four steps. Details of the synthetic procedures and nuclear magnetic resonance spectra of isolated compounds are given in the Supplementary Methods. RNA oligonucleotides were then prepared by solid-phase synthesis on CPG support  using 2ʹ-O-TOM-protected ribonucleoside phosphoramidites (100 mM in CH3CN) and ethylthiotetrazol  as activator, with 4 min coupling time. Rem-PA was used as freshly prepared solution (100 mM) in dry 1,2-dichloroethane, with a coupling time of two times 12 min. Detritylation was performed with 3% dichloroacetic acid in dichloromethane. Capping solutions contained 4-dimethylamino pyridine (0.5 M) in acetonitrile for Cap A, and acetic anhydride/sym-collidine/acetonitrile (2/3/5) for Cap B. Oxidation was performed with iodine (10 mM) in acetonitrile/sym-collidin/water (10/1/5). The oligonucleotides were deprotected with 25% NH4OH at 25 °C for 30 h, followed by 1 M TBAF in THF for 12 h, and purified by denaturing polyacrylamide gel electrophoresis.1ʹ-cyano-4-aza-7,9-dideazaadenosine , and HR-ESI-MS . An aliquot (200 pmol in 25 µL) was digested by snake venom phosphodiesterase  in the presence of bacterial alkaline phosphatase  in 40 mM Tris. pH 7.5, 20 mM MgCl2, and the resulting mononucleosides were analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry using an RP-18 column  and micrOTOF-Q III with ESI ion source operated in positive ion mode . Extracted ion chromatograms and UV absorbance traces at 245 nm confirmed presence of remdesivir.The purity and identity of the RNA oligonucleotides was analyzed by anion-exchange HPLC , 6 M Urea. Solvent B: 25 mM Tris-HCl (pH 8.0), 6 M Urea, 0.5 M NaClO11. The RNA scaffolds used for structural studies were comprisesd of two RNA strands. The first RNA strand forms the RNA template-product hairpin and lacks the last 15 nt at the 3ʹ end. The second strand contains the missing 15 nt sequence. Upon scaffold annealing, a complete RNA template-product is formed with a single nick in the product RNA. This strategy had to be used because the length of the remdesivir-containing oligonucleotides was limited due to technical reasons. RNA scaffolds for RdRp-RNA complex formation were prepared by mixing equimolar amounts of two RNA strands in annealing buffer  and heating to 75 °C, followed by step-wise cooling to 4 °C. RNA sequences for RMP at position -3 (structure 1) are: rUrUrU rUrCrA rUrGrC rArUrC rGrCrG rUrArG rGrCrU rCrArU rArCrC rGrUrA rUrUrG rArGrA rCrCrU rUrUrU rGrGrU rCrUrC rArArU rArCrG rGrUrA and rUrGrA rGrCrC rUrArC rGrCrG rRrUrG. RNA sequences for AMP/RMP at position -4 (structures 2 and 3) are: rUrUrU rUrCrA rUrGrC rArCrU rGrCrG rUrArG rGrCrU rCrArU rArCrC rGrUrA rUrUrG rArGrA rCrCrU rUrUrU rGrGrU rCrUrC rArArU rArCrG rGrUrA and rUrGrA rGrCrC rUrArC rGrC- rA/rR -rGrUrG. RdRp-RNA complexes were formed by mixing purified nsp12  with an equimolar amount of annealed RNA scaffold and threefold molar excess of each nsp8 and nsp7. After 10 min of incubation at room temperature, the mixture was applied to a Superdex 200 Increase 3.2/300 size exclusion chromatography column, which was equilibrated in complex buffer  at 4 °C. Peak fractions corresponding to the RdRp-RNA complex were pooled and diluted to ~2 mg/ml. For structure 2, an additional 0.36 nmol of annealed RNA scaffold were spiked into the sample prior to grid preparation. Three microliters of the concentrated RdRp-RNA complex were mixed with 0.5 µl of octyl ß-d-glucopyranoside  and applied to freshly glow discharged R 2/1 holey carbon grids (Quantifoil). The grids were blotted for 5 s using a Vitrobot MarkIV (Thermo Fischer Scientific) at 4 °C and 100% humidity and plunge frozen in liquid ethane.SARS-CoV-2 RdRp was prepared as described32 on a 300 keV Titan Krios transmission electron microscope (Thermo Fischer Scientific). Prior to detection, inelastically scattered electrons were filtered out with a GIF Quantum energy filter (Gatan) using a slit width of 20 eV. Images were acquired in counting mode (non-super resolution) on a K3 direct electron detector (Gatan) at a nominal magnification of 105,000x resulting in a calibrated pixel size of 0.834 Å/pixel. Images were exposed for a total of 2.2 s with a dose rate of 19 e−/px/s resulting in a total dose of 60 e−/Å2, which was fractionated into 80 frames. Previous cryo-EM analysis of the SARS-CoV2 RdRp showed strong preferred orientation of the RdRp particles in ice11. Therefore, all data were collected with 30° tilt to obtain more particle orientations. Motion correction, contrast transfer function (CTF)-estimation, and particle picking and extraction were performed on the fly using Warp33. In total, 8004, 11,764, and 7043 movies were collected for structures 1, 2, and 3, respectively.Cryo-EM data were collected using SerialEM33 1.0.9 to cryoSPARC34 2.15. After ab initio refinement of five classes, an intermediate map from the previous processing of EMD-1100711 was added as a 6th reference, and supervised three-dimensional (3D) classification was performed. 