The unprecedented emergence and spread of SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, underscores the need for diagnostic and therapeutic technologies that can be rapidly tailored to novel threats. Here, we show that site-specific RNA endonuclease XNAzymes – artificial catalysts composed of single-stranded synthetic xeno-nucleic acid oligonucleotides (in this case 2’-deoxy-2’-fluoro-β-D-arabino nucleic acid) – may be designed, synthesised and screened within days, enabling the discovery of a range of enzymes targeting SARS-CoV-2 ORF1ab, ORF7b, spike- and nucleocapsid-encoding RNA. Three of these are further engineered to self-assemble into a catalytic nanostructure with enhanced biostability. This XNA nanostructure is capable of cleaving genomic SARS-CoV-2 RNA under physiological conditions, and when transfected into cells inhibits infection with authentic SARS-CoV-2 virus by RNA knockdown. These results demonstrate the potential of XNAzymes to provide a platform for the rapid generation of antiviral reagents. RNA viruses have been responsible for large-scale epidemics and pandemics throughout the last few centuries. Here, the authors show the design, synthesis and screening of artificial RNA endonuclease XNAzymes capable of cleaving genomic SARS-CoV-2 RNA and self-assembling into enzymatic nanostructures inhibiting cellular viral replication. However, various challenges remain for these technologies, including issues of specificity, biostability, immunogenicity, target site accessibility, delivery, and the potential for viral mutational escape.Nucleic acid therapeutic technologies are proving to be crucial platforms for rapid response to emerging viral threats14 retargeted to viral RNAs17, including catalysts targeting the RNA genome of SARS-CoV coronavirus18, no antiviral DNAzyme has achieved clinical approval. A key concern is their dependence on unphysiological concentrations of divalent metal ions: the 10-23 catalytic core has been found to require high concentrations of magnesium (>10 mM) in order to fully fold into its active state19 whereas in intracellular free Mg2+ concentrations (0.5–1 mM) DNAzyme activity is severely reduced20. Although RNA knockdown effects can be measured in vivo when DNAzymes are exogenously delivered into cells, similar effects are observed in the absence of an active 10–23 catalytic core22, suggesting this is likely due to induction of RNase H (i.e. antisense mechanisms) and/or cytotoxic effects23.Modular nucleic acid catalysts capable of sequence-specific RNA transesterification – ribozymes and DNAzymes – offer an alternative platform for the generation of an almost unlimited variety of precision antivirals. However, despite the development of a range of variants of the classic ’10–23’ DNAzyme25) expand the capabilities of nucleic acid catalysts, either through modification of existing DNAzymes26  or the de novo evolution of fully-modified XNAzymes30. Recently, we described a modular XNAzyme, ’FR6_1’, composed of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA), originally selected to cleave a sequence in the RNA genome of the Zaire Ebolavirus, which has the ability to cleave long (>5 kb), structured RNA under physiological conditions in vitro and in vivo31.Synthetic alternative bases, sugars and backbones , over 6 million deaths worldwide33 and an estimated trillion-dollar reduction in productivity and economic growth. Despite the development of effective vaccines, waning immunity and the evolution of viral variants could mean SARS-CoV-2 will continue to pose a significant threat to human health for the foreseeable future35. Following the emergence of the first human cases in late 2019, there was a significant lag before the development of small molecule therapeutics such as the RNA-dependent RNA polymerase inhibitors remdesivir (Gilead) and molnupiravir (Merck), and variable efficacy in clinical trials37 suggest that continued discovery of novel antivirals and complementary modalities for tackling SARS-CoV-2 is necessary38. More broadly, the COVID-19 pandemic has underscored the need for expedited and streamlined drug discovery and development pipelines to prepare for future outbreaks39.RNA viruses have been responsible for numerous large-scale epidemics and pandemics throughout the 20th and 21st centuries, including catastrophic outbreaks of influenza, human immunodeficiency virus (HIV), coronavirus (SARS-CoV and MERS-CoV), flavivirus (Zika) and filovirus (Ebola) infection. Most recently, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)41. Thus far, therapeutic strategies have focused on the spike protein (S), which enables entry of the virus into the cell, the RNA-dependent RNA polymerase (RdRp) protein (nsp12), which copies the viral genome, the virally encoded proteases PL-PRO and 3CL-PRO (nsp3 and nsp5), the viral envelope protein E, the membrane protein M and the nucleocapsid protein N, which organises the genome in viral particles43. Direct targeting of the viral genome is an underexplored modality for the development of SARS-CoV-2 antivirals.In cells infected with SARS-CoV-2, the 29,903 nt genomic RNA is used to produce at least ten canonical subgenomic RNAs, which together serve as transcripts encoding ~12 open reading frames (ORFs) and ~26 viral proteins, 16 of which are derived from two polyproteins produced by ribosomal slippage during translation of ORF1a and ORF1ab in the genomic RNAHere, we describe proof-of-concept design, testing and assembly of variants of the RNA endonuclease XNAzyme FR6_1, enabling the rapid development of a series of artificial enzymes and a biostable catalytic nanostructure targeting RNA sequences spanning the genome of SARS-CoV-2, and capable of inhibiting authentic viral infection in cells.31. Substrate specificity arises from a combination of the complementarity of the binding arms for target RNA, as well as putatively unpaired substrate residues positioned in a three-residue bulge opposite the catalytic core—although FR6_1 tolerates almost any dinucleotide pair (except GG) at the cleavage site in this bulge, there is an absolute requirement for an A one nucleotide downstream31. Initially, we searched the RNA genome of SARS-CoV-2 (NCBI NC_045512.2) for sequences with similarity to the original target sequence of FR6_1, but with no similar (i.e. off-target) sequences in the human transcriptome. Two sequences in SARS-CoV-2 open reading frame (ORF) 1b were identified, residues 14804–14822 (’Sub_ORF1b’) (61% homology with Sub_Ebo) and 13684–13718 (’Sub_ORF1b2’) (56% homology with Sub_Ebo). Although initial variants of FR6_1 targeted to these sequences (and prepared using FANA nucleotides and polymerase D4YK44) had little or no activity under quasi-physiological conditions   and is predicted to have an architecture comprised of a catalytic core flanked by two guide strands or substrate-binding ‘arms’obs = 0.03 h−1 ± 0.01)   Fig. . The XNA−1) Fig. .46, targeted to the same RNA substrate sequences, as allowed by the DNAzymes’ cleavage site preferences  as expected, catalytic activities of all five DNAzymes were significantly reduced or abolished at quasi-physiological magnesium concentrations (1 mM Mg2+) and were clearly out-performed by the analogous XNAzymes  protein, an RNA-binding protein responsible for genome packaging, could inhibit oligonucleotide catalysts, we next examined XNAzyme-mediated RNA cleavage under quasi-physiological conditions in the presence of N protein up to 1.4 mM)  had to be introduced to improve biostability. An alternative approach is to sterically hinder the accessibility of 5′ and 3′ termini to exonucleases by incorporation into a nucleic acid nanostructure. We have previously shown that FANA is broadly compatible with DNA nanostructure designs48, so reasoned that an architecture used to improve DNAzyme stability by embedding catalysts into a simple self-assembling DNA polyhedron49 may be adapted to synthesise an analogous catalytic XNA nanostructure. We therefore appended additional FANA sequences on to Fz_CoV2_7b, Fz_CoV2_S and Fz_CoV2_1a, with regions of complementarity that enable assembly into a three-component 74.3 kDa nanostructure, ’TFz3’, in which each XNAzyme is presented on one of three single-stranded edges  compared with a single-stranded FANA component strand  Fig.  than the3 nanostructure, or an irrelevant XNAzyme (the Ebola-targeting FR6_1 ‘parent’ FANAzyme) with ex vivo SARS-CoV-2 genomic RNA under single-turnover and quasi-physiological conditions  , but not control XNAzyme (FR6_1)  genomic and/or subgenomic RNA, we next incubated the individual Fz_CoV2_7b XNAzyme, the assembled TFzNA) Fig. . CleavagNA) Fig. . After 53 nanostructure into reporter cells expressing a luminescent biosensor50, enabling quantitation of authentic SARS-CoV-2 infection 31, although in both cases, the final effective concentration of XNAzyme(s) inside cells was unknown. It is further unclear what proportion of the TFz3 nanostructures remain fully assembled inside cells, although the similar levels of XNAzyme activity between assembled nanostructures and unassembled components  as an additional control. We had previously found that a single point mutation in the FR6_1 catalytic core, fG27fA , was sufficient to render the XNAzyme catalytically inactive without significantly altering substrate binding31. We, therefore, synthesised equivalent [fG27fA] mutant versions of all three components of the nanostructure . As expected, this abrogated the inhibition of SARS-CoV-2 infection seen with the TFzure Fig. . Since dmes Fig.  could inure Fig.  with normalisation to host cell EIF2B2 mRNA , but not inactive TFz3i.To better understand the mechanism of XNAzyme inhibition of SARS-CoV-2 infection, we examined whether the effect was accompanied by specific knockdown of SARS-CoV-2 genomic and/or subgenomic RNA. Total RNA was extracted from cells infected with authentic SARS-CoV-2 following transfection with the TFzRNA Fig. . Consist3i to produce a measurable knockdown effect despite the fG27fA mutation(s), due to the potential for FANA-containing oligonucleotides to trigger RNase H-dependent cleavage of viral RNA51 (i.e. ‘antisense’-mediated knockdown), which can contribute to knockdown induced by FANAzymes, but is generally less specific31, and would be consistent with the small inhibitory effect of TFz3i observed in the infection assay  may simply be too small (<10%) to measure accurately—whereas, in the reporter assay, inhibition derived from this effect would be the result of simultaneous RNase H1-induction across all three SARS-CoV-2 RNA sites targeted by the three components of TFz3i.The discrepancy between the small inhibitory effect observed with the inactive TFzsay Fig.  and the say Fig.  suggests6 cells), with 60–70% of this effect attributable to specific XNAzyme activity . Although the retargeted enzymes exhibited variable catalytic rates which were lower than that of the parental FR6_1 XNAzyme  opposite the catalytic core. However, the variability in rates and cleavage plateaus between these XNAzymes suggests activity is also impacted by other sequences or secondary structures in their respective substrates and/or substrate-binding arms. One explanation could be differences in the stability of alternative RNA x FANA binding modes between XNAzyme variants - i.e. interactions between the substrate and core residues that could enable the core to adopt misfolded conformations, such that each XNAzyme has a distinct set of conformers in equilibrium, with different proportions of catalytically active and inactive folds. Consistent with this, embedding XNAzymes into the nanostructure design improved the proportion of maximal substrate cleaved (in the case of Fz_CoV2_S), presumably by constraining substrate binding arms into extended conformations, favouring the catalytically