The researchers showed their increased interest in linking human resource management (HRM) and corporate social responsibility (CSR) practices in recent studies. HRM is a critical factor in how CSR should be enacted, developed, and understood at a broader level to achieve organizational benefits. Hence, it is evident that current studies are asking for more studies on HRM–CSR nexus, and we argue that such a relationship is necessary and relevant. Probing more in this research stream, the current research investigates the impact of HRM and CSR on sustainable performance. More specifically, this study explores how 3-star, 4-star, and 5-star hotels achieve sustainable performance through HRM and CSR in the hospitality industry of the UK, Italy, and Pakistan. A stratified random sampling technique was used to select the hotels by collecting hotel details from Chambers of the Commerce United Kingdom, Italian Government Statistics, and Pakistan Hotel Associations for the UK, Italy, and Pakistan. Through a highly structured survey questionnaire, the data were collected from 438 UK, 520 Italian, and 354 Pakistani hotels. The results confirm the HRM–CSR–sustainable performance nexus in the hospitality industries of three countries. They show that HRM impact on CSR and sustainable performance is relatively stronger in five-star hotels followed by three-star and then four-star hotels. These results supported the resource-based view theory by providing strong evidence that HRM and CSR are essential resources for achieving sustainable performance and competitive advantage. In the twenty-first century, the global economy is driven by three major industries: tourism, telecommunication, and technology  agenda states itself as “an Agenda of the people, by the people, and for the people, and this will ensure its success” . Therefore, we recognize the dual role of employees as originators and recipients of SDGs creation and implementation.Over the past decade, the awareness of sustainable practices has increased. The emerging CSR–HRM literature toward the sustainable practices has addressed the relationship between CSR–HRM strategies and employee work behaviors  and Italy (2420) were randomly selected using a 1% margin of error and a 95% confidence interval strategy. All hotels were chosen from Pakistan as it contains fewer hotels as compared to their counterparts. To infer sample size from the literature, Tinsley and Tinsley (1987) also proposed the minimum sample size to have 5 to 10 respondents for each item, while Hair et al. (1998) suggested that the sample should be in between 5 and 20 respondents against each item. We measured HRM, CSR, and sustainable performance using 30 items; therefore, the adequate sample is in between 150 and 600.We send the questionnaire and cover letter to 1,208 UK hotels, 2,420 Italian hotels, and 475 Pakistani hotels. Considering the study’s objectives, a single respondent strategy was adopted to get precise information about HRM, CSR, and sustainable performance. After multiple follow-ups and deleting questionnaires with missing values and outliers , we successfully collected the data of 438 UK, 520 Italians, and 354 Pakistani hotels. A total of 1,312 questionnaires were received and exceeded the upper limit of minimum sample size.p = 0.342; χ2 = 1.017, p = 0.135), hence indicating that the sample is representative to the population.We used the Chi-square test to determine the sample representativeness to compare the sample to population ratio, as no official statistics about the number of employees working in the selected hotels. The Chi-square tests were insignificant , experience in current organization , designation , and experience in hospitality industry . These results indicate no issue of non-response bias.Non-response bias between early and late respondent was determined using Armstrong and Overton’s (1977) proposed method. The results showed no differences in age  issue. As Podsakoff et al.  proposedThe data were analyzed using SPSS 22.0 and AMOS 21.0. We performed confirmatory factor analyses and Cronbach’s alpha to determine discriminant validity, convergence validity, and reliability. Additionally, correlation and path analyses were employed for hypotheses testing, while multigroup analyses (MGA) were performed to identify the differences in three-, four-, and five-star hotels.2/df = 344.88/117, p = 0.001; SRMR = 0.04, RMSEA = 0.05; NFI = 0.95, IFI = 0.94, CFI = 0.95). Moreover, the factor loading of each item was greater than 0.60, and the composite reliability was 0.86.The scales used to measure HRM, CSR, and sustainable performance were extracted from previous studies and adapted for this study. Before administering the questionnaires at a large scale, a pilot study was conducted to determine the reliability and validity of the instruments, and results show acceptable values for reliability (< 0.70) and validity (< 0.60). HRM was measured on a 14-items scale, including questions related to cross-functional teams, remuneration systems, training, employee rights, safety procedures, well-being, recruitment, rules, and regulations extracted from previous studies . Additionally, the factor loading of each item was greater than 0.60, and the composite reliability of CSR was 0.84.The CSR-related initiatives of the hospitality industry are measured on a 9-items scale that includes questions on economic, environmental, and social development adopted from the previous study Turker, . EFA was2/df = 347.06/126, p = 0.001; SRMR = 0.05, RMSEA = 0.05; NFI = 0.94, IFI = 0.94, CFI = 0.96). All items were measured on a seven-point Likert scale where “1 = strongly disagree” and “7 = strongly agree.” The questionnaire also includes the demographic profile of the respondents, such as gender, position, and experience in the industry, and hotel characteristics like the number of rooms, hotel stars, and years of