Fibrosis after skeletal muscle injury is common in sports and can cause irreversible damage to the biomechanical properties of skeletal muscle. Long non‐coding RNAs (lncRNAs) have been validated to act as important modulators in the fibrosis of various organs. Here, we reported a novel lncRNA , which was highly expressed in skeletal muscle fibrosis. We demonstrate that lnc‐MFAT1 knockdown can reduce TGFβ‐induced fibrosis in vitro and attenuate skeletal muscle fibrosis after acute contusion in mice. Further study showed that lnc‐MFAT1 acted as a competitive endogenous RNA of miR‐135a‐5p. Besides, the miR‐135a‐5p inhibition obviously promoted TGFβ‐induced fibrosis in vitro via enhancing its target genes Tgfbr2/Smad4. Moreover, we discovered that lnc‐MFAT1 regulates Tgfbr2/Smad4 expression by sponging miR‐135a‐5p to exert competing endogenous RNA function, resulting in TGFβ pathway activation. In conclusion, our study identified a crucial role of lnc‐MFAT1‐miR‐135a‐Tgfbr2/Smad4 axis in skeletal muscle fibrosis, providing a promising treatment option against skeletal muscle fibrosis. Through a series of in vitro and in vivo experiments, we demonstrated that silencing lnc‐MFAT1 can alleviated skeletal muscle fibrosis after acute contusion. lnc‐MFAT1 functioned as a ceRNA of miR‐135a‐5p (miR‐135a) and subsequently regulated Tgfbr2/Smad4 expression. The present study identified a crucial role of lnc‐MFAT1‐miR‐135a‐Tgfbr2/Smad4 axis in skeletal muscle fibrosis, providing a promising treatment option against skeletal muscle fibrosis.22.1C57BL/6J mice aged 10 weeks were purchased from Vital Co. and were maintained in a 12‐h light/dark cycle with free access to food and water. All animal experiments procedures were carried out following the technical guidelines and approved by the Animal Care and Use Committee of Fudan University. The acute contusion‐induced mouse skeletal muscle fibrosis model of the right tibialis anterior (TA) was established following the previous method.2.212 AAV particles containing either a lnc‐MFAT1 or a null vector) were injected around the circumference of one side TA muscles with a 27‐gauge needle. A total of 15 μL of viral preparation were injected into each muscle. Twenty‐eight days later, all of mice were killed. TA was collected and analysed by real‐time RT‐PCR, Western blot, Masson staining and immunohistochemical analysis to determine the mRNA and protein expression of fibrosis indicators.Thirty‐six skeletal muscle fibrosis mice models were randomly assigned into three groups: acute contusion without injection (n = 12), acute contusion combined with injection of AAV‐NC (n = 12) and acute contusion in combined with injection of AAV‐MFAT1 (n = 12). Following established protocols with minor modifications, aliquots of 0.1 mL (1 × 102.32 at 37°C. Primary antibodies for Collagen1 (Col1), Vimentin (VIM), α‐SMA, TGFBR2, Smad4 and GAPDH were purchased . Both cells were maintained in DMEM (HyClone) containing 10% foetal bovine serum , penicillin (100 U/mL) and streptomycin (100 μg/mL). All cells were cultured in humid atmosphere containing 5% COed Table . Donkey ed Table .2.4The transfection was carried out using the Lipofectamine 3000 Reagent (Invitrogen) following the manufacturer's protocol. The small interfering RNAs against lnc‐MFAT1 (si‐MFAT1), negative control (si‐NC) and the pcDNA3.1 vector targeting lnc‐MFAT1 were all acquired from Sangon Biotech. To knockdown or overexpress miR‐135a, miR‐135a mimics, miR‐135a inhibitors and its negative controls were purchased from Sangon Biotech and were transfected into C2C12 cells. The sequences are listed in Table 2.5Total proteins were prepared in RIPA buffer (Beyotime) supplemented with protease and phosphatase inhibitor cocktail . Total protein quantity was determined using BCA protein assay . Then, samples were normalized and separated by 10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After that, the membrane was blocked with 5% skim milk in 1×TBST for 1 hour at room temperature and then immunoblotted in primary antibodies solution at 4°C overnight. After that, the membranes were washed in 1×TBST and incubated with goat anti‐rabbit IgG for 1 hour at room temperature. The proteins were visualized using ECL reagent (Beyotime) and imaged using Tanon‐5200 Multi Fluorescence imager. Antibodies used for Western blot are listed in supplemental Table 2.6Total RNA from cells was isolated using TRIzol reagent (Invitrogen). Afterwards, cDNA was synthesized using Advantage RT‐for‐PCR Kit (TaKaRa). qRT‐PCR was implemented with SYBR Green I Master (Roche) on a 7500 Real‐Time PCR System (Applied Biosystems).GAPDH and U6 were used as endogenous control for lncRNA/mRNA and miRNA, respectively.Expression fold change was calculated using the 2−ΔΔCt method. All primer sequences are listed in supplemental Table 2.7Cytoplasmic and nuclear RNA isolation were carried out with PARIS™ Kit (Invitrogen) according to the manufacturer's instruction. Then, qRT‐PCR was used for measurement of nuclear and cytoplasmic RNA. β‐Actin and U6 were used as cytoplasmic and nuclear controls, respectively.2.8The putative miR‐135a binding sites in lnc‐MFAT1, Tgfbr2‐3ʹ‐UTR or Smad4‐3ʹ‐UTR and the mutant binding sites were constructed and cloned to the pSI‐Check2 luciferase vector (Hanbio), immediately downstream of the luciferase gene. HEK293T cells were cotransfected with the pSI‐Check2 reporter plasmids and the miR‐135a‐5p mimics or miR‐135a‐5p inhibitor, respectively. Twenty‐four hours after transfection, the fluorescence intensity was detected with the Dual‐Luciferase Reporter Assay System (Promega). And the Renilla luciferase activity normalized to firefly luciferase activity. The experiments repeated independently three times, and the data are represented as mean ± SD.2.9After fixation for 24 hours in 4% paraformaldehyde, the tissues were dehydrated, embedded in paraffin and sectioned perpendicularly to the direction of the muscle fibre into 5‐μm‐thick slices. Then, slices were incubated for 12 hours in a‐SMA antibody followed by secondary antibody conjugated with HRP. Subsequently, immunohistochemical staining was conducted by 3,3′‐diaminobenzidine and haematoxylin detection. All histological slices were visualized with inverted light microscopy (Olympus) and digitalized with DP72 Manager (Olympus). Digital quantification of α‐SMA positive areas was conducted with software Image‐Pro (Meida Cybernetics).2.10C2C12 cells were seeded on glass slides in six‐well plates and incubated in DMEM until 80%‐90% confluent. After washed with PBS, cells were fixed with 4% paraformaldehyde for 10 minutes followed by permeabilization in PBS with 0.5% Trion‐X‐100 for 15 minutes and 3% BSA blockage for 30 minutes at room temperature. Immunofluorescence was then carried out with overnight Col 1 and α‐SMA antibodies incubation at 4°C and then with the corresponding secondary antibodies labelled with FITC for 45 minutes at 37°C. DAPI was added to stain the nuclei. Finally, images were taken with immunofluorescence microscope (Olympus).2.11The Cy3‐labelled lnc‐MFAT1 probe was purchased from RiboBio. The subcellular localization of lnc‐MFAT1 was evaluated using a FISH kit (RiboBio). Confocal section images were captured using a confocal laser scanning microscope . The specific target probe is listed in Table 2.12After 24 hours fixation in 4% paraformaldehyde, the tissues were routinely dehydrated, embedded in paraffin and sectioned at 5‐μm thickness. Sections were incubated at 37°C overnight, dewaxed and stained with Masson trichrome according to standard procedures.2.13In brief, 4 paired skeletal muscle fibrosis samples and normal control samples were collected. The expression of lncRNAs in skeletal muscle fibrosis was analysed using Gene Expression Hybridization Kit (Agilent technologies). Samples labelling and array hybridization were carried out following the Agilent One‐Color Microarray‐Based Gene Expression Analysis protocol (Agilent technologies) with minor modifications. Data were extracted with Feature Extraction software 10.7 (Agilent technologies). Raw data were normalized by Quantile algorithm, Gene Spring Software 12.6.1 (Agilent technologies).P < .05) were identified from microarray data. Finally, Hierarchical Clustering and combined analysis were performed to show the differentially expressed lncRNAs between the two groups using in‐house scripts.lncRNAs were chosen for further data analysis after quantile normalization of the raw data. Differentially expressed lncRNAs between the two groups . A significant P value  indicates a correlation between a pathway and the conditions. The recommend P value cut‐off is .05.The differentially expressed mRNAs were mapped with the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to investigate the biological functions using online bioinformatics tool DAVID6.8 . To determine statistical significance of differences between groups, the Student t test was performed. A two‐side 33.1The acute contusion‐induced mice skeletal muscle fibrosis model was established according to previous study.P < .05), the levels of differentially expressed lncRNAs in the samples were displayed using volcano plot  were shown in Figure http://asia.ensembl.org/), prediction software and the NCBI ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/); the results showed no protein‐coding capability for lnc‐MFAT1, indicating it a non‐coding RNA  and the ECM receptor interaction , both of which play critical roles in the fibrosis process  and overexpressed by transfecting pcDNA3.1‐MFAT1 in C2C12 cells. Then, compared with the negative control (si‐NC), the expression level of MFAT1 remarkably decreased or increased, respectively Figure . After t3.4Given the association between lncRNA function and its subcellular distribution, FISH and subcellular fractionation assays were used to identify the lnc‐MFAT1 as a cytoplasmic‐localized lncRNA Figure ,B, whichThe binding sites between lnc‐MFAT1 3′‐UTR and miR‐135a were predicted using bioinformatics analysis Figure . After t3.5http://www.targetscan.org/vert_72/), Tgfbr2 and Smad4 were selected for miR‐135a candidate prediction. The predicted binding sites between Tgfbr2 3′‐UTR, Smad4 3′‐UTR and miR‐135a were shown in Figure Combined with previous studiesA decrease in Col1, VIM and α‐SMA protein expression was noticeable after transfecting of with miR‐135a mimics Figure . Meanwhi3.6The qRT‐PCR and Western blot analysis demonstrated that the overexpression and knockdown of lnc‐MFAT1 increased and decreased Tgfbr2/Smad4 expression, respectively Figure ,B. Then,3.7An lnc‐MFAT1 knockdown mice model was built by adeno‐associated virus (AAV) injection to further study the effect of lnc‐MFAT1 on skeletal muscle fibrosis in vivo. The establishment procedure is shown in Figure Masson staining and immunofluorescence staining results showed that lnc‐MFAT1 knockdown inhibited skeletal muscle fibrosis development compared with that in the contusion and AAV‐NC group Figure . In addi4In this study, lnc‐MFAT1 was confirmed to be substantially up‐regulated in skeletal muscle fibrosis in vivo and in vitro, and its silence could attenuate the skeletal muscle fibrosis following an acute contusion in vivo and mitigate the proliferation of TGFβ‐induced C2C12 cells into myofibroblasts. Furthermore, it was predicted by the miRNA target prediction bioinformatics and confirmed by dual‐luciferase reporter assays that miR‐135a could simultaneously target lnc‐MFAT1 and Tgfbr2/Smad4. Silencing lnc‐MFAT1 attenuated, whereas inhibiting miR‐135a promoted, the expression of Tgfbr2, Smad4 and ECM markers. In addition, miR‐135a inhibition could remarkably restore the effects of lnc‐MFAT1 silence on Tgfbr2, Smad4 and ECM markers. In summary, our study verified that lnc‐MFAT1 could regulate skeletal muscle fibrosis by acting as a ceRNA to sponge miR‐135a through the miR‐135a‐Tgfbr2/Smad4 axis.Initially, the current study revealed that lnc‐MFAT1 was essential for maintaining skeletal muscle fibrosis both in vivo and in vitro. Multiple evidence showed that lncRNAs are aberrantly expressed in various organ fibrosis.In addition, the current study showed that lnc‐MFAT1 could regulate skeletal muscle fibrosis by acting as a miR‐135a ceRNA. It have been proposed that the biological function of lncRNA was closely related to its subcellular location.Further, Tgfbr2/Smad4 was proved to be target genes of miR‐135a and played an important role in mediating the fibrosis function of lnc‐MFAT1/miR‐135a. TGF‐β exerts its effects by binding to Tgfbr1/2, leading to phosphorylation of Smad2/3. Subsequently, activated Smad2/3 binds to Smad4 and translocates into the nucleus, which initiates TGF‐β signalling pathway.In summary, the newly identified lnc‐MFAT1 was found to be up‐regulated in skeletal muscle fibrosis tissues. Through integrating in vivo and in vitro experiments, we illustrated that lnc‐MFAT1 acts as an miR‐135a sponge to regulate Tgfbr2/Smad4, resulting in skeletal muscle fibrosis Figure . This stThe authors declare no competing financial interests.Jinrong Lin: Conceptualization (lead); Project administration (lead); Writing‐review & editing (lead). Xiaobao Yang: Conceptualization ; Project administration (supporting). Shaohua Liu: Methodology ; Project administration ; Writing‐review & editing . Zhiwen Luo: Methodology (lead). Qingyan Chen: Data curation ; Methodology ; Writing‐review & editing . Yaying Sun: Conceptualization ; Methodology . Zheci Ding: Methodology ; Writing‐original draft (supporting). Jiwu Chen: Conceptualization (lead); Writing‐review & editing (lead).Supplementary MaterialClick here for additional data file.