654k particles (37%) from the best class deemed to represent the polymerase were subjected to 3D refinement to obtain a 3.1 Å map. Half-maps and particle alignments were exported to M35 1.0.9, where reference-based frame series alignment with a 2 × 2 image-warp grid, as well as CTF refinement were performed for two iterations. Although the resulting map had the same 3.1 Å nominal resolution, the features of interest were significantly cleaner.For structure 1, 1.8 million particles were exported from WarpFor structure 2, 3.4 million particles were exported from Warp 1.0.9 to cryoSPARC 2.15. After ab initio refinement of 5 classes, the EMD-11007-related reference was added as a 6th reference, and supervised 3D classification was performed. To further clean up the resulting 881k particles (26%) from the best class deemed to represent the polymerase, another ab initio refinement of five classes was performed. Three of these classes and the EMD-11007-related reference were used for supervised 3D classification. 474k particles (54%) from the best class deemed to represent the polymerase were subjected to 3D refinement to obtain a 3.6 Å map. Half-maps and particle alignments were exported to M 1.0.9, where reference-based frame series alignment with a 2 × 2 image-warp grid, as well as CTF refinement were performed for two iterations to obtain a 3.4 Å map.For structure 3, 2.2 million particles were exported from Warp 1.0.9 to cryoSPARC 2.15. Initial unsupervised 3D classification in three classes was performed using the EMD-11007-related reference. To further clean up the resulting 1.1 million particles (23%), ab initio refinement of five classes was performed. Four of these classes and the EMD-11007-related reference were used for supervised 3D classification. 819k particles (70%) from the best class deemed to represent the polymerase were subjected to 3D refinement to obtain a 3.1 Å map. Half-maps and particle alignments were exported to M 1.0.9, where reference-based frame series alignment with a 2 × 2 image-warp grid, as well as CTF refinement were performed for three iterations to obtain a 2.8 Å map.11. For each of the structures 1–3, the model was first rigid-body fit into the density and subsequently adjusted in real-space in Coot36. Parts of the N-terminal NiRAN domain of nsp12, the N-terminal extension of nsp8a and the entire nsp8b molecule were removed due to absence or poor quality of density for these regions. Restraints for RMP were generated in phenix.elbow37 and the structures were refined using phenix.real_space_refine38 with appropriate secondary structure restraints. Model quality was assessed using MolProbity within Phenix39, which revealed excellent stereochemistry for all three structural models (Supplementary Table 40.Models were built using our previously published SARS-CoV-2 RdRp structure as starting model (PDB 6YYT [10.2210/pdb6YYT/pdb])Further information on experimental design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


High throughput RNA sequencing has revealed the existence of abundant circular RNAs (circRNAs) that are cell lineage-specific and have been implicated in human diseases. CircRNAs are resistant to exonuclease digestion, can carry genetic information of oncogenes, and are enriched in exosome to be transported from tissues into various body fluids. These properties make circRNAs ideal non-invasive diagnostic biomarkers for disease detection. Furthermore, many circRNAs have been demonstrated to possess biological functions in relevant cells, suggesting that they may also be potential therapeutic targets and reagents. However, our knowledge of circRNAs is still at an infant stage and far from being translated into clinics. Here, we review circRNAs in the disease setting of prostate cancer. We start by introducing the basic knowledge of circRNAs, followed by summarizing opportunities of circRNAs to be prostate cancer biomarkers, and discuss current challenges in circRNA research and outlook of future directions in translating current knowledge about circRNA into clinical practice. Although it was known for decades that there exist circular forms of RNA (circRNA) in virus, plant, fungi, bacteria, and mammalian cells , circRNACircular RNAs are synthesized by RNA spliceosome, the same splicing machinery to synthesize linear mRNAs but through a backsplicing process. During this process, the downstream splice donors are back spliced to the upstream acceptors . It shouBecause both circRNAs and linear mRNAs are processed from pre-mRNAs by the RNA splicing machinery, their relative expression levels could be either positively or negatively correlated pending upon the cell contexts. For a specific copy of an exon in the pre-mRNA, it can be processed into