Vldlr) in retinal neovascularization and hypoxia-induced factor 1 alpha (Hif1a). Moreover, functional enrichment showed that RNA editing enriched in FDM was primarily involved in response to fungicides, a potentially druggable process for myopia prevention, and epigenetic regulation. In contrast, RNA editing enriched in controls was mostly involved in post-embryonic eye morphogenesis. Our results demonstrate altered A-to-I RNA editing associated with myopia in an experimental mouse model and warrant further study on its role in myopia development.Myopia is one of the most common causes of vision loss globally and is significantly affected by epigenetics. Adenosine-to-inosine (A-to-I RNA) editing is an epigenetic process involved in neurological disorders, yet its role in myopia remains undetermined. We performed a transcriptome-wide analysis of A-to-I RNA editing in the retina of form-deprivation myopia mice. Our study identified 91 A-to-I RNA editing sites in 84 genes associated with myopia. Notably, at least 27 (32.1%) of these genes with myopia-associated RNA editing showed existing evidence to be associated with myopia or related ocular phenotypes in humans or animal models, such as very low-density lipoprotein receptor ( Myopia is among the most common refractive errors globally . As the Myopia is caused by a complex interplay between common genetic and environmental factors, yet the exact mechanisms are not fully understood . LinkageUnderstanding gene–environment interactions in myopia requires understanding the crucial role that epigenetics has in the process. Col1a1 promoter/exon 1 might reduce scleral collagen production and lead to myopia development (Form deprivation myopia (FDM) is experimental myopia induced by blocking the regular visual input to one eye with a diffuser or an opaque lens. FDM animal models are used to study the mechanisms of human myopia and the role of epigenetic factors in its development. FDM animal studies have identified several epigenetic markers and pathways associated with myopia and its progression. A recent study on FDM mice suggested that DNA methylation of the elopment .In the current study, we performed an epitranscriptome analysis to investigate RNA editing in the retina of a mouse model of formal deprivation myopia. Our findings show altered A-to-I RNA editing in an experimental myopia mouse model and warrant further study on the role of A-to-I RNA editing in the molecular mechanism of myopia.FASTQ files of RNA-Seq raw reads were retrieved from the Gene Expression Omnibus (GEO).The sequencing data were processed as previously described . SequencVariants were called to identify single nucleotide variations (SNVs) by using VarScan (version 2.4.3)  and filtProtein–protein interactive (PPT) network construction and gene function enrichment analysis were conducted using the STRING database . Items wp-values. For RNA editing sites with empirical GLM p < 0.05, Fisher’s exact test was performed to calculate Fisher’s P for intergroup comparisons of the total counts of the reference and alternative alleles between the FDM and control groups. The Benjamini-Hochberg method for FDR calculation was used to adjust empirical p-values for multiple comparisons. Differentially edited RNA editing sites were defined as GLM FDR < 0.2 or Fisher’s exact test FDR < 0.2. Principal component analysis (PCA) was performed using R (version 3.6.3).The intergroup editing levels of RNA editing sites were compared using the generalized linear model (GLM) and likelihood ratio test (LRT) to calculate the empirical GLM In all, 8,702 high-confidence A-to-I RNA editing events were observed in the RNA of 2,772 genes from all mouse retina samples . These iThe comparison of A-to-I RNA editing sites and genes between FDM and controls is shown in p-values are shown in A total of 91 A-to-I different RNA editing (DRE) sites in 84 genes were differentially edited between controls and FDM , with 46Trim9), p.S316G (c.946A > G) in zinc finger protein 397 (Zfp397), and p.S1973G (c.5917A > G) in nucleoporin 214 (Nup214). All these missense DRE sites showed decreased RNA editing in FDM.Among these DRE sites, three were missense DRE sites, namely, p.Y62C (c.185A > G) in tripartite motif-containing 9 (Iws1), polymerase (RNA) III (DNA directed) polypeptide H (Polr3h), RNA binding motif protein 25 (Rbm25), hypoxia-inducible factor 1 alpha subunit (Hif1a), and ring finger protein 40 (Rnf40)  .Tjp2), solute carrier family 35, member E2 (Slc35e2), cryptochrome 1 (Cry1), hypoxia-inducible factor 1 alpha subunit (Hif1a), poly(rC) binding protein 3 (Pcbp3), LMBR1 domain containing 2 (Lmbrd2), high mobility group 20A (Hmg20a), protein phosphatase 1B magnesium dependent beta isoform (Ppm1b), transient receptor potential cation channel subfamily M member 3 (Trpm3), CDP-diacylglycerol synthase 2 (Cds2), selenoprotein P (Selenop), trinucleotide repeat containing 6a (Tnrc6a), protein phosphatase 2 regulatory subunit B beta (Ppp2r2b), cell adhesion molecule 2 (Cadm2), geranylgeranyl diphosphate synthase 1 (Ggps1), and prominin 1 (Prom1).Our results showed that at least 27 of the 84 differentially edited genes were associated with myopia or related ocular phenotypes in humans or animal models . In partThe PPI networks were constructed, and enrichment analysis was performed for edited genes enriched in a specific group, i.e., with RNA editing sites upregulated or uniquely edited in FDM or controls. The results suggested that edited genes enriched in FDM were mainly involved in biological processes related to response to fungicides, regulation of epigenetics, such as oxidative DNA demethylation, histone methylation, and chromatin organization, and development and structure maintenance, such as photoreceptor cell maintenance, regulation of focal adhesion assembly, organelle transport along microtubules, and establishment of cell polarity. In contrast, edited genes enriched in controls were mainly involved in biological processes related to development and physiological function maintenance, such as post-embryonic eye morphogenesis, exonucleolytic RNA phosphodiester bond hydrolysis, postsynaptic membrane organization, cerebellar Purkinje cell layer development, cell junction maintenance, endothelial cell migration, regulation of receptor internalization, actin filament bundle assembly, and post-Golgi vesicle-mediated transport .Recent evidence has suggested the substantial role of epigenetics in myopia . Our curTjp2 encodes a protein that is a component of the tight junction barrier in epithelial and endothelial cells and is necessary for adequately assembling tight junctions. By using a GWAS approach, SNPs near TJP2, PCBP3, CADM2, and TRPM3 were identified to be associated with myopia or refractive errors , and lysine demethylase 5B (Kdm5b). It was noted that Hif1a could also be involved in response to econazole, which is another fungicide with anti-muscarinic effects  and MT3 (muscarinic acetylcholine receptor M4 receptor antagonists), have been reported to prevent experimentally-induced axial myopia . Further effects . SeveralThe current study demonstrates dramatic changes in RNA editing in the retinal epitranscriptome of an FDM mouse model. Our results were thus limited to the original study’s sample size, and further validation and functional analysis of RNA editing in additional mouse models are therefore needed in future studies.In conclusion, the current study provided initial evidence pointing toward a potential role played by A-to-I RNA editing during myopia development. Such findings thus warrant further studies on the role of RNA editing in myopia etiology.The original contributions presented in the study are included in the article/X-BP, Y-SH, and H-RP performed the data analysis and wrote the manuscript. Z-YW improved the data analysis pipeline. ZL and Y-YJ participated in the discussion. JW and J-HC conceived the project and planned the experiments. All authors contributed to the final manuscript.This study was supported in part by grants from the National Natural Science Foundation of China (No. 31671311), the “Six Talent Peak” Plan of Jiangsu Province (No. SWYY-127), the Innovative and Entrepreneurial Talents of Jiangsu Province, the Program for High-Level Entrepreneurial and Innovative Talents of Jiangsu Province, the Natural Science Foundation of Guangdong Province/Guangdong Basic and Applied Basic Research Foundation (2019A1515012062), the Taihu Lake Talent Plan, and the Fundamental Research Funds for the Central Universities (JUSRP51712B and JUSRP1901XNC), Wuxi Women and Children’s Health Research Program (FYKY202107).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.