We report the biological and structural characterization of umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs. Plant RNA viruses are frequently found in association with subviral RNAs that either share or do not share any sequence similarity with their partner RNA. All subviral RNAs are characterized by their inability to independently complete an infection cycle, which includes transmission to the next host plant. Subviral RNAs range substantially in size and complexity, from non-coding satellite RNAs (194 nt to about 500 nt) that depend on their helper virus for replication, movement and encapsidation . SubviraA more recent grouping of coding subviral RNAs is the coat protein-dependent RNA replicons, which encode their own RNA-dependent RNA polymerase (RdRp) but are dependent on a helper virus for at least encapsidation . UmbraviA recently recognized type of coat-protein dependent replicon is the tombusvirus-like associated RNAs (tlaRNA) . These iAnother group of coat protein-dependent replicons are similar to tlaRNAs in their coding capacity (ORFs 1 and 2) but their RdRps are reported to be more closely related to those of umbraviruses . As withOpuntia ficus indica fruit cactus plants with symptoms of pad swelling disease  ATP-labeled oligonucleotides. Plus-strand CYVaV ((+)CYVaV) probes were complementary to positions 2202–2239, 2464–2502 and 2655–2692. Minus-strand CYVaV ((−)CYVaV) probes were (+)-strand positions 325–342, 855–873, 1375–1392, 1895–1912 and 2416–2434. (+)PEMV2 probes were 2969–3004, 2731–2771 and 3229–3270. (+)OULV probes were complementary to positions 2950–2989, 2757–2796 and 2505–2554. (−)OULV probes were complementary to (+)-strand positions 404–423, 914–935, 1432–1452, 2099–2123 and 2597–2622.Total RNA was subjected to agarose gel electrophoresis and blotting, and membranes were hybridized with [γ-35S-methionine. The translation mixture was incubated at 25 °C for 45 min and then resolved on a 10% SDS-PAGE gel. The gel was dried and exposed to Fuji phosphorimager screen for 3 h. The screen was subsequently scanned by an Amersham Typhoon fluorescent image analyzer. The intensity of radioactive bands was quantified using ImageQuant TL 8.1 (GE Lifesciences). All experiments were repeated at least three times.Plasmids containing full-length CYVaV (pUC19-CYVaV) or PEMV2 (pUC19-PEMV2) were linearized with Hind III or SmaI, respectively, and served as templates for RNA transcription using T7 RNA polymerase. Quantification of the synthesized RNA was performed using a DeNovix DS-II FX spectrophotometer. For in vitro translation, 10 μL translation mixtures contained 5 μL wheat germ extract (Promega), 0.5 pmol RNA template, 0.8 μL 1 mM amino acids mix (without methione), 100 mM potassium acetate and 0.5 μL (5 μCi) 2, 1.2 mM spermidine, 1 U/µL RNase inhibitor (NEB) and 3 U/µL of T7 RNA polymerase. Synthesized RNA was treated with 1 U of RNAse-free DNAse (Promega) to remove the DNA template. DNase was removed by treatment with phenol:chloroform followed by lithium chloride precipitation of the RNA. RNA was quantified using a DeNovix DS-II FX spectrophotometer and the quality of the RNA was checked in a 1% TBE native agarose gel.pUC19-CYVaV was linearized using HindIII and linearized plasmid treated with phenol:chloroform followed by 3 M sodium acetate precipitation. Linearized pUC19-CYVaV was used to synthesized full length RNA via T7 polymerase in vitro transcription; briefly, 5 µg of linearized pUC19-CYVaV was incubated for 2 h in a 100 µL in vitro transcription reaction composed of 6 mM DTT, 0.6 mM rXTPs, 24 mM Tris pH 8, 15 mM NaCl, 4.8 mM MgCl4Cl and 11 mM MgOAc) and incubated at 37 °C for 20 min. The folded RNA was divided into two tubes (18.75 µL) with one treated with 3.3 µL of 100 mM N-methylisatoic anhydride (NMIA) in dimethyl sulfoxide (DMSO), and the other treated with DMSO only, for positive or negative samples respectively. The RNA samples were incubated at 37 °C for 45 min to allow interaction with the modifying agent. After modification, the RNA was precipitated with 0.1 volume of 3 M sodium acetate and 2.5 volumes of cold 95% ethanol. The RNA pellet was reconstituted in 0.5X TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0). RNA was quantified using a DeNovix DS-II FX spectrophotometer and the quality of the RNA was checked in a 1% TBE native agarose gel.Nine picomoles of RNA were diluted in 30 µL of water and denatured at 65 °C for 4 min followed by snap cooling on ice for 2 min. RNA was subjected to folding by adding 7.5 µL of 5X RNA folding buffer , 5 mM DTT, 0.5 mM dNTPs, 1U SSIII enzyme) or sequencing ladder mix  was added to the corresponding samples, which were incubated at 52 °C for 1 h. cDNA samples were treated with 1 µL of 4 M NaOH and heated at 95 °C for 5 min to degrade the RNA. Samples were then neutralized by adding 2 µL of 2 M HCl. Prior to cDNA precipitation, half of the sequencing ladder cDNA reaction (PET) was added to the corresponding SHAPE-modified (6FAM) cDNA sample ((−) DMSO or (+) NMIA/DMSO). The cDNA reaction mixtures were precipitated with 0.1 volumes of 3 M sodium acetate and two volumes of cold >95% ethanol. cDNA pellets were washed with 70% ethanol, air dried and resuspended in 15 µL of nuclease free water. The samples were then provided to Genewiz for fragment analysis. The cDNA fragmentation data obtained from Genewiz was scanned through Peak Scanner 2 (Invitrogen) to check for the presence of cDNA fragments in the data sets, and samples were analyzed using QuSHAPE software [https://rna2drawer.app/). To determine the secondary structure, mFOLD [SHAPE-modified RNA was subjected to cDNA synthesis using SuperScript III Reverse Transcriptase (Invitrogen) following the manufacturer’s recommendations. Briefly, 1 pmol of control or modified RNA was diluted in 8 µL of 0.5X TE buffer and 4 µL of 2.5 µM 5′ end labeled fluorescent primer (6FAM was used for SHAPE modified samples and PET was used for sequencing ladder samples). Tubes were incubated at 65 °C for 5 min followed by ice incubation for 5 min. Next, 8.5 µL of SHAPE enzyme mix (1X SSIII FS buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgClsoftware . Manual software , using te, mFOLD  was usede, mFOLD  and SHAP−∆∆Ct method.GFP reporters were agroinfiltrated with the p14 silencing suppressor and either mock or the dominant-negative Upf1 inhibitor of NMD as previously described . At 5 daGenome organization of viral and virus-like RNAs found in this report are shown in Arabidopsis thaliana protoplasts were inoculated with in vitro synthesized CYVaV transcripts containing the exact 5′ end and one non-template residue at the 3′ end, and total RNA was isolated 24 h later. Since efficient accumulation of PEMV2 in protoplasts is dependent on the presence of the ORF3 product p26 [To ensure that the CYVaV sequence reported in GenBank represented the complete full-length gRNA, an expression plasmid containing the reported sequence was generated to determine if CYVaV transcripts were capable of replication. duct p26 ,32, whicN. benthamiana plants (GDD). One possibility is that CYVaV was benefiting in N. benthamiana plants, but not Arabidopsis protoplasts, from the presence of p26. No CYVaV sgRNA was discernable in either protoplasts or infiltrated leaves, which differs from tlaRNAs, which all have a prominent sgRNA [Different results were obtained when CYVaV and PEMV2 were agroinfiltrated into leaves of young a plants E. Agroinnt sgRNA ,33.To begin resolving the taxonomic position of ulaRNAs, we generated a phylogenetic tree based on the complete RdRp aa sequences from ulaRNAs, tlaRNAs, umbraviruses and selected tombusviruses and betacarmoviruses. Initial trees generated using barley yellow dwarf virus  as an outgroup failed (BYDV was not an outgroup on the tree) and thus, the tree was mid-point rooted. The aligned viral sequences were checked for recombinants using the RDP4 package , which rThe RdRp region of ulaRNAs consists of conserved structural RNA elements (see below), and thus the RNA sequence is not only acting as an ORF. Therefore, a second phylogenetic tree was developed based on the full-length nucleotide sequence of the RdRp ORF B. Similacis-acting structures located at or near the 5′ and 3′ ends promote replication and translation [When the results of the two trees are considered together, they imply that the RdRp region of ulaRNAs might be undergoing differential selection pressures; one at the protein level and a second at the RNA level to maintain functional RNA structures. RNA structures throughout RNA virus genomes are engaged in functions important to the virus infection cycles. For umbraviruses and related betacarmoviruses and tombusviruses, distinctive nslation ,40,41,42nslation ,43, sgRNnslation ,46,47,48nslation ,49. Unstnslation  and as inslation ,53,54,55To better understand the evolutionary relationships among ulaRNAs and to start identifying regions in the genomes that are important for function, we used SHAPE RNA structure probing ,57 combiD1 contains the short 8 nt 5′ UTR and entire ORF1 of Class 2 ulaRNAs. CYVaV shares 68%, 59% and 58% sequence identify with OULV, EMaV and SULV in D1, respectively, making it the most conserved of the three domains. As shown in D1 stems 1 and 2 in CYVaV are short hairpins that are strongly supported by the SHAPE probing data. All ulaRNA stem 1′s contain the ultimate or penultimate 5′ nucleotide at the base of the hairpin , with alAUGG) is out of frame with most of ORF1, and ribosomes initiating translation at this AUG would terminate at an UAG stop codon after 41 amino acids. An in-frame AUG in a weaker Kozak context (AGGAUGA) is located at position 64, and this AUG is not present in the other Class 2 ulaRNAs. The exceptional nature of the SULV ORF1 translation initiation site suggests that additional information is needed to confirm its ORF1 initiation codon.SULV differs from the other Class 2 ulaRNAs in that the AUG that corresponds with the other class member’s start codons and is in an excellent Kozak context  comparable to locations in members of the Tombusviridae. An additional complementary sequence is located within the RSE between H1 and H2 in all ulaRNAs with the exception of PMeV2 . AlthougThe second conformation for the RSE region is a better fit for the SHAPE data and is also conserved among all ulaRNAs with the exception of PMeV2 . In thisFor PUV and SbaVA, the ORF1 stop codons  are similarly located 12 nt downstream from the slippery site within the stem of HA as with most of the Class 2 ulaRNAs. In addition, the PUV, BabVQ and SbaVA sequences, but not the PMeV2 sequence, can adopt a similar alternative conformation but with a three-way junction and two