operation. Moreover, all the scales are given in Appendix A.Lastly, sustainable performance was measured on a 7-items scale that includes questions on economic performance, social performance, and environmental-related performance  as compared to alternative models. Similarly, the measurement model for each hotel categorization shows adequate fitness. The reliabilities  and validities  show that all the values are greater than the minimum threshold as Hair et al. , HRM with sustainable performance , and CSR with sustainable performance  is higher for hotels with 5-star category followed by 3-stars and then 4-stars.Table p = 0.001) and sustainable performance . The results also confirm that CSR has positive relationship with sustainable performance . Therefore, the results confirm the H1, H2, and H3 in the overall data. Additionally, the mediating role of CSR in HRM and sustainable performance is also significant (p = 0.05), hence supporting H4.The hypotheses for this study were tested using structural equation modeling (SEM), and a summary of the results is shown in Table p = 0.001) and sustainable performance  is relative stronger for the hotels with 5-stars ranking. Surprisingly, three-star hotels have relatively stronger results for each relationship. Hence, the results also supported H5, H6, and H7.Table p = 0.001) and HRM impact on sustainable performance  in UK sample as compared to other countries. Additionally, CSR is more strongly related to sustainable performance in Pakistani sample .We run separate analyses to examine the cultural differences between the relationship of HRM, CSR, and sustainable performance. As shown in Table The goal of the present study was to improve our understanding of the role of HRM and CSR in maintaining the sustainable performance. We theoretically account for the implications of HRM and CSR strategies and activities on the generation of sustainable performance. We believe that HRM facilitates the employees in designing and executing of sustainable practices and that subsequently leads to higher economic, environmental and social performance, as supported by previous studies . Under RBV, both HRM and CSR are perceived as strategic assets that contribute to long-term profitability and competitiveness and build a strong reputation among consumers and employees’ minds. Hence, hoteliers need to pursue organizational schemes that replenish, support, and exploit HRM and CSR resources to achieve a competitive advantage over competitors.Despite enormous advantages to unearth HRM–CSR–performance nexus, few studies have empirically examined this relationship  as well. Additionally, the data were cross-sectional; therefore, longitudinal studies should be conducted to establish causal relationships. Lastly, future researchers should use control variables such as hotel star ranking, year of operations, and hotel size in the theoretical model.This research is an important study to examine the impact of HRM on CSR and sustainable performance and CSR’s influence on sustainable performance in the hospitality industry of the UK, Italy, and Pakistan. The data were analyzed using SEM on the overall sample and separately for each hotel category, i.e., three-star, four-star, and five-star hotels. Overall, the results supported all the hypotheses and confirmed that the impact of HRM on CSR and sustainable performance and CSR’s influence on sustainable performance is different in each hotel category. The association of HRM with CSR and sustainable performance is relatively stronger in five-star hotels, followed by three-star and four-star hotels. Theoritically, this study extends the existing literature on the relationship between HRM, CSR, and performance (Umrani et al.,


Here, we describe a high-throughput screen for such effects using employing fluorescence anisotropy in a 96-well microplate format. HBV genomic RNAs (gRNA) and short oligonucleotides encompassing PS1 were 5′ labelled with an Alexa Fluor 488 dye. Excess (with respect to stoichiometric T = 4 NCP formation) HBV core protein (Cp) dimers were titrated robotically into solutions containing each of these RNAs stepwise, using a Biomek 4000 liquid handling robot. The anisotropy values of these mixtures were monitored using a POLARstar microplate reader. NCP-like structures were challenged with RNase A to identify reactions that did not result in complete NCP formation. The results imply that ∼50% of the compounds prevent complete NCP formation, highlighting both PS-meditated assembly and the PS-binding compounds as potential directly-acting anti-virals with a novel molecular target. Importantly, this method allows high-throughput in vitro screening for assembly inhibitors in this major human pathogen.Multiple ssRNA viruses which infect bacteria, plants or humans use RNA Packaging Signal (PS)-mediated regulation during assembly to package their genomes faithfully and efficiently. PSs typically comprise short nucleotide recognition motifs, most often presented in the unpaired region of RNA stem-loops, and often bind their cognate coat proteins (CPs) with nanomolar affinity. PSs identified to date are resilient in the face of the typical error prone replication of their virus-coded polymerases, making them potential drug targets. An immobilised array of small molecular weight, drug-like compounds was panned against a fluorescently-labelled oligonucleotide encompassing the most conserved Hepatitis B Virus (HBV) PS, PS1, known to be a major determinant in nucleocapsid formation. This identified > 70 compounds that bind PS1 uniquely in the array. The commercially available 66 of these were tested for their potential effect(s) on HBV nucleocapsid-like particle (NCP) assembly  The PS-ligand assay described herein provides a novel 1Left to right – titration number, the cumulative Cp dimer concentration after titration, the Cp dimer stock used and the resultant RNA