RNA has been escaping the limelight for decades while it was used to decipher the genetic code , establiManuel et al. summarise the impact of alternative splicing on the proteome diversity in a comprehensive review also describing splicing contribution to the generation of noncoding and circular RNAs. They address the important challenges in the prevalence of functional alternative splicing, the artefact-prone difficulties it creates in genome annotations, and the linking between observed RNA splice-types and peptide datasets, including advances and limitations of the current technologies.A fundamentally distinctive feature of the RNA is its versatility. Consequently, we find the involvement of RNA function across all stages of gene expression. In eukaryotic and some prokaryotic cells, RNA-determined splicing is one of the cornerstone processes of gene expression. Splicing is driven by introns which likely originate from ancient retroelements (RNA retroviruses derivatives) but now are tightly controlled in the cells . RegulatAkinyi and Frilander enlist cooperation between the U2 and U12 types using the example of SNRNP48 and RNPC3 genes (encoding U11 and U12 proteins) where feedback-stabilised U11 and U12 interactions lead to the recognition of 3′splice site 1 and synthesis of a non-productive, nuclear-retained transcript–as opposed to the use of 3′splice site 2 that results in cytoplasmic export and translation. They further showcase examples of direct competition between U2 and U12 types , or cryptic activation of U2 splicing in a deficit of U12 machinery , and discuss implications in the context of spliceosomal diseases including Peutz-Jager Syndrome, spondyloepiphyseal dysplasia tarda and Cerebral palsy.Splicing picture was not complete until the alternative “minor” (type II) splicing apparatus has been identified . InitialHuang et al. feature the conservation and diversity of SYF2, an important splicing factor that interacts with cyclin D-type binding-protein 1, a cell cycle regulator at the G1/S transition. SYF2 has been characterised as essential or stimulatory in a variety of proliferative situations, including cancer  in an insightful review of these emerging RNA types. They highlight Terminus-Associated Short Non-coding RNAs (TASRs) and their antisense (aTASRs) varieties, Transcription Termination Site Associated RNAs (TTSa-RNAs), Transcription Boundary-Associated RNAs (TBARs), Terminus-Associated Long RNAs (TALRs) and 3′UTR-associated RNAs (uaRNAs), and explore evidence for their biogenesis, including the same and independent promoter models. Ni et al. further enlist organisation and discovery technology of several prominent TANRs and summarise the demonstrated TANR functions, including transcriptional interference, promoter and terminator juxtapositioning, transcription termination assistance, micro RNA sponging and sequence-directed RNA cleavage and modification. They note that functionally-relevant TANRs can originate also from long noncoding RNA genes, with MALAT1 prominently exemplified by its translation-activating MALAT1-associated small cytoplasmic RNA (MascRNA)  .Budrass et al. where they thoroughly describe a new micro RNA regulation network and intersect it with the protein control network of a matching complexity, as found for the chaperones Heat Shock Protein 40s   by micro RNAs of identical and different families, at one or multiple sites, opening new area of complex combinatorial regulatory opportunities.While individual micro RNAs may appear as less multifaceted regulators and interactors compared to the longer non-coding RNAs, a developing view is that micro RNAs function in networks, collectively targeting the entire pathways of the cells . Such neJ genes) . J-proteJ genes) . BudrassChukka et al. discuss eIF5B at the crossroads the ribosomal, transfer and messenger RNA interactions. They highlight canonical and most conserved eIF5B functions in initiator tRNA stabilisation on the ribosomal small subunit and subunit joining. Chukka et al. also provide an outlook into the less obvious eIF5B activities in checkpointing eukaryotic small subunit maturation, conveying initiation with the alternative initiator tRNA carrier eIF2A active in certain stresses and interacting with viral  and cellular  internal ribosome entry sites (IRESes). eIF5B action in upstream Open Reading Frame (uORF)-regulated genes is reviewed and its overall cell survival-promoting and thus, malignancy-maintaining role is brought into the focus as an attractive drug target.Decoding of the messenger(m)RNA into the proteins is the most crucial function performed by the RNA . TranslaSeixas et al. thoroughly present types of oxidative RNA damage in a mini review covering the most ubiquitous oxidating agents, RNA injuries including 8-oxo-7,8-dihydroguanosine, 8-oxo-7,8-dihydroadenosine, 5-hydroxycytosine and 5-hydroxyuridine, and their effects on mRNA, tRNA, ribosomal and micro RNA function. Seixas et al. emphasise the known passive (scavenging) and active (repair) RNA injury protection systems in prokaryotes noting open questions in comprehensive identification of these components across all life.Another interesting activity of RNA tightly linked to translation is that of a cellular protection and damage sensing. Oxygenation and oxidative environments present a substantial challenge to the nucleic acids-based life, and especially so to the RNA which can become oxidised in a diverse ways. It has been known that oxidised RNA induces translational errors and may be neurodegenerative or carcinogenic . Seixas tat mRNA, encoding the essential Tat transcriptional regulator of the virus. All viral transcripts share the initial 289 nt and thus the 59 nt of the highly structured trans-activation response (TAR) RNA element. TAR controls host translation via activation and suppression of Protein Kinase RNA-activated (PKR), and further activates viral transcription upon Tat binding  and High-mobility group box 3 (HMGB3) proteins affecting HIV infection the most. Using RNA modification protection, Khoury et al. located the SRP14 and HMGB3 binding sites nearby the tat start codon. Most intriguingly, SRP14 and HMGB3 negatively regulated latent and productive infection, while stimulating and repressing Tat synthesis, respectively. Khoury et al. propose SRP14 and HMGB3 alter the efficiency of the tat IRES, opening new depths in the lentiviral host interactions and additional pathways to manipulate HIV reactivation.RNA is often multifunctional in viruses where there can be certain restrictions on genome size, and in synthetic biology designs where vector and delivery limitations apply to the RNA length, together with the considerations of economy and cost . From it binding . Inspirenfection , Khoury etc., (Korniakova et al. present a new fluoride-sensitive vector design incorporating a fluoride riboswitch (Interestingly, HIV-1 RNA may contain other RNA regulators, riboswitches . Riboswietc., . Riboswietc., . In a brboswitch  in the rIt may not be an overstatement to name the RNA an ultimate molecular machine of life. RNA often performs in relatively straightforward ways built on direct molecular recognition through tertiary structure and basepairing, as happens during micro RNA target binding, and in distinct enzymatic reactions, as occurs during the intron lariat formation and excision. In the other cases, RNA performs as the structural and enzymatic core of conveying molecular machines such as the ribosome, where it uses chemical energy to process, transform and realise biological information. It is quite remarkable that the RNA can “work” with all types of biological macromolecules, be an enzyme and a substrate, carry and decode the genetic information, signal, receive and operate with chemical potentials, making it a “complete”, self-sufficient molecule. This self-sufficiency contains a value for novel synthetic biology designs, that is, being recognised in RNA vaccines and gene replacement therapeutics of more sophisticated construction, such as self-amplifying RNA . It also


Cardiovascular disease is one of the leading causes of mortality worldwide. Recent studies have shown that circular RNAs (circRNAs) have emerged as important players in the prevention and treatment of cardiovascular diseases. circRNAs are a class of endogenous noncoding RNAs that are generated by back-splicing and are involved in many pathophysiological processes. In this review, we outline the current research progress on the regulatory roles of circRNAs in cardiovascular diseases. Further, new technologies and methods available for identifying, validating, synthesizing, and analyzing circRNAs, as well as their applications in therapeutics, are highlighted here. Moreover, we summarize the increasing insights into the potential use of circRNAs as circulating diagnostic and prognostic biomarkers. Finally, we discuss the prospects and challenges of circRNA therapeutic applications for cardiovascular disease therapy, with a particular focus on developing circRNA synthesis and engineering delivery systems. Circular RNAs (circRNAs) are a class of covalently closed RNA sequences without 5′- cap and 3′-polyadenylate [poly(A)], most of which do not have protein-coding abilities . circRNAOn the basis of their sequence composition, circRNAs can be classified as exonic circRNAs, intronic circRNAs, and exon–intron circRNAs . circRNAGiven the diversity of circRNAs and their many individual distributions, circRNAs are expected to play many important cellular and molecular roles Fig. . circRNACardiovascular disease (CVD) is one of the leading causes of human death imposing a heavy economic burden on medical systems worldwide ,33. LargThe process that leads from studying circRNA through basic and clinical research to developing novel circRNA-based theranostics is a multistep and lengthy one Fig. . We will+ to Li+ in the RNase R reaction buffer is important for the efficient degradation of such linear RNAs [Because of lack of poly(A) tails and their low abundance in cells, circRNAs have long been ignored. With advanced deep sequencing technologies and enrichment methodologies, circRNAs are identified and annotated by mapping back-splicing junction (BSJ) sites in high-throughput sequencing data Table . Ribosomear RNAs . Besidesear RNAs . Althougear RNAs . Moreoveear RNAs .Experimental validation of circRNAs is essential before specific characterization and functional assays can be conducted Table . Both poThe subcellular localization of cellular circRNAs is important for determining their potential regulatory role, as it has been with other types of RNAs. Most of the exon-derived circRNAs are localized in the cytoplasm, while intron-containing circRNAs are mostly in the nucleus. RT quantitative PCR (RT-qPCR) and RNA fluorescence in situ hybridization (FISH) are the most widely used approaches to determining the subcellular localization of circRNAs Table . RT-qPCROnce the existence and subcellular location of circRNAs have been verified, the next logical step is exploring the biological function of the identified circRNA. Gain- and loss-of-function studies are commonly used approaches to determining the functional phenotypes of circRNAs in cells and animals Table . Many stcircRNA overexpression can also be achieved by several approaches. Currently, the most used method in cardiovascular research is generating a circRNA overexpression construct and then makes use of either plasmids to transfect cells or adeno/lentivirus delivery. In general, the overexpression vector can be constructed by inserting the cyclized sequences flanking with native intron sequences that are sufficient for cyclization . The useN6-methyladenosine (m6A) methylation-driven protein translation. circRNA translation and its clinical significance have been well described in a recent review; therefore, we will not elaborate here [Synthesis of circRNA in vitro and the provision of RNA-based therapies for CVDs will undoubtedly have a major impact on health care in the future Fig. . Normallate here . Engineeate here –58. T4 Date here . T4 RNA td) gene and Anabaena pre-tRNAs are most commonly used for circRNA circularization [Alternatively to enzymatic ligation by T4 ligases, using ribozymes sequences is another strategy to generate circRNAs. Specifically, group I and II introns have been applied to construct circRNAs in in vitro cyclization systems, also known as the permuted intron–exon (PIE) method ,59,60. Trization –59,61. Arization ,63. In gAs more and more circRNAs are identified, new databases are being developed to provide various types of information related to circRNAs Table . CircBascircRNAs have been found to be involved in the development of various human diseases. Here, we focus on the emerging regulatory and mechanistic roles of circRNA in CVDs, including cardiac fibrosis, myocardial infarction (MI), atherosclerosis, cardiomyopathy, and cardiac hypertrophy Fig. .Yap mRNA to YTHDF1 and YTHDF2, consequently, regulating the expression of YAP. Overexpression of circ-ZNF609 aggravates cardiomyocyte apoptosis and disrupts the cross-talk between Hippo-YAP and Akt signaling via downregulating YAP expression [The morbidity and mortality of MI remain high, despite the development of interventional coronary reperfusion approaches. Heart ischemia is associated with cardiomyocyte loss, a major contributing factor to cardiac dysfunction. Therefore, inhibiting cardiomyocyte apoptosis and promoting its survival are potential therapeutic approaches. To this end, the clinical significance of circRNA and its relationship with MI pathophysiology have been investigated in several studies. In one such study, Cdr1as overexpression was shown to promote mouse cardiac myocyte apoptosis and to exacerbate cardiac infarct size by targeting miR-7a . circNCXpression . The cirpression . circ-SNpression . CollectThe severity of cardiac fibrosis correlates with higher long-term mortality in patients with cardiac disease, especially heart failure. Cardiomyocytes, fibroblasts, and vascular cells in the heart are connected by a complex matrix, which helps to maintain the cardiac plasticity and structural integrity. When the heart is compromised, the balance between fibroblasts and cardiomyocytes is disrupted. An increase in collagen synthesis or a decrease in collagen degradation results in an excessive, diffuse accumulation of collagen in the interstitial and perivascular tissues. circRNA circNFIB is downregulated both in the heart after MI and in primary adult cardiac fibroblasts when treated with transforming growth factor-β. Conversely, overexpression of circNFIB inhibits primary fibroblast proliferation via sponging miR-433 . circHIPFascin, Lox, and Yes1 [It is now well established that atherosclerosis, endothelial cell dysfunction, vascular smooth muscle cell (VSMC) alteration, excess lipid, cholesterol accumulation, and inflammation are critical for the development of atherosclerosis . The occand Yes1 . circNRGand Yes1 . Mechaniand Yes1 . Overexpand Yes1 . cZNF292and Yes1 . NonetheCardiomyopathy is the progressive cardiac insufficiency caused by structural changes in the ventricle that ultimately impair cardiac function. Here, we will discuss mainly 3 types of cardiomyopathies: dilated cardiomyopathy, hypertrophic cardiomyopathy, and diabetic cardiomyopathy. Dilated cardiomyopathy is often characterized by an enlarged cardiac chamber and accompanied by systolic myocardial dysfunction, while hypertrophic cardiomyopathy is caused by genetic mutations and has a similar phenotype to pressure overload-induced cardiac hypertrophy. Diabetic cardiomyopathy is a diabetes-related cardiovascular complication that is accompanied by cardiomyocyte metabolic disorder, myocardial interstitial fibrosis, and cardiac dysfunction. A study that performed ribosomal depleted RNA sequencing from human hearts and RBM20-null mice found 6 circRNAs  that were significantly downregulated in response to dilated cardiomyopathy, while only one circRNA (cCAMK2D) was decreased in the heart with hypertrophic cardiomyopathy . In doxocircSlc8a1 suppresses pressure-overload-induced cardiac hypertrophy via endogenously sponging miR-133 [Pathological cardiac hypertrophy in patients with MI, valvular disease, and metabolic syndrome is initially characterized by reduced ventricular size and increased wall thickness, leading to ventricular dilatation, and impaired systolic function , eventually resulting in heart failure. circRNAs are implicated in cardiac hypertrophy via several pathways. Knocking down the highly abundant circRNA  miR-133 . circRNA miR-133 . Harness miR-133 . Another miR-133 . In summcircRNAs are abundant in blood, urine, and extracellular vesicles and exhibit high stability and differential expression in response to stress stimulus conditions . EmerginRNA-based therapies have emerged to be promising treatment strategies for many diseases and vaccine development ,135. OneEfficient delivery of RNA into cells and organs is a major challenge that needs to be addressed before RNA therapy can be applied clinically. RNA encapsulation mediated by viral or nonviral vectors represents the most common delivery strategy Fig. . SeveralTo deliver circRNAs in sufficient amount and in a safe manner, nonvirus delivery systems are also developed. Exosome and other cell-derived membrane vesicles and lipid nanoparticles (LNPs) are considered common carriers for RNA delivery . LNPs arIn this review article, we review the research progress and therapeutic applications of circRNAs in CVDs. circRNAs are involved in variety of cellular processes and are associated with many human diseases. In recent years, new technologies and methods for circRNA identification, validation, functional study, and therapy applications have been developed. In the cardiovascular system, however, the underlying regulatory mechanisms of circRNAs are still to be explored in depth. Most studies have focused on the function of circRNAs as miRNA sponge, while other functions, mechanisms, and pathways have been less studied. There is still a need for further in-depth mechanistic research to be carried out. Besides, given the increasing morbidity and mortality of CVDs, the need for development of new diagnostic and prognostic biomarkers is critical. The potential of circRNAs for biomarker development is enormous, as these molecules exhibit excellent stability in biofluids, evolutionary conservation, and various distinctive functional aspects. However, our knowledge on the biology of circRNAs is currently limited, making the application of circRNAs in clinical practice very challenging. Hence, further in-depth functional and structural studies to better understand the biology of circRNAs are required before these new therapeutic regimens can be routinely implemented.The aim of this review is to differentiate from previously summarized reviews regarding this topic by focusing on the research progress and challenges that have been faced in the development of circRNA-based therapy for the prevention of CVDs. With the advancement of RNA biology and RNA-related technologies, RNA-based drug design and drug delivery systems have achieved more diversified development, and RNA therapy has entered a new era of rapid development . BecauseThe growing understanding of the different roles of RNAs has led to the development of new RNA-based drugs. RNA therapy has the potential to be a powerful new therapeutic strategy to address previously untreatable diseases. One of the most attractive areas of circRNA research is the circRNA-based drug design for CVD therapy. With the progressive understanding of the role of circRNAs in CVDs and circRNA-mediated RNA therapy, a new way is paved for CVD diagnosis and treatment. However, there are still barriers to overcome. Many efforts have been made to synthesize circRNAs effectively. Currently, most circRNAs are generated directly in vivo by overexpressing strategies. However, these strategies harbor the danger of coproducing corresponding linear RNA as side product that can compromise the quality and purity of the newly produced circRNAs. To circumvent this problem, synthesized circRNA must be directly delivered into the target similarly to mRNA therapy. Moreover, the application of engineered circRNA synthesis strategies is usually limited because of low cyclization efficiency and excessive immune response stimulation caused by artificially introduced exogenous fragments. The development of improved circRNA production methodologies will enable further important research on the open questions about circRNA function and allow for the development of new circRNA-based technologies. The circRNA mode of delivery is another important challenge that needs to be overcome. For example, as mentioned earlier, nanoparticles can either inadvertently activate the immune system or sometimes be accumulated into other unwanted tissues causing side effects. Therefore, further research focusing on reducing the side effects of nanoparticles and improving their safety profile is essential to the clinical application of circRNA drugs.Vaccine development is another promising application of circRNA research. Although mRNA-based vaccines have now established themselves as a component of immunotherapy, progress in the clinical development of mRNA vaccines has been hampered by molecule instability and storage problems. Nonetheless, mRNA-based vaccines performed well during the coronavirus disease 2019 pandemic in preventing coronavirus disease 2019 variants . BecauseFunding: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to J.X.), National Natural Science Foundation of China (82020108002 and 82225005 to J.X. and 82270291 to L.W.), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to J.X.), the grant from Science and Technology Commission of Shanghai Municipality (21XD1421300 and 20DZ2255400 to J.X.), the “Dawn” Program of Shanghai Education Commission (19SG34 to J.X.), and ZonMw PSIDER grant (no. 10250022110004) to J.P.G.S. Competing interests: The authors declare that they have no competing interests.


Certain re-emerging alphaviruses, such as chikungunya virus (CHIKV), cause serious disease and widespread epidemics. To develop virus-specific therapies, it is critical to understand the determinants of alphavirus pathogenesis and virulence. One major determinant is viral evasion of the host interferon response, which upregulates antiviral effectors, including zinc finger antiviral protein (ZAP). Here, we demonstrated that Old World alphaviruses show differential sensitivity to endogenous ZAP in 293T cells: Ross River virus (RRV) and Sindbis virus (SINV) are more sensitive to ZAP than o’nyong’nyong virus (ONNV) and CHIKV. We hypothesized that the more ZAP-resistant alphaviruses evade ZAP binding to their RNA. However, we did not find a correlation between ZAP sensitivity and binding to alphavirus genomic RNA. Using a chimeric virus, we found the ZAP sensitivity determinant lies mainly within the alphavirus non-structural protein (nsP) gene region. Surprisingly, we also did not find a correlation between alphavirus ZAP sensitivity and binding to nsP RNA, suggesting ZAP targeting of specific regions in the nsP RNA. Since ZAP can preferentially bind CpG dinucleotides in viral RNA, we identified three 500-bp sequences in the nsP region where CpG content correlates with ZAP sensitivity. Interestingly, ZAP binding to one of these sequences in the nsP2 gene correlated to sensitivity, and we confirmed that this binding is CpG-dependent. Our results demonstrate a potential strategy of alphavirus virulence by localized CpG suppression to evade ZAP recognition. Togaviridae family that are transmitted by arthropod vectors [Alphaviruses are positive-sense, single-stranded RNA viruses of the  vectors . Alphavi vectors . Disease vectors . CHIKV h vectors . During  vectors . The geo vectors ,6, but n vectors . To deveFlaviviridae family, where ZAP can inhibit JEV but not YFV, DENV, or ZIKV [One viral strategy of increased virulence is the evasion of immune defenses, such as the type I (α/β) interferon (IFN) response. Production of IFN upon the cellular detection of viral invasion stimulates the expression of antiviral genes known as IFN-stimulated genes (ISGs). One important ISG product is zinc finger antiviral protein (ZAP), an RNA-binding protein that inhibits a broad range of RNA and DNA viruses . Specifi or ZIKV ,20. This or ZIKV ,22,23, benv gene and the ZAP sensitivity of HIV-1 strains [env gene [ZAP recognition and binding of viral RNA is essential for its antiviral activity . ZAP is  strains . This suenv gene . Other senv gene  and UpA env gene . FurtherRecently, we observed that Old World alphaviruses exhibit differential sensitivity to inhibition by individual ZAP isoforms: RRV and SINV are more sensitive to ZAP than CHIKV and its close relative, o’nyong’nyong virus (ONNV) . In