hairpins for PUV and SbaVA . WhereasSince most of the ulaRNA recoding sites are markedly different from those of umbraviruses, we were interested in determining the efficiency of the frame-shifting event and whether we had correctly identified the slippery site, which is unusually distal from the termination codon and the base of the RSE. Full-length transcripts of CYVaV and PEMV2 were synthesized in vitro and subjected to in vitro translation using wheat germ extracts. The CYVaV recoding site was surprisingly efficient, with a rate of frame-shifting nearly 6-fold higher than that of umbravirus PEMV2 B. MutatiD2 contains most of ORF2 and a portion of the 3′ UTR. This domain contains slightly less overall sequence similarity among the Class 2 ulaRNAs, with CYVaV sharing 62%, 55% and 52% identify with OULV, EMaV and SULV, respectively. Although the two inserts in OULV, SULV and EMaV in red,  protrudeAs described in Arabidopsis protoplasts. RNA was extracted after 24 h and analyzed by Northern blots. OULV gRNA accumulated to detectable levels in protoplasts, however no discernable sgRNA was present. The variant CCS in CYVaV was also converted to a canonical sequence by replacing the adenylate in position 2 with a guanylate . While A2069G was infectious and accumulated to near WT levels in protoplasts   is the location of the RdRp termination codon within an extended hairpin with a second long hairpin located just downstream (Structures 12 and 13 in ded p26) D, it is cis-acting NMD-resistant sequence, N. benthamiana leaves were agroinfiltrated with GFP reporter constructs containing either the 3′ UTR of PEMV2 (construct GFP-3′UTRPEMV) or the 3′ UTR of CYVaV (construct GFP-3′UTRCYVaV) (N. benthamiana leaves may be for a reason other than NMD protection (To determine if the 3′ UTR of CYVaV contains a TRCYVaV) A. ConstrTRCYVaV) B,C, as potection E. Since Although D2 has less conserved sequence overall compared with D1, Structure 8 (beginning at position 1040) and surrounding sequences  contains nearly 94% sequence identity between CYVaV and OULV . This reD3 contains the 3′ portion of the 3′ UTR and is the least conserved region among Class 2 ulaRNAs, with CYVaV sharing 44% sequence identity with OULV and 37% identity with EMaV. As described below, the SULV sequence is missing the 3′ terminal region of D3 and thus sequence identify was not determined. D3 begins with a series of short hairpins followed by a highly conserved structure containing short sequence stretches that are 100% conserved among Class 2 ulaRNAs (Structure 14) but not present in Class 1 and Class 3 ulaRNAs. At the 3′ end are elements that are similar to those found in most umbraviruses and carmoviruses. These features are detailed below.The most prominent feature of D3 is Structure 14 . The low1) connecting sequence in an H5 internal symmetrical loop (carmoviruses) or terminal loop (umbraviruses) with the 3′ terminal four residues [2) with sequence just downstream of H5. TCV and cardamine chlorotic fleck virus (betacarmovirus) as well as five umbraviruses including PEMV2, also contain a hairpin adjacent to H4b known as H4a, which forms an H-type pseudoknot (Ψ3) with sequence upstream of H4a [2 and Ψ3 form a ribosome-binding, T-shaped structure (TSS) that serves as a 3′ CITE [Carmoviruses and umbraviruses contain conserved features at their 3′ ends including (from 3′ to 5′): a 3′ terminal hairpin known as Pr, a spacer region, the penultimate hairpin known as H5, and a pseudoknot (Ψresidues ,73,74,75m of H4a ,73. Alto 3′ CITE ,71,75 (F1, H4b and H4a, whereas OULV is missing H4a (1 is unusual in not having the canonical GGGC/U:G/ACCC found in all carmoviruses and umbraviruses. None of the Class 2 ulaRNAs have the two pseudoknots necessary to form a TSS, and thus a TSS-type structure is unlikely to be present. The sequence complementary to the RSE sequence is located in the identical position between H5 and Pr in all Class 2 and Class 3 ulaRNAs. This is different from umbraviruses, where the sequence is always located in the Pr terminal loop. Although structures in comparable locations have not been identified for Class 1 ulaRNAs, Class 3 SbaVA contains Pr, H5, Ψ1 and H4b, similar to Class 2 ulaRNAs (CYVaV and EMaV contain Pr, H5, Ψsing H4a . EMaV Ψ1 ulaRNAs . In addi ulaRNAs , supportUnlike other viruses where selection pressure for different proteins could be determined ,77, the To investigate the evolutionary relationships based on conserved Class 2 RNA structures, cladograms were generated for the nucleotide sequences of groups of structures in relation to related regions from umbraviruses and Class 1 and Class 3 ulaRNAs. Group 1 consisted of Structures 1–6 A, Group Considering all the trees together , we propThe current model also accounts for the grouping together of Class 1 and Class 3 ulaRNAs based on RdRp sequence by suggesting that they originated from a common ancestor replicon. Based on weak homology between corresponding regions of Class 2 ORF5 and Class 3 ORF5-1, along with their different translation initiation and termination sites, we proposed that ORF5-1 in Class 3 SbaVA was acquired by a separate recombination event, which led to the divergence of Class 1 and Class 3 ulaRNAs. Acquisition of a more extensive ORF by a separate event would also account for the lack of Structures 12–14 in SbaVA. This model lays down a road map for future investigations into these novel virus-like RNAs.


Citrus tristeza virus (CTV), the largest non-segmented plant RNA virus, has several peculiar features, among which is the production of a 5′-terminal long non-coding RNA (lncRNA) termed low-molecular-weight tristeza 1 (LMT1). In this study, we found that p33, a unique viral protein that performs multiple functions in the virus infection cycle, specifically binds LMT1, both in vivo and in vitro. These results were obtained through the expression of p33 under the context of the wild type virus infection or along with a mutant CTV variant that does not produce LMT1 as well as via ectopic co-expression of p33 with LMT1 in Nicotiana benthamiana leaves followed by RNA immunoprecipitation and rapid amplification of cDNA ends assays. Further experiments in which a recombinant p33 protein and an in vitro transcribed full-length LMT1 RNA or its truncated fragments were subjected to an electrophoretic mobility shift assay demonstrated that p33 binds to at least two distinct regions within LMT1. To the best of our knowledge, this is the first report of a plant virus protein binding to a lncRNA produced by the same virus. The biological significance of the interaction between these two viral factors is discussed. Citrus tristeza virus (CTV), a member of the family Closteroviridae, is the largest non-segmented plant RNA virus that is responsible for two of the most destructive viral diseases of citrus: quick decline and stem pitting [ pitting ,5,6,7,8. pitting ,10,11,12 pitting . LMT2  and Prunus necrotic ringspot virus MPs to bind RNA was required for cell-to-cell movement [Poa semilatent virus MP, TGBp1, was needed for virus systemic movement [To date, at least 36 plant virus MPs were reported to bind nucleic acids, all in a sequence-non-specific manner . For insmovement ,33, whilmovement . Ultimathttps://www.genesilico.pl/meta2/, accessed on 6 October 2011) and InterPro  using the p33 amino acid (aa) sequence of the T36 isolate of CTV (EU937521).The prediction of RNA binding motif was performed with Genesilico Fold Prediction Metaserver . Cells collected from an over-night culture of a selected single colony were gently resuspended in a buffer containing 10 mM 2-(N-morpholino) ethane sulfonic acid , 10 mM MgCl2 and 150 mM acetosyringone at O.D.600nm = 1.0. After a 3 h incubation at room temperature without shaking, the suspension was infiltrated into six-week-old N. benthamiana plants using a needleless syringe.Agroinfiltration of constructs was conducted as previously described . BrieflyN. benthamiana by grinding in a lysis buffer  containing protease inhibitor cocktail . The homogenate was incubated on ice for 1 h with gentle shaking on the rocker and then clarified by centrifugation at 3000× g for 10 min at 4 °C. The supernatant (600 μL) was immunoprecipitated using GFP-Trap® or RFP-Trap® —anti-green fluorescent protein (GFP) or anti-red fluorescent protein (RFP) antibody-conjugated agarose beads (50 µL)—by incubating for 2 h on ice with gentle shaking. The supernatant described at this step was also used as an ‘Input’ sample for reverse transcription-PCR (RT-PCR) or immunoblot analysis. The binding reaction was washed three times as per the manufacturer’s instruction by centrifugation at 3000× g for 2 min at 4°C. The pellet of the agarose beads collected at this step was used as ‘Elute’ for further analysis. For the immunoblot analysis, total protein was extracted as described below. For RT-PCR analysis, total RNA was extracted using the TRIzol™ reagent  according to the manufacturer’s procedure. RNA extracts were resuspended in 50 μL of RNase-free water.Total protein was extracted from the infiltrated leaves of Total RNA extracts described above were measured using NanoDrop UV-Vis Spectrophotometer , diluted with RNase-free water to adjust concentration to 1 ng/µL, and used as a template for an RT-PCR reaction. RT-PCR was performed using SuperScript™ III One-Step RT-PCR System with Platinum Taq DNA Polymerase  in 20 µL volume . Complementary DNA (cDNA) synthesis was followed immediately by PCR amplification according to the manufacturer’s procedure with 30 cycles. Reaction products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide at 200 ng/mL.w/v) SDS, 20% (v/v) glycerol, and 0.01% (w/v) bromophenol blue) with dithiothreitol and boiled for 10 min. Mixtures were briefly centrifuged prior to loading, electrophoresed through 10% SDS-polyacrylamide gels, and electro-transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 1X TBS-T  containing 5 % (w/v) skim milk for an hour at room temperature prior to incubating with an anti-GFP, anti-RFP  or anti-CP antibody  polymerase . The first strand synthesis and 3′ RACE was performed as instructed  with a manufacturer-supplied reverse primer and a CTV-specific forward primer (GSP2). Both 5′ and 3′ RACE products were cloned into the manufacturer-supplied vector and selected clones (10 for 5′ RACE and 100 for 3′ RACE) were sequenced .The RNA isolated from the agarose beads at the “Elute” step of the RNA immunoprecipitation (RIP) assay was used for the amplification and the RACE assays  to determine the 5′ and 3′ ends of co-immunoprecipitated RNA. To determine the 5′ end, a random primer and manufacturer-supplied adapter oligos were used for the first-strand cDNA