active binding mode. Alternatively, differences in target accessibility due to substrate secondary structure may also impact rates. The comparatively slow rates of multiple turnover observed with previous variants of the FR6_1 XNAzyme31 and here  understanding the structure of the XNAzyme core, and XNA nanostructure assembly, in greater detail in order to optimise the folding landscape of retargeted enzymes; (3) improving turnover, perhaps through systematic evaluation of modifications to substrate-binding arms or other strategies58 and (4) extension of XNAzyme selection to alternative chemistries with beneficial physicochemical properties, such as ‘locked nucleic acids’ (LNAs), a chemistry used to develop ‘gapmer’ ASOs capable of 70–90% inhibition of SARS-CoV-26. It may also be possible, for example, to engineer XNAzymes from nucleotide analogue building blocks that are themselves antiviral2 in order to derive therapeutic efficacy from both the activity of the functional oligonucleotide but also the products of its degradation in vivo59.Future efforts could be directed toward; (1) improving methods for selecting optimal cleavage sites within long target RNAs - whilst we did not exhaustively screen sites within the SARS-CoV-2 genome in this study, in vitro selection for optimal sites is feasible61) will be adaptable to XNAzymes. Naked (gymnotic) delivery may also be possible, as shown for FANA antisense oligos62 and DNAzymes targeting RNA in the lung63. In this regard, nanostructures may offer enhanced uptake64. However, such approaches must contend with the need to deliver comparatively large doses when catalysts have limited capacity for multiple turnover. Furthermore, it is unclear whether viral genomic RNA, which is transcribed within double-membrane replication organelles65, would be accessible at all stages of viral infection and replication without, for example, conjugation to amphipathic compounds60.Optimisation of XNAzyme delivery is also a key outstanding problem. Here, we limited our study to the transfection of reporter cells in order to establish XNAzyme antiviral activity in vivo. It is reasonable to suggest that existing methods of delivery of functional oligonucleotides  ought to be a feasible general strategy to avoid the emergence of escape variants observed when single sites in genomic RNA of rapidly mutating RNA viruses are targeted by siRNAs, for example69.Our results nonetheless demonstrate that the cleavage capabilities of XNAzymes can be extended to genomic RNA and establish knockdown without substantial involvement of host effectors (such as RNase H), which we had previously found contribute to in vivo activityOur findings demonstrate the potential for modular RNA endonuclease XNAzymes to provide a platform technology for the rapid discovery of specific antiviral reagents. Although further development of catalyst designs and chemical composition are likely necessary for the clinical impact of this technology, as well as a more detailed understanding and optimisation of intracellular activity and pharmacokinetics, XNAzymes are well-placed to join the growing toolbox of nucleic acid therapeutics and technologies in preparation for rapidly emerging biological threats.44 was kindly provided by Philipp Holliger . Recombinant SARS-CoV-2 nucleocapsid (N) protein  was kindly provided by Jakub Luptak and Leo James . Ex vivo genomic SARS-CoV-2 RNA (purified from infected Vero cells) was obtained from Launch Diagnostics, UK (Vircell Amplirun coronavirus SARS-CoV-2 RNA control MBC137-R). HEK293T cells (authenticated by STR profiling) were kindly provided by Paul Lehner (University of Cambridge) and modified to generate the SARS-CoV-2 reporter cell line as detailed in ref. DNA and RNA (and chimeric DNA/RNA) oligonucleotides were synthesised by Integrated DNA Technologies (Belgium) or Sigma-Aldrich/Merck (USA). Polymerase D4K72.The SARS-CoV-2 virus used in this study was the lineage B viral isolate SARS-CoV-2/human/Liverpool/REMRQ0001/2020, a kind gift from Ian Goodfellow (University of Cambridge), isolated by Lance Turtle (University of Liverpool), David Matthews and Andrew Davidson (University of Bristol) from a patient from the Diamond Princess cruise shipDetailed characterisation of the FR6_1 FANA XNAzyme catalyst is provided in ref. 30, templated by 3′ biotinylated DNA templates   and polymerase D4KPartially hydrolysed RNA substrates (for use as electrophoresis size standards) were prepared by incubation at 65 °C in 20 mM sodium hydroxide (pH 12) for 4 min, then neutralised with 1 M Tris pH 7.2) or high magnesium buffer . Reactions were stopped by the addition of excess PAGE gel loading buffer  and analysed by urea-PAGE using 20% acrylamide gels. Fluorophore-labelled RNA substrates were visualised using an FLA-5000 scanner  and quantified using ImageQuant TL  or Fiji73. Pseudo-first-order reaction rates (kobs) under single-turnover pre-steady-state (Km/kcat) conditions were determined from timecourses; reactions were sampled and stopped at appropriate intervals by snap-freezing on dry ice in excess PAGE gel loading buffer and analysed as above. Quantification data from three independent replicates per time course were fit to a one-phase association model using Prism 9 (GraphPad)30.PAGE-purified XNAzymes (5 μM) or DNAzymes (5 μM), and short (35 nt) RNA substrates (1 μM) were annealed separately as described above and reacted at 37 °C in either quasi-physiological buffer , ’TFz_CoV2_7b’, ’TFz_CoV2_S’ and ’TFz_CoV2_1a’, or the inactive nanostructure (’TFz3i’), ’TFz_CoV2_7b_i’, ’TFz_CoV2_S_i’ and ’TFz_CoV2_1a_i’, were prepared using the relevant DNA templates (Supplementary Table 2), then purified using 50KDa ultra centrifugal filter concentrator (Amicon) as recommended by the manufacturer, with three 10 min wash steps with the same buffer.The sequences for the FANA nanostructure were adapted from components of a DNA design described by ref. 3 nanostructure or one of the single-stranded components (TFz_CoV2_7b) were annealed and purified as described above, then incubated  at 37 °C in 50% human serum (Sigma-Aldrich/Merck) for 48 h. Reactions were sampled and stopped at appropriate intervals by snap-freezing on dry ice in excess PAGE gel loading buffer. The proportion of undegraded full-length catalyst was determined by Urea-PAGE; gels were stained with SYBR Gold  and imaged as described above.TFz3 nanostructure  and incubated with ex vivo genomic SARS-CoV-2 RNA (~3000 copies/μl) in quasi-physiological buffer supplemented with 50 ng/μl total eukaryotic cell RNA (purified from RKO cells) and RNasein (1 U/μl) for 5 h at 37 °C. Reactions were purified using an RNeasy kit ) with additional RPE washes to remove XNAzymes (see ref. PAGE-purified XNAzymes (0.5 μM) or TFzsee ref. . Eluted https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html), residues 29188– 29210) using singleplex hydrolysis probe-based assays (performed in triplicate): 0.5 μM primers  and 0.125 μM probe  were used with the following cycling conditions: 95 °C 10 min, 40 x , 98 °C for 10 min. Data were analysed using Quantasoft Analysis Pro software  and Prism 9.2.0 . Statistical significance was determined using a paired t-test.Specific cleavage at the target site for XNAzyme FzCoV2_7b in ORF7b (residues 27873–27897 in the SARS-CoV-2 reference sequence (NC_045512.2)) was quantitated by ddPCR, and parallel reactions performed for normalisation to a non-target site in the N-protein gene , 100 units/ml penicillin, and 0.1 mg/ml streptomycin at 37 °C in 5% CO2. Cells were regularly screened and confirmed to be mycoplasma negative . PAGE-purified XNAzymes, TFz3 nanostructures , DNAzymes, siRNA or buffer alone were transfected into cells growing at ~60% confluency (detached using 0.25% trypsin EDTA) using a Neon electroporation system . 800,000 cells were used per transfection with 10 ul Neon tips and 2 × 20 ms pulses of 1400 V.HEK293T SARS-CoV-2 reporter cellsg for 5 min, and immediately frozen in aliquots at −80 °C. For the determination of MOIs, viral stocks were titrated in VeroE6 cells by 50% tissue culture infectious dose (TCID50) assays using standard methods.Viral stocks were prepared by passaging once in VeroE6 cells. In brief, VeroE6 cells were infected at a low multiplicity of infection (MOI) with the original viral stock and incubated for 72 h (by which time the cytopathic effect was evident). Virus-containing culture supernatants were then clarified by centrifugation at 600 × Quantification of SARS-CoV-2 replication in HEK293T reporter cells is detailed in ref. eIF2B2, using a singleplex assay: 0.9 μM primers ('eIF2B2_Fw' and 'eIF2B2_Rev') and 0.5 μM probe ('eIF2B2_Probe') were used with the following cycling conditions: 95 °C 10 min, 40 x , 98 °C for 10 min. Data were analysed using Quantasoft Analysis Pro software  and Prism 9.2.0 . Statistical significance was determined using a paired t-test.Total RNA from HEK293T SARS-CoV-2 reporter cells treated as above was extracted following lysis in RNeasy RLT buffer (Qiagen) supplemented with 2-mercaptoethanol (10 μl/ml) and purified using RNeasy kits  and quantified using ImageQuant TL  or Fiji73.Human RNase H1 assays were performed by incubating co-annealed RNA substrates (0.25 μM) and XNAzymes (0.25 μM) with 0.01 ug/ul recombinant human RNase H1  in 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 5 mM DTT, 1 mM MgClFurther information on research design is available in the Supplementary InformationReporting Summary


Skeletal muscle growth and development is a complicated process that is regulated at multiple steps and by numerous myogenesis genes. RNA editing represents one of the events at the post-transcriptional level, which contributes to the diversity of transcriptome and proteome by altering the nucleotides of RNAs. However, RNA editing events in the skeletal muscle of yaks are still not well defined. This study conducted whole-genome RNA-editing identification in skeletal muscle of yaks at embryonic stage (ES) and adult stage (AS). We found a total of 11,168 unique RNA editing sites, most of which were detected in the intergenic region. After annotation, we totally identified 2,718 editing sites within coding regions, among which 858 were missense changes. Moreover, totally 322 editing sites in the 3′ untranslated regions (UTR) were also predicted to alter the set of miRNA target sites, indicating that RNA editing may be involved in translational repression or mRNA degradation. We found 838 RNA editing sites (involving 244 common genes) that are edited differentially in ES as compared to AS. According to the KEGG enrichment analysis, these differentially edited genes were mainly involved in pathways highly related to skeletal muscle development and myogenesis, including MAPK, AMPK, Wnt, and PI3K-Akt signaling pathways. Altogether, our work presents the first characterization of RNA editing sites within yak skeletal muscles on a genome-wide scale and enhances our understanding of the mechanism of skeletal muscle development and myogenesis. Bos grunniens) is a vital species of livestock prevalent across the Qinghai-Tibet plateau and the adjacent alpine regions , such as the myocyte enhancer factor 2 (MEF2) family members , and stability of RNAs . Notablyhttps://www.ncbi.nlm.nih.gov/sra/). This dataset was developed from longissimus dorsi muscle tissues of 3 embryonic (90 days old) and 3 adult (3 years old) yaks. The Illumina HiSeq 2500 platform was employed to sequence cDNA libraries of these samples (2 × 150-bp paired-end read length).In order to investigate the RNA editing sites in muscle tissues of yak, six specific strand RNA-seq data (PRJNA550017) from our previous study  were dowBlood samples were gathered from yaks having the same genetic background as the study samples used for RNA-seq. All experiments and animal care