thisZinc finger nuclease-mediated ZAP knockout (KO) 293T cells (clone 89) and the parental wild-type (WT) 293T cells were generously provided by Dr. Akinori Takaoka at Hokkaido University, Sapporo, Japan . ZAP KO SINV-expressing enhanced green fluorescent protein (EGFP) (TE/5′2J/GFP), ONNV SG650 strain expressing EGFP  (ONNV-GFP), RRV T48 strain , RRV expressing EGFP , and CHIKV vaccine strain 181/clone 25  have been previously described ,40,41,42cis-acting RNA elements and nonstructural protein (nsP) genes in the viral plasmid backbones were obtained from SINV by digestion with XbaI (5′) and XhoI (3′) and from ONNV by digestion with AscI (5′) and NotI (3′), followed by gel extraction with the Zymoclean Gel DNA Recovery Kit . The subgenomic promoter, EGFP, structural protein (sP) genes, 3′ untranslated region, and poly (A) tail were amplified from SINV with primers to add 5′ AscI (5′-GTATGCGCGCGCCACCATGGTGAGCAAG-3′) and 3′ NotI (5′-gtttGCGGCCGCATTCCCCTCGAGGAATTCCC-3′) sites and from ONNV with primers to add 5′ NheI  and 3′ XhoI (5′-GTTTCTCGAGCCTCGATTAATTAAGCGGCCGC-3′) sites. Primers were synthesized by Integrated DNA Technologies and PCR was performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs). The resulting PCR products were digested and cloned into the corresponding viral plasmid backbones using T4 DNA ligase (New England Biolabs).The chimeric SINV/ONNV viruses were derived from TE/5′2J/GFP and ONNV-GFP. The Virus stocks were generated in BHK-21 cells as previously described . Titers cis-acting RNA elements and nsPs of SINV and ONNV were generated by restriction enzyme digestion as described in Cis-acting RNA elements and nsPs were amplified from RRV using the primers 5′-TCGCCACCTCTGACTTGAGC-3′ and 5′-GTTTACTGTTGTGAGCTGTATTAGATGAAGG-3′ and from CHIKV using the primers 5′-GCTCGATTTAGGTGACACTATAG-3′ and 5′-TATGGCTGATTGGTATTTAGGTAC-3. DNA templates for the transcription of biotinylated CpG-correlated window RNA were amplified using primers to generate templates of length 500 ± 2-bp  with a 5′ T7 promoter sequence , incubated for 1 h with streptavidin-HRP antibody  diluted 1:300 in blocking buffer, and then washed twice with wash buffer A (1:10 blocking buffer) for 30 min and twice with wash buffer B  for 5 min. Biotinylated RNA was visualized with ProSignal Pico ECL Reagent  on a ChemiDoc imager .Transcription of biotinylated genomic SINV RNA and firefly luciferase (Fluc) RNA was performed as previously described using Sp6 RNA polymerase (New England Biolabs) and the mMESSAGE mMACHINE T7 Transcription Kit  , respectsequence . The winsequence . BrieflyIn vitro RNA pull-down was performed as previously described . 0.4 pmoTotal protein was isolated by cell lysis using RIPA lysis buffer  supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche Life Science), followed by quantification with a colorimetric protein assay (Bio-Rad). Proteins were resolved by SDS-PAGE using Tris-glycine-SDS electrophoresis buffer  and 4–15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to nitrocellulose membranes (Bio-Rad). Immunodetection was performed with 1:5000 anti-ZAP  and 1:20,000 anti-actin-HRP . The ZAP primary antibody was detected with 1:20,000 goat anti-rabbit-HRP (Thermo Fisher Scientific). Proteins were visualized with ProSignal Pico ECL Reagent (Genesee Scientific) on a ChemiDoc imager (Bio-Rad).Total RNA was isolated by Quick-RNA kit (Zymo Research). An amount of 0.8 μg of input RNA was used as a template for reverse transcription by ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs) and random hexamers. RT-qPCR was performed with 5 μL of 8-fold diluted cDNA and primers targeting ISG15 or RPS11  using thhttps://github.com/nguyelea/alphavirus_zap_sensitivity_paper/blob/main/cg_sliding_window.py, accessed on 22 December 2022). In brief, the CpG observed/expected ratio was calculated by dividing the observed frequency of CpG dinucleotides within a window by the product of the frequencies of observed C and G mononucleotides within the same window. This calculation was performed on 500-bp sliding windows with a step size of 250-bp. The analysis was performed on the genomes of the wild-type alphavirus strains described in Sliding window analysis of alphavirus CpG content was performed using a custom Python script . The UpA observed/expected ratio was calculated by dividing the observed frequency of UpA dinucleotides by the product of the frequencies of observed U and A mononucleotides within each window.UpA content analysis of windows with CpG contents correlating to ZAP sensitivity was performed using a custom Python script  and the CpG contents of ONNV and CHIKV window 1 were enriched (CGhi mutants) . In brief, these scripts minimized or maximized the number of CpG dinucleotides in each sequence without altering overall GC content or maintaining amino acid coding sequences. Gene blocks of these mutants were synthesized by Integrated DNA Technologies and inserted into a Zero Blunt TOPO vector (Invitrogen) before amplification by PCR using primers to add a 5′ T7 promoter sequence (Custom Python scripts were used to design mutants in which the CpG contents of RRV and SINV window 1 were depleted (CGlo mutants)  . While tIFN-β treatment upregulates all isoforms of ZAP in 293T cells, with the short and medium isoforms being most dramatically upregulated . Thus, wFollowing 24 h of IFN-β treatment, we infected ZAP KO and WT 293T cells with GFP-expressing alphaviruses. The percentage of infected IFN-treated ZAP KO cells is similar across all viruses, although it is lower than that of infected untreated ZAP KO cells, demonstrating the antiviral effects of IFN-β A,C. TherWe also investigated the ability of endogenous ZAP in 293T cells to inhibit virion production of wild-type alphaviruses without GFP at different times post-infection. When we again pre-treated ZAP KO and WT 293T cells with 5 U/mL IFN-β for 24 h before infection, we found a significant decrease in RRV production at 48 h post-infection (h.p.i.) in WT compared to the ZAP KO 293T cells A. At 48 Our findings indicate that the pattern of differential alphavirus sensitivity to ZAP is not only observed with overexpressed, RFP-tagged ZAP isoforms as we previously demonstrated but also with endogenous ZAP in 293T cells both with and without IFN pre-treatment. Generally, it appears that RRV and SINV replication and virion production are more sensitive to inhibition by ZAP while ONNV replication and production are relatively ZAP-resistant. Interestingly, CHIKV production but not replication is relatively sensitive to ZAP, suggesting a novel mechanism for ZAP inhibition of CHIKV that is not at the step of viral translation.Given the importance of ZAP’s ability to bind viral RNA in its inhibition of virus replication, we hypothesized that ZAP-resistant alphaviruses may be able to evade ZAP recognition. Thus, we anticipated there would be greater ZAP binding to the RNA of alphaviruses that are more sensitive to ZAP inhibition  than to the RNA of those more resistant . To evaluate this hypothesis, we performed in vitro RNA pull-down assays in which we incubated lysates of ZAP KO 293T cells with induced expression of RFP-tagged ZAPS or ZAPL with biotinylated genomic RNA (gRNA) of RRV, SINV, ONNV, or CHIKV. We then immunoprecipitated the biotinylated RNA using streptavidin beads and probed for the presence of bound RFP-ZAPS or -ZAPL. As a control for ZAP background binding, we also assayed ZAP binding to biotinylated firefly luciferase (Fluc) RNA. The resultant ZAP immunoblots were quantified by ImageJ, and the intensity of each RNA-associated ZAP band was normalized to RRV gRNA-associated ZAP to account for variation in overall signal intensity across three independent experiments. In general, in comparison to ZAPS, less ZAPL is pulled down with all RNA, most likely a consequence of comparably lower ZAPL expression in the whole cell lysate input A,C. For The lack of correlation between alphavirus ZAP sensitivity and ZAP binding to alphavirus gRNA motivated us to narrow down the alphavirus genome region that confers sensitivity or resistance to ZAP. To this end, we generated chimeric viruses in which we swapped the nsP and sP gene regions of a ZAP-sensitive virus, SINV, and a ZAP-resistant virus, ONNV A. These We next used the in vitro RNA binding assay to probe if ZAPS and ZAPL binding to RNA of the nsP region correlates with alphavirus replication sensitivity to ZAP. We found significant enrichment in the binding of ZAPS to all alphavirus nsP RNAs over the Fluc control A,B. Howeenv gene correlates to HIV-1 ZAP sensitivity [Because we did not find a correlation between alphavirus ZAP sensitivity and ZAP binding to alphavirus nsP RNA, we hypothesized that there may be more localized areas within the nsP region that are more important for ZAP recognition. We decided to look at CpG dinucleotide content to identify the most likely candidate areas, given that ZAP has been demonstrated to selectively bind CpG dinucleotides in HIV-1 RNA, and that CpG content within a specific window of the HIV-1 sitivity ,35. Sincsitivity , here weAs done for alphavirus gRNA and nsP RNA, we assayed the in vitro ability of ZAPS and ZAPL to bind the RNA of the CpG-correlated windows. We excluded the Fluc RNA background control because it is significantly longer than the windows. As we expected based on CpG content, we observed a clear and significant increase in ZAPS and ZAPL binding to window 1 of the ZAP-sensitive viruses, RRV and SINV, over that of the ZAP-resistant viruses, ONNV and CHIKV , suggestWe next wanted to confirm that ZAP binding to window 1 is CpG-dependent. To this end, we generated mutants in which we depleted CpG content for the ZAP-sensitive viruses (referred to as CGlo mutants), reducing the observed/expected CpG contents of RRV window 1 from 0.970 to 0.357 and SINV window 1 from 0.864 to 0.446 . At the In this study, we sought to understand the viral determinants of alphavirus sensitivity to the restriction factor ZAP. We first assayed alphavirus sensitivity to endogenous ZAP at baseline and IFN-stimulated expression levels. We found that RRV is most ZAP-sensitive followed by SINV for both viral replication and production, while ONNV replication and production are both ZAP-resistant and CHIKV replication but not production is ZAP-resistant . We hypoWhen studying alphavirus restriction by endogenous ZAP, we found a similar pattern of replication sensitivity to ZAP as we observed with overexpressed individual ZAP isoforms: RRV replication is most sensitive to ZAP inhibition, while SINV replication displays a more intermediate phenotype, and ONNV and CHIKV replication are relatively resistant to inhibition by ZAP . Interesenv gene correlates with HIV-1 strain sensitivity to ZAP [cis-acting signal important in alphavirus genome translation, and that ZAP binding may shield or alter the signal. For example, ZAP was recently shown to block ribosomal frameshifting in SARS-CoV-2 by interfering with folding of an RNA element [We did not find a correlation between ZAP binding to genomic  or nsP  alphaviry to ZAP . It also element . ZAP bin element ,51, makiDespite ZAP’s known function as a CpG dinucleotide sensor, ZAP binding only correlates with CpG content for one of our tested windows . Our finSome viruses actively antagonize ZAP by interfering with its function or expression. While here we focused on the role of ZAP RNA binding in sensitizing alphaviruses to ZAP, future studies should also address the possibility that ZAP-resistant alphaviruses may possess ZAP antagonism activity. The ONNV nsP/SINV sP chimeric virus not only delivers and replicates a chimeric viral RNA sequence during infection but also produces chimeric viral proteins , so it iZAP-resistant alphaviruses may also evade ZAP inhibition through more indirect means, such as altering ZAP’s ability to interact with its co-factors. ZAP inhibition of alphaviruses requires its ability to interact with the E3 ubiquitin ligase TRIM25, as well as the ligase activity of TRIM25 . Thus, iIn conclusion, our work provides insight into a potential alphavirus virulence strategy of innate immune evasion. Given the re-emergence and spread of highly pathogenic alphaviruses like CHIKV, our study is critical and timely for understanding the determinants of alphavirus virulence, which will provide molecular targets for the development of attenuated vaccines and therapies to reinforce weak points of the immune response.