either one copy of circRNA or one copy of mRNA. Under this context, the levels of circRNAs and their counterpart mRNAs are negatively correlated. However, the RNA splicing process is tightly coupled with gene transcription initiation and elongation rates , and mulCircular RNAs possess several special features that make them ideal biomarkers for diseases. First, circRNAs are resistant to exonuclease degradation . They arMany circRNAs had been demonstrated to play important roles in promoting cancer cell proliferation, migration/invasion, anti-apoptosis, and differentiation , emphasizing that tumor-promoting circRNAs may be potential cancer biomarkers. The molecular functions of circRNAs had been reported to (1) regulate microRNA activity, (2) act as scaffolds or decoys for RBPs, or (3) serve as templates for cap-independent translation . It was Circular RNAs can also exert biological functions through forming complexes with proteins. For example, circFOXO3 was reported to form RNA-protein complexes with CDKN1A and CDK2, resulting in suppression of CDK2 activity and arrest of cell cycling . Circ-ANDrosophila head , to validate one of the circRNAs consisting of AR exon 2 whose expression was elevated by androgen deprivation conditions. Data from our lab had applied RISH to show that the exon 3 of the AR gene forms a circRNAs, called circ-AR3, that is highly expressed in benign and low Gleason primary tumors, but downregulated in high Gleason tumors and further reduced in CRPC (In prostate cancer (PCa), several circRNAs are promising to be potential diagnostic or prognostic biomarkers. Since the AR gene amplification and overexpression had been demonstrated to be one of the major mechanisms by which CRPC is developed , circRNA in CRPC . This co in CRPC . These fAlthough most of the PCa cells present adenocarcinoma (AdPC) phenotype with classic luminal epithelial morphology, emerging clinical evidence indicated that more aggressive subtypes of CRPC with AR indifferent phenotype become more prevalent, accounting for ∼15–20% of CRPC tumors . Many ofSeveral circRNAs had been studied for their molecular and cellular functions in PCa cells . Most ofAlthough high throughput sequencing is powerful for globally profiling circRNAs in tissues and cell models, it is not without limitations. During library preparations using polyA depletion, rRNA depletion, and RNAse R treatment protocols, there are still linear RNAs retained in the RNA samples . By contAnother challenge is that RNA samples used for RNA sequencing or microarray analyses are extracted from a homogenate of PCa tissue trunks containing not only cancer cells but also benign and stroma cells. This could compromise the accuracy of sequencing data analyses to identify tumor-specific circRNAs, since the detection of some lowly expressed circRNAs can be easily affected by the mixture of cell populations from different cell lineages. Even though all RNA samples had been processed with ribosome depletion and RNase R treatment, there is still a significant portion of none circRNAs remain. It is therefore critical to validate circRNAs identified by RNA sequencing or microarray in tissues from patients using techniques such as RISH, which will allow RISH signal to be evaluated together with the histology of tumor cells.Circular RNAs can form secondary structures of stem-loop RNA hairpins, and even three-dimensional structures complexed with RBPs . It is tProstate cancer specific circRNAs identified from tissues by RNA sequencing or microarray can provide new insights to develop non-invasive biomarkers in plasma, however, plasma circRNA levels are not always positively correlated with intratumoral circRNA levels. For example, we have shown that intratumoral circ-AR3 levels were reduced in high Gleason tumors and CRPC when compared with benign prostate and low Gleason tumors, while plasma circ-AR3 levels are increased in patients with high Gleason tumors and CRPC . MultiplOne solution to avoid the complicated factors that influence the release of PCa origin circRNAs into the bloodstream is to measure circRNAs directly from patient plasma by microarray or RNA sequencing. Such experiments had been performed in patients who have lung cancers  or gastrNew technologies enhance our understanding of the biology of RNAs in human cells. High throughput sequencing had revealed that there are aberrantly expressed circRNAs in PCa cells that have various biological functions. These findings have brought a plethora of opportunities to develop new diagnostic and prognostic biomarkers for PCa. Although we are still facing challenges, our accumulating knowledge from circRNA research will be eventually translated into clinical practice to benefit PCa patients.JL and XD developed the idea. JL and JF draft the manuscript. JQ and XD revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.