synthesis. The 5′ RACE product was amplified using the forward primer supplied by the manufacturer and a CTV-specific reverse primer (GSP1). To determine the 3′ end of the RNA, the isolated RNA was polyadenylated using Protein samples  were separated by electrophoresis through 10% SDS-PAGE and transferred onto a nitrocellulose membrane using semi-dry Trans-Blot apparatus . The membrane was incubated at room temperature in the RN (renaturation) buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 0.1% Triton X-100 and 1X Denhardt’s reagent ) for 20 EcoR I and Not I restriction enzyme recognition sites. The constructs were transformed into Rosetta strain (DE3)  competent cells. Protein expression induction was performed at OD600 nm 0.5–0.7 with 0.4 mM IPTG, and the cultures were then incubated at 20 °C for overnight. Pellets were resuspended in 1X PBS buffer  supplemented with 20 mM PMSF and sonicated to lyse the cells. The homogeneous cell lysate was centrifuged at 10,000× g for 30 min at 4 °C. The supernatant was collected and incubated for 1 h with 2 mL of 50% slurry of Glutathione Resin  before loading onto an empty EconoPac gravity-flow column . The resin was then washed with 10 mL 1X BS followed by elution with 10 mL elution buffer (50 mM Tris-HCl at pH 8.0 and 10 mM reduced glutathione). The elutes were concentrated using an Amicon protein concentrator . Proteins were then separated by 8% SDS-PAGE electrophoresis followed by Coomassie blue staining and destaining to estimate concentration using a BSA standard as reference. Purified GST:p33 and GST elutes were run on 12% Mini-PROTEAN® TGX™ Precast Gels  and electro-transferred to PVDF membrane. Western blot was carried out with 0.5 µg/mL anti-GST tag rabbit anti-tag polyclonal primary antibody  and 1:20,000 goat anti-rabbit IgG (H + L) (HRP) secondary antibody  after blocking with 5% non-fat milk in TBST. The PVDF membrane was treated with Clarity™ Western ECL Substrate  and visualized on Bio-Rad ChemiDoc™ XRS+ system with Image Lab™ software.The p33 or the transmembrane domain (TMD) lacking p33 (p33ΔTMD) coding sequences were amplified using P33-FW and either P33-RV or P33ΔTMD-RV  and were®-T Easy plasmid  and sequenced using a pair of M13 primers listed in Nde I  prior to transcription using T7 MEGAscript™ kit . Plasmids harboring the full-length LMT1 and fragment C were linearized by Nco I  before transcription using the SP6 MEGAScript™ Kit .A series of LMT1 clones was constructed. A fragment encompassing an extended full-length LMT1 (nt positions 1 to 780 in the CTV genome) and its truncated versions encompassing nt positions 201–400, 401–600, and 601–780  were amplified by PCR using set of primers provided in The indicated amounts of purified GST:p33ΔTMD or GST were incubated with 1 µM of an RNA transcript in 10 µL binding buffer  for 20 min at 28 °C with gentle shaking. Subsequently, the incubated mixture was electrophoresed in 6% non-denaturing PAGE using 1X TAE buffer and transferred onto Hybond™-XL nylon membrane  via a semi-dry transfer cassette  and was immobilized by a UV-crosslinker . The crosslinked membrane was blocked with Denhardt’s solution  for 30 min at 60 °C before adding a DIG-labeled probe. After overnight hybridization, the location of RNA transcripts was detected using DIG-Northern-Detection-system  per the manufacturer’s instructions. The Signals were captured by a ChemiDoc™ . The Hill equation was plotted using the default function in Prism version 8 .N. benthamiana plants through infiltration with an A. tumefaciens culture transformed with a binary vector carrying a fusion of the p33 ORF with that of GFP . To analyze the RNA bound to the GFP-tagged p33 protein, total RNA extracted from the beads at the final “Elute” step  in the N-terminal region of the protein , which iGFP:p33; ) positiostep see  was usedstep see A. All prstep see B; N/C. Istep see C.The results of the above experiment suggested that p33 could bind a specific RNA, which sequence corresponds to that of the 5′ region of the virus genome. As CTV generates both negative- and positive-stranded RNAs during replication ,10, the N. benthamiana leaves as described above and purified using GFP-Trap®. The purified proteins were verified via SDS-PAGE followed by silver staining and immunoblotting using the GFP-specific antibody. The northwestern blot analysis using DIG-labelled RNA probes comprising the 5′- or 3′-terminal ~1 kb-region of the viral gRNA . One set included a reverse primer annealing at the 660 nt position (R660) for detection of the LMT1 molecule and the viral gRNA, while the other set included a reverse primer binding at the 900 nt position (R900) that was designed to detect only gRNA. The shorter product (with R660) was amplified from the “Elute” RNA when GFP:p33 was co-expressed with either CTV-WT or transiently-expressed LMT1 . As p23 was expected to bind other viral RNAs, including gRNA, in addition to LMT1, we used two sets of primers mentioned above to analyze protein-bound RNA. As we expected, both R660 and R900 yielded amplification of the respective fragments when the RNA eluted from the immunoprecipitated RFP:p23 was used as the template in the RT-PCR reactions, confirming that, besides LMT1, p23 protein is able to bind viral RNA containing regions downstream of that encoding LMT1  of LMT1 involved in binding to the p33 protein, we conducted EMSA assays using a purified p33 protein and in vitro-transcribed RNA molecules generated from a set of constructs in which truncated fragments of the LMT1 sequence were cloned under the T7 or SP6 promoter. To purify p33, we first cloned a full-length p33 ORF into the pGEX-4T-1 expression vector fused with an upstream GST tag for protein expression in :p33ΔTMD  with eitTo examine the interaction, increasing amounts of purified GST:p33ΔTMD—as well as the GST protein alone—were incubated with in vitro-transcribed LMT1 RNA, separated on a non-denaturing gel, and transferred onto a nylon membrane. The membrane was hybridized with a DIG-labelled 5′ CTV probe described above see also A to deteTo identify the regions of LMT1 responsible for binding to p33, we first analyzed the LMT1 sequence using the mFOLD program, which predicted that the LMT1 secondary structure could be quite complex and have a number of clustered stem-loop structures  Supplem. For furCTV is one of the most intricate viruses with a complex biology. Among the peculiar features of CTV is the production of a viral lncRNA LMT1. The 5′-terminal sgRNA LMT1 was discovered more than two decades ago, however, for years, its role in virus biology has remained unknown . RecentlTomato bushy stunt virus (TBSV) replicase protein p33 that binds to an internal replication element of TBSV gRNA [Brome mosaic virus (BMV) protein 1a that specifically interacts with the box B motifs in the BMV gRNAs [Previously, many plant viral proteins were shown to bind RNA. A large number of these proteins are MPs, which interact with RNA in a sequence-non-specific manner, so they bind viral and non-viral RNA molecules with similar affinity (reviewed in ). Our eaBSV gRNA , RCNMV aBSV gRNA ,44, and MV gRNAs ,46. For MV gRNAs . This fiMV gRNAs . InteresMV gRNAs , and thiThe results of this work reporting binding of the CTV p33 protein, which is dispensable for virus replication, assembly, as well as cell-to-cell movement, to a viral lncRNA LMT1 stands out from known cases of plant viral protein-RNA interactions. LncRNAs were identified in a wide range of organisms, including prokaryotes, eukaryotes, and viruses, and represent RNA molecules with lengths more than 200 nts and no apparent protein-coding capacity . LncRNAsBeet necrotic yellow vein virus produces ncRNA3, a 500-nt long lncRNA with unknown function [Plant virus lncRNAs have not received the same amount of attention as their animal virus counterparts, and our understanding of their functions remains limited. function . Anotherfunction . Unlike function ,15, thesfunction ,64.Although viral lncRNAs often bind the host proteins, fewer cases of the interactions between the former RNAs with proteins produced by the virus are known. One of them is the binding of KSHV PAN RNA to several KSHV proteins, which mediates regulation of the lytic reactivation and, overall, viral gene expression . As for N. benthamiana and precluded citrus infection [According to our recent study, the lack of LMT1 production by a CTV mutant reduced virus invasiveness and systemic movement in the CTV experimental host nfection . Furthernfection ,27. It i


EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full‐length genomic flGAG‐POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG‐POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5’OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA‐DEPENDENT‐RNA‐POLYMERASE‐6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto‐unrecognized “translation‐dependent silencing” (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.Co‐evolution between hosts’ and parasites’ genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral‐like silencing response. To investigate this response’s activation, we studied the single‐copy element  Arabidopsis transposon EVADE (EVD) reveals a ribosome stalling event during EVD translation that correlates with the production of small RNAs involving RNA‐dependent RNA polymerase 6 (RDR6).Analyzing the initiation of RNA silencing of the  These harbor only mosaics of de‐methylated DNA while maintaining wild‐type (WT) MET1 and DDM1 functions  retroelement EVADÉ (EVD) in theTy1/Copia family, which is one of the most proliferative families in plants  akin to that mounted against plant viruses RNAs. However, despite their loading into the antiviral PTGS effectors ARGONAUTE1 and ARGONAUTE2 (AGO1/2), they do not suppress expression of EVDs increasingly more abundant genomic copies. This ultimately gives way to DCL3, instead of DCL4/2, to process the RDR6‐made long dsRNAs into 24‐nt siRNAs. In association with AGO4‐clade AGOs, these species guide RNA‐directed DNA methylation (RdDM) of EVD copies. Initially localized within the EVD gene body, it later spreads into the LTRs to eventually shut down the expression of EVD genome‐wide via TGS  in an AGO1‐dependent manner  shared by Ty1/Copia retroelements to optimize protein expression from their compact genomes  GAG‐POL isoform codes for a polyprotein processed into protease, integrase/reverse‐transcriptase RNase, and GAG nucleocapsid components. On the other hand, a spliced and prematurely terminated short (sh) GAG subgenomic isoform is solely dedicated to GAG production. Though less abundant than the flGAG‐POL mRNA, shGAG is substantially more translated  in reporter‐gene