However, undesirable effectsof Mg2+ such as hydrolysis of reactive RNA building blocksand degradation of RNA structures are likely to undermine its beneficialroles in ribozyme catalysis. Further, prebiotic cell-like compartmentsbounded by fatty acid membranes are destabilized in the presence ofMg2+, making ribozyme function inside prebiotically relevantprotocells a significant challenge. Therefore, we sought to identifyconditions that would enable ribozymes to retain activity at low concentrationsof Mg2+. Inspired by the ability of ribozymes to functioninside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lowerthe Mg2+ concentration required for ribozyme-catalyzedRNA assembly. Here, we show that the ribozyme-catalyzed ligation ofphosphorimidazolide RNA substrates is significantly enhanced in thepresence of the artificial crowding agent polyethylene glycol. Wealso found that molecular crowding preserves ligase activity underdenaturing conditions such as alkaline pH and the presence of urea.Additionally, we show that crowding-induced stimulation of RNA-catalyzedRNA assembly is not limited to phosphorimidazolide ligation but extendsto the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzedRNA ligation is also stimulated by the presence of prebiotically relevantsmall molecules such as ethylene glycol, ribose, and amino acids,consistent with a role for molecular crowding in primordial ribozymefunction and more generally in the emergence of RNA-based cellularlife.Catalytic RNAs or ribozymes are considered to be centralto primordialbiology. Most ribozymes require moderate to high concentrations ofdivalent cations such as Mg 2+ conditions and consequently may have playedan essential role in the origin and evolution of RNA-based primordiallife.Molecular crowding enables ribozyme-catalyzedRNA assemblyunder low Mg RNA assembly processes (ligationand polymerization) that generate complex folded RNA structures weretherefore likely to have played an important role in the propagationand evolution of the earliest living cells. Ribozymes usually requiredivalent cations like Mg2+ to access their functional foldsand perform catalysis. Mg2+ facilitates RNA folding bypartially neutralizing the negatively charged RNA backbone and oftenparticipates in catalytic interactions within the ribozyme activesite.4 Although essential to RNA function, Mg2+ can also be detrimental. Mg2+ catalyzes RNA backbonehydrolysis, thereby disrupting functional structures. It also acceleratesthe hydrolysis of intrinsically reactive RNA building blocks suchas phosphorimidazolides that would have been important for primordialRNA assembly.7 Additionally, Mg2+ is generally detrimentalto the integrity of prebiotic cell-like compartments bounded by fattyacids, which are commonly used models of primordial cell membranes.This incompatibility between ribozyme function and the stability ofprotocell membranes poses a significant challenge for efficient RNAcatalysis within fatty acid protocells.5The catalytic repertoire of RNA lies atthe foundation of the RNAworld hypothesis, which posits that early life used RNA as both thegenetic material and enzymes (ribozymes).5 Ligase andpolymerase ribozymes that use 5′-triphosphorylated oligoribonucleotidesand nucleoside triphosphates as substrates, respectively, exhibithigh Mg2+ requirements. For example, the Mg2+ concentration at which the half-maximum ligation rate was achieved,[Mg2+]1/2, of the first of its kind, class Iligase is 70–100 mM,8 and polymeraseribozymes derived from the class I ligase have an optimal [Mg2+] of ∼200 mM.10 We previously reportedribozymes that catalyze the ligation of RNA oligomers 5′ activatedwith a prebiotically plausible, 2-aminoimidazole (2AI) moiety. 2AI-activatedRNA monomers/oligomers are useful substrates for nonenzymatic RNAassembly; therefore, these “2AI-ligase” ribozymes providecontinuity between chemical and enzymatic RNA ligation.6 Most of these 2AI-ligases were inefficient atlow Mg2+ concentrations (<4 mM); however, we identifieda single ligase sequence that had a significantly lower Mg2+ requirement ([Mg2+]1/2 ≈ 0.9 mM).11 Although ribozymes with reduced Mg2+ requirements clearly exist, they are apparently relatively uncommonin the RNA sequence space. We have therefore searched for a more generalsolution that would have enabled ribozymes to operate in low-Mg2+ environments such as freshwater ponds or within protocellsbounded by prebiotic fatty acids.12 Mechanismsthat stimulate ribozyme activity at low [Mg2+] would lowerthe evolutionary threshold for the emergence of such molecules inthe RNA world.RNA assembly would have driven primordial genetics and generatedthe catalytic diversity required to sustain RNA-based primordial life;therefore, ribozymes that catalyze RNA ligation or polymerizationwere crucial to primordial biology. Such ribozymes have been identifiedthrough in vitro evolution.2+ concentrations to function in vitro, naturallyoccurring ribozymeshave evolved to function in the presence of 0.5–1 mM free Mg2+ within cellular environments.13 This lower Mg2+ requirement is thought to be a consequenceof the crowded cellular environment. In addition to cellular structureslike organelles, the intracellular milieu is crowded with moleculesthat range from biopolymers like nucleic acids and proteins to smallermolecules including amino acids, nucleotides, sugars, amines, andalcohols, which collectively occupy up to 30% of the cellular volume.14 The presence of these molecules introduces a variety of physicaland chemical forces that alter the properties of cellular RNAs.17 Volume excluded by macromolecules decreases the conformational entropyof unfolded RNA (an effect commonly referred to as “macromolecularcrowding”) and consequently promotes RNA folding and RNA function.Unfavorable interactions between the solvent-exposed RNA backboneand low-MW species in the cellular milieu also induce folding to minimizethese interactions. A decrease in dielectric constant may favor RNA–Mg2+ association due to the diminished solvation of free Mg2+, which can stimulate RNA folding and catalysis. A decreasein water activity caused by cosolutes may favor the formation of RNAfolds with reduced solvent-exposed surface area that is accompaniedby water release.Although ribozymes usually require moderate tohigh Mg17 Biophysical studies using small-angle X-ray scattering(SAXS) and single-molecule Förster resonance energy transfer(smFRET) demonstrated that molecular crowding induces RNA folding.This effect is most pronounced in the low-Mg2+ regime,where folded structures are not usually predominant.20 Enhanced folding in crowded solutions is often reflected in modestto significant increases in catalytic rates.17 Ribozymes in the RNA world may have evolved in similarly crowdedenvironments within either primitive cellular compartments or confinedmicrospaces on the Earth’s surface, which may have allowedthem to function at low concentrations of Mg2+.21Investigations into RNA structure and functionin solutions artificiallycrowded with cosolutes like polyethylene glycol (PEG) have revealedfavorable effects of crowding on RNA function.2+ and the preservation of ribozyme activity under harsh reactionconditions such as alkaline pH or urea-induced denaturation. We proposethat the stabilization of catalytic RNA folds in prebiotic crowdedenvironments could provide a general means of enabling ribozyme-catalyzedRNA assembly in diverse environments including those with low availabilityof Mg2+.Here, we demonstrate the beneficial effects of molecular crowdingon ribozyme-catalyzed RNA assembly, which includes the stimulationof ribozyme ligase activity at low millimolar concentrations of Mg2+ concentrations below 4 mM.6 For example, ligation proceeded to ∼30% in 3 h at4 mM Mg2+, but yields were reduced to 8%, 2%, and 1% at3, 2, and 1 mM Mg2+, respectively. This ribozyme exhibitsa corresponding reduction in activity in the low-Mg2+ regimewith only 5–15-fold rate enhancement over background at 2–3mM Mg2+ compared to the ∼300-fold enhancement observedat 10 mM Mg2+.6 We used polyethyleneglycol (PEG) to generate a crowded environment in vitro. PEG is chemicallyinert and available in a wide range of MWs, which allowed us to simulatethe presence of a variety of small molecules and biopolymers thatcould have been present in prebiotic milieus. We also included ethyleneglycol (EG) in our studies in addition to PEGs of various MWs . EG can be synthesized abiotically23 and is one the larger molecules detected in interstellar medium.25 We first screened various concentrations of EG, PEG 200, PEG 400,PEG 1000, and PEG 8000 to identify optimal crowding conditions forligase 1 activity in the presence of 1 mM Mg2+ and 100mM Tris-HCl, pH 8 EG. Similar stimulation in ligation was observed in 30% (w/v) PEG200, 30% (w/v) PEG 400, 19% (w/v) PEG 1000, and 19% (w/v) PEG 8000at 1 mM Mg2+ with ∼50% ligation after 3 h, whichis comparable to the 60% ligation observed in solution at 10 mM Mg2+ with no crowding agents (2+ (from 0.7to 1.3 h–1) were also comparable to the rate observedin the absence of crowders at 10 mM Mg2+ (∼1.5 h–1)  , previouCl, pH 8 . We obseg agents 1B and 1C1.5 h–1) 1D. Ligat1.5 h–1) . This di2+ dependenceof ribozyme-ligaseactivity, we measured ligation rates as a function of Mg2+ concentration in the presence of 10% EG (low-MW additive), 30% PEG200 (low-MW additive), and 19% PEG 1000 (high-MW additive). [Mg2+]1/2 was significantly lowered in the presenceof crowding agents  identified from our previous in vitro selection experiment.6 Althouartrend .2+ concentrations thatare compatible with fatty acid-based protocell membranes. As ligase1 and ligase 2 are inactive at low Mg2+ in the absenceof crowding agents, these ribozymes cannot be used to capture thedetrimental effects of denaturants or the protective effects of crowdingin the presence of denaturants under these low-Mg2+ conditions.Therefore, we used a previously reported 2AI-ligase  that is functional under these conditions for the following experiments.11 First, we tested RNA ligation by ligase 3 inthe presence of urea, which is an effective denaturant of RNA andalso an important precursor molecule in the prebiotic syntheses ofribonucleotides and amino acids.28 As expected,ligation rates in the background of 1 mM Mg2+ decreasedby ∼6-fold in the presence of 1 M urea, and ligation was furtherreduced in the presence of 2.5 M urea . We tested ligation at pH 10 with different crowders. Low-MWcrowders like EG and PEG 200 showed no benefit; however, the lossof ligase activity at pH 10 was less pronounced in the presence ofhigh-MW crowders PEG 1000 and PEG 8000 with only a 2.6-fold and 2.3-foldreduction in kobs, respectively, relativeto their values at pH 8  in its thermal stability inthe presence of high-MW crowder, PEG 1000 , which enabledRNA-catalyzed RNA polymerization within Mg2+-resistant1-palmitoyl-2-oleoylphosphatidylcholine (POPC) vesicles.33 Similarly, Takahashi et al. demonstrated theaddition of up to 5 nucleotides by the tC9Y polymerase ribozyme inthe presence of 10 mM Mg2+ upon addition of 20% PEG 200.34 We tested the ability of the 38–6 polymeraseribozyme10 to extend a 10 nt RNA primeron a 21 nt RNA template in the presence of 5 mM Mg2+ insolutions containing low- or high-MW PEGs. Negligible extension beyond+4 was observed in the absence of crowding agents; however, smallamounts of full-length products (+11) were detected in the presenceof