C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of  Neural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion. The connectivity of neuronal circuits is established through properly guided axons which form functional synaptic connections. The growing axon is guided to its target by the motile, actin-based growth cone at the tip of the growing neurite. Growth cone response to extracellular guidance cues allows the axon to extend, retract, turn and branch, regulated by the reorganization and dynamics of the actin and microtubule cytoskeletons of the growth cone .C. elegans and vertebrates, the conserved laminin-like UNC-6/Netrin guidance cue and its receptors UNC-40/DCC and UNC-5 direct dorsal-ventral axon outgrowth  LGX: rhgf-1, lqIs170 [rgef-1::vab-10ABD::gfp]. Chromosomal locations not determined: lqIs279 [Punc-25::ebp-2::gfp] by integration of lqEx809, lhIs6 [Punc-25::mCherry], lqIs296 [Punc-25::myr::unc-5], lqIs312 [Punc-25::rho-1(G14V)] by integration of lqEx1043, lqIs314 [Punc-25::rho-1(T19N)] by integration of lqEx1070. Extrachromosomal arrays were generated using standard gonadal injection , lqEx1131, lqEx1132, lqEx1133 and lqEx1134 , OX347 . Multiple (≥3) extrachromosomal transgenic lines of Punc-25::ebp-2::gfp, Punc-25::rho-1(G14V) and Punc-25::rho-1(T19N) were analyzed with similar effect, and one was chosen for integration and further analysis.Experiments were performed at 20°C using standard chniques . Mutationjection  and inclwild-type, and average of 16 of the 19 commissures of the VD/DD axons are distinguishable, as commissural axons sometimes run together as a bundle and cannot be resolved. For these experiments, 100 animals were scored for an average total of 1600 axons. In unc-5 mutants, another guidance metric was used. “% failure to cross lateral midline” were axons that failed to extend dorsally past the lateral midline. Significance of difference was determined by Fisher’s Exact Test.In VD growth cones were imaged and quantified as previously described . BrieflyC. elegans [The F-actin binding domain of VAB-10/spectraplakin fused to GFP has been used to monitor F-actin in  elegans , 69. We  elegans . To contC. elegans cells including neurons [unc-25 promoter driving expression of ebp-2::gfp in the VD/DD neurons. In growth cones, a faint fluorescence was observed throughout the growth cone, resembling a soluble GFP and allowing for the growth cone perimeter to be defined. In addition to this faint, uniform fluorescence, brighter puncta of EBP-2::GFP were observed that resembled the EBP-1::GFP puncta described in other cells and neurons. For each growth cone, the perimeter and filopodia were defined, and the EBP-2::GFP puncta in the growth cone were counted. For each genotype, the puncta number for many growth cones (≥25 unless otherwise noted) was determined. Puncta number displayed high variability within and between genotypes, so box-and-whiskers plots (Graphpad Prism) were used to accurately depict this variation. The grey boxes represent the upper and lower quartiles of the data set, and the “whiskers” represent the high and low values. Dots represent major outliers. Significance of difference was determined by ANOVA.EBP-2::GFP has previously been used to monitor microtubule plus ends in other  neurons –72. We crho-1 coding region was amplified by PCR and inserted behind the unc-25 promoter in a plasmid (primer and plasmid sequences available upon request). A “sense” and “antisense” orientation relative to the unc-25 promoter was isolated. An equimolar mixture of the sense and antisense plasmids was used to construct transgenic animals. These transgenic animals were predicted to express both sense and antisense RNAs driven by the unc-25 promoter in the VD/DD motor neurons, which was expected to trigger a double-stranded RNA response in these cells (RNAi).We used a cell-specific transgenic RNAi approach as described previously . Fragmen


Here, we performed a whole-transcriptome study of the influence of synthetic snoRNA analogs with various modifications on gene expression in human cells. Moreover, we confirmed the role of PKR in the recognition of snoRNA and snRNA analogs using the short hairpin RNA (shRNA) technique. We believe that the data obtained will contribute to the understanding of the role of nucleotide modification in ncRNA functions, and can be useful for creating the agents for gene regulation based on the structure of natural snoRNAs and snRNAs.Short nuclear regulatory RNAs play a key role in the main stages of maturation of the precursors of the major RNA species. Small nuclear RNAs (snRNAs) form the core of the spliceosome and are responsible for the splicing of pre-mRNA molecules. Small nucleolar RNAs (snoRNAs) direct post-transcriptional modification of pre-rRNAs. A promising strategy for the development of non-coding RNA (ncRNAs) mimicking molecules is the introduction of modified nucleotides, which are normally present in natural ncRNAs, into the structure of synthetic RNAs. We have created a set of snoRNAs and snRNA analogs and studied the effect of base modifications, specifically, pseudouridine (Ψ) and 5-methylcytidine (m O-methylation, while box H/ACA snoRNAs are responsible for pseudouridylation of rRNA nucleotides that form functional active centers of the ribosome ppp[5′]G trimethylated cap analog  . To obtain RNAs with partial substitution of a modified nucleotide for its canonical equivalent, the transcription reaction was performed in a reaction mix in which the ratio of one particular modified NTP relative to the corresponding unmodified NTP was 20% (m5CTP), 30% (ΨTP), or 50% (both m5CTP and ΨTP).Artificial RNAs were synthesized by in vitro transcription with T7 RNA polymerase  in the reaction mixture containing 2.5 ng/μL of purified PCR product, 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2, 10 mM Tris-HCl (pH 7.5), and 0.1 mM CaCl2. Finally, RNA transcripts were purified via ion-pair reversed-phase high-performance liquid chromatography  (pH 7.5); eluent B — 0.05 M solution of TEAA in a 20% solution of acetonitrile (pH 7.5)) on a Milichrome A-02 liquid chromatograph  with a multi-wavelength UV-detector using a 2.0 × 7.5 mm column with the ProntoSIL-120-5-C18 sorbent (Econova) and precipitated by ethanol with 0.6 M NaOAc. RNA concentrations were determined spectrophotometrically on Nanodrop (Thermo Fisher Scientific) and verified using “Qubit” fluorometer with an RNA BR Assay Kit (Thermo Fisher Scientific). RNA integrity was verified on 2.5% agarose or 8% denaturing polyacrylamide gels using visualization by SYBR Green (Thermo Fisher Scientific) or 5′-[32P]-label . Synthetic RNAs were stored in diethylpyrocarbonate (DEPC)-treated water solution at −70 °C. RNA sequences were verified with reverse transcription and subsequent Sanger cDNA sequencing, with the product being analyzed by SB RAS Genomics Core Facility (ICBFM SB RAS). The presence and amount of modified monomers were verified by HPLC-MS/MS and HPLC-UV of nucleosides after enzymatic digestion of artificial RNAs . The synthetic RNA solutions were treated with DNase I (Thermo Fisher Scientific) and alkaline phosphatase (Thermo Fisher Scientific) to remove DNA templates and 5′-triphosphate moieties. The ncRNA analogs were isolated with phenol/chloroform extraction using Lira Reagent (Biolabmix). To obtain snoRNA and snRNA analogs with 5′-monophosphate , artificial RNAs were treated with the 20 U T4 polynucleotide kinase (Biosan) in the buffer containing 0.1 mM ATP, 10 mM DTT, 2.5 mM MgClial RNAs .5CTP and 30% ΨTP contained 13–15% m5C and 25–28% Ψ; analogs obtained in the mixture with 50% m5CTP and 50% ΨTP contained 50–52% m5C and 52–54% Ψ.Synthetic RNA analogs (1.0–5.0 µg) were digested with 0.65 mg/mL of ribonuclease A (Thermo Fisher Scientific), 4.0 µg/mL nucleases P1 , 0.2 mg/mL phosphodiesterase I (Sigma-Aldrich) and 2 U alkaline phosphatase (Thermo Fisher Scientific) per 1 µg RNA in 10 mM Tris-HCl (pH 8.0), 5.0 mM MgCl2, 0.1 M KCl, and 0.02% Triton X-100, 0.1 mg/mL BSA at 37 °C for 5–20 h. The deactivation of enzymes was carried out by heating at 75 °C for 10 min and chloroform extraction. The aqueous phase containing nucleosides was collected and was dried in Eppendorf vacuum concentrator 5301 . The pellet was dissolved in DEPC-treated water. Samples were analyzed with HPLC-UV  on a Milichrome A-02 liquid chromatograph (Econova) or HPLC-MS/MS (as described below). Control nucleosides were obtained from corresponding nucleoside triphosphates (Jena Bioscience) under the same conditions. Using HPLC-UV, it was found that synthetic RNAs obtained in the mixture with 20% mm/z of the transmitted parent ion, m/z of the monitored product ions for modified nucleosides was: Ψ, 1.7 min, m/z 245→125; m5C, 2.5 min, m/z 258→126. The identities of individual nucleosides were established by comparison with standards obtained (as described above) and using literature data [During HPLC-MS/MS, nucleosides were resolved on an Eksigent ekspert microLC 200 system  with a Zorbax 300 SB-C18 (4.6 mm ID × 150 mm) column  by elution with the following gradient of acetonitrile in water: 0.2 mL/min flow rate, 40 °C, eluent A—water, eluent B—acetonitrile: 100% A for 2 min, 1–20% B for 2–6 min, 20–30% B for 6–8 min. The HPLC column was coupled to an AB Sciex 3200QTRAP mass spectrometer  with an electrospray ionization source. The ions were detected in positive ion mode. The retention time, ure data ,45. The l-glutamine, and 40 μg/mL gentamicin (Sigma-Aldrich). RNA transfections were carried out with Lipofectamine RNAiMAX (Invitrogen) cationic lipid delivery vehicles. For Lipofectamine RNAiMAX transfections, the required amount of synthetic RNAs  and 1.5 μL reagent per 1 mL of culture medium were first pooled and then diluted in 15 μL of PBS. This mixture was incubated for 15 min at room temperature before being dispensed to the culture medium. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 24 h. At time points of 24 h, total RNA was isolated from the cells using Lira Reagent (Biolabmix) according to the manufacturer’s protocol. The accumulation of synthetic RNAs into cells was confirmed by RT-PCR using specific primers and BioMaster RT-PCR SYBR Blue (Biolabmix).Human breast adenocarcinoma cells, MCF-7, obtained from Russian Cell Culture Collection of Vertebrates  were cultured in Iscove’s Modified Dulbecco’s Media (IMDM)  supplemented with 10% fetal bovine serum (FBS) , 10 mM p-value for multiple-testing q < 0.05) were applied to the gene enrichment analysis powered by the Enrichr platform [Whole transcriptome analysis with sequencing was performed using polyA fraction of total cellular RNA on an Illumina HiSeq 1500 platform by Genoanalytica Company . Sequencing data (fastq formatted reads) were applied to the “classic” RNA-Seq workflow, which includes read mapping with TopHat (hg19), and assembly with Cufflinks (RefSeq). The comparison of expression levels of genes and transcripts in RNA-Seq experiments was carried out with CuffDiff . The lisplatform .qRT-PCR reaction was performed with BioMaster RT-PCR SYBR Blue  by LightCycler 96 System . The primers used in the study are presented in the Thermal denaturation experiments of 1.0 μM concentration of RNA were carried out in PBS buffer (pH 7.0) using Cary 300 Bio  spectrophotometer. Heating and cooling of the samples were performed in the range 5–95 °C, with the temperature change rate of 1 °C/min. Absorbance at 260, 270, and 300 nm was measured. Absorbance at 300 nm was used as a baseline and was subtracted from the two other denaturation curves . The mel3 cells were plated onto 96-well plates and incubated at 37 °C with 5% CO2. After 24 h, the cells were transfected with 10 nM artificial RNAs (complexed with Lipofectamine RNAiMAX). After incubation for 24 h, MTT -2,5-diphenyl-2H-tetrazolium bromide) (Sigma-Aldrich) was added to the cells at a final concentration of 0.5 mg/mL and the plates were incubated at 37 °C for 3 h. The medium was removed, followed by the addition of 0.15 mL of DMSO to each well. The plates were read at 570 and 620 nm using an Apollo LB912 plate reader . Cell viability was determined as the absorbance at 570 nm with reference to 620 nm and expressed as a percentage of control (control cells were incubated with Lipofectamine RNAiMAX only) ±SD for triplicate independent experiments.To estimate the influence of the synthetic RNAs on MCF-7 human adenocarcinoma cell viability and proliferation we used MTT assay. Aliquots (150 mL) containing 2.5 × 10The proliferation rate and survival of A549 human cells with shRNA-mediated PKR, RIG-I, and MDA5 knockdown as well as cells expressing scrambled shRNA (scr-shRNA), under snoRNA and snRNA analogs transfection were analyzed using the iCELLigence RTCA System . Cells were plated in 8-well E-plates (ACEA Bioscience) at a density of 5000 cells per well in a total volume of 200 µL of IMDM, and were monitored in real-time mode . After tt-test (data are presented as means ± SD). Differences were considered statistically significant at p-value < 0.05.The statistical significance of the differences in experiments was determined using the two-tailed Student’s 5C, as well as in the structure of the 5′-terminal nucleotide, namely, the synthetic RNAs bore 5′-OH, 5′-monophosphate, or trimethylated cap  U25 snoRNA analogs without modifications and analogs modified by complete substitution of Ψ for U or mp-value < 0.05, Fold change ≥ 4) in MCF-7 cells , respectively. As demonstrated in p-value ≤ 5.0 × 10−10, see p-value = 3.93 × 10−4; adj. p-value = 3.62 × 10−2) and IRF3  transcription factors. Interestingly, the group containing 43 genes specific for the non-modified synthetic RNA transfection was shown to be regulated by two factors mentioned above and, in addition, by NF-κB1  and HDAC2 , suggesting the possibility that the epigenetic mechanisms of gene regulation were involved after transfection. Moreover, the sets of genes activated by each of analogs contained predicted targets of miR-146a-5p , which is known as a negative regulator of innate immune activation  to 27-fold (m5C) and 18-fold (Ψ); the expression of IFIT3 decreased from 35-fold (NM) to 25-fold (m5C) and 13-fold (Ψ). Even though the modified analogs caused a similar effect, it is evident that complete substitution of Ψ for U resulted in the most significant effect on suppression of the cellular innate immune response.Together with a decrease in the total number of activated genes, a diminution of the individual activation level of the key genes involved in the innate immune response was observed after using modified U25 RNA analogs. The differences in the gene expression were confirmed by qRT-PCR . The mos5C and Ψ modifications to decrease the immune-stimulating activity of synthetic ncRNAs, we obtained modified analogs of human U12 snRNA and U35a snoRNA with additional trimethylated m32,2,7G cap similar to those in native ncRNAs [5C, respectively. We showed that the differences in IFIT3 and OAS1 activation observed between the intact and modified RNAs were more significant in the case of these U35a and U12 analogs, which were longer than U25 RNA analogs , RIG-I, and MDA5 knockdown  were observed after 16 h of transfection with unmodified analogs, although modification with Ψ delayed the same effect to 30 and 24 h for U35a and U12 analogs, respectively.It was found that knockdown of To clarify the structural effects caused by modified nucleotides, we studied the thermodynamic stability of U35a snoRNA and U12 snRNA analogs. First, using the ViennaRNA Package 2.0 , we simuSynthetic RNAs are known to activate the pathways of the innate immune response upon transfection into human cells . Activat5C-modified U25 snoRNA analogs. As expected, the strongest effect was caused by the analog without modifications, while m5C and Ψ substitutions led to a decrease in the interferon-dependent cellular response. We found that observed gene expression changes induced by each of three types of U25 RNA analogs were regulated by interferon-dependent IRF9 and IRF3 transcription factors. Specifically, the group of 43 genes, which were upregulated in the cells transfected with the unmodified U25 analog, included an additional transcription factor, NF-kB. Analysis of known activation mechanisms involved in immune-stimulating pathways allowed us to propose that modifications mainly affected RNA interaction with RIG-I and PKR. To better understand the mechanisms of suppression of the innate immune response, we synthesized m32,2,7G-capped, Ψ-, and/or m5C-modified analogs of other short ncRNAs, namely, U35a snoRNA and U12 snRNA. U35a was selected from the list of the box C/D snoRNAs that mediated the metabolic stress response after entering the cytoplasm induced by saturated free fatty acids. We assumed the possibility of transferring the function of this snoRNA not only to the cytoplasm, but also outside the cell [5C/Ψ-modifications, while TLR3 is still activated by a modified RNA. Considering our results of thermal denaturation experiments, we can speculate that the main role of RNA modifications in decreasing the immune response is connected with the significant increase in the thermodynamic stability of secondary structure elements of synthetic RNAs and, as a consequence, impaired rearrangements in the RNA-receptor complex that are necessary for the activation of signal transduction through the adapter proteins. In support of this hypothesis, a recent study has demonstrated that Ψ modification inhibits binding of minimally structured RNA to specific proteins due to the reduced RNA flexibility [PKR in the recognition of snoRNA and snRNA analogs. We found that the suppression of PKR expression significantly reduced the cytotoxic effect of unmodified RNA. Modification of ncRNA analogs, especially the simultaneous complete substitution of Ψ for U and the capping, allowed minimization of the antiproliferative effect and the difference between control cells and the PKRkd line, showing that these modifications were able to inhibit PKR-dependent pathways. Downregulation of MDA5 and RIG-I genes was not found to significantly change the sensitivity of human cells. Our data are in good agreement with general concepts that RIG-I and MDA5 recognize RNAs with 5′-triphosphate and dsRNAs, respectively, and with a recent work describing the interaction of some snoRNAs with PKR [We observed activation of the innate immune response in human cells upon transfection with non-modified and Ψ- or mthe cell . U12 snRthe cell ,63. At txibility . Using twith PKR ,42,59. OOn the one hand, our data might reveal the role of minor nucleotides in native small ncRNAs. On the other hand, the results can be used in the development of artificial regulators of gene expression based on the structure of short regulatory RNAs. Recent data indicated that RNA modifications could serve as regulators that tune the intracellular transport and guide the metabolism of the nuclear forms of short ncRNAs, for example, the release of these RNAs into the cytoplasm without activation of the innate immune response. Interestingly, Ψ sites have been found in many snoRNAs with the help of high-throughput sequencing ,65,66. BRegarding the gene therapeutic relevance, it should be noted that activation of the innate immune response is usually considered as an undesirable side effect. However, immune-stimulating properties, as well as off-target mechanisms of si/shRNAs, may also have therapeutic implications and can be used in combination therapy ,76,77,785C nucleotides provided a reduction in the level of non-specific immune activation. Moreover, we showed that PKR can be activated not only by unmodified snoRNAs and snRNAs, but also by modified RNAs without a cap. Our data can be useful in the development of therapeutically relevant RNA agents for gene regulation based on the structure of snoRNA and snRNA species.We have demonstrated that snoRNA and snRNA analogs activate a significant innate immune response upon transfection into human cells, while incorporation of Ψ and m