settings. Given that Ty1/Copia retroelements share a PCPA‐based genome expression strategy  recapitulate the restriction of EVD siRNA to the 3’ part of the shGAG sequence  as opposed to rdr6 (siRNA‐deficient) background  to a more advanced (F11) epi15 inbred generation  mRNAs in the Arabidopsis line 35S:GFP‐int/terEVD‐GUS  or a fully deleted intron (35S:ΔiEVD) −) mRNAs are actively degraded by RQC, both of which explain their general absence from polysomes  display varying degrees of polysome association, some within the range of protein‐coding genes + mRNAs as opposed to nascent transcripts or splicing intermediates, cDNA from the same RNA samples was synthesized using exclusively oligo‐dT to capture polyadenylated RNAs only. This approach generated comparable results  deposition enhances translation of mRNAs even when tethered to intron‐less transcripts  RDR6 recruitment.The analysis revealed strikingly distinct nucleo‐cytosolic distribution patterns for the full‐length osol Fig , flGAG‐Pions Fig D. To valults Fig , indicatet al, et al, et al, Arabidopsis XRNs, XRN2 and XRN3 are nuclear, whereas XRN4 is cytosolic and, hence, mediates co‐translational mRNA decay  was conducted on the same RNA to monitor gene expression , presumably reflecting the higher transcript abundance. However, ACT2 does not spawn siRNAs  displaying the characteristic triplet periodicity  coinciding with this peak  of Arabidopsis transcripts displaying the most intense stalling events  in which two prominently covered and consecutive codons (pos. 235–236) accounted for ~40% of footprints  in a region corresponding, surprisingly, to the shGAG 3’UTR enhanced translation of a linked luciferase ORF  to 5’OH ends and was used previously to map co‐translational RNA cleavage fragments in yeast  requires a 3’ polyA tail to generate cDNA. Thus, the fact that the technique failed to detect the shGAG 5’OH cleavage fragments could indicate that they are indeed mostly poly(A)−. Preliminary PAGE‐based analyses as conducted for the 3’ ends of ribothrypsis products in mammalian cells ‐ fraction isolated from 35S:EVD in the rdr6 background  the disproportionate translation efficacies of each mRNA, (ii) the shGAG‐centric distribution of translation‐dependent EVD‐derived siRNAs, and consequently, (iii) the contrasted sensitivity of each isoform to cytosolic PTGS. Splicing‐coupled PCPA probably underlies most, if not all, of features (i–iii) because they were recapitulated with the GFP‐int/terEVD‐GUS construct containing the shGAG intron and proximal PCPA signal  and spawns RDR6‐dependent siRNAs , hyl1‐2, rdr6‐12, xrn2‐2, xrn3‐3, xrn4‐3 plants are all derived from the Col‐0 ecotype  were described previously : 35S:EVDwt (#167119), 35S:EVDmU1 (#167121), 35S:EVDΔI (#167120), 35S:GFP‐GUS (#167122) and 35S:GFP‐EVDint/ter‐GUS  from cytoplasm (supernatant). Cytoplasmic fractions were cleared by centrifugation at 14,000 g for 1 min. in a new tube and the resulting supernatant set aside. Nuclear pellets were rinsed by inverting the tube 3–5 times without disturbing the pellet with 1 ml of 1× PBS, 0.5 mM EDTA. Nuclei were spin for 15 s at 1,300 g before gently removing the wash solution. Nuclei pellets were resuspended by pipetting in 200 μl of nuclear lysis buffer . For isolation of total RNA and protein from the different fractions, samples were mixed 1 volume of acid PCI . In addition, nuclear fractions were further homogenized after addition of PCI by passing the sample through a 21‐gauge needle with a 1‐ml syringe. All steps were carried on ice or centrifuged at 4°C. Buffers were freshly prepared in advance and chilled on ice before use.For each sample, twice 250 mg of 3‐week‐old seedlings grown in ½ strength (2.2 g/l) Murashige and Skoog medium  was ground to fine powder in liquid nitrogen and homogenized in 575 μl of lysis buffer  and 1× cOmplete protease inhibitor cocktail (Roche)). Lysates were gently mixed and incubated on ice for 10 min. before being filtered through one layer of Miracloth. 400 μl from each lysate was recovered and one set aside as Total. The second set of cell lysate were gently overlaid on top of 1 ml of cold sucrose buffer ) in protein low binding 1.5‐mL tubes  by slowly pipetting against the side of the tube. Samples were centrifuged at 3,500 RNA was extracted from frozen and ground tissue with TRIzol reagent  and precipitated with 1× vol. of cold isopropanol. For RNA extraction from cyto‐nuclear fractionations, 20 μg of glycogen  and 0.1× vol. of sodium acetate 3 M pH5.2 was mixed with recovered aqueous phases after PCI before RNA precipitation with 1× vol. of cold isopropanol. DNA was extracted using the DNeasy Plant Mini Kit  according to manufacturer’s guidelines.g at 4°C). Proteins were precipitated from the phenol phase (including those from PCI) by the addition of 5 volumes of 0.1 M ammonium acetate in methanol. Precipitated proteins were collected by centrifugation for 30 min , washed twice with ammonium acetate in methanol and resuspended in resuspension buffer .Protein of frozen and ground tissue was homogenized in extraction buffer , and cOmplete EDTA‐free protease inhibitor cocktail . Water‐saturated and Tris‐buffered phenol (pH 8) was added to an equal volume and samples were agitated for 5 min. Phases were separated by 30‐min centrifugation −) was separated on a denaturing 4% polyacrylamide–urea gel, transferred to a HyBond‐NX membrane by electroblotting and UV‐crosslinked. For low‐molecular‐weight RNA analysis, 10–40 µg of total RNA was separated on a denaturing 17.5% polyacrylamide–urea gel, transferred to a HyBond‐NX membrane by electroblotting and chemically crosslinked  and oligo probes were radiolabeled by incubation of PNK  in the presence of [γ‐32P]‐ATP. Membranes were hybridized with these probes in PerfectHyb hybridization buffer  and detected on a Typhoon FLA 9500  laser scanner. Oligonucleotides used for probe generation are listed in Appendix Table For high‐molecular‐weight RNA analysis, 5–10 µg of total RNA was separated on a 1.2% agarose MOPS‐buffered gel with 2.2 M formaldehyde. RNA was partially hydrolyzed on gel with 5× gel volumes of 0.05N NaOH for 20 min. Gel was washed twice for 20 min with 20X SSC, transferred overnight by capillarity to a HyBond‐NX membrane  and UV‐crosslinked for fixation. For high‐molecular‐weight RNA analysis by PAGE, 1–40 µg of RNA et al, Proteins were separated on SDS‐polyacrylamide gels, transferred to Immobilon‐P PVDF membranes  by electroblotting and incubated with antibodies in 1× PBS with 0.1% Tween‐20 and 5% nonfat dried milk. After incubation with HRP‐conjugated secondary goat antibody against rabbit or rat primary antibodies (Sigma), detection was performed with the Clarity Max Western ECL substrate  on a ChemiDoc Touch imaging system (BIO‐RAD). Affinity‐purified antibodies were used at the specified dilutions: GAG  and cDNA was subsequently synthesized with the Maxima First‐Strand cDNA Synthesis Kit , or RevertAid cDNA Synthesis Kit with Oligo(dT) . qPCRs were run on a LightCycler480 II (Roche) or a QuantStudio5 (Applied Biosystems) machine with the SYBR FAST qPCR Kit (KAPA Biosystems). Ct values were determined by the 2nd derivative max method of minimally two technical replicates for each biological replicate. Relative expression values were computed as ratios of Ct values between targets of interest and + from non‐poly(A) RNA was performed from 75 μg of Trizol‐extracted total RNA from floral buds, using the DynabeadsTM mRNA Purification Kit (Ambion Cat#.61006) following the manufacturer’s instructions. Non‐polyA RNA was precipitated from the DynabeadsTM‐unbound fraction and resuspended in the same volume (200 μl) as the poly(A)+ RNA fraction. Efficiency of the separation was confirmed by running aliquots of each fraction on a 1% agarose gel to monitor efficient depletion of rRNA in poly(A)+ fractions before downstream analysis.Isolation of poly(A)EVADE transcript were mapped by a modified 5’ RACE method. Total RNA isolated from a pool of 2‐ to 3‐week‐old plants extracted by standard protocols (See nucleic acid extraction section) was taken for RNA ligations after DNase I treatment . RNA adapters with a 5’ inverted dT modification . The ligated RNA was converted to cDNA with RevertAid first‐strand cDNA synthesis kit  and a primer specific to the EVADE transcript , 1 mM EDTA, 400 mM ammonium acetate, 0.5% (w/v) SDS), and collected by precipitation with equal volumes of isopropanol. RNA was quantified using the Qubit™ RNA HS Assay Kit (Thermo Scientific) and subsequently cloned using the Small RNA‐Seq Library Prep Kit (Lexogen). Sequencing was performed on an Illumina HiSeq 4000 machine.Small RNA sequencing of 2, 2% (v/v) polyoxyethylene (10) tridecyl ether, 1% (v/v) de‐oxycholic acid, 1 mM DTT, 10 unit/ml DNase I (Thermo Scientific), 100 μg/ml cycloheximide). Precleared solutions were incubated with 650 U RNase I (Ambion) for 45 min at 25°C. Nuclease digestion was stopped by the addition of 10 μl SUPERase In RNase Inhibitor (Ambion). Resulting monosomes were purified by ultracentrifugation of the lysate on a sucrose cushion , 100 mM KCl, 5 mM MgCl2, 10 μg/ml cycloheximide, 10 units/ml RiboLock (Thermo Scientific) and cOmplete protease inhibitor cocktail (Roche) for 4 h at 250,000 g in 4°C. RNA was extracted using the TRIzol RNA extraction described above and treated with 10 U PNK (Thermo Scientific) for 30 min. Ribosomal RNA depletion was performed using the RiboMinus Plant Kit (Thermo Scientific) and libraries were generated as above, except that the 25–32 nt RNA fraction was excised from the denaturing polyacrylamide gel prior to RNA ligation.For RIBO‐seq libraries, frozen inflorescence tissue was ground in digestion buffer (100 mM Tris·HCl (pH 8), 40 mM KCl, 20 mM MgClet al .Arabidopsis genome with STAR  mapped against the TAIR10 et al, et al, Arabidopsis genome and transcriptome sequences. Quality control of the RIBO‐seq libraries was performed with the riboWaltz (Lauria et al, et al, RIBO‐seq libraries were analyzed as follows. Reads were trimmed of adapter sequences with bbduk as above. Reads mapping to rRNA loci using Bowtie2 (Langmead & Salzberg, EVD 5’OH ends, MT and VB‐B investigated EVD copy number in rdr6 and RNA isoform distribution in cyto‐nuclear fractions. MAS, AP, and MDN performed nanoPARE. LL conducted seed counting. SO and MAS performed computer analyses. SO and MT performed statistical analyses. AM‐O, SO, and OV analyzed the data and wrote the manuscript.AM‐O, SO, and OV conceived and designed the study. SO and AM‐O performed most experiments. RR cloned and sequenced the AppendixClick here for additional data file.Source Data for AppendixClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.