PEG 200 or PEG 1000 after 24 h. The prominent +1 extension productincreased from 24% without crowding agents to 33% and 40% in the presenceof PEG 200 and PEG 1000, respectively. While only 26% of the primerwas extended in the absence of crowding agents, 37% and 43% of theprimer was extended in the presence of PEG 200 and PEG 1000, respectively and 3.8% (w/v) increased ligation yieldfrom ∼1% to ∼11% and ∼26% after 3 h in the presenceof 1 mM Mg2+ with kobs valuesof ∼0.4 and ∼0.5 h–1, respectively(2+. All of the above amino acids were found to stimulateligation regardless of their concentrations with yields of 25–35%after 3 h (2+ without any additive (2+ even in the presence ofhigh concentrations of Na+ (300 mM),6 low concentrations of monovalent counterions in reactionscontaining 2.5–20 mM amino acids are unlikely to cause thispronounced rate stimulation, and the amino acids must be playing adirect role.While our observationson the effects of molecular crowding agents on ribozyme activity arepromising, the above results were obtained with prebiotically irrelevantsynthetic PEG molecules with the exception of EG. Therefore, we exploredthe potential of prebiotically relevant small molecules for stimulatingribozyme-ligase activity. Considering the importance of simple sugarsin a pre-RNA/RNA world and the stabilizing effect of ribose on fattyacid membranes, we decided to explore the effect of ribose on ligase1 ribozyme activity.ectively4. We alsfter 3 h . As lowefter 3 h 4. The pradditive 4B. As li2+ byribose or amino acids is not clear. Aliphatic alcohols such as methanol,ethanol, propanol, 2-methoxyethanol, and propane-1,3-diol stimulatehammerhead catalysis at 1 mM Mg2+ by decreasing the dielectricconstant of the solution, thereby enhancing interactions between theribozyme and Mg2+.31 Ribose-mediatedenhancement of ribozyme-catalyzed RNA ligation could be a result ofsimilar solution-level effects. The beneficial effect of amino acidstoward ribozyme activity has been previously observed for RNA self-cleavage.It was proposed that the increase in ribozyme activity resulted fromstructural compaction of the RNA, which allowed greater sampling ofits catalytic fold.40 This assertion wassupported by thermal denaturation and SAXS studies. Amino acids maystimulate RNA folding by altering solvent properties like dielectricconstant or water activity.17 Additionally,as amino acids can weakly chelate Mg2+, the chelated aminoacids may form a layer on the RNA surface, increasing the local concentrationof Mg2+, which may lead to improved folding and catalysis.41 Regardless, this ability of prebiotic small molecules to facilitateribozyme-catalyzed RNA assembly presents a “systems”-levelsolution for lowering the Mg2+ requirement for this centralprocess in primordial biochemistry.The mechanism for ribozyme activation at low Mg2+ in bothnonenzymatic- and ribozyme-catalyzedRNA replication coupled with its ability to accelerate RNA degradationand destabilize fatty acid protocells presents a puzzle for the emergenceof RNA-based cellular life. Therefore, exploring scenarios that mitigatethis “Mg2+ problem” is of critical importance.The low Mg2+ requirement for natural ribozymes that functionwithin crowded cellular environments inspired us to study molecularcrowding as a general solution to the Mg2+ problem in thecontext of ribozyme-catalyzed RNA assembly. Our results show a dramaticstimulation of ribozyme-catalyzed assembly of 2AI-activated RNA oligomersand nucleoside triphosphates at low millimolar Mg2+ byprebiotically relevant amino acids, ribose, ethylene glycol, and polyethyleneglycols of various MWs (200–8000). The beneficial effects ofamino acids, ribose, and ethylene glycol are especially notable sincethese molecules can be synthesized abiotically and therefore werelikely to have been present in early Earth environments. The 3–10-foldlower Mg2+ requirement for ligase ribozymes in the presenceof such solutes likely stems from enhanced RNA folding in “crowded”solutions as the corresponding nonenzymatic ligation reaction wasnot affected by crowding. Stimulation of catalytic activity in thepresence of molecular crowding has been reported for other ribozymes.17 Since the crowding-induced enhancement of RNAassembly was largely independent of crowder size, ribozyme foldingcould be favored by an interplay of both enthalpic forces arisingfrom interactions between the RNA surface and the crowder and entropicforces arising from volume exclusion.17 In most cases where both low-MW and high-MW crowders affect macromolecularfunction, it is extremely difficult to delineate the individual contributionsof volume exclusion and the various enthalpic forces that are alwaysat play.17 Further studies may help isolatethe effects of these distinct thermodynamic forces.The crucial role of Mg2+ regime, crowding offers modest to significantprotection to ligase ribozymes under various denaturing conditionsrelevant to early Earth environments. The ability to function underconditions that favor the disruption of RNA secondary structure couldhave been important for rapid RNA-catalyzed RNA replication, whichrequires the separation of newly synthesized RNA strands from theirRNA templates while preserving catalytic RNA structures.We demonstratedthat in addition to enabling RNA ligation in thelow-Mg2+ concentrations,presents a path to reconcile ribozyme function with the stabilityof protocell membranes made of fatty acids. Protocells crowded withprebiotic small molecules like sugars, alcohols, and amines and polymericspecies such as short oligonucleotides or polypeptides could potentiallysupport a wide range of ribozyme activities under the low Mg2+ concentrations required for maintaining membrane integrity. Thisis particularly interesting in the context of our earlier observationthat prebiotically relevant small molecules including ribose alsoreduce RNA leakage from fatty acid vesicles.11 The combined effect of enhancing ribozyme function under low Mg2+ conditions and stabilizing protocell membranes against Mg2+ suggests a potential role for these prebiotic moleculesthat is separate from their roles as components of the building blocksof life. By providing a general mechanism to activate RNA catalysisat low Mg2+ concentration, molecular crowding expands therange of environments in which ribozymes can function to less saltyenvironments such as freshwater bodies12 and increases the likelihood of the emergence of active ribozymesfrom the RNA sequence space.21 Suboptimalsequences that would otherwise not be selected in low-Mg2+ environments could emerge in crowded milieus, potentially creatingneutral mutational pathways that would facilitate ribozyme evolutionand therefore increase the catalytic diversity of the RNA world.EfficientRNA assembly, at low MgO-methyl modifications to reducetranscriptional heterogeneity at the 3′ end of the RNA42 , 30 U/mLRNase inhibitor murine (NEB), 2.5 U/mL thermostable inorganic pyrophosphatase(TIPPase) (NEB), a 4 mM concentration of each NTP, 30 pmol/mL DNAtemplate, and 1U/μL T7 RNA Polymerase (NEB) and were incubatedfor 3 h at 37 °C. DNA template was digested by DNase I (NEB)treatment, and RNA was extracted with phenol–chloroform–isoamylalcohol (PCI), ethanol precipitated, and purified by denaturing PAGE.Ligation templates, FAM-labeled primers, and ssDNA were purchasedfrom Integrated DNA Technologies.Ribozymeswere prepared by in vitro transcription of PCR-generated dsDNA templatescontaining 2′-he RNA42 . Transcr6The 5′-monophosphorylatedoligonucleotide corresponding to the substrate sequence was activatedby incubating it with 0.2 M 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide and 0.6 M 2-aminoimidazole for 2 h at room temperature. The reaction was washed with water inAmicon Ultra spin columns (3 kDa cutoff) 4–5 times (200 μLof water per wash) and purified by reverse-phase analytical HPLC usinga gradient from 98% to 75% 20 mM TEAB  versus acetonitrile over 40 min.2, and crowding agents. All reactions were performedat room temperature unless mentioned otherwise. Aliquots were quenchedwith 5 volumes of quench buffer  and analyzed by denaturing PAGE. Gels werestained using SYBR Gold,43 imaged on anAmersham Typhoon RGB instrument , and analyzed in ImageQuantIQTL 8.1. Intensities corresponding to the ligated product were normalizedto account for the difference in size between the 95 nt precursorband and the 111 nt product band. Kinetic data were nonlinearly fittedto the modified first-order rate equation, y = A(1 – ekx–), where A represents the fraction of active complex, k is the first-order rate constant, x is time, and y is the fraction of ligated product in GraphPad Prism 9.For nonenzymatic ligation, a 5′-FAM-labeled RNA primer correspondingto the last 8 nt of the ribozyme sequence was used instead of theribozyme, and the gel was directly imaged.Ligation reactions contained 1 μMribozyme, 1.2 μM RNA template, and 2 μM 2-AI-activatedRNA substrate in 100 mM Tris-HCl pH 8.0, the indicated concentrationsof MgCl2+].All ligationassays under denaturing conditions were performed with the ligase3 ribozyme, which retains activity under low [Mg2. The substrate was addedimmediately after the addition of MgCl2 to initiate ligation.Ribozyme and template were heatedat 95 °C for 2 min in the absence of any buffer and cooled toroom temperature. CAPS buffer (pH 10 or 11) was added to a final concentrationof 100 mM in the absence or presence of crowding agents (19% PEG 1000or 19% PEG 8000) and 1 mM MgClReactions with or withoutcrowding agents  were incubated at 55 °C after initiating ligationby adding the substrate.2 to minimize degradation at high temperaturesrequired for refolding. The substrate was added immediately afterthe addition of MgCl2 to initiate ligation.A 10 M concentrationof urea was added to final concentrations of 1 or 2.5 M after refoldingin the presence of crowding agents (30% PEG 200 or 19% PEG 1000) and1 mM MgCl2 was addedto final concentrations of 5 and 200 mM, followed by a 0.5 mM concentrationof each NTP. Reactions were incubated at 17 °C for 24 h, and1 μL aliquots were quenched with 7 μL of quench buffer. Reactions were analyzedby denaturing PAGE. Gels were imaged on an Amersham Typhoon RGB instrument and analyzed in ImageQuant IQTL 8.1.A FAM-labeledRNA primer (80 nM), RNA template (100 nM), and polymerase ribozyme(100 nM) were heated in the absence and presence of crowding agentsand 25 mM Tris·HCl pH 8 at 80 °C for 30 s and cooled to17 °C at a gradient of 0.1 °C/s. MgCl44 Briefly, 0.5 μM ribozyme was incubated at 95 °Cfor 2 min in 10 mM sodium cacodylate buffer (pH 7) and refolded inthe absence or presence of crowding agents (10% ethylene glycol or19% PEG 1000) in the presence of 1 mM MgCl2 by heatingthe solution to 55 °C for 10 min followed by cooling to roomtemperature for 10 min. A Cary UV–vis multicell Peltier spectrophotometerwas used for melting experiments. Absorbance was recorded at 260 nmevery minute between 20 and 90 °C. Data was normalized with respectto “buffer only” sample in each case, which containedall components in the experimental sample except RNA. Derivative plotsof normalized data (dA/dT) vs T) and melting temperatures (Tm) were obtained by the instrument’s default software.UV melting experimentswere performed to determine the thermal stability of the ligase 1ribozyme in the absence of presence of low- and high-MW crowding agentsaccording to the protocol used by Struslon et al.