Integration of proteomic data with functional and imaging analysis revealed that Rab1b and ARF5, two ER-Golgi members, are RNA-binding proteins that colocalize with picornavirus IRES-RNA reporters in human cells. Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES–binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment. Internal ribosome entry site (IRES) elements promote internal initiation of translation using cap-independent mechanisms . DespiteBeyond internal initiation of translation, little evidence for the involvement of the IRES in other steps of the viral cycle has been reported. A role for the poliovirus IRES in RNA encapsidation was reported based on the different genome stability and encapsidation efficiency of RNA replicons carrying chimeric IRES elements . Similartrans-Golgi independently of its GTP-binding state . Whereas Rab1b is a regulator of coat complex protein I (COPI) and COPII ER-Golgi transport pathway depending upon its GTP-binding state , ARF5 isng state . It is wng state . Yet, thng state .Beyond the identification of RNA-binding proteins by proteomic approaches, we have found that the IRES transcripts bind directly with purified Rab1b and ARF5, revealing a previously unknown RNA-binding capacity of these proteins. RNA-FISH studies showed that mRNA carrying the IRES element displayed a cluster arrangement relative to mRNA lacking the IRES. Remarkably, IRES-containing RNAs colocalized with Rab1b and ARF5 to a higher extent than cap-RNA. However, in support of the different role in IRES-dependent translation, a dominant-negative form of Rab1b decreased IRES function, whereas ARF5 silencing stimulated IRES activity. In sum, our data show that both ARF5 and Rab1b exhibit RNA-binding capacity, suggesting a role for domain 3 of the IRES in RNA localization into specific cellular compartments.The large size of the picornavirus IRES region (450 nt) compared with other IRES elements prompted us to investigate whether this RNA region harbors motifs involved in additional RNA life steps, overlapping with internal initiation of translation. The FMDV IRES element is organized in domains, designated 1, 2, 3, 4, and 5 . The cenTo obtain transcripts with stabilized secondary structure, cDNAs were inserted into pBSMrnaStrep vector , which aDataset 1). Only factors identified in both replicates with more than two peptides (FDR < 0.01) were considered for computational studies (R2 ≥ 0.81) (Dataset 1). To eliminate potential false positives, the factors associated with the control RNA were subtracted from the overlap of the biological replicates identified with each subdomain. Following application of these stringent filters, the number of proteins remaining with SL3a was 156, 143 for SL3abc, 214 for SL123, and 158 for D3 (Dataset 1). Representation of these data in a Venn diagram (bioinfogp.cnb.csic.es/tools/venny/) revealed that the number of proteins specific for each subdomain was higher than those shared among transcripts  . The avenscripts . Further10 score of proteins bound to subdomain SL3a against SL3abc . Of interest, overrepresentation of the ER-Golgi transport network was statistically significant in all transcripts, ranging from P = 3 × 10−4 to 5 × 10−5 . Beyond proteins known as IRES-binding factors , ribosomal proteins (RPS25), and translation factors (eIF3I), we also identified ER-Golgi transport factors  than SL3abc (Kd ∼ 2.16 nM)   and S4B.2.16 nM) , althoug2.16 nM)  to 3A. H2.16 nM)  and S4B,To further analyze the RNA-binding specificity of these proteins, we used a control probe differing in sequence and predicted secondary structure as a control. None of them yielded a band shift at the protein concentration used with the IRES transcripts . FurtherNext, we wished to compare the interaction of these factors to PCBP2 and Ebp1, two proteins known to interact with domain 3 , which wOverall, the RNA–protein binding results match with the proteomic identification . AccordiTo analyze the functional implication of these factors on IRES activity, we relied on siRNA-mediated approaches to reduce the cellular level of these proteins. Silencing of Rab1b did not alter relative IRES activity compared with a control siRNA using bicistronic constructs , or monoAs mentioned earlier, both Rab1b and ARF5 are members of the ER-Golgi network . AccordiP = 3.7 × 10−18)  . This reNext, considering the role of Rab1b in ER-Golgi transport , cells cThese data prompted us to analyze RNA juxtapositioning with GFP-Rab1b DN protein, which induced Golgi disorganization and decreased IRES activity . In contcis-Golgi, we analyzed the vicinity of IRES-luc RNA with ARF5, an integral member of trans-Golgi (P = 0.003). Manders’ coefficient M2 denoted a trend similar to M1 for all proteins cis-Golgins-Golgi , reinforproteins . TherefoTaken together, we conclude that both Rab1b and ARF5 are involved on the IRES-driven RNA localization on the ER-Golgi area of the cell cytoplasm, although they exert different effects. Rab1b stimulates translation, whereas ARF5 diminishes IRES-dependent translation.The data presented herein represents the first instance of the characterization of IRES interactions with ER-Golgi factors, reinforcing the importance of exploring novel RNA–protein interactions to understand host–pathogen interface. Here, we describe a robust RNA–protein interaction approach, which allows detecting ribonucleoprotein complexes associated with specific subdomains of the IRES element. In this manner, a number of RBPs were selected, including known IRES interacting factors , validatFollowing uncoating, the first intracellular step of picornavirus life cycle requires translation of the viral genome, which is governed by the IRES element. We hypothesize that interplay between host factors and viral RNA motifs could be an integral part of the regulation of viral RNA function, and as such, can be studied in the absence of infection. In accordance with this hypothesis, our data show that the IRES-containing mRNA exhibited a cluster arrangement, whereas the cap-luc RNA showed disperse punctate cytoplasmic location , suggestConcerning the implication of IRES subdomains in directing RNA to specific subcellular locations, we selected two factors involved in ER-Golgi trafficking, Rab1b and ARF5, for further characterization. Rab1b is a key regulatory protein involved in COPI and COPII transport , whereastrans-Golgi, hence interfering IRES-driven translation.The finding that Rab1b and ARF5 proteins interact directly with the IRES was unprecedented. No report of their RNA-binding capacity was available despite anterograde transport pathway participates in the life cycle of various RNA viruses, including picornaviruses and flaviviruses . In the Further supporting the notion that specific members of the anterograde transport pathway mediate IRES recognition, as shown here by Rab1b, several members of the anterograde and retrograde transport were identified in the proteomic approach , althougtrans-Golgi, hence interfering IRES-driven translation , SL3abc (nt 151–225), SL123 (nt 137–246), and D3 (nt 86–299) of the FMDV IRES  were genOligonucleotides.Table S1 32P-CTP (500 Ci/mmol). RNA integrity was examined in 6% acrylamide, 7 M urea denaturing gel electrophoresis. RNAs SL3a, SL3abc, SL123, D3, and the control RNA were isolated from fresh bacterial cell lysates, as described (In vitro transcription was performed as described . When neescribed . The intescribed .2), RNA (20 pmol) for 30 min at RT in a rotating wheel. The bead–RNA complexes were collected in the tube wall standing on the magnet 3 min. The supernatant was removed and followed by three washes with binding buffer to eliminate unbound RNA. The pellets were resuspended in 20 μl PBS before adding S10 HeLa cells protein extract (100 μg), 2 nM yeast tRNA, 1 mM DTT in binding buffer , and incubating for 30 min at RT in a rotating wheel. Aliquots (1%) were taken at time 0 as input samples. Beads were washed three times with five volumes of binding buffer and left for 5 min at RT. The proteins were eluted in SDS buffer and resolved by SDS–PAGE.Streptavidin-aptamer–tagged RNAs coupled to streptavidin-coated magnetic beads were used to purify proteins interacting with the IRES transcripts . BrieflyMass spectrometry (LC/MS–MS) was performed as described . Two indP values for the overrepresented nodes were used to compute the average statistical significance of the network.The Biological Networks Gene Ontology application (BiNGO) was used to assess the overrepresentation of proteins associated with SL3a, SL3abc, SL123, and D3 transcripts and to determine the statistical significance of overrepresented proteins relative to a complete human proteome . The resEscherichia coli BL21 transformed with plasmids pET-28aLIC-ARF5 (Addgene plasmid# 3557) and pPB-N-His-Rab1b (abm# PV033914) grown at 37°C were induced with IPTG and purified as described , Rab1b (GAUCCGAACCAUCGAGCUGUU), and a control sequence (siRNAcontrol AUGUAUUGGCCUGUAUUAGUU) were purchased from Dharmacon. HeLa cells were treated with 100 nM siRNA using lipofectamine 2000. Cell lysates were prepared 24 h post-transfection in 100 μl lysis buffer . The protein concentration in the lysate was determined by Bradford assay. Equal amounts of protein were loaded in SDS–PAGE to determine the efficiency of interference. Commercial antibodies were used to detect ARF5 (Abnova), Rab1b , and Tubulin (Sigma-Aldrich). Appropriate secondary antibodies were used according to the manufacturer instructions. Protein signals were visualized with ECL. Quantification of the signal detected was performed in the linear range of the antibodies.6 were resuspended in 37.5 mM NaCl, 10 mM Hepes, pH 8.0, before adding the plasmid of interest (5 μg) and salmon sperm DNA (20 μg). After the pulse, cells were plated on glass coverslips in six-well dishes, 0.3 × 106 cells/well, in 2 ml DMEM supplemented with FCS 10%. 