procedures were carried out according to the guidelines of the Animal Administration and Ethics Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Permit No. SYXK-2014–0002). DNA extraction kit  was utilized to extract genomic DNA from blood in line with the specific instructions. DNA libraries were constructed with the use of the MGIEasy Universal DNA Library Prep Kit  following the BGI's standard preparation protocol. Paired-end sequencing (2 × 150-bp) was carried out on an MGISEQ2000  at Frasergen Bioinformatics Co., Ltd .https://broadinstitute.github.io/picard/) using the function MarkDuplicate. We then mapped the clean reads from RNA-seq data to the genome sequence of domestic yak (LU_Bosgru_v3.0) using the HISAT2 (2.1.0)   was used (2.1.0) . The res (2.1.0) .In order to detect the RNA editing sites, the REDItoolDnaRna.py in REDItools v1.0.4  was util® Green Master Mix  and 7 μl of ddH2O. The PCR program was set as follows: 95°C, 2 min,  30 cycles and 72°C, 5 min. The PCR products were then sequenced by Sanger sequencing.To validate the reliability of RNA editing sites identified in this study, three editing sites were randomly selected for PCR validation. Six independent samples used for PCR validation were collected at the same developmental stage as those used for RNA-Seq. Total RNA was extracted using the animal tissue RNA isolation kit  following the manufacturer's protocol. Genomic DNA was isolated using the animal tissue Genomic DNA Kit . Total RNA was used for reverse transcription by PrimeScript™ RT reagent kit with gDNA Eraser . Equal amounts of cDNA were mixed. The primer details are presented in We used RNAhybrid   and miRaP-value < 0.05 were labeled as significant.For an RNA editing site in a given sample, the RNA editing level was calculated as the ratio of the reads supporting the edited base to the total number of reads detected on this site. To identify RNA-edited sites related to muscle development, we performed Tukey's Honest Significant Difference approach to screen significantly differently edited sites between ES and AS. Sites with P-value < 0.05.The genes with RNA editing sites located in exonic regions were extracted for functional enrichment analysis. To explore the functions of these putative RNA editing sites, we carried out gene ontology (GO) analysis based on the G: Profile web approach . KOBAS 3http://srv00.recas.ba.infn.it/atlas/)  . In totalongissimus dorsi muscle, we collected six strand-specific RNA sequencing samples from both the ES and AS. After trimming the adaptor sequence and low-quality reads, we obtained 282,913,382 total clean reads for ES and 363,479,946 total clean reads for AS. The total mapped ratio between the reads and the reference genome of all samples ranged from 88.19 to 92.58% , totally eight conserved RNA editing sites were identified (COPA) gene resulted in missense substitution.To investigate the conservation of RNA editomes between human and yak, upstream and downstream sequence of editing sites was aligned using nucleotide BLAST tools. With a rigorous filtering threshold  belongs to the myosin heavy chain (MYH) family and is predominantly expressed in distinct muscle developmental stages. Missense mutations in the MYH3 gene have been reported to give rise to human muscle development disorders (SUN1) belongs to the linker of nucleoskeleton and cytoskeleton (LINC) complex, which plays a critical role in myotube formation (MYO18B) is the new nontraditional myosin heavy chain that is mostly expressed in human cardiac and skeletal muscle  encodes an important extracellular matrix (ECM) protein which is mainly expressed in the basement membrane of skeletal muscle .XW, PY, and CL conceived the research and drafted the manuscript with comments from all authors. XW, MC, and XM conducted the experiments and analyses. JP, LX, and XG participated in experiments and revised the manuscript. All authors approved the final version.This study was supported by the National Natural Science Foundation of China (32102500), Agricultural Science and Technology Innovation Program (25-LZIHPS-01), Gansu Province Science Foundation for Youths (20JR5RA576), Foundation for Innovation, Groups of Basic Research in Gansu Province (20JR5RA580), and Central Public-interest Scientific Institution Basal Research Fund (Y2022LM10).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite its early discovery, poliovirus and other related IRES elements of type I are poorly characterized, and it is not yet clear which host proteins  are required for their full activity in vivo. Here we discuss recent and old results devoted to type I IRESes and provide evidence that Poly(rC) binding protein 2 (PCBP2), Glycyl-tRNA synthetase (GARS), and Cold Shock Domain Containing E1  are major regulators of type I IRES activity.The phenomenon of internal initiation of translation was discovered in 1988 on poliovirus mRNA. The prototypic  Recognition of a suitable start codon  is followed by the dissociation of initiation factors and 60S subunit joining to form the 80S complex [Most cellular mRNAs are translated by the so-called mechanism ,2, which7G cap and hence cannot be translated via the classical “scanning” mechanism. One prominent alternative mechanism of translation initiation, first described for poliovirus (PV), is internal ribosome entry: complex RNA structural elements located in the 5′UTR are able to recruit the components of the translation machinery and direct initiation without scanning of the entire 5′UTR. Such elements are called Internal Ribosome Entry Sites, or IRESes. In the case of poliovirus, the IRES element is composed of four structural domains—from domain II to VI, excluding domain III [743 (position corresponding to the PV Mahoney strain). In addition, translation from the universally conserved upstream AUG codon located in the stem of domain VI (known as “cryptic” AUG586) gives rise to the short protein UP, which enhances virus release and is important for virus propagation in intestinal organoids [All known eukaryotic viruses rely on the cellular translation machinery to translate their mRNAs. Some viral mRNAs do not bear an mmain III ,7,8. It rganoids .trans-acting factors, ITAFs) for their activity [Currently, viral IRES elements are divided into four (or five) classes or types which are remarkably different both in RNA secondary structure organization and in molecular mechanisms which allow them to direct translation internally ,11,12,13activity ,15. CleaAny RNA inside the cell is expected to form a complex with RNA-binding proteins, which is then called ribonucleoprotein (RNP). In the case of an IRES element, it is expected that some IRES interacting proteins can modulate its activity. This idea stems from the observation that translation of poliovirus mRNA is inefficient in the rabbit reticulocyte lysate (RRL) cell-free system but can be strongly enhanced by the addition of HeLa cell crude lysate or its fractions ,17,18,19The major challenge is how to prove if a particular protein actually modulates IRES activity. All currently available biochemical approaches have their own, sometimes serious, limitations. Perhaps the most prominent technique to address the impact of individual proteins on IRES-mediated translation was implemented in 1996, when Pestova, Hellen and Shatsky succeeded in reconstituting 48S initiation complexes from purified components on the IRES of encephalomyocarditis virus (EMCV), a picornaviral type II IRES . This ap+, Na+, Mg2+, Ca2+ etc.) in the binding buffer can have important effects on RNA-protein interactions and on intra- and intermolecular RNA-RNA interactions, but it is very hard or impossible to precisely control the concentrations of these ions due to the usually unknown corresponding concentrations that come from the cell lysate itself. Thirdly, the perhaps most important limitation is the fact that negative control RNA can, in principle, bind genuine ITAFs, which may be discarded during data evaluation just because of the wrong choice of the negative control. Finally, as the sensitivity of mass spectrometry increases dramatically, it becomes very challenging to select proteins of interest for validation  yield hundreds of proteins).The main pitfall of the reconstitution system from purified components described above is its reliance on prior knowledge of which factors should be included in the reconstitution mixture. Thereby, the maximally achieved activity of the system using known components is artificially defined as 100%, not knowing what the maximal activity of the RNA in an appropriate cellular context could be. Another widely used approach to detect specific RNA binding proteins is the affinity purification of RNPs from cell lysates with immobilized RNA of interest. Usually, some non-specific RNA is included as a negative control in order to filter out non-specific RNA-protein interactions. Despite its utility, there are obvious and important limitations. Firstly, excess RNA bait can lead to the purification of major RNA-binding proteins with relaxed selectivity that perhaps would not bind the RNA of interest when a limited amount of RNA is present in the cell. Secondly, it is reasonable that concentrations of various ions , but this is beyond the scope of this review. PCBP2, along with PCBP1 and hnRNP K, corresponds to the major poly(rC)-binding proteins. The common feature of PCBPs is the presence of three RNA-binding hnRNP K homology (KH) domains which are separated by linker sequences. PCBPs are multifunctional proteins implicated at multiple levels of regulation of gene expression ,26. In aThe link between PCBP2 and poliovirus translation was established in 1996 when PCBP2 was identified as a protein that can interact with domain IV of the poliovirus IRES . HeLa cePCBP2 likely plays a role as an RNA chaperone in IRES-mediated translation by modulating and stabilizing the structure of the central large domain IV of the PV IRES. PCBP2 binds close to the conserved GNRA tetraloop, and in the cryo-EM structure of the PCBP2-domain IV complex, this GNRA is protruded from the area where KHI and KHII domains are located . InteresApart from modulation of the RNA structure, PCBP2 can also be implicated in the recruitment of other proteins to the viral mRNA. For instance, it has been demonstrated that PCBP2 can interact with poly-A binding protein (PABP) , and thiIt should be mentioned that it is controversial whether and to what extent PCBP1 and PCBP2 can substitute for each other both at the “translation control” site (domain IV) and at the “replication control” site (cloverleaf). As the focus of this review is on IRES-mediated translation, we will just mention that in the reconstitution system using purified components, both PCBP1 and PCBP2 allow efficient 48S complex formation .CSDE1, also known as UNR (Upstream of N-RAS), is a ubiquitously expressed gene that codes for an RNA binding protein implicated in posttranscriptional regulation of gene expression  mRNA that is remarkably unstable. It was initially demonstrated that two distinct sequence elements are responsible for rapid mRNA degradation by various pathways, and one of the instability determinants, mCRD, is located in the protein-coding region [One example is found for g region . Interesg region . In 2000g region . In a fog region . Importag region . Thus, icis-acting element found in the PABPC1 mRNA 5′leader that is important for translation repression. Repression of PABPC1 mRNA translation involves the binding of PABP to the adenine-rich autoregulatory sequence (ARS) in the 5′-untranslated region of its own mRNA [6–8 separated by conserved pyrimidine nucleotides. It appeared that the ARS binds not only PABP itself but also two additional RNA binding proteins, insulin-like growth factor 2 mRNA binding protein 1 (IMP-1) and UNR [Another