Loop-mediated isothermal amplification (LAMP) is a method of nucleic acid amplification that is more stable and resistant to DNA amplification inhibitors than conventional PCR. LAMP multiplexing with reverse transcription allows for the single-tube amplification of several RNA fragments, including an internal control sample, which provides the option of controlling all analytical steps. We developed a method of SARS-CoV-2 viral RNA detection based on multiplex reverse-transcription LAMP, with single-tube qualitative analysis of SARS-CoV-2 RNA and MS2 phage used as a control RNA. The multiplexing is based on the differences in characteristic melting peaks generated during the amplification process. The developed technique detects at least 20 copies of SARS-CoV-2 RNA per reaction on a background of 12,000 MS2 RNA copies. The total time of analysis does not exceed 40 min. The method validation, performed on 125 clinical samples of patients’ nasal swabs, showed a 97.6% concordance rate with the results of real-time (RT)-PCR assays. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time. The SARS-CoV-2 virus, belonging to Betacoronavirus genera, caused the COVID-19 coronavirus infection pandemic, which is continuing as of May 2021. The disease has spread throughout the world, and an urgent need has emerged for the for SARS-CoV-2 diagnostic tests. Most widely used test systems are based on real-time (RT)-PCR, requiring 1.5–2 h to obtain the final results. Meanwhile, the sharply increased pressure on diagnostic laboratories and the limited number of thermocyclers has made the time of analysis one of the factors limiting the number of tests that can be performed in a single day. In other words, techniques that are faster than qPCR are necessary to increase the throughput capacity of laboratories.During the last several decades, the explosive development of molecular biology has brought about a vast amount of different approaches for the amplification of nucleic acids. Many of these rely on the amplification of DNA at a constant temperature, also known as isothermal amplification  ,5,6,7,8.The advantages of LAMP make it a promising method for the development of SARS-CoV-2 diagnostic tests. In 2020, several LAMP-based tests were designed; some of them have been certified for clinical use ,22,23,24Multiplex amplification is the simultaneous amplification of two or more DNA fragments in the same tube, which enables the concurrent detection of several pathogens and cuts testing costs. Multiplexing allows for the simultaneous amplification of internal reaction controls or internal controls in DNA or RNA isolation, which allows researchers to monitor the quality of NA isolated from samples and the performance of reagents for reverse transcription and amplification. The nature of the amplification mechanism makes the multiplexing of LAMP a challenging task. Thus, DNA polymerases employed for LAMP lack 5′–3′ exonuclease activity , forestaHere, we have developed a method of SARS-CoV-2 RNA detection employing multiplex reverse-transcription loop-mediated isothermal amplification. The method couples multiplex real-time RT-LAMP with melting curves analysis, allowing for the addition of MS2 phage as an internal control in all test procedures.We designed the multiplex RT-LAMP for the simultaneous detection of two RNA molecules, the fragment of SARS-CoV-2 RNA (GenBank ID NC_045512.2) and MS2 phage RNA (GenBank ID NC_001417). The latter served as an internal control for RNA isolation when added to the clinical samples before the isolation procedure.As targets for the LAMP, we selected conserved regions of SARS-CoV-2 genomic RNA, encoding E and N proteins, and two regions of MS2 phage genomic RNA. The primers were designed according to the recommendations on the primerexplorer.jp website. In total, we made two primer sets for the LAMP-based detection of SARS-CoV-2 and MS2 .As DNA standards for the evaluation of LAMP efficiency, we constructed plasmids based on pBlueScript II SK (+) vector and 200-bp SARS-CoV-2 and MS2 genome fragments. As RNA standards, we used RNA fragments obtained by the in vitro transcription of SARS-CoV-2 plasmid standards or MS2 genomic RNA. All standards were quantified by means of droplet digital PCR using the QX200 platform .5, two copies per reaction). The results are presented in For the evaluation of real-time LAMP efficiency with the selected primers, we used serial dilutions of plasmid DNA . Using serial dilutions of plasmid DNA, we compared Tt values and melting curves for monoplexes of CoV-2-E and MS2 and the duplex. We also tested the amplification efficiency of the MS2-CoV-2-E duplex with the CoV-2-E plasmid on the background of the MS2 plasmid (2000 copies per reaction). The results of the comparison are presented in We determined an optimal concentration of the control MS2 RNA. We compared the amplification efficiency of CoV-2-E primers or the MS2-CoV-2-E duplex using serial dilutions of CoV-2-E RNA on the background of MS2 RNA . The height of the corresponding peak on the melting curve served as an assessment criterion of the amplification efficiency. The results of the comparison are presented in The limit of detection (LoD) was defined as the lowest amount of SARS-CoV-2 RNA reliably detectable in a single reaction. We evaluated the LoD of the multiplex LAMP using various amount of CoV-2-E RNA, ranging from 100 to 10 copies per reaction, on the background of 12,000 copies of MS2 RNA. For each concentration, LAMP with the MS2-CoV-2-E duplex was performed in 20 technical repeats in a single run. The reaction was defined as “positive” when the peak corresponding to CoV-2-E RNA was presented on the melting curve . It shou6 MS2 phage particles before RNA purification. RNA was isolated from the samples using an AmpliSens® RIBO-prep kit , then was tested by two methods: real-time RT-PCR with primers recommended by the WHO [Multiplex MS2-CoV-2-E LAMP was validated on clinical samples of nasal swabs taken from 125 patients from CNMT ICBFM SB RAS. All patients participating in the study signed an informed consent form. Each clinical sample was spiked with 6*10 the WHO  and multSince the discovery of the novel SARS-CoV-2 coronavirus in December of 2019, dozens of studies have been published on LAMP-based testing for SARS-CoV-2 detection ,37,38. MIn the context of laboratory diagnostics, real-time detection of the pathogen provides a quantitative and semi-quantitative analysis of infectious agents. However, the option of multiplexing is equally significant, i.e., the single-tube detection of several targets saves time, consumables and samples for analysis. Multiplexing allows for the introduction of qn internal control for nucleic acid isolation and amplification, thus lowering the probability of false-negative results originating from the incorrect processing of clinical samples.A wide spectrum of methods is available for the detection of LAMP results, from intercalating fluorescent dyes to turbidimetry. However, LAMP multiplexing remains a complicated task, requiring a solution. Here, we developed a multiplexing technique using fluorescent intercalating dye and melting curves analysis. This approach has been widely applied in PCR and has also been used for LAMP in two works ,39. ThusDuring the in vitro amplification of nucleic acids, the simultaneous amplification of several fragments can be hampered due to unequal reaction efficiencies for different fragments or the formation of primer dimers ,41. ThisThe characteristics of the developed duplex LAMP assay for SARS-CoV-2 RNA detection are similar to previously published analogous tests based on monoplex LAMP. The limit of detection for reported monoplex LAMP tests lies in the range of 300–10 copies of viral RNA per reaction; the concordance with PCR results was 92–95%. Therefore, the developed approach demonstrated its feasibility and can be further used to detect other nucleic acids related to other pathogens.It is worth noting that LAMP, and the approach to multiplexing used in this study, possess some limitations. The large amount of the amplified product makes LAMP reactions a potential contamination source for lab equipment, requiring careful handling and proper waste disposal. LAMP requires more primers than PCR: six primers instead of two, and in higher concentrations. The length of BIP/FIP primers is about 40–50 nt. These two factors lead to higher requirements for the quality and quantity of synthesized oligonucleotides. LAMP also requires Bst-like DNA polymerases, which are less available compared to Taq polymerase and its analogues, which are routinely employed in PCR.LAMP is known to be more specific than PCR due to its use of six primers instead of two. Although high specificity itself makes the test more robust and less prone to false-positive results, the detection of mutated pathogens by means of LAMP could be challenging. The SARS-CoV-2 genome is known to bear multiple mutations that could be located in the primers’ binding sites and affect the sensitivity of tests. Thus, including more target regions in the multiplex LAMP for SARS-CoV-2 detection could prevent possible false-negative results caused by mutations in primer-binding sites.Multiplexing of LAMP is a complex task, which limits the number of pathogens for a single-tube analysis. The LAMP multiplexing method chosen in this study requires a careful selection of primers and the adjustment of melting temperatures of amplified fragments to exclude the overlapping of melting peaks. Therefore, LAMP, as well as PCR, possesses its own requirements and limitations, and the choice of testing method should be based on the careful consideration of the technique’s features and the facilities of the diagnostic lab.We have developed a test for the detection of SARS-CoV-2 RNA based on multiplex loop-mediated isothermal amplification. Fragments of SARS-CoV-2 RNA and MS2 phage RNA were detected in a single-tube format; MS2 phage served as an internal control for RNA isolation and amplification. Multiplexing was based on the melting curves analysis of the amplification products. The limit of detection for multiplex LAMP was 20 copies of SARS-CoV-2 RNA per reaction. Testing of 125 clinical samples showed a 97.6% concordance rate with real-time RT-PCR results. The developed multiplexed LAMP can be employed as an alternative to PCR in diagnostic practice to save personnel and equipment time.Clinical samples  were taken from patients in the Center of New Medical Technologies ICBFM SB RAS. Plasmids with genome fragments of SARS-CoV-2 and MS2 phage, prepared using a traditional restriction-ligation cloning method, in vitro synthesized RNA fragments and MS2 phage RNA served as the control samples. For full descriptions of the cloning procedures, in vitro transcription, MS2 phage growth and MS2 phage RNA purification, see the The concentrations of DNA and RNA in the obtained standards were refined by means of a digital PCR, using the QX200™ Droplet Digital™ PCR System  according to the manufacturer’s instructions. The reactions were carried out in a total volume of 20 µL, containing the DNA under examination (approximately 103 copies per 20 µL), 1× ddPCR master-mix , 300 nM oligonucleotide primers and probes: E_Sarb_F/R/P for CoV-2-E, N_Sarb_F/R/P for CoV-2-N  and MS2-Real-time RT PCR was performed in a CFX 96 thermocycler  in a total reaction volume of 20 µL, containing 1× PCR buffer 2SO4, 0.05% Tween 20, 2.5 mM MgCl2), 0.3 µM primers and 0.1 µM probes: E_Sarb_F/R/P for CoV-2-E and N_Sarb_F/R/P for CoV-2-N , 1 unit The reaction mixture for LAMP (20 µL) contained 1× reaction buffer for Bst-polymerase 2SO4, 150 mM KCl, 0.1% Tween-20, 2 mM M MgSO4), 1.4 mM each of dNTP, 0.2 µM each of external primer (F3/B3), 0.6 µM loop primers (LF/BF), 1.6 µM internal primers (FIP/BIP) , DNA or The limit of detection of the multiplex LAMP was assessed by varying the amount of RNA template in the reaction mixture. Multiplex LAMP was performed with 1× CoV-2-E primers and 0.5× MS2 primers under the conditions described above. The amount of CoV-2-E RNA template in the reaction mixture was 100, 50, 20 or 10 copies, on the background of 12,000 copies of MS2 phage RNA. Multiplex LAMP was performed in 20 technical repeats; CoV-2-E RNA amplification was registered as a characteristic peak on the melting curve of the amplification products. The limit of detection was defined as the concentration of CoV-2-E RNA that provided the appearance of a melting peak for CoV-2-E amplification products in all technical repeats.Analytical specificity was evaluated in silico by checking the homology of primers with the genomes of most widespread human viral pathogens causing respiratory infections. The comparison was made using the BLAST algorithm on the NCBI website.® RIBO-prep kit  according to the manufacturer’s protocol. Each clinical sample was spiked with 6 × 106 MS2 phage particles before RNA purification. Purified RNA was tested in the presence of SARS-CoV-2 RNA using real-time PCR and multiplex LAMP.The clinical sensitivity and specificity of the multiplex LAMP were assessed using clinical samples  taken from the patients of CNMT ICBFM RAS. RNA was isolated from clinical samples using an AmpliSens