30 h after transfection, the cells were fixed for 10 min in 4% methanol-free formaldehyde in PBS at RT. Cells were permeabilized for 10 min at RT in PBS containing 0.2% Triton X-100, followed by 30 min in 3% BSA, TBS. After blocking, the cells were incubated with antibodies diluted in PBS containing 1% FBS and 0.1 Triton X-100. Golgi was stained with anti-GM130 mouse polyclonal antibody (1:500) for 1 h at 37°C in a humidifying chamber. The cells were washed three times with PBS before incubation with the secondary antibody Alexa Fluor 555–conjugated donkey anti-mouse antibody (1:500) for 1 h in the dark at RT. The nucleus was stained with DAPI (1 μg/ml). Finally, the cells were washed three times with PBS, mounted on slides in Vectashield mounting medium and imaged.HeLa cells were electroporated using Gene Pulser Cuvette (0.4 cm) 200 V, 950 µFA and 480 Ω. Briefly, 4 × 10For imaging experiments, HeLa cells growing in coverslips were fixed 30 h post-electroporation for 10 min in 4% methanol-free formaldehyde in PBS at RT. Next, the cells were permeabilized for 10 min in PBS containing 0.3% Triton X-100 at RT in a humidifying chamber. Coverslips were transferred to 24-well dish with wash buffer . Washed cells were transferred to the humidifying chamber and incubated overnight in the dark with the RNA probe diluted in 2× SSC, 10% formamide, and 10% dextran sulphate at 37°C. The probe blend labeled with Quasar 570 dye targeting luciferase RNA (Stellaris RNA-FISH) was used (250 nM). Finally, the coverslips were washed twice with wash buffer, adding DAPI in the second wash. The samples were mounted on slides in Vectashield mounting medium and imaged. Transfections and hybridization assays comparing different constructs were conducted side by side.D [refractive index] = 1.518 [23°C]) using a 14-bit Hamamatsu 9100-02 EM-CCD High Speed Set cooled CCD camera (Hamamatsu Photonics) with Metamorph 7.10.1.16 (Molecular Devices) image acquisition software. The following filter sets were used: DAPI for detection of DAPI, GFP for detection of GFP, and TRITC for detection of Quasar 570 Dye. Each Z-slice was exposed for 20–50 ms, except for Quasar 570, which required 2 s. After deconvolution from about 60 z-sections, 0.3 μm spacing, the images were analyzed by local background subtraction and thresholding using Huygens Software (Scientific Volume Imaging). Each Z-series was collapsed and rendered as a single max-intensity projected image using ImageJ v1.51u  and channel 2 for green (GFP proteins). The region of interest was analyzed using Coloc 2 plugin and Manders’ correlation coefficient  as folloP values for different distribution between two samples with the unpaired two-tailed t test. Differences were considered significant when P < 0.05. The resulting P values were graphically illustrated in figures with asterisks.We computed


MicroRNAs hold great promise as biomarkers of disease. However, there are few efficient and robust methods for measuring microRNAs from low input samples. Here, we develop a high-throughput sequencing protocol that efficiently captures small RNAs while minimizing inherent biases associated with library production. The protocol is based on early barcoding such that all downstream manipulations can be performed on a pool of many samples thereby reducing reagent usage and workload. We show that the optimization of adapter concentrations along with the addition of nucleotide modifications and random nucleotides increases the efficiency of small RNA capture. We further show, using unique molecular identifiers, that stochastic capture of low input RNA rather than PCR amplification influences the biased quantitation of intermediately and lowly expressed microRNAs. Our improved method allows the processing of tens to hundreds of samples simultaneously while retaining high efficiency quantitation of microRNAs in low input samples from tissues or bodily fluids. Sequencing provides an unbiased approach to miRNA biomarker discovery unlike qPCR or array analysis, which depend on validated probes and/or primers5. Until recently, cost was a substantial hurdle to large-scale screens by sequencing. That barrier has come down with increases in sequencing efficiency and the ability to multiplex samples. Now, it has become reasonable to sequence thousands of patient samples. However, accuracy and reproducibility remain major issues. The reasons for these issues are not entirely clear, but likely lie at the level of sample collection, RNA isolation, and library production.MicroRNAs (miRNAs) play a critical role in regulating gene expression in mammals. They have been found not only in tissue biopsies, but also in bodily fluids. A number of studies have pointed to miRNAs as biomarkers with potential utility in diagnosis, prognosis and treatment9. All small RNA library production protocols involve the addition of an adapter or linker to each end of the RNA fragment of interest. The 3′ adapter enables reverse transcription, and together with the 5′ adapter, DNA amplification of the library as a whole. The 5′ adapter also provides the touch down sequence for the sequencing primer. 5′ and 3′ adapter ligation can lead to sequencing bias for specific miRNAs. In particular, the RNA ligases used for adapter ligation exhibit both sequence and structural preferences11. The addition of a barcode in either adapter can worsen this problem11. A common solution to the barcode contribution to ligation bias is to introduce it at the PCR step12. However, adding the barcode at this later step requires processing all samples individually all the way through the PCR amplification step, making the processing of large sample sets costly and tedious. PCR bias is yet another concern given that some sequences can be more efficiently amplified than others. For example GC content can effect PCR amplification14. The addition of unique molecular identifiers (UMIs) can address this issue, but are rarely used in small RNA sequencing17.Major issues plaguing small RNA library production include adapter ligation bias, adapter dimer contamination, PCR amplification bias and barcode bias18. This many-to-one sample handling strategy allows for the processing of dozens to even hundreds of samples simultaneously. However, the barcode and fixed adapter ends introduces a high risk of ligation bias and, therefore, we introduced optimizations to reduce this bias. Also, we optimized methods to reduce contamination of libraries with self-ligated adapters, significantly enhancing the number of usable reads following sequencing. Finally, by introducing unique molecular identifiers (UMIs), we show that preferential PCR amplification of specific miRNA does not appear to be a significant contributor to miRNA bias. Instead, stochastic capture of miRNAs at very low input concentrations requiring high PCR cycle numbers does lead to poor and irreproducible calling of miRNA expression levels, especially of intermediately and lowly expressed miRNAs. Together, our optimized high-throughput method of library production results in the highly efficient and reproducible capture of small RNAs for downstream sequencing.Here, we aimed to optimize a sequencing library strategy that is high throughput yet minimizes ligation and PCR bias. We started with a method originally described by Tuschl and colleagues, which introduces a barcode into the 3′ adapter allowing pooling of samples after the very first step of library production18, we included two alterations that have been shown to improve ligation efficiency in other protocols  in all three biological samples; Plasma 2, Plasma 3 and Cell1 Fig. , again dExamination of the miRNAs in the high input samples revealed that the optimized protocol identified over a hundred more miRNA species than the original protocol in each case . Thus, reduction of adapter contamination for low input RNA samples vastly improves quality of resulting small RNA libraries.21. The inclusion of random nucleotides at the ligated ends of the adapters allowed us to evaluate this bias, including which nucleotides and nucleotide positions within ligated adapters have the greatest influence on ligation bias for any particular miRNA. When averaged across all miRNAs, there was little evidence for a strong nucleotide bias at any position , we asked whether differential PCR amplification exists and if so, how great is its impact. First, we evaluated whether the 12 random nucleotides provided a great enough diversity of sequences to cover the potential number of input miRNAs at our sequencing depth of over four million reads. If all positions are represented equally there are over 16 million combinations of the four bases at twelve positions, over 65 thousand at eight positions and 256 combinations at four positions. However, ligation bias reduces these numbers. Thus, to empirically determine how many random Ns are required to capture the full complement of input miRNAs, we evaluated the impact of increasing the number of random Ns on either side of the insert and collapsing resulting reads  or Agilent Bioanalyzer RNA 6000 Pico kit ( < 50 pg/μL). The 5′ and the 3′ adapters included four degenerate bases at the ligation site of each. In addition to the blocking modification already present on the 3′ adapter18, a blocking amino modification was added to the 5′ end of the 5′ adapter. The concentration of each adapter was then titrated using the low input plasma samples to minimize adapter dimer contamination. These changes increased the efficiency of miRNA capture in the library five-fold.There are few commercially available miRNA sequencing kits optimized for low-input RNA samples. The most commonly used kits  recommend 1 ug of total RNA input. Trilink Cleantaget al. recently showed that the addition of four degenerate bases to adapters of the TruSeq protocol greatly improved miRNA capture22. However, their library production strategy is based on the individual preparation of samples. Our use of the many to one sample approach greatly simplifies the building of libraries from large numbers of samples. In addition, it enables the combination of many low input samples.Unbiased miRNA capture across samples was achieved regardless of nucleotide preferences by particular miRNAs. Consistent with our findings, Giraldez Importantly, we show that increasing the number of PCR cycles to compensate for low sample input results in high duplication rates and loss of information for lowly and intermediately expressed species. Thus, combining multiple low input samples through early barcoding not only increases efficiency of library preparation, but also reduces artifacts associated with high PCR cycle number. A disadvantage of this pooled preparation is the potential for over-representation or under-representation of reads from a subset of samples. This can especially be the case when it is difficult to quantify and normalize RNA levels between samples prior to building of libraries. However, we have found it straightforward to rescue the under-represented samples, by repeating their sequencing in a separate pool with other lowly represented samples.Our optimizations allow for robust screening of miRNAs from low-input