functional role of UNR/PABP has been found in a own mRNA . The ARS and UNR . While b and UNR  suggests and UNR . While tmsl-2 mRNA is dependent on the UNR-PABP interaction. In Drosophila, msl-2 mRNA is translationally repressed in females by the sex-lethal (SXL) RNA binding protein, which binds to two cis-acting elements in the msl-2 mRNA 5′ and 3′ UTRs [cis-acting elements repress msl-2 translation by different mechanisms [msl-2 mRNA, while the SXL binding site in the 5′UTR blocks scanning of residual PICs that escaped the above recruitment inhibition. Interestingly, the interaction of SXL with the SXL binding sites in the 3′UTR appears to be dependent on interaction with UNR and PABP. It was demonstrated that 3′UTR-bound SXL promotes the binding of UNR to an adjacent nucleotide sequence, and UNR acts as a translational corepressor [msl-2 mRNA—instead, the ribosome recruitment is impaired [Also, the UNR-mediated translation repression of  3′ UTRs ,57. Theschanisms : the SXLepressor ,48,59. Depressor  showed timpaired .Finally, UNR can bind directly to a Renilla luciferase reporter mRNA containing an unstructured 200 nucleotides 5′UTR by virtue of its cold shock domains 2 and 4, and UNR can interact with eIF4G and PABP, thereby increasing the PABP-eIF4G interaction in cells . Moreove−/− murine embryonic stem (ES) cells, translation driven by type-I HRV and poliovirus IRESes was selectively suppressed in comparison to wt cells, whereas the activity of type-II IRESes (EMCV and FMDV) was not affected [Regarding UNR requirements for type-I IRES translation, pioneering research has been performed in Richard Jackson’s laboratory. Historically, it was demonstrated that HeLa ribosome salt wash could be fractionated by ion exchange chromatography into two components, A-type and B-type activity, each of which, when added individually to a reticulocyte lysate system, could stimulate type-I IRES-mediated translation . First, affected . Importaaffected . In our affected , stronglSTRAP, also known as UNRIP (UNR-interaction protein), which is the second protein component of type-B activity fraction, is also enriched in poliovirus polysomes . While tMany tRNA synthetases have diverse moonlighting functions in all kingdoms of life . Some ofWhy do the type-I IRESes appear to be selectively regulated by GARS and, given the evolutionary conservation of its “gly anticodon,” not by other tRNA synthetases? One possible reason is that GARS is not part of the cytoplasmic multi-tRNA synthetase complex (MSC), which contains eight tRNA synthetases  and three additional multifunctional proteins  ,71. TherIn the following, we summarize the information about the structural organization of type-I IRESes and map interactions of three different ITAFs along with canonical initiation factors . In thisThe poliovirus IRES domain II shows interaction with UNR, and since the second UNR binding site is located at the apical part of domain V, it seems likely that RNA-protein interactions can result in the spatial approximation of these two distal RNA structures in a translation-competent RNP. Moreover, the GARS binding site on domain V is located very close to that of UNR, and we may propose that GARS can stimulate UNR binding. Interestingly, also the upstream domain II of the type-I IRES may contain a second, weaker GARS binding site (unpublished observation). As a class II tRNA synthetase, GARS is a dimer, with the two tRNA anticodon stem-loop binding sites at the outside of the dimer pointing in opposing directions . This spPABP may even add to the activity of this multi-component complex on the poliovirus IRES. It has been reported that PV IRES-driven reporter translation in vitro is more strongly dependent on the presence of poly-A tail than the type-II EMCV IRES-driven translation . InteresTo summarize, several lines of evidence lead us to conclude that at least three ITAFs are important for poliovirus IRES activity. Previous studies established that PCBP2, UNR and GARS all can efficiently bind to poliovirus IRES, and their binding sites have been mapped to specific IRES domains. Moreover, these factors are selectively recruited to polysomes of poliovirus-infected cells. The apparent discrepancy with the results obtained so far from reconstituted in vitro-systems, where only PCBP2 has been shown to be sufficient for complex formation, can be explained by the following reasons. First, to our knowledge, the combination of all four factors  has never been tested in vitro. Second, a truncated eIF4G fragment rather than the full-length eIF4G or even the heterotrimeric eIF4F complex has been used in the in vitro-system for practical reasons, mainly originating from the difficult purification of full-length eIF4G protein. Therefore, the reconstituted system, as used so far, may not recapitulate the pioneer rounds of viral translation initiation when eIF4G is not yet cleaved. Third, poly-A and PABP were not included despite the fact that PV IRES activity is strongly dependent on poly-A and PABP in vitro. Finally, all ITAFs were prepared as recombinant proteins, which may not be fully active, either because of improper folding or due to the lack of eukaryote-specific post-translational modifications. Without questioning the utility of the extremely useful method of reconstitution from purified components, we argue that establishing the composition of IRES translation initiation complexes in living cells (after transfection of the RNA) or at least in a cell lysate where all required components are present is needed to better understand the mechanism of IRES-mediated translation.


ATR) and tet methylcytosine dioxygenase 2 (TET2) genes were significantly enriched in lymphatic metastases (p ≤ 0.05). Alterations in these two genes could be considered metastasis-related driving events. Mutated ATR and TET2 might play an active role in the metastasis of lymph nodes with NSCLC. More case enrollment and long-term follow-up will further verify the clinical significance of these two genes.Lymphatic metastasis influences clinical treatment and prognosis of patients with non-small-cell lung cancer (NSCLC). There is an urgency to understand the molecular features and mechanisms of lymph node metastasis. We analyzed the molecular features on pairs of the primary tumor and lymphatic metastasis tissue samples from 15 NSCLC patients using targeted next-generation sequencing. The potential metastasis-related genes were screened from our cohort based on cancer cell fraction. After filtering with gene functions, candidate metastasis-related events were validated in the MSK cohort with Fisher’s exact test. The molecular signature and tumor mutational burden were similar in paired samples, and the average mutational concordance was 42.0% ± 28.9%. Its metastatic mechanism is potentially a linear progression based on the metastatic seeding theory. Furthermore, mutated ataxia telangiectasia mutated and Rad3-related ( Lung cancer is the leading cause of cancer-related mortality worldwide, and more than 90% of lung cancers were diagnosed at II or higher stage . DistantSome researchers were focusing on the heterogeneity of primary sites and metastatic lymph nodes in given gene alterations or expressions  or the mHere, we comprehensively investigated the genetic divergence between primary tumor and lymph node metastasis with paired tissue samples of NSCLC patients and innovatively explored the lymph node metastasis-driving genes following the metastatic seeding idea rooted in the tumor clonal evolutionary theory. A total of 15 paired primary-metastasis samples were performed with targeted panel sequencing. Furthermore, smoking status was recorded for all enrolled cases. Of these 15 cases, 12 were smokers including current smokers and former smokers, and three patients were nonsmokers. All tissue samples were obtained before any systemic therapy. Our aims included the following: 1) to determine whether the lymph node metastasis of NSCLC has distinct molecular characteristics in terms of single nucleotide variants (SNVs) from their primary tumors; 2) to identify “metastasis-related driving genes” that enabled metastasis of lymph nodes.http://gdc-portal.nci.nih.gov). Mutation data in “maf” files were downloaded from The Cancer Genome Atlas  database, which included 492 lung squamous cell carcinoma (LUSC) and 567 lung adenocarcinoma (LUAD) samples.Primary and matched lymph node metastatic site tissues were collected from 15 NSCLC patients at the Fourth Medical Center of PLA General Hospital. Any cases included met the following criteria: patients were diagnosed as NSCLC with lymph node metastasis. This project was approved by the institutional review board of our hospital. All enrolled patients signed the informed consent forms before sample collection. To verify “metastasis-related driving genes,” the Lung_MSK_2017 NSCLC cohort was obtained from the Genomic Data Commons (GDC) data portal (Genomic DNA extraction and library preparation with TruSight™ Oncology 500 (TSO 500) Library Preparation Kit  were performed following protocols described previously . The libde novo mutational signatures inference from nonnegative matrix factorization (NMF) in our cohort. The hierarchical clustering analysis was performed to classify lung cancer samples into two subclusters based on signature contribution proportions. We then mapped these two de novo signatures to the known COSMIC signatures by the Pearson correlation analysis.The COSMIC  mutational signatures analysis was performed to discover the mutational signature difference between primary lesions and lymph node metastases . The R pATR and TET2 was analyzed by Maftools.First, the cancer cell fraction (CCF) of somatic mutations in paired samples was calculated using the CCF function of the “cDriver package” . Second,p values were two-tailed, and p values <0.05 were considered statistically significant.Then R package “maftools package”  was applTo clarify the molecular features of lymphatic metastasis, we recruited 15 advanced lung cancer patients in clinical stage II ∼ IV . Of the TP53, 56%), Kirsten rat sarcoma viral oncogene homolog , and epidermal growth factor receptor  mutations were among the top 10 somatic alterations  in primary tumors and 2 to 23  in metastases . The moserations . Notablyerations . With coerations . Mutatioerations . Despiteerations , which werations .To explore inter-tumor heterogeneity, molecular alterations identified were classified into shared (mutations in both primaries and metastases) and private (mutations in unique primaries or metastases) groups . Shared As a developing biomarker for NSCLC, TMB presents organ-specific properties . TMB wasOur results suggested the TMB of lymphatic metastases  was lower than primary tumors  but without statistical significance due to the limited samples. Moreover, a few smoking NSCLC patients exhibited inter-tumor heterogeneity.https://cancer.sanger.ac.uk/cosmic/signatures_v2) were made, which was useful to uncover the pathological and clinical relevance of these two signatures. These seven paired P-LN samples and CN_9-LN were clustered to the S1 signature matched COSMIC signature 1, which is found in all cancer types and correlates with the age of cancer diagnosis , we performed a secondary screen to limit the SNVs to metastasis-driving genes, which can promote or drive solid tumorigenesis. Thus, 17 potential metastasis-related driving genes were produced and their mutation frequencies in our cohort and the Lung_MSK_2017 cohort are shown in Taking advantage of the matched P-LN samples in our cohort, we attempt to search for metastasis-related events that were first seemed in metastatic lymph nodes or changed from subclone (in the primary tumor) to clone (in metastasis) . Then 55ATR and TET2 (2/17 of candidate metastasis-related genetic features) were