concentration in each well after said titration.Depository data:Fluorescence Anistropy.xlsxLeft to right; plates 1 to 3 – the compound used, and the associated raw anisotropy value.Normalised anisotropy Changes.xlsxLeft to right; plates 1 to 3 – the compound used, and the normalised anisotropy value .Averages and errors.xlsxLeft to right – the compound used, and the average normalised anisotropy value of the triplicate plates and the associated standard error of the mean.light green 0.6-1, suggestive of efficient NCP assembly; light yellow = 0.3–0.6, suggestive of some Cp dimer: RNA binding; and light red = 0-0.3, suggestive of poor Cp dimer: RNA binding. Assay was performed in triplicate, and the average anisotropy change shown with associated standard error of the mean available in Depository data: averages and errors.xlsx, for PS1 and gRNA reassemblies, respectively.Anisotropy change is displayed here by the colour fill in each well. Left to right: 22.1HindIII restriction site was purchased from Genscript. gRNA was transcribed from HindIII-linearised plasmid using a T7 polymerase HiScribe kit (New England Biolabs), supplementing the reaction with 5´ amino-GMP (Jena Bioscience), according to the manufacturer's protocol. The PS1 RNA oligonucleotide A pUC57 plasmid encoding for the gRNA substrate positioned between a 5´ T7 promotor sequence, and 3´ 2.2E. coli (New England Biolabs) from a pET28b plasmid containing the Cp gene Cp was expressed in BL21 (DE3) competent 2.3gRNA and PS1 oligonucleotides were heat-annealed by heating to 70 °C, cooling slowly to room temperature in a buffer containing 10 mM MES pH 7.0, 25 mM NaCl and 1 mM DTT. RNA substrates were then diluted to working concentrations of 1.1 / 16.5 nM respectively in a buffer containing 25 mM HEPES pH 7.5, 250 mM NaCl and 5 mM DTT.178 µL of RNA substrate was titrated using a Biomek 4000 liquid handling robot (Beckmann Coulter) into the wells of a 96 well plate  and allowed to equilibrate at room temperature for 30 mins. 2 µL DMSO ± 10 µM compound 1 - 66 were added and a further equilibration step performed.Purified Cp dimer was then titrated using the Biomek 4000 liquid handling robot stepwise into the RNA substrates as detailed in Fluorescence anistropy was monitored throughout using a POLARstar Omega plate reader (BMG Labtech) and normalised with respect to control reactions in the absence of compound ± Cp dimer .Fardokht Abulwerdi: Initial small molecule screen against PS1; Farzad Fatehi:In silico modelling of compound effects in vivo; Iain Manfield: Compound:RNA binding affinity methodology; Stuart Le Grice, John S. Schneekloth Jr.: SMM methodology, supervision and editing; Reidun Twarock: Conceptualization and supervision; Peter G. Stockley: Conceptualization, supervision and editing; Nikesh Patel: Investigation, formal analysis, conceptualization, fluorescence anisotropy methodology, writing and editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


N6 methylation of adenosine (m6A), N1 methylation of adenosine (m1A), N7-methylguanosine (m7G), 5-methylcytosine (m5C), 2′O-methylation (2′-O-Me or Nm), pseudouridine (5-ribosyl uracil or Ψ), and adenosine to inosine RNA editing (A-to-I editing). We then show how they relate to different types of lung disease. This paper hopes to summarize the mechanisms of RNA modification in chronic lung disease and finds a new way to develop early diagnosis and treatment of chronic lung disease.Chronic lung diseases are highly prevalent worldwide and cause significant mortality. Lung cancer is the end stage of many chronic lung diseases. RNA epigenetics can dynamically modulate gene expression and decide cell fate. Recently, studies have confirmed that RNA epigenetics plays a crucial role in the developing of chronic lung diseases. Further exploration of the underlying mechanisms of RNA epigenetics in chronic lung diseases, including lung cancer, may lead to a better understanding of the diseases and promote the development of new biomarkers and therapeutic strategies. This article reviews basic information on RNA modifications, including  Chronic lung diseases include chronic obstructive pulmonary disease (COPD), pneumonia, idiopathic pulmonary fibrosis(IPF), asthma, and lung cancer . Despite1A, and then detects m1A on the mRNA . Am. AmN6 mecytosine . The m6Aversible ,42. The function . The m7Gnd tRNAs ,45,46. Rn miRNAs . The m7Gd ncRNAs . It is iigration ,50,51. 2igration ,53. Of aigration . Ψ can cigration . Ψ couldhttps://www.ncbi.nlm.nih.gov/geo, accessed on 27 November 2022). The data related to chronic lung disease in the GEO database is mainly related to m6A modification. The m6A levels in cisplatin-resistant A549 cells were up-regulated compared to A549 cells . In addition, multiple data sets were compared and screened for m6A markers in LUAD cells . There are also RNA-seq expression profiles associated with writer/eraser perturbation. For example, expression profiles after METTL3 knockdown in A549 and H1299 cells (GSE76367), ALKBH5 knockdown in PC9 cells (GSE165453), and YTHDF1 or YTHDF2 knockdown PC9 cells (GSE171634).High-throughput sequencing data has a key impact in the study of RNA modification. These sequencing data are available on public websites, including the NCBI-Gene Expression Omnibus database (NCBI-GEO) . Specialized reversible RNA modification databases include m6Avar [6A-TSHub [6A [6ASNP [6A modification. The m6AVar database is a comprehensive database for studying m6A-related variants that may affect m6A modification. It will explain the influence of variants through the function of m6A modification. The m6AVar can be used to predict potential modification sites of m6A in chronic lung disease. According to the m6AVar database, three m6A-related genes  are reliable prognostic indicators for lung adenocarcinoma (LUAD) patients and are potential therapeutic targets [6AVar in that it mainly collects related