biofluids of tens to hundreds of patients simultaneously. These methods should allow for increasingly accurate quantification of miRNAs in blood and tissue samples enabling the discovery of novel biomarkers of disease. Future improvements in RNA extraction and size selection methods will further enhance the utility of this method for small RNA biomarker discovery.Blood samples were collected, and plasma prepared by standard clinical protocol between 2015 and 2016. Informed consent was obtained from all subjects. All samples were de-identified therefore this study was exempt from review by the UCSF Committee on Human Research.Cellular RNA was collected from PC3 cells that were obtained from ATCC (ATCC® CRL-1435) and cultured following recommended conditions. RNA was prepared by lysing the cells in Trizol followed by purification using the miRNeasy kit (Qiagen 217004) Biological replicates of PC3 extractions are referred to as Cell1, Cell2. Concentrations were determined by nanodrop (Thermo Fisher Scientific). Low-input RNA was collected from human plasma that was collected following standard clinical procedures using EDTA-coated tubes (Becton Dickinson). The RNA was prepared by thawing the plasma on ice, then following the micro miRNeasy kit (Qiagen 217084) with the change of adding 5x the volume of QIAzol lysis reagent to the plasma and using a final elution volume of 7 μl of RNase/DNase free water per 200 μl input plasma. Nanodrop One (Thermo Fisher Scientific), Bioanalyzer RNA 6000 Pico kit (Agilent) or Qubit (Life Technologies) analysis was employed to assess RNA quantity and quality. RNA from 200 μl of plasma was inconsistent or undetectable by any method. Biological Replicates of plasma samples from independent blood draws of healthy volunteers are referred to as Plasma1, Plasma2 and Plasma3.et al. . Input RNA for the cellular sample was 500 ng total RNA while input for the plasma samples was total RNA from 200 μl plasma unless otherwise noted. Sequencing adapters were synthesized by Integrated DNA Technologies using the codes provided in SupplementaryTable 2.xlsx. 3′ adapters were polyadenylated as described in the detailed protocol available in supplementary methods. The adapter concentrations were titrated as noted in the figure legends. 3′ adapters were used at 0.05, 0.2 or 0.85 μΜ while 5′adapters were added at 0.33, 1.0 or 3.3 μΜ. The experiments using the optimal conditions were done using 0.05 μΜ 3′ adapter, 0.33 μΜ amino-modified 5′ adapter. Fragments of the correct size were PAGE purified after each ligation step and after the PCR amplification. Unless otherwise noted, PCR was performed for 14 cycles. The 14-cycle and 24-cycle libraries presented in Fig. Small RNA-seq libraries were made following the general workflow of Hafner al. Fig. 18. Input24 to demultiplex and trim adapters, sequences were then aligned using STAR (params = ‘–genomeLoad NoSharedMemory–outReadsUnmapped Fastx–outSAMtype BAM SortedByCoordinate–outSAMunmapped None–outFilterMismatchNmax 0.05–outFilterMatchNmin 16–outFilterScoreMinOverLread 0–outFilterMatchNminOverLread 0–outFilterMultimapScoreRange 0–outFilterMultimapNmax 1–limitBAMsortRAM 1044427000’) or Bowtie v1.1.2 (-n 0 -l 18 –best) to a mature miRNA and other small RNA genomes were downloaded from miRbase26, and the human genome index curated by Illumina for bowtie or the comprehensive GENCODE annotations (Release 27 (GRCh38.p10) at https://www.gencodegenes.org/releases/current.html) for STAR. Reads were mapped sequentially to first remove all sequences inherent to library production , then mature miRNA, other small RNA, finally to the human genome. The remaining reads were labeled “Unmapped”. Analyses were performed and plots generated using custom R scripts in R studio28 and the tidyverse, forcats, ggseqlogo packages that are available for download on the CRAN website, https://cran.r-project.org. miRNA count data available in SupplementaryTable 1.xlsx; all data has been submitted to the GEO repository (GSE109162). Scripts are available upon request.The miRNA-Seq analysis data were preprocessed using CutAdapt v1.8Supplementary InformationDataset 1Dataset 2


Persicaria minor, to identify cleavage sites in small RNA libraries in control (mock-inoculated) and Fusarium oxysporum treated plants. The degradome library consisted of both control and treated samples which were pooled together during library preparation and named as D4. The D4 dataset have been deposited at GenBank under accession number SRX3921398, https://www.ncbi.nlm.nih.gov/sra/SRX3921398.Degradome sequencing referred as parallel analysis of RNA ends (PARE) by modifying 5′-rapid amplification of cDNA ends (RACE) with deep sequencing method. Deep sequencing of 5’ products allow the determination of cleavage sites through the mapping of degradome fragments against small RNAs (miRNA or siRNA) on a large scale. Here, we carried out degradome sequencing in medicinal plant,  Specifications TableValue of data•Persicaria minor, where the genome information still unavailable.This data is the first degradome library in •Degradome technique is the latest technology for determination of cleavage site of small RNA.•Identification of miRNA-target interaction is vital in understanding the biology of the miRNA regulatory mechanism.1The small RNA data in the article 22.1P. minor explants were cultured and grown as mentioned in the previous work P. minor plants that consist of mock-inoculated and Fusarium-treated were prepared. The fungus treatment was carried out as mentioned in 2.2Plant RNA Reagent  were used to isolate total RNA from both samples according to recommended protocol. Then, both RNA samples were pooled together in one tube and named as D4. Quality control and library preparation for D4 was carried out according to previous report 2.3https://sourceforge.net/projects/skewer) Trimming of adapter index sequences were carried out using Skewer software (


To replicate and disseminate, viruses need to manipulate and modify the cellular machinery for their own benefit. We are interested in translation, which is one of the key steps of gene expression and viruses that have developed several strategies to hijack the ribosomal complex. The type 1 human immunodeficiency virus is a good paradigm to understand the great diversity of translational control. Indeed, scanning, leaky scanning, internal ribosome entry sites, and adenosine methylation are used by ribosomes to translate spliced and unspliced HIV-1 mRNAs, and some require specific cellular factors, such as the DDX3 helicase, that mediate mRNA export and translation. In addition, some viral and cellular proteins, including the HIV-1 Tat protein, also regulate protein synthesis through targeting the protein kinase PKR, which once activated, is able to phosphorylate the eukaryotic translation initiation factor eIF2α, which results in the inhibition of cellular mRNAs translation. Finally, the infection alters the integrity of several cellular proteins, including initiation factors, that directly or indirectly regulates translation events. In this review, we will provide a global overview of the current situation of how the HIV-1 mRNAs interact with the host cellular environment to produce viral proteins. Flexibility and adaptability are both essentials when facing difficult situations; such an assumption retains all its meaning when it comes to the viral kingdom. Indeed, viruses, unlike other pathogens, such as bacteria, require a host to replicate and spread. One of the key steps of the viral cycle, which is also completely dependent on the host, is protein synthesis and always occurs with cellular ribosomes. In a very interesting way, the host’s innate immune response, the first line of defense against viral infection, targets the actors of translation to stop protein synthesis. In addition, most of viral messenger RNAs (mRNAs) contain many different RNA motifs that are essential for replication but are not compatible with a conventional mechanism of translation. Therefore, viruses have to modify and adapt to produce proteins by implementing different strategies to circumvent the host cell’s defenses and the RNA barriers.Lentivirus genus from the Retroviridae family and the etiologic agent of the acquired immunodeficiency syndrome. It is a good representative of this viral adaptability with more than 50 viral RNA transcripts produced by alternative splicing [The type 1 human immunodeficiency virus (HIV-1) is a member of the splicing ,3 that asplicing .Through this review, we will first and briefly describe the mechanisms by which some viral elements intervene to suppress activation of the first line of defense against viral infections at the translational level. Next, we will see that alternative splicing of the primary transcript generates a multitude of mRNAs that contribute to regulate the translational efficiency. Lastly, we will focus on the unspliced genomic RNA. Indeed, translation of this transcript can use both a classical translation initiation mechanism and internal recruitment of ribosomes in order to adapt to the cellular modifications imposed by infection.In higher eukaryotes, translation initiation requires association of the mRNA to the ribosomal subunits. This event is orchestrated in a timely manner by a set of proteins that allows progression through the different stages. Initiation begins with the attachment of the small 40S ribosomal subunit with associated factors to the mRNA and progression of the ribosome in a 5′ to 3′ direction to find and recognize the initiation codon. The elongation stage allows peptide bond formation in the ribosomal peptidyl transferase center between incoming amino acids and the nascent peptide until ribosomes reach a stop codon. This codon arrests protein synthesis and induces the release of the neo-synthesized peptide. Recycling of the ribosomal subunits constitutes the last and final step of protein synthesis. The initiation step is complex and highly regulated by at least 12 eukaryotic initiation factors (eIFs), which catalyze several processes such as the binding of the initiator methyonyl-transfert RNA (Met-tRNAi) to the small ribosomal subunit, ribosomal recruitment at the 5′end, ribosomal scanning, and initiation codon recognition. Translation