found significantly enriched in metastatic lymph nodes compared to primary tumors (p < 0.05). We also tried to explore whether lymphatic metastasis has a different mutational domain or cluster from the paired primary tumor, but the results were disappointing  and ATR  were significantly higher in lymphatic metastasis than primary lesions, which considered as metastasis-related events. To exclude the influence of different pathological subtypes of NSCLC, we analyzed the frequencies of 17 metastasis-related genes in LUAD and LUSC from the TCGA database. We also remarkably noticed the limitation in our study that there is no CNVs analysis involved in our research. However, unpaired but large samples cohort analysis suggested that distant molecular mutational features may drive metastasis of specific organs  in lymphatic metastasis than in primary tumors (3%) with lung cancers, but there is no difference in ATR alteration sites between primary and lymphatic lesions. The ATR protein, first identified as a mammalian ortholog of Mec1 in 1996 . In one case report, mutated ATR and BRCA2 were thought to contribute to extensive metastasis with clear renal cell carcinoma  catalyzing 5-methylcytosine (5 mC) into 5-hydroxymethylcytosine (5 hmC) in DNA  in DNA . No evidg cancer . Another control . In thisSeveral restraints should be acknowledged. First, we collected the primary or metastatic tissue from one region instead of multi-regions. To abate the interference of intratumor heterogeneity, it is ideal to validate our results with multiregional samples . Second,ATR and TET2 are likely to play a crucial role in NSCLC lymph nodes metastasis. This finding would deliver new insights into the process of lymph node metastasis and provide suggestive information for developing novel approaches to monitor prognosis, formulate treatment strategy, and further refine pathological staging of lymph nodes.In summary, our study suggested that the metastatic process of lymph nodes is the result of the linear progression, and


Moreover, Gene Ontology analysis demonstrated that these identified ncRNAs were associated with several essential biological processes known to be related to cellular stress responses. In conclusion, we systematically established functional regulatory networks of lncRNA/circRNA-mRNA, lncRNA/circRNA-miRNA-mRNA and lncRNA/circRNA-RBP to perceive the potential interactions and biological processes during cellular stresses. These results provided insights in ncRNA regulatory networks of stress responses and the basis for further identification of pivotal factors involved in cellular stress responses.Mammalian cells employ various adaptive responses to cope with multiple stresses to maintain homeostasis. Functional roles of non-coding RNAs (ncRNAs) in response to cellular stresses have been proposed, and systematical investigations about the crosstalk among distinct types of RNAs are required. Here, we challenged HeLa cells with thapsigargin (TG) and glucose deprivation (GD) treatments to induce endoplasmic reticulum (ER) and metabolic stresses, respectively. Ribosomal RNA (rRNA)-depleted RNA sequencing (RNA-seq) was then performed. Characterization of the RNA-seq data revealed a series of differentially expressed long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) with parallel changes responsive to both stimuli. We further constructed the lncRNA/circRNA-mRNA co-expressing network, competing endogenous RNA (ceRNA) network in the lncRNA/circRNA-miRNA-mRNA axis, and lncRNA/circRNA-RNA binding protein (RBP) interactome map. These networks indicated the potential  Eukaryotic cells possess an extraordinary capacity to adapt to extra- or intracellular stimuli such as endoplasmic reticulum (ER) stress, heat shock, UV light, oxidative stress and nutrient starvation . To copevia three distinct stress sensors: IRE1α, ATF6, and PERK , one circRNA (CDR1as), and two miRNAs (miR-7 and miR-671) by using a panel of mouse knockouts  with regulatory roles in physiology and diseases . Lines oferation . Additioferation . Long ncferation . LncRNAsferation . The lncor SART3 . The lncsplicing . The lnc failure . Circula failure . Most ci failure . The cirnd SIRT1 . CircACCxidation . Accumulxidation . For exanockouts . Additiopressors . The welal roles . Althougal roles , identifNumerous studies have focused on one particular cellular stress , and to http://www.atcc.org) and authenticated by short-tandem-repeat (STR) profiling. They were cultured under standard conditions with DMEM  containing 10% FBS , and 1% penicillin/streptomycin  at 37°C with 5% CO2. HeLa cells were determined with a PCR-based method and DAPI staining to ensure no contamination of mycoplasma.HeLa cells were purchased from the American Type Culture Collection  for 6 h. For glucose deprivation, cells were cultured with DMEM without glucose  at 37°C for 6 h.Total RNA was extracted by TRIzol reagent  according to the manufacturer’s instructions. The concentration and quality of extracted RNAs were verified by Nanodrop and gel electrophoresis, respectively. Libraries were constructed by the TruSeq Ribo Profile Library Prep Kit  according to the manufacturer’s instructions. In brief, 10 μg total RNA was depleted rRNA with the Illumina Rio-Zero Gold Kit  and next purified for end repair and 5′ adaptor ligation. Then, the reverse transcription was performed with random primers containing the 3′ adaptor and randomized hexamer sequences. Finally, complementary DNA (cDNA) was purified and amplified with a Thermal Cycler. The PCR products of 300–500 base pairs (bp) were purified, quantified and stored at −80°C before sequencing. The libraries were subjected to 150-nt paired-end sequencing generating a depth of 50–100 million read pairs with an Illumina Novaseq platform (Novogene).p-value < 0.05. The DE mRNAs were determined by DEseq2 with the cutoff . For circRNA prediction, find_circ and CIRI2 were applied to identify high-confidence BSJs with default parameters. Only circRNA candidates predicted by both pipelines were used for further investigations and CIRI2-annotated BSJ reads (BSJ reads ≥2) were used to calculate circRNA levels. The DE circRNAs were determined by DEseq2 with the cutoff .For data processing, the adaptors were trimmed with Cutadapt to obtain clean reads. The data quality was then checked with FastQC and the low-quality  reads were removed. The remaining reads were subsequently aligned to the human reference genome (hg19) with Bowtie2 (-v 1). For linear RNAs including lncRNAs and mRNAs, the corresponding reads were counted with BEDtools and read per million (RPM) was used to calculate levels for lncRNAs and mRNAs. LncRNAs with an average RPM ≥0.1 were used for further analysis and the DE lncRNAs were determined by DEseq2 with a criterion of fold change ≥2 or ≤0.5 and XBP1 and FST mRNAs, amplification was performed with 30 cycles. Real-time quantitative PCR (RT-qPCR) was carried out with GoTaq SYBR Green qPCR Master Mix  on a QuantStudio Applied Biosystems (Thermo) according to standard procedures. All amplification curves reached the stationary stage before 35 cycles and the readings of the Ct value were obtained at the exponential stage. ACTB mRNA was used as an internal control. All PCR products were sequenced for confirmation and all primer sequences were included in cDNA was synthesized from 500 ng total RNA with the GoScript Reverse Transcription System  according to the manufacturer’s protocol. For RT-PCR gels of PRDM1 3′ UTR containing the binding sites of miR-9-5p and SOX12 3′ UTR containing the miR-744-5p binding sequences were PCR-amplified from the cDNA of HeLa cells and then inserted into the Firefly luciferase reporter vector pGL3-control  between XbaI  and FseI  double-digested sites. Small interfering RNAs (siRNAs) targeting lncSLC25A1, TINCR and circBANP BSJ were synthesized by GenePharma . Transfection of plasmids and siRNAs was performed with Lipofectamine 2000  according to the manufacturer’s protocol. Oligonucleotide sequences for primers used in plasmid construction and siRNAs are included in All plasmids for the luciferase reporter system were constructed with recombinant methods . p-value < 0.01. The constructed network consisted of 29 lncRNAs, 20 circRNAs, and 131 mRNAs, and was visualized with Cytoscape (https://cytoscape.org/).The lncRNA/circRNA-mRNA co-expression network was constructed according to the expression levels in our dataset. Briefly, 51 lncRNAs, 39 circRNAs, and 279 mRNAs sensitive to TG and GD treatments were used to construct the network with a criterion of Spearman R ≥ 0.95 or ≤ -0.95 and https://www.targetscan.org/vert_72/). Briefly, mRNAs, lncRNAs, or circRNAs with at least two putative binding sites for the individual miRNA were used to construct the network.The ceRNA network was constructed based on the lncRNA/circRNA-miRNA-mRNA axis. The lncRNA-miRNA, circRNA-miRNA, and mRNA-miRNA interactions were predicted with TargetScanHuman . After transfection for 48 h, cells were lysed with passive lysis buffer on ice for 20 min and the luciferase activity was performed with Dual Luciferase Reporter Assay System Kit  according to the manufacturer’s protocol. The Firefly luciferase activities were measured and normalized to Renilla luciferase activities (F/R).Briefly, 1×10http://rbpmap.technion.ac.il/). The lncRNA/circRNA-RBP interaction was determined by a criterion of the individual RNA sequence containing more than 2 motifs for RBP of interest . The RNA-RBP interactome map consisted of 46 lncRNAs, 17 circRNAs and 77 RBPs, and was visualized with Cytoscape.The RNA-RBP network was constructed based on the lncRNA-RBP and circRNA-RBP interactions predicted by RBPmap  with the corresponding insertion into the T vector  as a template. The probe was then labeled with Alexa Fluor546, by using the ULYSIS Nucleic Acid Labeling Kit , which added a fluor on every G of the probe to amplify the fluorescence intensity. The primers for the antisense probe amplification were included in FISH was carried out as previously described with minor modifications . RNA proPublished eCLIP-seq data were obtained from Gene Expression Omnibus (GEO) database under the following accession numbers: GSE91952 (EIF4G2), GSE126263 (MSI1), GSE71096 (SRSF10), GSE69153 (RC3H1) and GSE107768 .http://cbl-gorilla.cs.technion.ac.il)  . For datThe physiological experiments were carried out in triplicates (N = 3), and statistical analysis of the data was performed with the two-tailed Student’s t-tests. Data were present as the mean from three independent experiments with SEM. The RNA-seq was performed with four replicates and statistical analysis for analysis was calculated by DEseq2.XBP1s and FST mRNAs, and found that XBP1s and FST detected by RT-PCR with specific primers were significantly increased upon TG and GD treatment, respectively  depleted RNA sequencing (RNA-seq) of human HeLa cells treated with TG (an ER stress inducer) or GD (a metabolic oxidative stress inducer). XBP1s is a well-characterized marker for ER stress and Follistatin (FST) is upregulated in response to GD . We firsectively . Then, wectively . To idenectively  and findectively  were appectively . CircRNAectively . Principectively , indicatp-value < 0.05) upon TG and GD treatments, respectively  upon TG and GD treatments, respectively  and 193  DE lncRNAs were identified  of lncRNAs, circRNAs and their co-expressed DE mRNAs  for the 51 DE lncRNAs and 39 DE circRNAs identified in both stresses according to our RNA-seq data (trans roles of lncRNAs and circRNAs on the co-expressed mRNAs. RNA interference (RNAi) is a widely used approach to deplete lncRNA/circRNA of interest, although there are multiple other methods (TINCR silencing resulted in significantly decreased expressions of its co-expressed mRNAs (ERN1 and SOX12) in HeLa cells (lncSLC25A1 knockdown decreased its co-expressed targets (SOX12 and ATOH8), and circBANP depletion downregulated its co-expressed targets (ERN1 and ATF4)  . The heand ATF4) . Gene Onnd ATF4) .via sequestering miRNAs and thus regulating mRNA targets, we constructed a ceRNA network among the DE lncRNAs, circRNAs, and mRNAs respond to both ER and metabolic stresses. 