Klebsiella pneumoniae strains with different virulence, a total of 13 RNA editing events are identified. The RNA editing event of badR is focused, which shows a significant difference in editing levels in the two K. pneumoniae strains and is predicted to be a transcription factor. A hard‐coded Cys is mutated on DNA to simulate the effect of complete editing of badR. Transcriptome analysis identifies the cellular quorum sensing (QS) pathway as the most dramatic change, demonstrating the dynamic regulation of RNA editing on badR related to coordinated collective behavior. Indeed, a significant difference in autoinducer 2 activity and cell growth is detected when the cells reach the stationary phase. Additionally, the mutant strain shows significantly lower virulence than the WT strain in the Galleria mellonella infection model. Furthermore, RNA editing regulation of badR is highly conserved across K. pneumoniae strains. Overall, this work provides new insights into posttranscriptional regulation in bacteria.Millions of adenosine (A) to inosine (I) RNA editing events are reported and well‐studied in eukaryotes; however, many features and functions remain unclear in prokaryotes. By combining PacBio Sequel, Illumina whole‐genome sequencing, and RNA Sequencing data of two  Klebsiella pneumonia, it is reported that adenosine to inosine RNA editing on badR gene regulates quorum sensing and has a certain regulatory effect on the growth and virulence of bacteria. The conclusion suggests that in addition to the mutation of the genome, it is necessary to consider posttranscriptional regulation in future research on bacteria.Through the analysis of RNA editing regulation in  The most prevalent type of RNA editing in metazoans is the conversion of adenosine to inosine (A‐to‐I) catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes. Recent studies have provided functional evidence for a series of editing events such as events in coding regions recoding amino acid messages, events located near exon‐intron boundaries modulating splicing strength, and some intergenic editing events implicated in the biogenesis and targeting of microRNAs and piwi‐interacting RNAs. An increasing amount of work has also led to the discovery and identification of RNA editing sites in fungi. However, RNA editing regulation in bacteria has rarely been reported. Recently, Bar‐Yaacov et al. provided evidence of RNA editing in Escherichia coli on hokB, which encodes a toxin that confers antibiotic tolerance, that increases as a function of cell density and enhances the toxicity of the protein. Furthermore, the authors found that tRNA‐specific adenosine deaminase (TadA) was also identified as the catalyzing enzyme of A‐to‐I editing on mRNA. In addition, Nie et al. identified A‐to‐I editing events of fliC, and they discovered that the S128P editing event is induced by H2O2 and demonstrated its importance in bacterial pathogenicity and adaptation to oxidative stress. These studies reported mRNA editing events in bacteria and proved the important function of RNA editing regulation. It is still unclear whether bacterial growth, virulence, or stress responses are regulated by RNA editing in other bacterial strains such as Klebsiella pneumoniae (K. pneumoniae).RNA editing is a posttranscriptional process that introduces differences between RNA and its corresponding DNA template.1 WithK. pneumoniae, an important pathogen, can cause a wide range of infections including pneumonia, urinary tract infection, bloodstream infection, and liver abscess. Moreover, some K. pneumoniae strains have various virulence factors, such as rmpA, rmpA2, iucABCD, and iroBCDN, which confer hypervirulence. This hypervirulent K. pneumoniae leads to more serious infections and higher mortality rates. However, there were also some strains that had a highly virulent phenotype but no hypervirulence genes and the reason for this remains unclear. To date, most studies on the virulence of K. pneumoniae have focused on the DNA level, and there are few reports on the transcription level. In this study, we developed a strict pipeline for RNA editing identification in bacteria by using PacBio genome sequencing data, Illumina whole genome sequencing (WGS) data, and strand‐specific RNA Sequencing (RNA‐Seq) data. By applying this pipeline, we profiled and identified RNA editing events in one hypervirulent isolate and one classic strain of K. pneumoniae. Specific editing events were identified and quantified between the two strains, and their regulatory effects on bacterial growth and virulence were demonstrated.22.1K. pneumoniae, we developed a new strict pipeline that was a modified and improved version of our previous pipeline. Taking advantage of the long reads of the PacBio third‐generation sequencing platform, we obtained the complete genome sequence of K. pneumoniae R16 (a hypervirulent strain) (more details and the refined genome structure have been published in our previous work). Then, WGS was performed by Illumina on the DNA of K. pneumoniae strain R16, and RNA‐Seq in strand‐specific mode was performed on the same strain. WGS and RNA‐Seq data, which have higher sequencing abundance and lower sequencing error rates, were mapped to the assembled reference genome, and stringent parameters were applied to identify RNA editing events that can manifest themselves as base differences between the DNA and RNA sequences. Making full use of the strand‐specific mode of RNA‐Seq, we separated reads into different transcriptional directions for more accurate identification of RNA editing events. In total, we found 13 RNA editing sites in K. pneumoniae R16. 12 of them were A‐to‐G, and 7 were within known ORFs (Table 2) All editing sites within ORFs were predicted to recode a tyrosine (Tyr) encoded by the TAC codon into a cysteine (Cys) encoded by the TGC codon, presenting stronger consistency than other bacterial species (Table  3) All A‐to‐G editing events were embedded within a five‐base‐long motif TACGA, with the edited A at the second position  and compared it with the hypervirulent strain (R16). By applying the RNA editing identification pipeline to the RNA sequencing data of QD110, 11 A‐to‐G RNA editing events were found, and 10 of them were also detected in strain R16  family, which is critical for bacterial cells to respond to chemical signals. HokA, belonging to the Hok family of host‐killing toxins, is the toxic component of a type I toxin‐antitoxin (TA) system. Interestingly, the function of RNA editing events on hokB, which is a homologous gene of hokA, was previously confirmed in E. coli. All the above results demonstrated that RNA editing regulation occurs on some key genes in K. pneumoniae, suggesting that it may play an important role. The differential RNA editing levels between the two strains also suggested that these editing events might have an impact on the differences in cell growth or virulence between the hypervirulent and classic K. pneumoniae strains.To further explore whether RNA editing has a regulatory effect on the bacterial growth or virulence of strain QD10 and co regulon.24 Bad2.3K. pneumoniae strains  substitution, induced by RNA editing, on the biological function of the BadR protein, and the result showed “Deleterious” with a PROVEAN score of −5.555. Furthermore, protein modeling was performed for Y99 and C99 separately to detect the influence of RNA editing on protein structure . The variation in position 99 may help to accommodate relative shifts of the two DNA‐binding lobes of the dimer that might occur upon DNA‐binding; furthermore, this variation might have an influence on target preference.Among all the genes that underwent RNA editing events with significantly different editing levels between hypervirulent and classic s Figure , we focus Figure , which w  to mimic complete editing at this site . Moreover, the editing level of badR decreased almost tenfold as cells grew from the logarithmic phase to the stationary phase , indicating that the RNA editing of badR was detrimental to sustaining cells through the infertile condition. The ability of bacteria to withstand adversity is also an important factor in the survival of the host. In the Galleria mellonella infection model, the mutant strain badR‐99Cys showed lower virulence than the WT strain .As BadR is predicted to be a transcription regulator and Y99C might influence target selection, we further asked how the change can affect the transcriptome profile of n Figure 3A. Inter, Figure 29 werbadR gene in K. pneumoniae with stimulated by high temperatures, high salinity, and oxidation, which are the most common situations bacteria deal with. The results showed that there was no significant change in RNA editing level at this site , which may suggest that the RNA editing event at this site might be not a transcriptional regulation that is widely activated by stress.Considering that bacteria are easily stimulated by external environmental factors, resulting in a stress response process, we examined the variation of RNA editing level of badR, the A‐to‐G editing site of position 5 325 069 on virF also had a significantly different RNA editing level between the two strains  and virB, which are activators of the virulence regulon. Thus, we also mutated the genomic virF using the CRISPR/Cas9 system in the QD110 strain, with the codon TGC encoding a Cys hard‐coded into the DNA (virF‐31Cys). To examine the influence of this editing event on K. pneumoniae, we compared the growth of the WT strain and mutant strain, and no significant difference was detected . In the G. mellonella infection model, the mutant strain virF‐31Cys also showed similar virulence to that of the WT strain .Beyond s Figure . VirF is regulon.24 Thu2.4badR, we performed multiple sequence alignment of BadR with a representative nonredundant set of orthologs from 66 bacterial species (764 strains), and we found the interplay between the TAT and TAC codons at position 99  on K. pneumoniae, we simulated the effect of 100% editing on cell transcriptional maps by mutating the genome of the classic strain QD110. Based on RNA‐Seq analysis, a series of cellular quorum sensing genes  were downregulated in the mutant strain. These Lsr family genes are involved in AI‐2 importation and phosphorylation, which are associated with the intracellular signal transduction of stress responses such as biofilm formation and drug resistance; this result suggests that the RNA editing event in badR may affect intracellular signal transduction and cellular density changes. The wild‐type strain QD110 reached a higher limit in the cellular population than the mutant strain, accompanied by a decrease in the RNA editing level on badR , which had previously been used to create the badR 100% edited mutation system . We carried out ten mutation experiments using seven different complementary sgRNA sequences including the sgRNA that was used to successfully build the badR‐Y99C strain  for identification confirmation using MALDI‐TOF MS . G. mellonella survival assays and human neutrophil assays were used for the virulence assessment of R16 and QD110.The nd QD110.18−1) at 37 °C. RNA and DNA were isolated from cultures at the middle of the logarithmic phase (OD600 in a 1‐cm cuvette of ≈0.6–0.8) for transcriptome sequencing and genome sequencing. Four replicates were performed for the identification of RNA editing.RNA and DNA of the R16 and QD110 strains were purified using the GeneJET RNA Purification kit (Thermo Fisher Scientific) and Wizard Genomic DNA Purification kit (Promega), respectively, according to the manufacturer's protocol. Cultures were grown on LB supplemented with ampicillin  following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample amplification. Finally, PCR products were purified (AMPure XP system), and libraries were analyzed for size distribution by an Agilent2100 Bioanalyzer and quantified using real‐time PCR. For RNA‐seq, a total amount of 2 µg RNA was first treated with a Ribo‐zero rRNA removal kit . Subsequently, sequencing libraries were generated using rRNA‐depleted RNA with a VAHTS Strand‐specific mRNA‐seq Library Prep Kit with dUTP‐mode for Illumina  following the manufacturer's recommendations. The DNA and RNA libraries constructed above were sequenced by the Illumina Nova Seq platform, and 150 bp paired‐end reads were generated with an insert size of ≈350 bp. PacBio Sequel‐seq was completed in the previous work.18 and circularization was achieved by manual comparison and removal of regions of overlap. The final genome was further confirmed by remapping Illumina reads using BWA (0.5.9) and Pilon (v.1.13). This assembled genome was published in the previous work. Then, RNA‐seq reads were aligned to this reference genome by BWA with default parameters, from which only uniquely mapped reads were retained. In addition, potential duplications were removed using Picard MarkDuplicates (2.18.7). Uniquely mapped RNA‐seq reads were then divided into two groups according to their Flag information in SAM format: reads transcribed from the plus strand and those from the minus strand. Single‐nucleotide variation (SNV) calling was separately performed for the two groups of reads using Samtools mpileup (v0.1.16). Reads harboring SNVs within 5 bp of both ends were discarded in SNV calling due to read‐end‐biased sequencing errors. A candidate RNA‐editing site was required to be homozygous, with 95% of the covered reads supporting the major allele