efficiency is modulated by both RNA structures, RNA modifications, and by the interplay with eIFs and a large number of cellular proteins. Each mRNA is unique in its folding and modifications, which requires an adaptation of ribosome recruitment to these characteristics. Thus, it does not come as a surprise that translation initiation can be mediated by several different mechanisms. [All eukaryotic mRNAs contain an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5′ to 5′ triphosphate linkage called the cap structure, which is required for mRNA processing and nuclear export, but also for stability and translation efficiency .Most mRNAs initiate translation via a cap dependent and scanning mechanism , which sAUGG (referenced as the “Kozak sequence”) has been reported to favor start codon recognition [Initiation generally occurs at an AUG codon, which is recognized by the Met-tRNAi with perfect codon/anticodon annealing. The Met-tRNAi has a high affinity for eIF2●GTP and forms the ternary complex (TC). TC binding to the 40S subunit is strongly enhanced by eIF1, eIF1A, and eIF3 to form the 43S pre-initiation complex (PIC). eIF3, which is the largest of all initiation factors, is composed of 13 subunits and interacts with eIF4G to bridge the 43S PIC to the mRNA cap-binding complex ,12,13. Oognition ,17. Onceognition ,18,19.Most of the time, the first AUG codon encountered during ribosomal scanning of the 5′ UTR is used to initiate translation. However, the efficiency of codon selection depends on several parameters. Hence, the nucleotides sequence surrounding the initiation codon, called the “Kozak context” ,17, playPicornaviradae, Flaviviradae, Dicistroviridae, and Retroviridae [IRESes have been first identified in the genomic RNA of two members of the picornaviral family in the late eighties ) ,26. Picooviridae , and in oviridae . The mecoviridae ,33. IRES6A) on mRNA is a post-transcriptional modification mediated by a regulated interplay between m6A methyltransferases (named writer), demethylase (named eraser) and m6A-specific binding protein  [6A. This modification is reversible by at least two eraser proteins, which are FTO and ALKBH5 (for fat mass and obesity-associated and AlkB homolog 5 respectively) [6A. YTHDF1 is associated with a stimulation of the translation efficiency of its mRNA target while YTHDF2 promotes mRNA degradation [6A modification, enhances translation of the target mRNA [6A residues located in 3′ UTR promotes mRNA circularization, via an interaction YTHDF1/eIF3 or METTL3/eIF3, that enhances translation. N6-methyladenosine (m reader) . The met reader) ,47,48 foctively) ,50. Therctively) ,52 with ctively) . This occtively) , and conctively) . The rearadation . The mecradation . In a saget mRNA ,57. This6A residues in their 5′ UTR [6A-induced ribosome engagement site . A large part of these mechanisms of translation regulation occurs at the initiation step and focuses on two factors eIF2 and eIF4E. The activity of eIF2 is modulated by phosphorylation at serine 51 on the alpha subunit. As a consequence, the P-eIF2α binds with high affinity to the guanine nucleotide exchange factor eIF2B which reduces the recycling of inactive eIF2●GDP into active eIF2●GTP. The low amount of available and active eIF2●GTP in the cell inhibits the global level of translation ,61. SeveThe cap binding factor eIF4E is also regulated by phosphorylation or by sequestration by a family of small proteins that specifically bind to it and that are called 4E-binding proteins (4E-BP). Indeed, eIF4E binds to a protein motif that is common to both eIF4G and 4E-BP and the affinity of the interaction between eIF4E and 4E-BP is controlled by a series of phosphorylation events . Hypophotrans-activation response (TAR) element that plays a major role in stimulating the transcription of viral RNAs in the presence of trans-acting transcription (Tat) protein [The HIV-1 is a retrovirus that replicates via reverse transcription of its RNA genome into double-stranded DNA flanked by two long terminal repeats (LTRs). This proviral DNA is imported in the nucleus and integrated into the host genome. Then, all viral mRNAs are transcribed by the cellular type II RNA polymerase into capped and polyadenylated transcripts. As only one transcription start site exists on the HIV-1 genome, the ≈9700 bp primary viral transcript is alternatively spliced to generate a broad spectrum of viral mRNAs. All viral transcripts share the first 289 nucleotides (nt) located upstream of the 5′ major splice donor. The first 59 nt of HIV-1 form a very stable hairpin structure called the  protein . However protein ,70,71,72 protein ,70,71,72 protein  and the  protein ,75. Inte protein ,77. TRBP protein ) and TRB protein ,80,81. T protein . ADAR1 a protein ,84. For  protein ,86,87.Viral protein production depends on two main parameters: the intracytoplasmic concentration of viral mRNAs and their relative translational efficiency. The full-length unspliced genomic mRNA has two fates during viral replication: it serves as genomic RNA, which is encapsidated into new viral particles, and also plays the role of messenger RNA to produce the Gag and Gag-Pol polyproteins in the cytoplasm of the host cell. This ≈9700 bp primary viral transcript contains, at least, 5 donor and 8 acceptor sites used to generate more than 50 different transcripts  that canenv gene  and includes many structured RNA regions : (i) thetrans-activation of viral RNA transcription and therefore for viral replication. However, the presence of this hairpin structure at the 5′ end of the unspliced HIV-1 transcript is an unequivocal barrier to ribosome recruitment at the cap. Indeed, insertion of TAR at the 5′ end of a reporter construct is sufficient to suppress RNA expression in in vitro translation systems like rabbit reticulocyte lysate (RRL) or Xenopus oocytes [The TAR structure is essential for  oocytes ,113,114. oocytes . Thus, tThe La protein was originally identified as the major antigen for certain autoimmune diseases such as the systemic lupus erythematosus and the Sjögren’s syndrome . NuclearThe cellular protein TRBP was discovered for its ability to bind the HIV-1 TAR RNA  and to iStaufen1 is a dsRNA-binding protein involved in multiple post-transcriptional gene-regulatory processes such as nuclear export, alternative splicing , mRNA trThe RNA helicase A  is a highly conserved (Asp-Glu-Ala-Asp) DEAD-box protein involved in many different facets of cell metabolism such as RNA processing , innate 7GTP-sepharose column independently from eIF4E suggesting that the helicase could bind directly the 5′ cap structure of mRNAs [The DEAD-box polypeptide 3 (DDX3) is a cellular ATP dependent RNA helicase whose expression is stimulated by the production of the Tat protein . Meta-anof mRNAs ,142. AllIt is noteworthy that the proportion of active eIF4E is reduced during HIV-1 infection see , leadingThe nuclear cap binding complex (CBC) is composed of two cap binding proteins (CBP) CBP80 and CBP20 that interact with the 5′end of the mRNA into the nucleus and participate to the first round of mRNA translation in the cytoplasm . SucroseThe unspliced HIV-1 mRNA also interacts with the human peroxisome proliferator-activated receptor-interacting protein with methyltransferase domain (PIMT) that is able to hypermethylate the guanosine of the cap structure to a trimethylguanosine (TMG), which is associated with an increase of unspliced mRNA export and translation . This inTo resume, many cellular helicases promote translation of the unspliced mRNA but their relative requirement during infection remains unclear. Do they co-operate and are they temporally regulated? Once recruited and attached to the mRNA, the PIC is able to scan the 5′ UTR through the RNA structures as demonstrated by artificial insertion of AUG codons in the 5′ UTR . It was As we have seen, translation of the unspliced HIV-1 mRNA has to adapt to specific viral constraints such as (i) the export of unspliced transcripts via an alternative pathway, (ii) the presence of stable RNA structures at the 5 ‘end, and (iii) the requirement for non-canonical initiation factors. In addition, translation of the unspliced RNA takes place during the late stages of infection  when celStructural modeling by selective 2′-hydroxyl acylation analyzed via primer extension (SHAPE) of the entire HIV-1 unspliced mRNA has confirmed that the 5′ UTR was highly structured . AlthougThe virally encoded HIV-1 protease, which is required for processing of Gag and Gag-Pol polyproteins during maturation, can also modulate cellular and viral translation. In the course of viral infection, this protease can cleave some cellular proteins ,162,163 Another viral protein, Vpr, has also been involved in the control of HIV-1 translation. Vpr has pleiotropic functions during infection since it participates in reverse transcription and nuclear import, LTR transactivation, apoptosis, and cell cycle progression . Since 1Viral IRESs are generally long and structured RNA regions that can interact with PIC components; this is generally evidenced by the use of a bicistronic vector in which the putative IRES sequence is placed in the intercistronic region. Brasey and colleagues have demonstrated in HeLa cells that the 5′ UTR of the unspliced HIV-1 genomic RNA exhibits IRES activity . The minR362Q mutant [Translation initiation can occur either at an AUG located immediately at the 3′ border of the IRES as in the case of EMCV, HCV, and CrPV IRESs, or several nucleotides downstream of the ribosome binding site as it is the case for the poliovirus IRES, and in this case, will require additional ribosomal scanning to reach the initiation site ,196. ThuQ mutant  or hippuQ mutant .trans-acting factors that remain to be determined. The involvement of cellular proteins in HIV-1 translation was first evidenced by the fact that Hela cell extracts prepared from cells arrested in the G2/M phase were necessary to support initiation in vitro ,182. In Packaging of the genomic RNA is one of the last stages of viral infection and begins with the interaction of the neo-synthesized Gag polyprotein with the encapsidation signal (Psi) located in the 5′ UTR ,201. As The regulatory Rev protein has also been involved in HIV-1 translation. Indeed, it was