23 circRNAs, 2 lncRNAs, 48 miRNAs, and 32 mRNAs composed of the ceRNA regulatory network (circBANP interacted with miR-9-5p to derepress the PRDM1 level (circBANP mediated by siRNA significantly reduced the luciferase activity of Firefly with the PRDM1 3’ UTR region containing the miR-9-5p binding site (lncSLC25A1-miR-744-5p-SOX12 axis exhibited a similar result according to our experiments (One of the molecular mechanisms for lncRNAs and circRNAs is to act as competing endogenous RNAs (ceRNAs), in which lncRNAs and circRNAs bind with miRNAs and decrease the corresponding binding of miRNAs on mRNA targets . In orde network . Among 3 network . Then, w network . To furtM1 level . Knockdoing site . The lnceriments . Althougvia RBPmap, a tool mapping the interacting RBPs for RNAs of interest  displayed that linc00612 mostly localized to the cytoplasm, and a small portion resided in the nucleus in HeLa cells  metastasis and docetaxel chemoresistance via serving as a ceRNA against miR-422a to improve the FOXQ1 level , splicing , RNA stability , and translation . Cellular localization of an ncRNA is critical for its functionality. Linc00612 mostly localizes to the cytoplasm, which may be associated with its role in regulation of translation. Meanwhile, a small portion of linc00612 resides in the nucleus, which may be responsible for its regulatory roles in transcription and splicing. CircSTAU2 interacts with splicing-related proteins  and RNA stability-related proteins . Linc00612 and circSTAU2 both interact with SRSF10 and PABPC4, implying that both proteins may be key factors in response to cellular stresses.RNA-protein interaction is another functional mechanism of RNAs . Increasectively . Linc006r cancer . Linc006We would also like to point out several limitations of this study. We cannot rule out that the DE lncRNAs/circRNAs identified under the cellular stresses tested have cell-type specificity. Although we have found a series of DE lncRNAs and circRNAs in cellular stress responses, investigations about functions of individual ncRNA of interest, and the molecular mechanism are required. Moreover, the lncRNA/circRNA regulatory networks need further validation with more experimental explorations.In summary, our study has identified a number of DE lncRNAs and circRNAs responsive to ER and metabolic stresses, and has constructed associated regulatory networks to provide novel insights for functional and mechanistic explorations of ncRNAs under cellular stresses.


High-resolution manometry (HRM) is a diagnostic tool used to evaluate esophageal motor function and diagnose motility disorders. A standardized protocol is used to make an accurate diagnosis based on the Chicago Classification. Some existing literature suggests that incomplete or imperfect manometry tests are common, however; there remains a paucity of data to evaluate risk factors for failure to help clinicians determine when a study may be difficult to perform.Our goal was to quantify how often failed tests occurred and determine specific factors that may be associated with failed HRM.We retrospectively evaluated records for HRM tests performed over 1 year at our academic centre. Based on clinical experience, we identified several factors that may be associated with the success of HRM testing including the following: indications and symptoms leading to testing, patient’s age and biological sex, previous esophageal manometry history, previous esophageal/gastric surgery, previous septal repair/deviated septum, history of significant nausea/vomiting, history of anxiety/depression, history of irritable bowel syndrome, and medication use . We then compared patients with successful HRM vs. unsuccessful HRM with regard to our pre-specified risk factors.29 HRM tests were unsuccessful from a total of 152 that were performed (19% failure rate). Reasons for failure included the inability to pass the probe through LES (55%) and the inability to tolerate the manometry probe for a minimum of 10 saline swallows (45%). After separating the failed cases from successful tests, both groups had a similar distribution of age and sex. Specific symptoms and indications did not have a significant association with unsuccessful tests. A previous history of failed manometry was associated with unsuccessful HRM . Conversely, PPI usage was associated with fewer failed HRM tests . Other medical history or medication use was not found to be associated with testing failure in our study.HRM is useful for diagnosing esophageal motility disorders, but incomplete tests are common. Although this study did not identify any factors in a patient’s medical history that could be used to predict failure in patients who have never had testing, further investigations may identify if PPI therapy can make HRM testing more tolerable. Additionally, the association between previous failed HRM and repeat failures suggests that endoscopic probe placement techniques should be considered instead of retrying conventional probe placement.NoneNone Declared


Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death globally and liver transplantation (LT) can serve as the best curative treatment option. However, HCC recurrence after LT remains the major obstacle to the long-term survival of recipients. Recently, immune checkpoint inhibitors (ICIs) have revolutionized the treatment of many cancers and provided a new treatment strategy for post-LT HCC recurrence. Evidence has been accumulated with the real-world application of ICIs in patients with post-LT HCC recurrence. Notably, the use of these agents as immunity boosters in recipients treated with immunosuppressors is still controversial. In this review, we summarized the immunotherapy for post-LT HCC recurrence and conducted an efficacy and safety evaluation based on the current experience of ICIs for post-LT HCC recurrence. In addition, we further discussed the potential mechanism of ICIs and immunosuppressive agents in regulating the balance between immune immunosuppression and lasting anti-tumor immunity. With almost 906,000 new cases and 830,000 deaths in 2020, liver cancer has become the third leading cause of cancer death worldwide . HepatocIn the past decades, numerous risk factors have been identified for HCC recurrence, including the pre-transplant alpha-fetoprotein levels, tumor number and size, etc. Therefore, some criteria, such as Milan criteria , UniversPost-LT HCC recurrence progressed with a predominant pattern of extra-hepatic metastases, including lung, bone and abdominal lymph nodes . For the2+/Calcineurin/nuclear factor of activated T-cells (NFAT) pathway, reduce the secretion of interleukin-2 (IL-2) and interferon-γ (IFN-γ), and contribute to long-term allograft survival (in vitro and in vivo studies showed that CNIs could enhance the expression of transforming growth factor-β (TGF-β) and promote the proliferation of cancer cells  inhibitors and calcineurin inhibitors (CNIs) . Immunossurvival . Howeversurvival , 21. Furer cells , 23.Mycophenolate mofetil (MMF) is an anti-metabolite purine antagonist and its application in LT began in the late 1990s . Given tNowadays, mTOR inhibitors (rapamycin), such as sirolimus and everolimus, have been reported to be anti-recurrence/metastasis and improve the prognosis of patients who underwent LT for HCC . Using mde novo malignancies after LT  than that in anti-PD-1 (8/32) and anti-CTLA-4 (1/4) groups. However, due to the limited cases, the current evidence is not certain to conclude that anti-PD-L1 therapy is relatively safe for post-LT HCC recurrence. Second, a longer interval from LT to initial ICIs treatment and a lower dose of ICIs might be related to a lower incidence of rejection. We found that patients without graft rejection after ICIs treatment have a longer interval from LT to drug exposure (4.65 yr vs. 2.52 yr), which is consistent with the previous studies . Given tAs described above, ICIs showed clinical benefits for the treatment of HCC recurrence but increased the risk of transplant rejection Figure 12, IL-10 and TGF-β) and induce the death of cytotoxic T cells through the increased expression of immune checkpoints, such as PD-1 and CTLA-4 , macrophages and dendritic cells (DCs), played vital roles in promoting a tolerogenic microenvironment . These cd CTLA-4 . Specifid CTLA-4 –70.+ T cells and increase the granzyme B expression by enhancing the mTOR signaling, further activating the immune system  could mediate immune response in the pro-inflammatory microenvironments and maintain tolerance in organ transplant models . Differe+ Tregs in the liver grafts, contributing to tolerance induction and further prolonging the survival time of recipients   . Recentlltration , 93.We speculate that there could be the following possible reasons. Firstly, PD-L1 is mainly expressed on antigen-presenting cells (including macrophages and DCs) and tumor cells, therefore, PD-L1 antibodies always target these cells, unlike PD-1 antibodies, which directly target T cells to completely block T cell exhaustion. However, macrophages and DCs could also inhibit the activation of T cells by expressing other immune checkpoints, such as TIM-3 and LAG-3 , 95. SecTo reduce the risk of graft rejection, the combination therapy of ICIs and immunosuppressive agents was proposed, which has attracted great attention recently. Herein, + and CD8+ T cells and the serum of IFN-γ, which were already proved to be helpful for the prediction of graft rejection in kidney and lung transplantation. Finally, once the acute graft rejection occurred, treatments such as ICIs withdrawal, high high-dose steroids and thymoglobulin should be taken immediately to improve patients’ outcomes. Further studies about the mechanism of the crosstalk of ICIs and immunosuppressive agents are necessary to improve the therapeutic effect for post-LT HCC recurrence.In this review, we summarized the existing research on the immunotherapy of post-LT HCC recurrence and discussed the experience of using ICIs in this setting. We believed that it’s better to adopt a steroids-free and mTOR-based regimen in patients with post-LT HCC recurrence instead of the CNIs. Compared to CsA and TAC, sirolimus and everolimus showed a promising role in anti-tumor with mild side effects. Additionally, based on the available data and cases mentioned above, we recommend that physicians should consider cautiously before the application of ICIs. The risks and benefits of ICIs-based immunotherapy must be fully assessed individually, depending on the circumstances of each patient. There are several factors should be taken into account to minimize the risks of graft rejection. Firstly, before the ICIs treatment, negative PD-L1 expression in liver biopsy and increased length of time from LT may contribute to lowering the risk of rejection. Secondly, compared to PD-1 and CTLA-4 inhibitors, PD-L1 therapy is a promising strategy to reduce the risk of graft rejection in post-LT HCC recurrence. Thirdly, the combination protocol (ICIs plus mTOR inhibitors) is a potential strategy to balance cancer immunology and graft tolerance. Moreover, close monitoring of immune status is mandatory during the ICIs therapies, such as the number of CD4QL and JJ participated in research design. JJ, HH, and RC participated in the writing of the paper. JJ and YL participated in data analysis. JJ, HH, and QL participated in reviewing and editing the manuscript. All authors contributed to the article and approved the submitted version.