variants affecting m6A modification. However, RMVar has more comprehensive functions than m6AVar. RMVar also contains other RNA-modified variants. The databases of genetic variation in RNA modification also include RMDisease and m6A- TSHub. RMDisease integrates the predictions of 18 different RNA modification prediction tools and a large number of experimentally validated RNA modification sites and identifies single nucleotide polymorphisms (SNPs) that may affect eight types of RNA modification. Most of the m6A-associated cancer variants are tissue- and cancer-specific. The m6A-TSHub consists of four core components, namely m6A-TSDB, m6A-TSFinde, m6A-TSVar, and m6A-CAVar. The m6A-TSDB platform can be used to retrieve m6A sites in normal lung tissue and lung cancer cell lines. Then, m6A-TSVa can be used to explore the influence of lung tissue variation on m6A by integrating tissue-specific m6A. Finally, m6A-CAVar can be used to screen for cancer variants affecting m6A in lung tissue. Through these databases, we can screen out the gene variants associated with RNA modification in chronic lung disease for further verification.With the further study of RNA modification, databases containing various information are emerging. These databases, which have received widespread interest and use, in turn, provide the basis for further research about the function of RNA modification. The existing RNA modification databases can be divided into biochemical RNA modification databases, comprehensive reversible RNA modification databases, specialized reversible RNA modification databases, and RNA editing databases . Biochem6A-Atlas , m6A2tarA2target , m5C-Atl5C-Atlas , m7GHub s [7GHub  and RNA s [7GHub . RMBase e m6Avar , m6A-TSH6A-TSHub , CVm6A [SHub [6A , RMVar [SHub [6A  and RMDiSHub [6A . RNA ediSHub [6A , RigorouSHub [6A , DatabasSHub [6A , and REDSHub [6A . There aA [6ASNP  and ConsA [6ASNP . Genetic targets . RMVar i6A is regulated by three types of proteases, methyltransferases , RNA-binding motif protein 15/15B (RBM15/15B), zinc finger CCCH type containing 13 (ZC3H13), virlike m6A methyltransferase associated , cbl proto-oncogene like 1 (CBLL1), and Wilms’ tumor-associated protein (WTAP), demethylases  and alkB homolog 5 (ALKBH5)), and readers (including YTH domain family 1/2/3 (YTHDF1/2/3), YTH domain containing 1/2 (YTHDC1/2), insulinlike growth factor 2 mRNA binding protein 1/2/3 (IGF2BP1/2/3), and heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1)), and is reversible and can be dynamically regulated [6A levels. Reversible m1A methylomes are achieved by the dynamic modulation of m1A RNA-modifying proteins, including m1A methyltransferases such as tRNA methyltransferase 6 noncatalytic subunit (TRMT6)-TRMT61A complex, TRMT10C, TRMT61B and nucleomethylin (NML), m1A demethylases such as ALKBH1, ALKBH3 and FTO, and m1A-dependent RNA-binding proteins such as YTHDF1/2/3 and YTHDC1 [7G in mammals is catalyzed by the compounds METTL1 and WD repeat domain 4 (WDR4), a complex that facilitates the installation of m7G in tRNA, miRNA, and mRNA [7G. Among them, RNMT is the catalytic subunit, and RAM is the regulatory subunit, which plays the role of activation. Williams–Beuren syndrome chromosome region 22 (WBSCR22) and TRMT112 are responsible for regulating m7G in rRNA. The main function of these regulatory mechanisms is to add m7G to the target RNAs, thereby mediating many key biological processes by modulating RNA production, structure, and maturation [5C is reversibly regulated by methyltransferases, including DNA methyltransferase  and NOP2/Sun RNA methyltransferases (NSUNs), and demethylases, including ten-eleven translocation (TETs), and reader proteins including YTHDF2, Aly/REF Export Factor(ALYREF) and Y-box binding protein 1(YBX1) [Three proteins have been found to regulate RNA modification. The first is an enzyme that introduces modified nucleotides into RNA during post-transcriptional RNA modification; the second protein interacts with the modified nucleotides; the third protein removes the modification labels . The metegulated ,80. Thesd YTHDC1 . The modand mRNA ,83. RNA turation . The m5C 1(YBX1) ,86. Ther 1(YBX1) ,88. In a 1(YBX1) . Fibrill 1(YBX1) . Ψ is pr 1(YBX1) . A-to-I  1(YBX1) .6A can affect the RNA stability, localization, turnover, and translation efficiency of genes, thereby regulating cellular processes such as cell self-renewal, differentiation, invasion, and apoptosis, and is even critical for skeletal development and homeostasis [6A. This complex enhances m5C methylation, which synergistically promotes p21 expression and affects oxidative stress-induced cellular senescence [6A-related regulators promote the progression of atherosclerosis by regulating macrophage polarization and inflammation. WTAP and METTL14 also can affect the phenotypic regulation of vascular smooth muscle cells (VSMCs) via m6A modification [6A RNA methylation generates compensatory cardiac hypertrophy, whereas decreased m6A causes cardiomyocyte remodeling and dysfunction [6A modification plays an important role in controlling the generation and self-renewal of hematopoietic stem cells and in mediating the development and differentiation of T and B lymphocytes from hematopoietic stem cells [6A, can maintain the homeostasis and maturation of natural killer (NK) cells, and positively regulate the antitumor and antiviral activities of NK cells. Its deletion significantly impairs NK cell antitumor and antiviral activity in vivo [6A methylation is also associated with NK cell homeostasis and antitumor immunity [6A is associated with apoptosis, autophagy, pyroptosis, ferroptosis, and necrosis [5C mediates cell proliferation, differentiation, apoptosis, and stress response [RNA modifications have a key influence in the regulation of many fundamental biological processes associated with chronic lung disease . The meteostasis ,94. The nescence . In