shown that Rev can interact with the A loop of the DIS structure ,151 and As previously mentioned, the HIV-1 protease inhibits cap-dependent but not IRES-mediated initiation driven by the EMCV IRES ,166,167.Seminal studies have shown that the Gag coding region could exert a negative effect on the activity of the 5′ UTR IRES ,188,204 Interestingly, the HIV-1 matrix coding region can promote internal initiation of the ribosomes, both in vivo and in vitro , and thi6A modifications are needed. A focus on the impact of m6A residues located in the RRE showed that methylation controls the interaction between RRE and the Rev protein to enhance nuclear export of unspliced and partially spliced mRNAs. m6A residues were found in the sequence spanning nucleotides 170 to 420 (from the +1 of transcription of the LAI strain) that include the PBS, DIS, and Psi elements, and the beginning of the Gag coding region, but their functions have not yet been characterized [6A editing only in the last 1.4kb of the unspliced mRNA. This difference with the work of Lichinchi and collaborators could be attributed to the use of different kinds of cells (MT4 T vs CEM-SS cells) or HIV-1 strains (LAI vs NL-4.3), but also from differences in experimental settings. These methylated adenosines interact with the reader proteins (YTHDF 1 to 3) in infected cells, and overexpression of the YTHDF proteins correlates with an enhancement of both viral transcription and viral proteins expression [6A residues in both UTRs, in the Rev reading frame and the Gag coding region, and all these are recognized by the YTHDF reader proteins [6A residues on the unspliced mRNA. Change in the expression of writers and/or eraser proteins modulates the viral mRNAs’ translation [6A residues and the YTHDF family also negatively regulates the replication, but not at the translational level, of other RNA viruses such as HCV and Zika virus .6A residues, most probably address the viral RNA to either a translational or degradation pathway, but the mechanistic and determinants remain to be characterized. The identification of methylated adenosines inside the HIV-1 unspliced mRNA has just emerged and these nucleotide modifications appear to differentially regulate expression of the unspliced mRNA, but also other steps of the viral replication. Changes in the expression pattern of the writer and eraser proteins have consequences on viral protein level and infectivity ,216. In All viral mRNAs share a common region of 289nt from the +1 transcription to the major splice donor site, which includes TAR, poly(A), PBS, and DIS RNA elements. Thus, alternatively spliced transcripts differ by the RNA segment comprised between the SD and their cognate AUG codon; as a consequence, they exhibit 5′ UTRs with different lengths and structures ,220,221,The knock-down of DDX3 expression in HIV1 (NL-4.3) infected Jurkat T cells’ reduced translation of Gag-p55, but also that of Nef and Vif proteins without affecting their cytoplasmic mRNA level . SimilarAUG, Vpu-AUG codon is underlined). The Vpu-AUG codon, which lies in an unfavorable context, is poorly recognized and most of the ribosomes scan down to the downstream Env-AUG codon [The reading frame coding for the Env protein is the only ORF that is not preceded by a splice acceptor site. As a consequence, and due to differential alternative splicing, 16 natural bicistronic mRNAs are produced. All of them are composed of the Vpu ORF followed by Env but they differ by their 5′ UTR ,224. TraUG codon ,226. A mUG codon ,221,224 In contrast to other viruses, such as HCV, EMCV, or poliovirus, for which translation relies exclusively on IRES-mediated initiation, the unspliced HIV-1 genomic RNA can use three different mechanisms of ribosomal recruitment:(i) Similar to cellular mRNAs, ribosome attachment can occur at the cap structure and it requires different RNA helicases  to promote unwinding of the 5′ end TAR structure. However, how these RNA helicases cooperate in the course of the viral cycle remains a question to be addressed. Similarly, how the cap structure of the viral transcripts is recognized and which cellular proteins intervene in the process is also a matter of debate. Indeed, during viral infection, translation of cellular mRNAs is reduced due to the sequestration of eIF4E by 4E-BP induced by Vpr, which renders eIF4E unavailable . Thus, t(ii) The PIC can also interact with the IRES in the 5′ UTR and the IRES in the matrix coding region. Here again, the cellular and viral proteins involved in these processes are not fully understood. Technically, it is very difficult to measure the ratio of ribosomes delivered to the cap structure versus ribosomes that are loaded onto the IRES. This may be possible in the future through the development of inhibitors specifically targeting only one of the two mechanisms (cap or IRES). Interestingly, as both DDX3 and Rev can bind to structures located in the IRES ,150,151,(iii) Finally, an additional level of complexity came with the discovery of the recent implication of methyladenosines that can also promote direct ribosomal entry. These modifications could also modify the RNA structure  and may In conclusion, translational control of HIV-1 is a very complex process with multiple strategies to utilize or detour the cellular machinery. Although a lot of work has already been done to decipher some of the molecular mechanisms, some issues remain to be addressed and future work should clarify how the virus juggles with all these mechanisms during the course of viral replication.


Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal “hand-shaped” polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two “helicase” domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication. Rotaviruses are of great medical significance because they cause gastroenteritis in children. Here the authors provide insights into the mechanism of viral mRNA transcription by determining the in situ cryo-EM structures of a working rotavirus’ RNA-dependent-RNA polymerase, which is of interest for antiviral drug design. The gradual emergence of DNA-based life forms from the RNA world has been hypothesized to be punctuated by major leaps, including RNA replication, RNA-dependent RNA transcription, and RNA reverse transcription to synthesize DNA2. Although ribozymes are rare in the modern world, recent discoveries3 have supported the theory that the first RNA-dependent RNA polymerase (RdRp) was likely a ribozyme6. In the modern DNA-protein world, proteins have evolved to be the preferred polymerases that catalyze DNA replication and RNA transcription, including RNA-dependent RNA transcription occurring in viruses and cells. The first atomic structure of a polymerase (Escherichia coli Polymerase I) revealed a characteristic core shaped like a right hand7. Crystal structures of viral RdRps9, such as those in poliovirus10, bacteriophage phi611, animal reovirus12, and rotavirus13, also have cores similar to that of DNA polymerases. A similar core structure also exists in telomerase reverse transcriptase (TERT)14. The conserved function of the core is to take a single-stranded nucleotide template and amplify it to a double-stranded product. These polymerases are specialized by both the addition of peripheral domains surrounding the core and the binding of regulatory factors at different time points of polymerization. In the spatial dimension, polymerases that carry out DNA replication (such as DNA polymerase III) contain an exonuclease as a peripheral domain to proofread the dsDNA product; those involved in RNA transcription  possess endonuclease and cap-binding peripheral domains to direct the primer into the active site15. In the temporal dimension, this specialization can be further reflected by various regulatory factors, which form various complexes with the polymerase at different stages of polymerization. For example, the RdRp of bacteriophage Qβ recruits host translation elongation factors to form replicase holoenzyme16.DNA replication and RNA transcription are two of the three steps of Crick’s central dogma governing cellular life13 have been resolved previously by X-ray crystallography, all of which have a cage-like structure with a cap-binding site and four channels: template entry, NTP entry, template exit, and transcript exit. However, many purified RdRp only shows binding affinity to RNA/NTP substrates and limited polymerization activity17, leaving the spatial context unknown. Additionally, previous studies19 on active reovirus polymerases also failed to show the complete trajectory of the template or transcript RNA, thus leaving unclear the function of potential RNA-interacting peripheral domains . Previous research into these structures has also left unclear the temporal context of these polymerases that undergo conservative transcription (in which the nascent strand is the transcript). Some dsRNA viruses that conduct conservative transcription cannot achieve full polymerase activity by itself. For example, the inner capsid shell protein (CSP) is required for rotavirus’ RdRp to be active in vitro20. On the other hand, for some dsRNA viruses that conduct semi-conservative transcription, in which the nascent strand is part of the dsRNA genome , RdRp is completely functional for replication in vitro. However, exactly how CSP regulates23 RdRp’s activities in rotaviruses remains unknown. Also, unlike other RdRps that conduct semi-conservative transcription, reovirus’s RdRp can conduct both replication and transcription and switch between the two states directly after polymerization. In essence, a virus must be actively running to understand the temporal context, which is very difficult to do through X-ray crystallography.In order to fully understand these specialization processes, detailed in situ structures of polymerases in its active states are needed. However, there have been issues with obtaining the correct spatial and temporal contexts for these structures. Reoviruses have long served as model organisms for studying viral RdRp and RNA conservative transcription. Structures of Reovirus RdRp with various RNA substratesReoviridae family, rotaviruses are of particular interest for several reasons. In terms of medical significance, they cause diarrhea responsible