type (DNA filter). RNA SNVs with a homozygous genotype were included in an initial list of RNA‐editing sites and were further subjected to a stringent filtering protocol: i) At least five RNA‐Seq reads with ≥2 nucleotides sequenced with high PHRED base quality (≥25) were required to support the variant form. ii) SNVs displaying more than one mismatch type were discarded. iii) For candidate RNA‐editing sites, BLAT alignment filtering was performed to eliminate SNVs potentially caused by misalignment to paralogs or pseudogenes; iv) A Strand Bias Filter, RNA‐editing sites exhibiting strand bias in read distribution  or supported by <2 reads on either of the two strands were excluded. All editing sites were finally validated by Sanger sequencing with the primers listed in Table De novo assembly of the genome was performed using HGAP3 within SMRT Link v5.0.0 with PacBio sequencing data. Gap closing was completed by PBJelly,2 and http://rna.tbi.univie.ac.at/cgi‐bin/RNAWebSuite/RNAfold.cgi) to predict the RNA secondary structure with minimum free energy. To further demonstrate the local sequence motif for TadA recognition, the surrounding sequences of editing sites were put into MEME (https://meme‐suite.org/meme/tools/meme) to identify the conserved motif.To examine the RNA secondary structure, RNA sequences of 35 bases upstream and downstream from the edited site were extracted. Sequences were then entered into the RNAfold website . The model 5aiq had the highest sequence identity (52.59%) to BadR among the MarR family members whose apo crystal structure has been released on RCSB PDB. Also, this model had a relatively high GMQE score . Sequence alignment was performed for BadR and 5aiq  and cloned into pSGKP‐spe between BsaI restriction enzyme cutting site using the Gibson Assembly kit  pBadRSGKP‐spe. Repair template sequences were amplified and overlapped using PrimeSTAR Max DNA Polymerase (TAKARA). The primers are listed in Table pCas9KP‐apr were transformed into QD110 competent cells using a Gene Pulser Xcell system (Bio‐Rad), and colonies were selected with 50 µg mL−1 apramycin. Then, 200 ng pBadRSGKP‐spe and 2 µg concentrated repair template sequence were transformed into QD110‐pCas9KP competent cells. Colonies were selected with 100 µg mL−1 spectinomycin and identified by Sanger sequencing  | > 1 and FDR < 0. 05. Enrichment analysis of KEGG was performed on DEGs using the clusterprofiler package (version: 3.18.1) treating expressed genes (RPKM >1) in any samples in WT and mutant strains under the K. pneumoniae  as background annotations. The significance of KEGG signaling pathways was set at a p‐value <0.05.To identify DEGs between WT and mutant strains, strand‐specific RNA‐seq was performed with three replicates for each strain. Differential expression analysis was completed using the DESeq2 package (version: 1.24.0) in Vibrio harveyi strain BB170 luminescence was introduced to calculate AI‐2 activity as described in previous research.K. pneumoniae WT and mutant strain badR‐99Cys were incubated in AB medium to induce AI‐2 production for 0, 2, 3, and 4 h at 37 °C, respectively. K. pneumoniae cells were removed by passing through 0.2 µm syringe filters. BB170 cells diluted in AB medium were co‐cultured with K. pneumoniae cell‐free cultures for a mixture ratio of 9/1 (v/v) at 30 °C for 6 h to calculate AI‐2 activity. The light production at a wavelength of 490 nm of BB170 was analyzed at the 3rd hour when the lowest bioluminescence was produced by the control. The induction of bioluminescence was calculated as the light output of the sample divided by the light output of the control.badR‐99Cys were cultured at rest in 96‐well plates at an OD600 nm of 0.02 . The experiments were repeated three times. The optical density was measured by the microplate reader  every hour from 0 to 28 h. The cellular growth curve was drawn using GraphPad Prism software, and the Mann–Whitney test was used for comparisons between the wild‐type and mutant strains.The wild‐type strain and mutant strain badR‐99Cys were collected from cultures by centrifugation at 3000 rpm for 5 min. Then, the cells were washed twice and suspended at 108 cells/mL in saline. Each G. mellonella was injected with a 10 µL suspension, with an infection dose of 106 cells (n = 20). The death timepoint of each G. mellonella was observed at 8, 12, 15, 18, 24, 36, and 48 h after infection and there are 15 larvae for each group. Experiments were repeated three times. The survival curve of G. mellonella infection was drawn using GraphPad Prism software. The log‐rank (Mantel–Cox) test was used to compare survival rates between the wild‐type and mutant strains.Cells of the wild‐type strain and mutant strain badR was examined with stimulated by high temperatures, high salinity, and oxidation. QD110 strain was cultured in LB medium overnight and then diluted 100 times with LB broth containing nothing, 0.5 m KCl , or 5 mm H2O2 (oxidation). All the strains were incubated at 37 °C, except high‐temperature stimulation at 48 °C, by a shaker culture system until reach the logarithmic growth phase (OD600 = 0.6–0.8). Three biological replicates were conducted for each condition and then RNA sequencing was applied to all samples.Variation of RNA editing level of https://www.uniprot.org/blast/) was used with the amino acid sequence of BadR in strain R16 as a query and “Microbial proteomes” as the target database. The alignment results were filtered with an e‐value of 1e‐5, identity of 80%, and coverage of 80%. A nonredundant set of orthologs with one BadR sequence per species was constructed. The corresponding nucleic acid sequences were downloaded from the European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/browser/home). Multiple sequence alignments were conducted with ClustalW.To examine the amino acid composition at position 99 of badR, the BLAST tool on UniProt ( The evolutionary tree was inferred by using the maximum likelihood method and the Tamura‐Nei model. The tree with the highest log likelihood (−4935.73) was shown. Initial tree(s) for the heuristic search were obtained automatically by applying the neighbor‐joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura‐Nei model and then selecting the topology with a superior log likelihood value. The tree was drawn to scale, with branch lengths measured in the number of substitutions per site (next to the branches). The proportion of sites where at least 1 unambiguous base was present in at least 1 sequence for each descendent clade was shown next to each internal node in the tree. This analysis involved 15 nucleotide sequences. There were a total of 1558 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.The 16S ribosomal RNAs were used to build a genus phylogenetic tree to visualize the amino acid composition in BadR's position 99 in an evolutionary context. The 16S ribosomal RNA from one representative from each genus (Table 35 Thet‐test were used to identify significant differences where appropriate.Statistical analyses were performed using the Statistical Package for Social Sciences. Data were presented as mean ± SD. Experiments were performed with a minimum of three replications. Mantel–Cox test, Mann–Whitney U test, Fisher's exact test, and Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


KRAS proto‐oncogene, GTPase (KRAS)‐mutant metastatic colorectal cancer (mCRC) because the underlying mechanism remains obscure. Based on bioinformatic analysis, this study aims to elucidate a potential gene target for which an approved drug is available, and to reveal the function as well as the underlying mechanism of the candidate gene. Here, we identified that ryanodine receptor 2 (RyR2) expression was upregulated in KRAS‐mutant mCRC, and that this promoted cancer cell metastasis. S107, an approved drug to inhibit calcium release from RyR2 in the clinic, inhibited cancer cell metastasis both in vitro and in vivo. High expression of RyR2 predicts poor survival in our patient cohort. CRC patients with serosa invasion and vascular tumor thrombus are characterized by high RyR2 expression. Analysis of expression profiles upon RyR2 knockdown and inhibition, revealed a set of metastasis‐related molecules, and identified BTB domain and CNC homolog 1 (BACH1) as the main transcription factor regulated by RyR2. RyR2 regulates cellular reactive oxygen species (ROS) levels, which activates nuclear factor erythroid 2‐related factor 2  and HMOX1 expression, and thus BACH1 accumulation. Collectively, this study provides evidence that the RyR2/ROS/BACH1 axis may be a potential intervention target for CRC metastasis.There is no targeted therapy for  RyR2 was identified as a regulator of colorectal cancer metastasis. RyR2 expression raised intracellular Ca2+ concentration and promoted cellular ROS levels, which in turn activated Nrf2 activity followed by BACH1 accumulation. The BACH1 accumulation led to the start‐up of the metastasis progression of cancer cells.Through bioinformatics analysis combined with molecular strategies,  RyR2 isdow Fig. . Transwe KD Fig. . While wD‐1 Fig. .RyR2 (R2474S) and is orally available to prevent cardiac arrhythmias and raise the seizure threshold [2+]i enhancement triggered by 5 μm ATP  revealed enrichment in cancer metastasis (68 genes), brain‐ or heart‐biased expression (35 genes), transcription regulation (23 genes), ROS related function (21 genes), cytoskeleton or extracellular matrix (ECM) (18 genes), ion channel or transporters (13 genes) . To this end, we identified 37 genes downregulated by RyR2 KD and S107 treatment that showed a higher expression in metastatic CRC tissues, and 71 genes upregulated by RyR2 KD or S107 treatment that showed a lower expression in metastatic CRC tissues  Fig. . To furtues Fig. .CKMT1A, CKMT2 and MAOA as well as metastasis‐associated KISS1, KISS1R, MLF2 and EGLN3 were consistent with our RNA‐seq results  TF enriched by DEG following RyR2 KD; (ii) TF enriched by DEG following S107 treatment; (iii) TF enriched by DEG derived from TCGA data, by comparing low and high RyR2 expression samples  was not changed following RyR2 KD in SW480 cells  or pPYK2(Tyr402)   Fig. . In addiII) Fig. . We idenCH1 Fig. . Thus, wBACH1 revealed its regulation by antioxidants and Nrf2 [RyR2 KD or inhibition. RyR2 KD did indeed significantly decrease cellular ROS level in both SW480 and HCT116 cells  by Nrf2‐mediated BACH1 degradation. We also found downregulation of HMOX1 expression after RyR2 KD or inhibition  in humans [RyR2 through enhancement of the binding affinity between RyR2 and calstabin2 [RyR2. The ability of S107 to inhibit cancer cell metastasis and ROS production was consistent with the results of RyR2 KD. Furthermore, mice treated with S107 at 30 mg·kg−1 via intraperitoneal injection for 2 weeks presented with a healthy appearance. As S107 was commercially available, we wondered whether this drug could be repurposed to intervene with CRC metastasis.Multiple congenital n humans . S107 walstabin2 . We founOrai1 and STIM1, which mediate extracellular calcium influx into ER via store‐operated calcium entry (SOCE), were critical for breast cancer cell metastasis [RyR1‐3 and IP3R1–3) [RyR2 expression predicts a poor prognosis in CRC  . AlthougCRC Fig. . High Ry from ER , and suble cells , glucosele cells  and ROS le cells , Interfele cells . The dision Fig. .BACH1 is well established as a promoter of cancer metastasis, especially in non‐small cell lung cancers with NFE2L2 mutation [BACH1 promotes CRC cell metastasis [BACH1 is a member of the Cap ‘n’ Collar and basic region leucine zipper family of TF and it acts as a transcription repressor of target genes [RyR2 KD decreases the cellular BACH1 level, and thus dysregulation of BACH1‐regulated metabolism and metastasis genes. BACH1 does not interact with canonical calcium‐related molecules, including calmodulin, CaMKII and CaMKIV, indicating that BACH1 is not a first‐line effector of cellular calcium change. The crosstalk between calcium and BACH1 sheds further light on the regulation of cancer metastasis by calcium.mutation , 15. Evitastasis , 26, 27.et genes . We founNrf2 are frequently reported [Intimate relationships between cancer metastasis and mitochondrial activity, ROS and reported , 32, 33.reported . However5RyR2 is a potential therapeutic target for intervention of CRC metastasis.Through bioinformatics analysis and experimental assays, our results provide evidence that The authors declare no conflict of interest.TC, XW conceived the project. TC, XiZ performed most of the experiment. XD conducted some molecular experiments. JF analyzed some data. XuZ, DX, XW supervised this study. TC wrote the report.https://publons.com/publon/10.1002/1878‐0261.13350.The peer review history for this article is available at Fig. S1. RyR2 inhibition did not affect cellular growth.Click here for additional data file.Fig. S2. RyR2 inhibition decreased CRC liver metastasis in vivo.Click here for additional data file.Fig. S3. RyR2 did not interact with calcium‐related molecules.Click here for additional data file.Fig. S4. RyR2 inhibition increased cellular heme level.Click here for additional data file.Fig. S5. Overall survival and disease‐free survival analysis of ITPR and RYR.Click here for additional data file.Table S1. Relationship between expression of RYR2 in CRC and clinicopathologic features.Click here for additional data file.Table S2. Materials and primers used in this study.Click here for additional data file.