the fication . Enhancefunction . An increm cells . YTHDF2, in vivo . METTL3-immunity . In addinecrosis , and m5Cresponse .6A modification plays an important role in tumorigenesis, proliferation, and metastasis. Active m6A regulators in lung cancer are related with the activation of multiple signaling pathways, such as DNA replication, RNA metabolism, epithelial–mesenchymal transition (EMT), cell cycle, cell proliferation and apoptosis, energy metabolism, inflammatory response, drug resistance, tumor metastasis and recurrence [6A regulates tumor metabolism by directly regulating nutrient transporters and metabolic enzymes or indirectly by controlling key components in metabolic pathways [6A modification contributes to the progression of malignant tumors and affects patient prognosis. Some m6A-related mRNA markers have also been shown to be independent prognostic biomarkers in patients with different types of cancer [6A methylase METTL3 is abnormally activated in cisplatin-resistant nonsmall-cell lung cancer (NSCLC) cells. METTL3 enhances YAP mRNA translation by introducing YTHDF1/3 and eIF3b into the translation initiation complex and increasing YAP mRNA stability by regulating the MALAT1-miR-1914-3p-YAP axis. This induces NSCLC drug resistance and metastasis [6A demethylase discovered to promote NSCLC cell growth by demethylating USP7 mRNA or MZF1 mRNA transcripts and increasing their stability and transcriptional levels [6A-modified PDK4 mRNA positively regulates the glycolysis of cells by binding to IGF2BP3, thereby promoting the development of cancer. While METTL14 knockout can reverse the function of IGF2BP3 and play a tumor suppressor role [6A modification also affects the mediating tumor metastasis process. When lung cancer brain metastasis (BM) occurs, miR-143-3p was upregulated in paired BM tissues compared with primary lung cancer tissues. METTL3-mediated m6A modification induces the maturation of miR-143-3p, which induces lung cancer invasion and angiogenesis via suppressing the expression of the target gene vasohibin-1 [6A modification in LUAD. METTL14-mediated HCG11 inhibits the growth of LUAD by targeting large tumor suppressor kinase 1 (LATS1) mRNA through IGF2BP2 [7G methyltransferase METTL1 and WDR4 complex is significantly elevated in lung cancer, which can promote lung cancer cell growth and invasion and negatively correlate with patient prognosis. Impaired m7G tRNA modification in the absence of METTL1/WDR4 results in decreased proliferation, colony formation, cell invasion, and tumorigenicity of lung cancer cells [7G-let-7 miRNA can inhibit lung cancer cell metastasis by reducing the expression of target oncogenes, including high mobility group AT-hook 2 (HMGA2), RAS, and MYC, at the posttranscriptional level [5C levels can be used as a cancer marker. For example, in lung squamous cell carcinoma (LUSC), the upregulation of m5C-related NSUN3 and NSUN4 is related to poor patient prognosis [5C levels can be found in circulating tumor cells from lung cancer patients [FAK mRNA by binding to FAK. FAK blocks the ADAR-induced invasiveness of LUAD cells [RNA epigenetic modifications have been broadly reported in lung cancer development and other chronic lung diseases . The moscurrence . Reprogrpathways . Aberranf cancer ,106. Rectastasis . FTO is l levels ,109. Thesor role . In addisor role . The m6Aohibin-1 . The lnc IGF2BP2 . The m7Ger cells . Highly al level . Studiesrognosis . In LUADrognosis . Abnormapatients . The stupatients . The rs9patients . FinallyAD cells . A-to-I AD cells .6A RNA methylation regulators is abnormal in COPD, among which the mRNA expressions of IGF2BP3, FTO, METTL3, and YTHDC2 show a tight association with the occurrence of COPD. IGF2BP3, FTO, METTL3, and YTHDC2 have obvious correlations with various important genes enriched in signaling pathways and biological processes that promote the development and progression of COPD [2.5) is an important cause of COPD. METTL16 may regulate sulfate expression through m6A modification, thereby participating in PM2.5-induced microvascular injury and promoting the development of COPD [5C modification may negatively affect normal lung metabolic activities by upregulating gene expression levels in the lungs of mice exposed to PM2.5 [STAT2 mRNA m6A [6A modification was upregulated in the bleomycin (BLM)-induced pulmonary fibrosis mouse model, FMT-derived myofibroblasts, and lung samples from IPF patients. Silencing METTL3 can inhibit FMT by reducing m6A levels. KCNH6 is involved in the m6A-regulated FMT process. m6A modification regulates KCNH6 expression through YTHDF1 [6A methylation has also been implicated in the pathogenesis of asthma, and YTHDF3 has an effect on eosinophils for severe asthma, which can guide future immunotherapy strategies [7G has an important value in predicting the prognosis of IPF patients and early diagnosis of IPF patients [In addition to studies on lung cancer, the effects of RNA modification in other chronic lung diseases such as COPD, pneumonia, asthma, and pulmonary fibrosis have also been explored . Exposur of COPD . Exposur of COPD . In addito PM2.5 . LncRNA mRNA m6A . Myofibrh YTHDF1 ,130. ALKh YTHDF1 . The m6Arategies . Increaspatients .6A modification plays a multifunctional role in normal and abnormal biological processes, and its regulatory proteins can act as therapeutic targets for cancer and are expected to be biomarkers for overcoming drug resistance [6A modification. METTL3 facilitates the translation of a large subset of oncogenic mRNAs and has direct physical and functional interactions with translation initiation factor 3 subunit h (eIF3h). METTL3-eIF3h interaction is required for oncogenic transformation. The depletion of METTL3 inhibits tumorigenicity and sensitizes lung cancer cells to bromodomain-containing protein 4 (BRD4) inhibition [METTL3 mRNA, reduce its expression, and further inhibit NSCLC cell