We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element.  I,2. I1,2]elements . It is telements ,15,16,1713C-NMR emerged as a key technique to study intrinsically disordered proteins (IDPs) -ATP was accomplished with T4 polynucleotide kinase (NEB). Labeled RNA was separated from unincorporated [γ-32P]-ATP by column purification (NucAway) and adjusted with binding buffer  to 0.03 pmol/μL. A master mix containing tRNA, 32P-labeled SL4 RNA, and reaction buffer was prepared and then mixed with dilutions of the NTR or NTD, respectively, to achieve the indicated protein concentrations. Binding was performed for 10 min at RT in 20-μL reaction volume in the presence of 0.6 μg tRNA from baker’s yeast (Sigma), 3 nM 32P-labeled SL4 RNA, 25 mM potassium phosphate, 150 mM KCl, pH 6.5, and 1 mM MgCl2. After the addition of 3 μL loading buffer , the RNP complexes were resolved by PAGE  at 80 V for 75 min at RT. Gels were fixed and dried and subsequently exposed to a phosphor imager screen and visualized using a GE Typhoon laser scanner under “phosphorimager” settings.Radioactive EMSAs were performed according to  with the13C-detected 13C-15N CON (2D CON) experiment. Due to the very different structural and dynamic properties of the globular NTD domain and the flanking disordered regions, with the chosen setup, the NMR signals of the NTD are very weak or absent in the 2D CON. This allows to selectively pick up information about the disordered regions of NTR, yielding well-resolved NMR spectra, which reveal also information about seven proline residues (1H-detected 1H-15N HSQC (2D HN) experiment. The latter allows monitoring of most of the residues belonging to the folded domain, while those of the flexible regions suffer from extensive spectral overlap or line broadening (15N) common to the two spectra. Moreover, the 2D HN can be collected with high S/N without increasing the experimental time, just exploiting the relaxation delay of the 2D CON experiment. The NMR spectra obtained through this approach on NTR are reported in The interaction of NTR with 5_SL4 (referred to as RNA hereafter) was studied through the residues . It thusoadening . The comNMR spectroscopy reveals at the residue level the importance of the two disordered regions for the interaction with RNA. This is already evident when a sub-stoichiometric RNA concentration  is added to NTR A. InspecA zoom into the IDRs can be achieved through the analysis of the 2D CON spectrum. This allowed us to monitor most of the residues belonging to the highly flexible IDRs. As an example, 32RSGARSKQRRPQGLP46, and six arginine residues in the 177RGGSQASSRSSSRSRNSSRNSTPGSSR203 region  has completely different properties. This region possesses a peculiar amino acid composition (–225 has Differently from the two arginine-rich regions involved in the interaction with RNA (32–46 and 177–203), the 216–225 region does not present positively charged amino acids but has a highly hydrophobic nature resulting from branched-chain amino acids such as leucine [thus referred to as the poly-leucine (poly-L) region]. This hydrophobic stretch of 8 amino acids flanked by two negatively charged residues (Asp 216 and Asp 225) is likely to be engaged in transient interactions with other portions of NTR. A comparison of chemical shifts observed for the isolated NTD with those of the same nuclei in the NTR construct supports this hypothesis, and the insertion of a spin-label at position 211 indeed confirms a cross-talk between the IDR and the NTD domain . Of noteSummarizing, the present results indicate that electrostatics is the main driving force for molecular recognition and the arginine-rich regions, that were found to be perturbed at the early stages of the titration, are key players to promote binding with the negatively charged RNA backbone ,56. InteThe experimental investigation of the highly dynamic properties of N is by no means a trivial task but is of crucial importance to identifying novel approaches to interfere with SARS-CoV-2. Several insights have been recently obtained on its dynamic heterogeneity , on the 13C-detected NMR experiments such as the 2D CON allow us to access residue-resolved information on IDRs also when part of a multi-domain protein. They can be added to any high-resolution investigation performed through NMR, often based on the analysis of 2D HN NMR spectra only. The mr_CON//HN approach allows their simultaneous acquisition, providing a complete picture at residue level not only for the flexible regions but at the same time for the globular NTD domain. This complementary information is highly valuable as it reflects all components in their native context.In conclusion, The NMR data, supported by EMSA data, demonstrated that the flanking disordered regions of the SARS-CoV-2 NTD initiate and enhance the binding of the protein to RNA. They revealed specific tracts of the IDRs involved in the interaction within a multi-domain, cleavage prone, structurally and dynamically complex protein as NTR is.This represents a first step necessary to unravel the detailed molecular determinants of the N protein for specific RNA encountering and subsequent complex formation, e.g., during viral genome packaging. It paves the way for further studies with increasingly complex protein constructs, ultimately with the full-length protein, as well as with other relevant elements of the SARS-CoV-2 RNA.


Lying can be defined as stating a deliberate falsehood with the intent to deceive. It is part of our everyday life but it can be pathological, without motivation and a symptom of psychiatric illness. Although pathological lying has been debated for a century, it remains a controversial issue in Psychiatry.We aim to perform a review regarding pathological lying and related issues.We performed an updated review in the PubMed database and GoogleScholar using the terms “pathological lying”, “compulsive lying”, “mythomania” and “pseudologia fantastica”. The included articles were selected by title and abstract. We also consulted reference textbooks.We described the difference between normal and pathological lying and debated the different types of pathological lying, such as compulsive lying, mythomania and pseudologia fantastica.Recognizing lying is crucial for a skilled patient interview and distinguishing between pathological and non pathological lying may be decisive for an accurate differential diagnosis.


The clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein Cas) system is a powerful and highly precise gene-editing tool in basic and applied research for crop improvement programs. CRISPR/Cas tool is being extensively used in plants to improve crop yield, quality, and nutritional value and make them tolerant to environmental stresses. CRISPR/Cas system consists of a Cas protein with DNA endonuclease activity and one CRISPR RNA transcript that is processed to form one or several short guide RNAs that direct Cas9 to the target DNA sequence. The expression levels of Cas proteins and gRNAs significantly influence the editing efficiency of CRISPR/Cas-mediated genome editing. This review focuses on insights into RNA Pol III promoters and their types that govern the expression levels of sgRNA in the CRISPR/Cas system. We discussed Pol III promoters structural and functional characteristics and their comparison with Pol II promoters. Further, the use of synthetic promoters to increase the targeting efficiency and overcome the structural, functional, and expressional limitations of RNA Pol III promoters has been discussed. Our review reports various studies that illustrate the use of endogenous U6/U3 promoters for improving editing efficiency in plants and the applicative approach of species-specific RNA pol III promoters for genome editing in model crops like Arabidopsis and tobacco, cereals, legumes, oilseed, and horticultural crops. We further highlight the significance of optimizing these species-specific promoters’ systematic identification and validation for crop improvement and biotic and abiotic stress tolerance through CRISPR/Cas mediated genome editing. A synthetic promoter should be optimized for precise specificity, immediate inducibility, versatile applications, and efficient editing  are reported to be conserved among species . HoweverPlutella xylostella for enabling genome editing in non-model organisms  expression in vector-based RNA interference , and in rganisms . Howeverrganisms . The U3 rganisms . The U6 rganisms , while trganisms .Arabidopsis (AtU3 and AtU6) and rice (OsU3 and OsU6) promoters used specifically for dicots and monocots, respectively  genes along with some small nuclear RNA (snRNA) genes, while the third class of polymerase, RNA Pol III, transcribes small, non-coding RNA set of genes like 5S rRNA and tRNA. RNA pol III transcribed genes which are involved in cellular metabolic processes like t-RNA processing, mRNA splicing, and protein synthesis . The RNAsequence . There asequence . The thisequence . The typsequence .In plants, the two basal promoter elements required for Pol III transcribed snRNA genes are the −70 bp highly conserved plant snRNA gene-specific element, USE (consensus RTCCCACATCG) and −28 to −30 bp TATA-box  Marshal. The U6 U3 and U6 snRNA genes. These Pol III promoters are generally used in expression of small nuclear RNA, short hairpin RNA, and guide RNA in the CRISPR/Cas9 genome editing system  are transcribed by RNA Polymerase II, while genes like H1, U3, and U6 snRNAs are transcribed by RNA Polymerase III . The geng system . In mostg system . These Pg system . Thus, sg system .CRISPR/Cas9 plant genome editing system uses two sets of RNA polymerases. Expression of Cas9 gene under RNA polymerase II promoter while the sgRNA cassette is driven by the RNA Polymerase III (U6 or U3 promoters) . The twoThe transcription initiation leads to the polymerase complex formation in the promoter region. Protein factors or TFs that recognize the sequence motifs of RNA polymerase III transcribed genes are well studied in yeast and animals. The multi-subunit complex for activation of RNA polymerase III promoter includes TFIIIC, A and B boxes, TFIIIB, and TATA-binding protein (TBP) in yeast and vertebrates . The recThe tCAAca sequence is another core promoter element in fungi, and plant Pol III transcribed genes are involved in TSS selection by RNA Polymerase III. In this tCAAca sequence, the uppercase letters indicate the least variable positions, and the first A is the TSS . The shaHomo sapiens U6 snRNA)  . The RNA6 snRNA) .Type 1 promoters of RNA polymerase III require two internal sequence elements for efficient transcription, an A block located from +50 to +70 and a C block from +80 to +90, and an intermediate element (IE) between blocks A and B . Type 2 Transcription with RNA polymerase III takes 3 general steps: initiation, elongation, and termination. Positioning of eukaryotic RNA polymerase III enzyme to the transcription start site (TSS) requires many transcription factors that work synergistically. To initiate transcription on type 1 promoters, the RNA polymerases III complex relies on a different set of transcription factors  as it has less affinity for promoter sequence elements . BrieflyIn the case of type 3 promoters , assembly of a transcription factor on the promoter sequence occurs upstream of the transcription start site (TSS). Here SNAPc , an activating protein complex, binds to the upstream promoter element, proximal sequence element (PSE) to promote the TFIIIB recruitment and RNA polymerase III loading for transcription initiation. In this case, TFIIIB consist of TBP (TATA-box binding protein), BDP1 (B double prime 1), and BRF2 (B-related factor 2) .RNA polymerase III has a high and steady small nuclear RNA transcriptional activity, accounting for approximately 40% of total RNA, which is validated by the fact that Pol III promoter has a primary role in RNA-guided genome editing strategies like CRISPR/Cas technology . The guicis-regulatory element sequences of a promoter that exist in nature are used as fundamental blocks for synthesizing these artificial promoters. These can be created using rational design or ligation  and alcohol-O-acetyltransferase genes.A synthetic promoter is a sequence of DNA which is artificially designed in order to regulate the expression of the target gene. The ligation . Synthetchnology . Hao et e sgRNAs . These se sgRNAs . The same sgRNAs . They pre sgRNAs . Also, ipromoter . Löbs etThe plant species-specific Pol III promoters like U6 and U3 have been extensively used for increased sgRNA levels to achieve efficient editing using the CRISPR/Cas technology. In the last two decades, there have been several reports addressing the use of species-specific U3/U6 promoters in targeting certain traits in cereals, legumes, oilseeds, and horticultural crops, the details of which are discussed hereunder. Endogenously identified species-specific RNA Pol III promoters to enhance the genome editing efficiency are represented in A. thaliana genes viz., phytoene desaturase (PDS3), flagellin sensitive (AtFLS2), and the Nicotiana benthamiana ortholog of AtPDS3 (NbPDS). Moreover, the authors targeted two members of the Arabidopsis RACK1 (Receptor for Activated C Kinase 1) family with multiple sgRNAs expressed under the U6 promoter, thereby ensuring targeted mutagenesis and gene knockout , liguleless1 (LIG) and acetolactate synthase (ALS2). Under the expression of maize U6 promoter, the in vitro transcribed gRNAs and the purified Cas9 protein were pre-assembled to initiate the targeted mutagenesis  and TaMTL  (β-kafirin (major grain storage protein). Using SbU6_2.3 resulted in 80.0% of the mutation rate in the β-kafirin gene. Thus, endogenous pol III promoter employment leads to a higher and more efficient CRISPR/Cas editing system. (romoters . Moreove editing . Svitashagenesis . Very reneration . In anotolipase) . In the  system. .Medicago truncatula, M. truncatula through root hair transformation, where the target guide RNA was expressed under the Arabidopsis U6 promoter (phytoene desaturase (MtPDS) gene using a sgRNA under the effect of the native MtU6 promoter (symbiosis receptor-like kinase (SYMRK) gene is crucial for nodule and arbuscular mycorrhizal symbiosis in legumes. Targeted disruption of three targets of exon 2 of the VuSYMRK in Cowpea (Vigna unguiculata) through the CRISPR/Cas9 system was carried out by SYMRK gene was also targeted for mutagenesis in Lotus japonicus along with three homologous leghemoglobin loci , the designed guide RNAs of which were placed under the effect of LjU6-1 promoter (Medicago sativa L. The expression of the sgRNAs targeting the PDS and PALM1 (encoding a Cys(2)His(2) zinc finger transcription factor) genes was driven by the MtU6 Polymerase III promoter  (M. truncatula U6.6 Polymerase III promoter. Glutamine synthase (GS1) and chalcone-flavanone isomerase (CHI20) genes under the effect of the Arabidopsis U6 promoter (Brassica napus), ALCATRAZ (ALC) gene. The sgRNA was placed under the control of the Arabidopsis U6-26 promoter, where a single target sequence generated four alc mutant alleles in an edited T1 plant  using sgRNAs, driven by the Arabidopsis U6 promoter and M. truncatula U6 promoter, respectively (LPAT) aids the catalysis of fatty acid chains into 3-phosphoglycerate, thereby enhancing oil production. The BnLPAT2 and BnLPAT5 genes from B. napus were targeted using the Arabidopsis U6-26 promoter to drive the sgRNA expression further establishing their role in oil biosynthesis  and GmAGO7b (Glyma.02G111600) using the Arabidopsis U3 or U6 promoters to drive expression of each gRNA (Genome modification in soybean was demonstrated by 1g07220) . The sinpromoter . To addrT1 plant . Contemppromoter . The samectively . The enzynthesis . In anotach gRNA .via Agrobacterium tumefaciens mediated transient expression of phytoene desaturase (PDS) gene in N. benthamiana. They further transformed the CRISPR/Cas9 construct in potato to make stable mutations in the StIAA2 gene encoding an Aux/IAA protein in potato (Cucumis sativus L.) by eIF4E gene (eukaryotic translation initiation factor 4E), was demonstrated through Cas9/sgRNA editing. Two sgRNAs, expressed under the effect of Arabidopsis U6 promoter, were designed to target two sites of the eIF4E gene. A successful CRISPR/Cas9 editing of the flavanone-3-hydroxylase (F3H) gene was performed in the carrot. Two single-guide RNA (gRNAs) was expressed in the CRISPR/Cas9 vectors under the effect of the Arabidopsis U3 promoter for obstructing the biosynthesis of anthocyanin (n potato . A contehocyanin .SlAGO7) through Agrobacterium-mediated transformation. The sgRNAs, expressed under the effect of the Arabidopsis U6 promoter, were in duplicates in order to create large and well-defined deletions. The mutant plants had needle-like or wiry leaves as compared to the compound leaves in wild-type tomatoes (phytoene desaturase (PDS) gene was confirmed by the use of four sgRNAs that functioned under the effect of Arabidopsis U6 promoter, which resulted in an approximately 13.6% editing efficiency  genes were successfully knocked-out in apple. Two guide RNAs were expressed with U3 and U6 apple promoters for targeted editing. Successful editing in the MdPDS gene was confirmed by distinctive albino phenotype in about 85% of the edited lines, while early flowering was observed in 93% of the edited lines where the MdTFL1 was targeted (auxin biosynthesis (TAA1) and auxin response factor 8 (ARF8) genes of wild strawberry Fragaria vesca was achieved by RAS-PDS1 and RAS-PDS2), expressed under the rice U3 promoter, created albino phenotype and abnormalities in growth of the edited plants (Vitis vinifera U3 and U6 promoters and two UBQ promoters in precise targeting of grape phytoene desaturase (PDS) gene was established by TMT1 and TMT2 (tonoplastic monosaccharide transporter) demonstrated the successful editing in grapes  with four sgRNA under the control of the Arabidopsis U6 promoter  gene using the CjU6 promoter to obtain the albino phenotype (Hevea brasiliensis via CRISPR/Cas9 system. This study targeted the PDS and flowering time (FT) related genes. The first report of an immature embryo plant regeneration system and genetic transformation system in Phyllostachys edulis, a monopodial bamboo species using two PeU3 promoters and targeting the PePDS1 and PePDS2 genes. The usage of endogenous pol III promoters led to higher editing efficiency (35%–39%) than editing with the OsU3 promoter  formation of snRNA Pol III promoters in plants thus, this area of research needs to be explored to understand more of the transcriptional factors and regulatory elements. The review highlights the U3 and U6 promoters and their application in various plant systems. Recent studies show that the use of endogenous RNA Pol III promoter that transcribes single or multiple guide RNAs in CRISPR/Cas9 system have increased the editing efficiency. Therefore, the researchers, presently are aiming to identify the species-specific U3/U6 promoters and to broaden the understanding of transcriptional assembly for more specific and efficient genome editing.