E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing  Base editors can induce transcriptome-wide RNA off-target editing independent of gRNA. Here, the authors engineer ABEmax variants with minimized RNA off-target activities. E.coli TadA (ecTadA) and a laboratory-evolved E.coli TadA (TadA*) with a Cas9 (D10A) nickase (Cas9n), can induce efficient A-to-G or T-to-C conversions with very low levels of unwanted mutations or insertions2. ABE is designed based on the native structure of homodimerized ecTadA, which can deaminate an adenosine within a transfer RNA (tRNA)3, with an evolved TadA* being capable of deaminating genomic DNA adenosines1. Although ABEs show no detectable guide RNA-independent DNA off-target editing5, both ABEs and cytosine base editors (CBEs) can induce tens of thousands of A-to-I (inosine) or C-to-U (uracil) RNA edits transcriptome-widely in guide RNA-independent manners in human cells7. Engineered CBE and ABE variants bearing rAPOBEC1 mutations6 or TadA/TadA* mutations9, have been recently reported with reduced RNA off-targeting activities. In these studies9, GATK HaplotypeCaller, a tool for evaluating germline single nucleotide polymorphisms (SNPs) and indels10, is employed for analyzing RNA A-to-I edits. It is worth noticing that RNA edits with 0–10% efficiency was not able to be recovered by this tool9, suggesting a possible underestimation of RNA off-targets, therefore, driving us to further examine ABE-induced off-target editing of cellular RNAs in depth. In this work, according to structure-guided principles, we successfully engineered ABEmax and mini ABEmax (TadA* fused with Cas9n) variants to generate engineered ABEs (eABEs) that retained DNA on-target editing activities while largely decreased RNA editing activities.Adenine base editors (ABEs), which is originally designed by fusing a wild-type 9, we reasoned that MuTect2, a GATK tool for sensitive detection of somatic point mutations in heterogeneous cancer samples11, might be more appropriate than HaplotypeCaller (for detection of euploid germline SNPs) for detection of RNA edits12 , demonstrating that these optimized ABE variants still retained a relatively large number of RNA edits  and tRNA as well as the alignment of the conserved amino acid sequences between ecTadA and SaTadA with high similarity13, showing that the amino acids possibly responsible for interaction with tRNA were conserved between the two types of TadA  gene containing tRNA-like CTACGAA sequence, which has been reported to be highly edited by ecTadA at RNA levels14, into a CMV promoter-driven vector. Then, this reporter was co-transfected with an sgRNA targeting HEK site 3 and ABEmax or its engineered variants, and the A-to-G editing efficiencies in ecHokb cDNA (reversely transcribed from mRNA) or genomic DNA (gDNA) were determined by targeted deep sequencing on ecHokb cDNA or gDNA amplicons. It showed that both ABEmax and 2xTadA induced highly efficient RNA but not DNA editing within ecHokb locus. Notably, we identified three variants  with substantially decreased RNA editing activities, especially R153P with most reduced RNA edits comparable to the negative Cas9n control  or mini ABEmax9 (mini del153). As expected, we demonstrated that compared with ABEmax or mini ABEmax, the RNA off-targets induced by del153/del153* and mini del153 were largely decreased, and there were as few as 291 (MuTect2) or 98  RNA A-to-I edits for mini del153 group 9. The results showed that our eABE variants, del153/del153* and mini del153, induced much fewer RNA off-targets by both MuTect2 and HaplotypeCaller tools under the same experimental conditions  with reported variants possessing reduced RNA off-targeting activities, including ABEmaxAW (E59A/V106W*)6, del153/del153* and mini del153 variants-induced RNA A-to-I edits were decreased to only dozens or hundreds of off-targets when using HaplotypeCaller . Oligos are showed in Supplementary Table 07721. OlHEK293T and U2OS cells were purchased from ATCC and cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) (v/v)  and 1% Penicillin Streptomycin at 37 °C with 5% CO2.For deep sequencing samples, HEK293T cells were seeded on 24-well plates (JETBIOFIL) and transfected at ~70% confluence with editors (628 ng) and sgRNAs (373 ng) using Lipofectamine LTX  according to the manufacturer’s protocol. GFP positive cells were harvested from fluorescence-activated cell sorting (FACS) 48 h after transfection.For RNA sequencing samples, HEK293T cells were seeded on 6 cm dish (JETBIOFIL) and transfected at ~70% confluence with editors (4 µg) and sgRNA-expressing plasmids (2 µg) using Lipofectamine LTX  according to the manufacturer’s protocol. GFP signal positive cells of top 15% were harvested from fluorescence-activated cell sorting (FACS) 48 h after transfection.Genomic DNA of HEK293T and U2OS cells was extracted using phenol-chloroform method. For RNA extraction, cells harvested from FACS were immediately treated with TRIzol reagent , according to the manufacturer’s instructions.Target sites were amplified with primers listed in Supplementary Table The libraries were sequenced on an Illumina HiseqXten-PE150, at a depth of ~20 million reads per sample. The reads were mapped to the human reference genome (hg38) by STAR software (Version 2.5.1); annotation from GENCODE version V30 was used. After removing duplication, variants were identified by GATK . For MuTect2 method, variants were filtered with FilterMutectCalls. For HaplotypeCaller method, variants were first filtered with QD  <2, then all variants were verified and quantified by bam-readcount with parameters -q 20 -b 30. The depth for a given edit should be at least 10x and these edits were required to have at least 99% of reads supporting the reference allele in the wild-type samples. Finally, only A-to-G edits in transcribed strand were considered for subsequent analysis. Motif or sequence logo was analyzed by WebLogo (v3.6.0) for RNA edits. The downloaded data subjected to RNA off-target analysis from four published papers were listed in Source Data for Supplementary Figures. Detailed information for called mutations was provided in in a Source Data file .http://www.rcsb.org/structure/2b3j) by PyMol . TadA from Staphylococcus aureus (SaTadA) was shown as cartoon model in gray and the RNA bound was shown as stick model rendered by elements, with the Zn2+ ion as green sphere. The residues critical for the RNA binding of TadA were shown in ball-and-stick model and labeled with single-letter codes in red.A structural model for TadA-RNA complex was generated using coordinates from PDB ID 2B3J , and original data presented in supplementary figures were presented in a Source Data file . Statistical analyses and graphing were carried out by using GraphPad Prism 8.0. Comparisons of mean values were analyzed by Student’s Further information on research design is available in the Supplementary InformationReporting Summary


Over the past 20 years, a variety of approaches have been employed to discover chromosome-encoded sRNAs in a multitude of bacterial species. However, although largely improved bioinformatics tools are available to predict potential targets of base-pairing sRNAs, it is still challenging to confirm these targets experimentally and to elucidate the mechanisms as well as the physiological role of their sRNA-mediated regulation. Here, we provide an overview of currently known  Whereas cis-encoded sRNAs are transcribed from the same genetic locus in opposite direction to their single target RNA and are, therefore, completely complementary to this target RNA, trans-encoded sRNAs are transcribed from a promoter somewhere else on the bacterial chromosome and are only partially complementary to their target RNAs. Therefore, they can only form partial duplexes with their—often multitude of—target RNAs. In both cases, target RNA binding results in inhibition or activation of target gene expression. Cis- and trans-encoded sRNAs employ a variety of mechanisms of action for posttranscriptional gene regulation [Small regulatory RNAs (sRNAs) that act by base-pairing constitute the main class of posttranscriptional regulators in bacteria. They can be divided into gulation ,2,3 whicA second class of sRNAs acts by protein binding. It comprises the ubiquitous 6S RNA that binds the RNA polymerase by mimicking an open promoter and helps to shift transcription from vegetative to stationary phase promoters (rev. in ), sRNAs cis-encoded base-pairing sRNAs, i.e., bona fide antisense RNAs, were discovered in E. coli plasmids in 1981 and intensively studied as regulators of replication , conjug(rev. in ). Only iectively . Since tectively ), and coectively ). Hitherectively ). In addrference , and somB. subtilis were performed in 2009 and 2010 [trans-encoded sRNAs. However, in contrast to Gram-negative bacteria like E. coli or Salmonella enterica [B. subtilis sRNAs with identified targets is still very limited. In this review, we summarize our current knowledge on all cis- and trans-encoded sRNAs from B. subtilis, for which targets have been identified and physiological functions elucidated. We do not include protein-binding sRNAs or riboswitches in our review.Two transcriptomics approaches for the prediction of small RNAs in and 2010 ,14, whicenterica , the numcis-encoded sRNAs and their corresponding targets are known in Bacillus subtilis  systems are genetic modules comprising a toxin gene and the counteracting antitoxin. These systems are widespread among bacteria and archaea—Both on the chromosome and on plasmids or phages to ensure the stable inheritance of DNA elements. In type I TA systems, the antitoxin is a (rev. in ). A furt and its antitoxin RatA [txpA overexpression drastically impairs growth. The toxin contains a trans-membrane domain followed by charged aa and likely disturbs membrane integrity by pore formation. The antitoxin RatA, encoded on the opposite strand, overlaps the txpA transcript by ca. 120 nt at the 3′ end. The txpA RNA–RatA complex is a target for RNase III. This role in toxin mRNA degradation makes RNase III essential for B. subtilis [txpA translation efficiency but exclusively promotes the toxin RNA degradation. The txpA/RatA system is located on the skin element, which is excised during the sporulation process and could prevent loss of this region even during a longer period of vegetative growth to preserve the ability to sporulate [The xin RatA . In the subtilis . Bindingporulate . MoreovebsrG/SR4 system is encoded on the SPβ prophage region [sr4 leads to cell lysis after overnight incubation at 37 °C [bsrG mRNA, forming a perfect duplex of 123 bp. SR4 is bifunctional: it induces i) RNase III-dependent degradation of the bsrG mRNA and ii) a refolding around the RBS (ribosome binding site) that inhibits translation initiation [bsrG mRNA regions [bsrG mRNA comprises a YUNR motif in a loop, a sequence previously shown to be able to form a U-turn [The e region . The toxe region . Deletioat 37 °C . The anta U-turn  that faca U-turn .bsrE/SR5 system is located on the prophage-like element P6 in the chromosome [bsrE from a multicopy plasmid causes cell lysis on agar plates and inhibits growth significantly [bsrE by 114 nt at the 3′ end. Complex formation of both RNAs leads to degradation of bsrE mRNA by RNase III [bsrG/SR4, no direct translational inhibition of bsrE mRNA by SR5 was observed. The interaction pathway between bsrE mRNA and SR5 was investigated in detail [bsrE and sr5 is regulated differently in response to a multitude of stress factors. The otherwise very stable bsrE mRNA is sensitive to ethanol and alkaline stress as well as heatshock, while SR5 amounts differ under acid and alkaline stress, iron limitation, and anaerobic conditions. Interestingly, SR5 is rapidly degraded under oxygen deficiency, which allows bsrE expression. Therefore, this TA system could play a role in the response of B. subtilis to anaerobic conditions [The romosome . The toxficantly . The antNase III . In contn detail : the ininditions .yonT/yoyJ/SR6 system is very special, because it contains two toxin genes that are regulated by the same antitoxin. YonT (59 aa) is a very strong toxin, consisting of an N-terminal transmembrane domain and a highly positively charged C-terminal region. The deletion of the antitoxin gene sr6 causes cell lysis. YoyJ (83 aa) is a very weak toxin with two transmembrane domains. In the absence of SR6, yoyJ overexpression is detrimental to the cells. The antitoxin SR6 uses two distinct mechanisms to repress yonT and yoyJ: SR6 and yonT mRNA interact at their 3′ ends inducing RNA degradation and, therefore, toxin inactivation by RNase III [yoyJ mRNA involves both 5′ ends and does not influence the yoyJ mRNA stability. Instead, SR6 inhibits the yoyJ translation by blocking the RBS [sr6 promoter is much weaker than the yonT promoter, which is unfavorable to generate an excess of the antitoxin. However, this is compensated for by an unusually stable antitoxin. The TA system is located on the SPβ prophage region of the chromosome and, like bsrE/SR5, both RNAs are impacted by several stress conditions.The Nase III . The int the RBS . SurprisyonT mRNA under heatshock or manganese stress suggests a role of the system in the cellular stress response [An excess of response .bsrH/as-bsrH system is a potential type I TA system [bsrH mRNA interacts with the antitoxin as-bsrH at the 3′ end and is degraded by RNase J1 [txpA/RatA system.The A system ,29. The RNase J1 . The sysyabE. S25 is transcribed under control of the extracytoplasmic sigma factors σM and σX in response to cell envelope stress, e.g., the presence of antibiotics. The amount of yabE RNA is reduced during S25 expression, which may be due to RNase III-dependent degradation [The non-coding RNA S25 is an antisense RNA regulating the gene radation . The fungdpP gene on the opposite strand. GdpP is a membrane-localized c-di-AMP specific phosphodiesterase [D-dependent, suggesting a role in or during motility regulation. The expression of S1559 reduces the amount of cellular GdpP but does not influence motility or antibiotic resistance [The sRNA S1559 is transcribed convergently to the esterase , which isistance . The mecB-dependent promoter. The transcript was detected after phosphate stress, is of heterogeneous length, and is complementary to the cwlO gene. CwlO is an extracellular D,L-endopeptidase-type autolysin involved in the cell wall synthesis and mainly active in the exponential growth phase [cwlO transcript is very unstable. How the antisense RNA influences the cwlO expression and the biological role of this regulation are so far unknown. The only effect observed was a slightly faster entry into exponential growth after resuspension in fresh medium [The antisense RNA S1326 is transcribed from a σh medium .B-dependent antisense RNA is S1290, which is complementary to opuB mRNA. OpuB is a choline importer required to maintain the osmotic homeostasis under high salinity conditions. The transiently transcribed S1290 acts by transcriptional interference to repress a fast and strong opuB induction after osmotic shock. This delays the opuB expression in favor of the expression of opuC, which encodes an alternative but more general osmoprotectant import system. Therefore, S1290 is involved in fine-tuning the salt stress response [Another σresponse .rpsD transcription and on the other hand is an mRNA encoding the small protein SR7P (39 aa). This small protein interacts with the glycolytic enzyme enolase, thereby promoting its recruitment to RNase Y to enhance the RNase Y activity on at least two substrate RNAs, rpsO mRNA, and the 5′ UTR of yitJ mRNA [in cis to reduce the amount of rpsD mRNA, most likely by transcriptional interference [B-dependent transcription of sr7 is strongly induced under ethanol stress [The sRNA SR7 S1136) is a dual-function antisense RNA that on the one hand interferes with itJ mRNA . ConsequitJ mRNA ,36. SR7  is a duarference . RpsD , for a 20- to 30-fold repression of sr1 transcription [ahrC mRNA [rocABC and rocDEF and the transcriptional repressor of the arginine biosynthesis genes [ahrC mRNA, designated A to G in SR1 and A’ to G’ in ahrC mRNA, located in the central portions of both RNAs [ahrC RBS and inhibits translation initiation by a novel mechanism: SR1 induces structural changes 20 to 40 nt downstream of the ahrC RBS that obstruct binding of the ribosomal 30S subunit [SR1 was the first subtilis . A compublotting . The sr1 by CcpA . Whereascription ,48. CcpNcription . The firhrC mRNA , encodinis genes . SR1 shaoth RNAs . Binding subunit .ahrC RNA complex formation [ahrC mRNA [ahrC mRNA but does not promote their interaction. Instead, it is required for ahrC translation by opening a secondary structure 5′ of the RBS. By contrast, the RNA chaperone CsrA that also binds to both RNAs facilitates their interaction by slightly altering the ahrC mRNA structure around the G’ region to support SR1 binding [Although the G/G’ interaction is decisive, the complementary regions B/B’ to F/F’ also contribute to SR1/ormation . SR1 barhrC mRNA ,46, excltrans-encoded sRNA that acts by a base-pairing mechanism, SR1 is an mRNA that codes for a small protein of 39 aa [B. subtilis), promotes the interaction of GapA with RNase J1, and enhances the enzymatic activity of RNase J1 on two substrate RNAs [In addition to being a of 39 aa . This prate RNAs . The SR1ate RNAs . So far,ate RNAs ,54 , dicarboxylate transporter (dctP), the extracytoplasmic thioreductase resA, leuABCD (leucine biosynthesis), and menaquinolcytochrome c oxidoreductase qcrA genes [gltAB mRNA , FbpB (48 aa), and FbpC (29 aa) postulated to be RNA chaperones [lutABC mRNA has been shown, which allows the direction of iron to higher priority target proteins [lutABC mRNA levels were affected approximately two-fold by both FsrA and FbpB with FbpB having a higher effect on RNA levels. Only small effects on the LutA, LutB, and LutC protein levels were observed. Whereas FsrA might directly repress lutABC translation by a base-pairing interaction, FbpB might facilitate the FsrA-lutABC RNA interaction since the role of FbpB could be bypassed by modest upregulation of fsrA. Alternatively, FbpB might recruit an RNase for lutABC mRNA degradation. So far, experimental evidence for any of these mechanisms is still lacking. For the repression of sdhCAB mRNA and citB mRNA by FsrA, all three Fbp proteins seem to be dispensable as has been shown by a proteomics study with fsrA and fbp mutants [Whereas the functionally related Fur-regulated sRNA RyhB from aperones . Howeverproteins . The lut mutants .B. subtilis and S. aureus. Transcription of roxS is activated in response to nitric oxide by the two-component system ResDE [+/NADH balance by temporarily turning down part of the TCA (tricarboxylic acid) cycle. So far, four direct RoxS targets have been experimentally confirmed: ppnKB mRNA encoding an NAD+/NADH kinase, sucCD mRNA encoding succinyl-CoA synthase [yflS encoding one of four malate transporters transcribed under control of MalKR that senses malate [acsA encoding acetyl CoA synthase [ppnKB mRNA . The latter is required for inhibition of sucCD translation but can also efficiently regulate ppnKB. For acsA, fourfold-reduced mRNA levels in the presence of RoxS have been observed [yflS, RoxS disrupts a structure at the 5′ UTR that obstructs ribosome binding to activate translation. Furthermore, it stabilizes yflS mRNA about 3.6-fold against 5′-3′ exoribonucleolytic degradation by RNase J1 [yflS mRNA is independent of its protection against 5′-3′ exoribonucleolytic degradation by RNase J1 [sucCD mRNA can be uncoupled [RoxS  is, so far, the only base-pairing sRNA that is conserved between em ResDE  and inhiem ResDE . The repsynthase , yflS ens malate , and, resynthase . In all nKB mRNA C and sucobserved , but effRNase J1   in a ΔrosA strain corroborated the hypothesis that RosA sequesters RoxS [B. subtilis grown in LB medium yielded a 1:1 RoxS/RosA ratio, indicating that even a complete out-titration of RoxS by RosA would be possible. The RosA effects on RoxS target mRNA levels were substantiated by proteomics confirming altered protein levels for PpnKB, SucC, SucD, AcsA, and YrhF in the absence of RosA. Interestingly, RoxS also plays a role in RosA processing, affecting at least the abundance of the 128 nt RosA species.Recently, a sponge RNA, RosA (regulator of sRNA A) was identified that is transcribed from a σsm, CcpA . Northerers RoxS . AccordiAnother interesting question raised by the authors is whether the RosA/RoxS duplex might be a reservoir for RoxS or the truncated RoxS(D) species, from which the sRNA can be recycled for base-pairing with new target RNAs for which it has a greater affinity.B. subtilis under oxidative respiration conditions was demonstrated by co-cultivation of a wild-type and a ΔrosA strain: the ΔrosA strain was rapidly outcompeted due to the RoxS- and FsrA-mediated reduction of TCA cycle enzyme levels, which did not allow the mutant to generate ATP as quickly as the wild-type [The CcpA-dependent RosA regulation places RoxS that is controlled by malate via transcription factor Rex at the key position of central metabolism in response to different carbon sources. The importance of RosA for ild-type .rosA strain would not allow RoxS to respond quickly to altered environmental conditions and that, therefore, RosA might be required to rapidly downregulate RoxS activity first by neutralizing the C-rich regions required for target RNA binding and afterwards by promoting RoxS degradation [Why does RoxS, which is regulated by two transcription factors, ResD and Rex, need an additional level of posttranscriptional control by a sponge RNA? The authors argue that the long half-life of 30 min in a Δradation .The RosA–FsrA interaction involves RosA GRR2 as well, which binds to CRR2 of FsrA, as also shown in vitro by EMSAs. Proteomics revealed the expected alterations in the amount of proteins encoded by FsrA target mRNAs, among them CitB, SdhA, and CitZ.Whereas the currently known sponge RNAs from Gram-negative bacteria are either mRNAs  or derivRosA contains a seven codon ORF that was associated with ribosomes in a ribosome profiling study . Since tB. subtilis intergenic regions. However, so far it has not been visualized in Northern blots. This sRNA is only transcribed during the exponential growth phase under control of σD. Compensatory mutations introduced into predicted complementary loop regions revealed that RnaC base-pairs with abrB mRNA at the RBS and the first six codons to modulate the cellular level of transition state regulator AbrB [abrB RNA interaction that did not require Hfq was shown to support degradation of abrB mRNA and proposed to inhibit its translation. However, in ΔrnaC strains grown in LB medium, only very small alterations (≈33%) of abrB mRNA and AbrB levels were detected. The authors observed an enhanced cell-to-cell variation of AbrB levels, which finally resulted in growth rate heterogeneity within one population of logarithmically growing cells. They hypothesized that the subpopulations of fast- and slow-growing B. subtilis cells reflect a bet-hedging strategy for enhanced survival of unfavorable conditions since slowly growing cells are less susceptible to harsh environmental conditions [RnaC/S1022 was first identified in a microarray screen of tor AbrB  (Figure nditions .B. subtilis [cis-encoded and five trans-encoded sRNAs have been characterized in detail. For each of the trans-encoded sRNAs, several interaction partners have been identified and biological functions in different areas elucidated. However, for the majority of the sRNAs, targets have yet to be identified and perhaps new mechanisms of action have yet to be unraveled. In addition, the recent discovery of the sponge RNA RosA [Although more than 100 sRNAs have been discovered by transcriptome analyses in subtilis ,14, and subtilis ,38,63, oRNA RosA  indicateB. subtilis does not seem to rely on two general RNA chaperones to promote the majority of sRNA-target RNA interactions or to stabilize sRNAs but seems to employ several RNA-binding proteins depending not only on the corresponding sRNA, but the specific sRNA/target RNA system, as illustrated by SR1/ahrC and SR1/kinA. A global approach is required to find out if the function of CsrA is confined to the SR1/ahrC system or if it plays a broader role in B. subtilis similar to Hfq and ProQ in Gram-negative bacteria like E. coli or Salmonella enteric [B. subtilis (see Bacillus subtilis chromosome like a number of eukaryotic siRNAs.In contrast to Gram-negative bacteria,  enteric . Moreove enteric ,58 are iilis see , but an