proliferation [6A methylome is a new mechanism for drug resistance in cancer, such as tyrosine kinase inhibitors (TKIs) [MZF1 mRNA level, and MZF1 gene silencing significantly inhibited the cell viability and invasion of LUSC [6A. Knockdown of ALKBH5 under this condition significantly inhibited cell invasion by upregulating the m6A level of FOXM1 mRNA and reducing its translation efficiency [6A modification destabilizes SLC7A11 mRNA and accelerates mRNA decay upon recognition by YTHDC2, thereby affecting cystine uptake and contributing to antitumor activity [6A modification-related inflammatory cytokine interleukin 37 (IL-37) has received extensive attention for the treatment of lung cancer. IL-37 inhibits the proliferative capacity of LUAD cells by regulating RNA methylation. Meanwhile, overexpression of IL-37 decreased the expression of ALKBH5 and thus can also be used to treat NSCLC patients [6A modification reduces gefitinib resistance (GR) in NSCLC patients via the FTO/YTHDF2/ABCC10 axis [In recent years, RNA modification has been identified as a novel regulatory mechanism in controlling cancer pathogenesis and treatment response/resistance. The msistance . METTL3 hibition . METTL3 feration . The dyns (TKIs) . FTO is  of LUSC . Two FTO of LUSC . Under tficiency . Downregactivity . The m6Apatients . GefitinC10 axis . The dev6A regulators are upregulated in cancer and play a role in promoting tumor growth. These regulators, including METTL3, METTL14, and WTAP, and their key targets are associated with the clinical characteristics of various cancer patients, which may provide new possibilities for early cancer diagnosis [6A targeted drugs with traditional chemotherapy drugs or PD-1/PD-L1 inhibitors has great therapeutic prospects [6A methylation [5C deletion of mitochondrial RNA in tumor cells can reduce the metastasis and invasion of cancer cells. This means that when cancer patients undergo clinical treatment, they can inhibit the metastasis and spread of cancer cells by inhibiting m5C modification in mitochondria, thus increasing clinical benefits. As an enzyme responsible for modifying RNA, NSUN3 is only used to modify the formation of m5C, so it is a very promising drug target [In conclusion, this article reviews the recent advances in the function and molecular mechanism of RNA epigenetics in the progression of chronic lung diseases. RNA epigenetics is expected to be a research tool for the development of new diagnostic biomarkers with clinical value. Enzymes involved in regulating RNA modification can be new targets for the treatment of chronic lung diseases. Most of the miagnosis . The comrospects . The mechylation . YTHDF1 hylation . The m5Cg target . RNA modIn the future, precision medicine based on RNA epigenetics may target individual patients with chronic lung diseases for diagnosis and treatment. However, the function of these chemical modifications in both coding and noncoding RNAs remains in its infancy, and collaborative efforts are still needed to establish a clear link between RNA epigenetics and chronic lung diseases. At present, many new methods of RNA modification have emerged, among which Direct RNA sequencing is the representative one. Through the unique way of the library building and relying on the algorithm, the collection of RNA modification information at the single base level can be achieved. The development of RNA modification quantitative map databases (such as DirectRMDB) based on this sequencing method also undoubtedly opens another direction for RNA modification detection . In brie


Most of the human genome, except for a small region that transcribes protein-coding RNAs, was considered junk. With the advent of RNA sequencing technology, we know that much of the genome codes for RNAs with no protein-coding potential. Long non-coding RNAs (lncRNAs) that form a significant proportion are dynamically expressed and play diverse roles in physiological and pathological processes. Precise spatiotemporal control of their expression is essential to carry out various biochemical reactions inside the cell. Intracellular organelles with membrane-bound compartments are known for creating an independent internal environment for carrying out specific functions. The formation of membrane-free ribonucleoprotein condensates resulting in intracellular compartments is documented in recent times to execute specialized tasks such as DNA replication and repair, chromatin remodeling, transcription, and mRNA splicing. These liquid compartments, called membrane-less organelles (MLOs), are formed by liquid–liquid phase separation (LLPS), selectively partitioning a specific set of macromolecules from others. While RNA binding proteins (RBPs) with low complexity regions (LCRs) appear to play an essential role in this process, the role of RNAs is not well-understood. It appears that short nonspecific RNAs keep the RBPs in a soluble state, while longer RNAs with unique secondary structures promote LLPS formation by specifically binding to RBPs. This review will update the current understanding of phase separation, physio-chemical nature and composition of condensates, regulation of phase separation, the role of lncRNA in the phase separation process, and the relevance to cancer development and progression. Human genome sequencing came up with a massive surprise that only less than 2% of the genome is translated and the remaining ∼98% does not encode a protein, creating loopholes in the central dogma. This non-coding part of the genome was considered “junk or dark matter,” with the exceptions of rRNAs, tRNAs, snRNAs, and snoRNAs . It was Moreover, a bridge between LLPS and lncRNA has been formed, giving a new meaning to the subcellular localization of lncRNA, where it can perform its functions. It has been shown that phase condensates have a dynamic character, and this property can give lncRNAs the ability to perform their