for up to half a million children deaths annually24. Rotaviruses also display significant biochemical simplicity, as their RdRp does not have a separate NTPase protein bound as in other reoviruses; thus, the working mechanisms of rotavirus’s RdRp can be studied clearly.Cryo electron microscopy (cryoEM) offers opportunities to address both these issues, as it enables the structural characterizations of in situ structures in transient, active states. Here, we report the in situ near-atomic resolution structures of RdRp before and during transcription in rotavirus double layered particles (DLP). Compared to other viruses in the 25. The final localized reconstruction from sub-particles reached 3.6 Å resolution, which showed RdRp (VP1) interacting with both RNA and inner capsid proteins (VP2)  under active transcribing conditions  Fig. . An atomd RdRp VP interactdRp Fig. . The 5′ dRp Fig. . Based o28. In addition to resolving densities of genomic RNA and mRNA in action, our in situ structures differ from previous rotavirus’s RdRp crystal structures13 in the following aspects: we resolved two protein fragments  and identified large conformational changes in three fragments   near the cap-binding site. This HLH subdomain extends towards the genomic (+) strand in DOS  strand must approach and recognize some region of the RdRp during transcription. In DOS, the cap-binding site of the N-terminal domain Fig.  in RdRp DOS Fig.  and retrDOS Fig. . The N-t13. This strand is then guided by this subdomain through a bottleneck towards the palm  in TES RNA then immediately pairs with complementary NTP in the active site between the fingers and the palm. The incoming NTPs are in position to form a backbone with the 5′ end of the nascent RNA ; it is slightly deformed by CSP but remains retracted due to the unexpected refolding of neighboring CSP-B1s’ N-terminal arm27 (residues 73–92) outside the RdRp  in the palm RNA strand traverses the template entrance towards the active site Fig. . In DOS,TES Fig. , confirmRNA Fig. . The pri1 and narrows the transcript exit channel  and residues 968–979 of C-HB (wedge helix)] ] Fig.  compete dRp Fig. . SimultadRp Fig.  and refo2 attaches to a hydrophobic pocket next to the structured HLH subdomain in TES but detaches from this pocket as the HLH subdomain becomes flexible in DOS  inserts into the template exit channel in DOS, but moves away in TES to allow (−)RNA to exit. This C-terminal plug is close to the priming loop in DOS and potentially influences the priming loop’s approach to the nascent NTP during initiation  form different transcriptional complexes with RdRp Fig.  through DOS Fig. . This heTES Fig. . In contion Fig. . Thus, t30. Because the RdRp’s N-terminal domain interacts with string-like RNA and is close to the thumb, we renamed the N-terminal domain of RdRp the N-terminal “thumbpick” domain. In TES, the C-terminal bracelet not only exhibits functional helicase activity, but also redirects the two RNA strand products to exit through their respective channels. In redirecting RNA strand products, the C-terminal region also helps reorganize the nascent genomes. These peripheral domains allow RdRp to operate in a continuous fashion during transcription RNA undergoes a near U-turn . Both termini effectively function as minimalistic helicases and are essential for conservative transcription. In DOS, the N-terminus is capable of splitting the dsRNA genome with only around 330 residues; this domain recognizes and interacts with 5′ consensus bases (GGC) of (+)RNA at the cap-binding site (CBS), so that the subsequent 6-base-long A/U-only box can be more efficiently split by the neighboring HLH subdomain. As a result, the newly-isolated (−)RNA attaches to the nearby ssRNA recognition site on the fingers Fig. . This A/ion Fig. . In DOS,urn Fig.  in RdRp 2 locks the HLH subdomain to prevent further undesirable interactions with the genome during elongation; this same amphipathic helix in CSP-A4 locks C-HB and blocks the template exit channel as an inhibiting factor in DOS. This supports previous findings that rotavirus’s RdRp–CSP interactions are crucial for polymerization activity32. It is also consistent with previous suggestions28 that aquareovirus CSP’s N-terminal region can form different transcriptional complexes with the polymerase at different time points.Not only do the N and C terminal domains regulate the genome, but they may also provide interfaces for potential association of transcription factors. This regulation of transcription factors further specializes the protein’s function. In rotavirus, the amphipathic helix in CSP-AUnderstanding the polymorphic nature of the C-terminal domain also yields insights into viral replication Fig. . Without11), in which the cap is not bound during transcript elongation. However, φ6 phage’s RdRp differs quite drastically from rotavirus’ in their terminal domains: the RdRp of φ6 has no N-terminal domain, and its C-terminal domain is shorter (65 a.a.) and is suspected to prime polymerization11 rather than to split and rearrange RNA products. It is possible that in semi-conservative transcription, the transcript is split from the dsRNA genome by a different mechanism; therefore, in φ6 phage, we do not see N- and C-terminal structures similar to those of rotavirus and other reoviruses that conduct conservative transcription.Based on the observation that the capped end of dsRNA leaves RdRp during TES and re-associates with RdRp at cap-binding site in DOS, we propose that the other end of the dsRNA genome  is close to the capped end in DOS. When elongation starts, the entire dsRNA strand is pulled towards the RdRp so that the tail end will leave RdRp, leaving enough space to accommodate the reannealed capped end. At the end of the elongation step, the capped end follows the tail end and circles back to RdRp again. The capped end can then bind to the nearby cap-binding site and start a new transcription cycle, much like an Ouroboros. In this model, the cap is not always bound to the cap-binding site, so there are no undesirable kinks or sharp U-turns on the dsRNA genome during elongation. This model is also more consistent with other RdRps that conduct semi-conservative transcription  of 3 were harvested at 100% cytopathic effect. Cell lysate was generated by freezing and thawing twice. The lysate was treated with 50 mM EDTA (pH 8) followed by incubation for 1 h at 37 °C. After centrifugation, the pellet was resuspended in TNC buffer  supplemented with 0.1% Nonidet P-40, and 50 mM EDTA (pH 8) and trichlorotrifluoroethane was added. The aqueous phase was separated by centrifugation, and DLPs were isolated by equilibrium ultracentrifugation at 100,000 × g in a CsCl gradient for 18 h. A band containing DLPs was collected, diluted in TNC buffer, and pelleted through a sucrose cushion (15% sucrose prepared in TNC buffer) by ultracentrifugation at 110,000 × g for 2 h. Finally, particles were resuspended in 10 mM Tris–HCl, pH 8 prior to either transcription reaction or plunge-freezing.Simian rhesus rotavirus (RRV) double-layered particles were purified from rotavirus-infected cells as described elsewhere2) for 5 min at 37 °C prior to plunge-freezing for cryoEM.For the transcription reaction, purified DLPs were incubated in transcription buffer  parameters were determined with CTFFIND437 for both datasets.High quality cryoEM images were then collected in an FEI Titan Krios 300 kV electron microscope, equipped with a Gatan K2 direct electron detector and a Gatan Quantum energy filter. The microscope was carefully aligned and the coma-free alignment was performed to align the beam tilt immediately before the data collection. As detailed in Supplementary Fig. 38. Virus particles’ center and orientations were refined with Relion39 with icosahedral symmetry i2  applied. The final resolution of the resulting icosahedral reconstruction at FSC > 0.143 is 3.6 Å.For DOS, particles were automatically boxed with ETHAN25 to focus on particle vertices . First, the icosahedral reconstruction is rotated to follow an icosahedral symmetry i3  and particle orientations were adjusted accordingly39  differs from each other by an increment of 72°). Third, all sub-particles were then subjected to RELION 3D classification by asking for 16 classes with the “skip_align” option  that processes the RELION star file. By taking the star file from 3D classification as input, this script analyzed order of group  placements of the five entries of each sub-particle and find its best match to the 5 possible circular permutations of the ideal group list. If the best match has less than two outliers out of the five groups, this sub-particle will be retained with permuted orientation; otherwise, this sub-particle will be discarded. For example, the result group list “B,C,D,X,A” best matches one-time permuted ideal list “B,C,D,E,A” with one outlier so this sub-particle would be retained with one rotation of 72°, but result group list “B,C,D,X,E” matches permuted ideal list “B,C,D,E,A” with two outliers so this sub-particle would be discarded. A new star file was created with the retained sub-particle and their orientation assignments. A RELION local classification with limited range of angle search (relion parameter--sigma_ang 3) was then performed to select the major conformation ; backbone tracing error (residues 804–821 in CSP); newly-resolved asymmetric features ; conformational changes introduced by RdRp’s docking on CSP ; large conformational changes between different states ; and in situ RNA features . For those discrepancies, we manually traced the backbone in all-alanine mode in Coot and then mutated them into the correct sequence. RNA in DOS was built with conserved sequences m7GpppGGC at the 5′ end of the coding strand and its complementary strand, while RNA in TES was built with repetitive AU polynucleotides. The models in both states were then refined by the PHENIX real-space refine function and validated by the wwPDB validation server44.The atomic models of RRV’s RdRp and CSP were built with Coot43. The sequence is visualized by ESPRIPT45.Visualization of the atomic model, including figures and movies, is made with UCSF ChimeraFurther information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Reporting Summary