regulatory roles rapidly and transiently. Several functional lncRNAs have been known for years, but what makes them unique if they participate in phase condensation will be discussed in this review. It will also be discussed how condensates aid the lncRNAs in playing a pivotal role in fine-tuning many physiological and pathological activities. The content below will also talk about the connection between LLPS and lncRNA in cancer.The presence of a lipid bilayer helps in compartmentalization and allows eukaryotic cells to organize biochemical reactions in space and time. But one fundamental question remains: how do cells maintain biomolecular interactions and homeostasis in the cytoplasm and nucleus. One way to coordinate spatiotemporal regulation is to control the localization of specific biomolecules at one place while excluding others, thereby creating a heterogeneous environment. Work from past years has shown the existence of such functional membrane-less compartments in the nucleus and cytoplasm. These compartments include the pericentriolar matrix which plays a role in microtubule nucleation , the nucin vitro and in vivo approaches have provided a solid foundation for understanding the structural and functional aspects of condensates. LLPS can be described as “a thermodynamically driven, reversible, de-mixing process wherein, above a threshold macromolecule concentration, components separate into coexisting dense and dilute liquid phases with different solute concentrations”  in a solution. If the interaction between X and Y is energetically more favorable than that between X and X or Y and Y molecules, then the solution will be homogenously mixed, and the system’s entropy will be maximum. However, suppose X and X molecular interaction is energetically favorable over X and Y interaction, in that case, X will get de-mixed from neighboring Y molecules after reaching a critical threshold concentration. Entropy, which generally favors the homogenous distribution of X and Y molecules, gets counteracted by energetically favorable interactions between X and X molecules. The living system also undergoes similar changes where interactions between different molecules lower the energy, driving them to get phase-separated .π–π, π–cation, cation–anion, dipole–dipole, hydrogen–hydrogen, and hydrophobic forces that influence the spatiotemporal arrangement of the molecules in condensates  spherical, due to interfacial tension between two phases; 2) can undergo fission and fusion processes; 3) have diffuse nature, thereby allowing molecular exchange from the nearby environment to support biological function; and 4) can wet other surfaces upon contact .In a living cell, forces that favor phase separation are mediated by the cell’s biopolymers, such as proteins and nucleic acids. Condensate components such as protein and RNA can be of two types: scaffolds and clients. Scaffold molecules have higher valency, thereby acting as drivers of phase separation, providing a platform for other proteins. These molecules, abundant in condensates, decide the condensate threshold, and their removal likely prevents condensate assembly. Client molecules are recruited for condensation based on their relative stoichiometry with the scaffold. They perform their function by interacting with scaffolds and other client molecules and are generally dispensable for phase separation. For example, in paraspeckles, a nuclear phase condensate, NEAT1  lncRNA acts as a scaffold, while the RNA binding protein, FUS (fused in sarcoma), is a client protein  Banani . The cenTwo important features that govern phase separation are the presence of IDR and modular domains. The IDR is a region of amino acids that exhibit low-sequence complexity, are present in a heterogeneous ensemble of conformations, and do not fold into a well-defined three-dimensional structure. They have a higher polar and charged amino acid ratio, including glycine, serine, glutamine, proline, glutamic acid, lysine, and arginine. Even though they have low hydrophobic amino acid content, aromatic amino acids such as tyrosine and phenylalanine are generally interspersed in these regions. These characteristics fulfill the multivalent nature required for phase separation. To phase separate, IDRs can interact with other IDRs of the same protein and/or other proteins. For example, in paraspeckles, RBM14 (RNA binding motif 14) and FUS interact via their IDRs, namely, prion-like domain . These IStudies on phase separation reveal the presence and importance of RNA in condensates. RNA can interact with other RNAs and proteins by virtue of its flexible and variable properties. 1) The most fundamental property imparting multivalency is the negative charge of the RNA backbone through which it can mediate electrostatic interactions with other molecules. Also, the single-stranded nature of RNA exposes its bases and phosphate group to interact with multiple positively charged amino acids or molecules. 2) Flexibility also favors attaining multiple confirmations such as G-quadruplexes, hairpin loops, and helix motifs which provide protein or RNA binding sites. RBPs containing RRMs can recognize these structures. 3) Longer lengths of RNAs like long non-coding RNAs  can prov6-methyladenosine modification, can also impact condensate assembly. These modifications can influence phase separation in multiple ways. 1) The modifications give the potential for dynamic and adaptive nature to the RNA. 2) It affects the sequence information and has a substantial impact on the structure of RNA molecules. 3) RNA modifications also affect the processing of RNAs. These changes altogether mediate adaptive RNA–RNA and RNA–protein interactions. These factors cumulatively make condensation an adaptive process and only happen upon receiving specific cues from the environment nearby. Thus, the relative stoichiometry of condensate components controls their dwell time within condensate and consequently fine-tunes functions performed by them. , the presence of a membrane, molecular chaperones, and other active processes. PTMs affect the physio-chemical nature of amino acids, thereby affecting the strength and multivalency of molecules. Membrane surfaces affect the concentration of molecules by either hampering diffusion or promoting transport or crowding of condensate molecules. Similar to PTMs of proteins, modifications of RNA bases alter essential aspects of RNA functions. These posttranscriptional modifications, such as Nby them. .LncRNAs are emerging as important players in the formation of biomolecular condensates. Other RNAs such as mRNAs, rRNAs, and tRNAs have been shown to play important roles in various condensates such as stress granules and P bodies , but decSeveral features within these anchors have been shown to aid them in organizing a phase condensate. One of these features is their length. LncRNAs being longer than other non-coding RNAs such as miRNA can provide a larger platform for binding RBPs (RNA-binding proteins), as with NORAD lncRNA that has 18 Pumilio binding motifs , providiThe presence of lncRNAs within a condensate makes it less viscous, thus giving a more liquid-like nature compared to the increased protein component, which imparts a solid nature to the condensate . This prFor any protein or RNA, reaching a threshold concentration for phase separation is necessary . TherefoIn addition, several reports suggest that inhibiting lncRNAs either by knockdown, antisense oligonucleotides, RNases, RNA pol II inhibitors, or deleting their protein-interacting regions hinders condensate formation, thereby negatively affecting the normal function of the condensate and its components . This agSome of the lncRNAs reported to be the mainstay in phase separation  are brieMajSAT lncRNA is transcribed from heterochromatin-abundant repetitive elements with which a nuclear matrix (NM)–associated protein, SAFB (scaffold attachment factor B), interacts via its R-/G-rich region. This association forms foci in DAPI-dense regions of the nucleus (heterochromatin region), where SAFB mediates pericentromeric heterochromatin (PCH) stabilization. Downregulation of MajSAT lncRNA with antisense oligonucleotides leads to dissociation of the SAFB condensate, thereby causing weakening of PCH foci as indicated by H3K9me3 staining .6A (N6-methyladenosine) modification, as characterized by methylation-inscribed nascent transcript sequencing (MINT-seq)  causes a reduction in YTHDC1 binding to the enhancer, thereby reducing the gene expression. In vitro analysis of YTHDC1/m6A-eRNA suggested that YTHDC1 cannot form the condensate alone.Enhancer RNAs (eRNAs), as the name suggests, are long non-coding RNA transcribed from enhancer regions and have a crucial role in transcriptional regulation. They have a heavy deposition of mINT-seq) . These mThe role of phase condensates in cell signaling is beginning to be deciphered. SNHG9  lncRNA is now known to regulate the Hippo pathway  by physiNORAD (non-coding RNA activated by DNA damage) lncRNA is localized in the cytoplasm and sequesters Pumilio proteins (PUM1 and PUM2) in a condensate. Pumilio proteins are RNA-binding proteins that are posttranscriptional repressors and cause translation inhibition and degradation of several mRNAs that include important regulators of mitosis. Negative regulation of PUM proteins by NORAD prevents abnormal mitosis and maintains genomic stability in mammalian cells. NORAD is a perfect example of RNA imparting multivalency in biomolecular condensate as it has 18 PUM response elements (PRE) where PUM proteins bind. A decrease in PREs leads to reduced phase separating ability and requires higher PUM concentration to form condensates . KnockdoLncRNAs such as TERRA (telomeric repeat-containing RNA) are known to be a part of ALT-associated pro-myelocytic leukemia bodies (APBs) clustered on the telomeres. Along with TERRA, other DNA replication and repair proteins such as BLM (helicase) and RAD52 (RNA-templated DNA repair) are present in APBs. These bodies mediate telomerase-independent telomere maintenance known as alternative lengthening of telomeres (ALT) in cancer cells, where TERRA acts as a template for telomeric DNA synthesis. Knockdown of associated proteins leads to a reduction in APBs , but theDouble-strand breaks (DSBs) in DNA are sensed by the MRE11-RAD50-NBS1 (MRN) complex, which recruits RNA polymerase II at the damaged sites. This recruitment leads to the synthesis of dilncRNAs (damage-induced long non-coding RNAs) , which aAs mentioned earlier, lncRNAs provide binding platforms for various RBPs, but it is not necessary that a particular lncRNA interacts with only a single type of RBP. Various features of lncRNA, especially the repetitive nature, give them the liberty to bind multiple RBPs. One such versatile long non-coding RNA is HSAT III (highly repetitive satellite III) which is now known to provide architecture to two distinct types of nuclear stress bodies under thermal stress. First is nSB-S, in which SAFB associates with HSAT III for the transcription of heat shock proteins. The second is nSB-M, where HNRNPM (heterogeneous nuclear ribonucleoprotein M) interacts with HSAT III and affects pre-mRNA splicing. Knockdown of HSAT III with antisense oligonucleotides causes the disappearance of both types of stress bodies .Over the years, people have been trying to understand the role of cancer driver mutations and their role in cancer progression. Interestingly, it was found that specific mutation that leads to oncogene activation also results in the formation of super-enhancers, which is a type of phase condensates that are formed by a clustered DNA element . Many reLong-noncoding RNAs are dysregulated in cancer and have an important role in cancer development and progression. It is challenging to completely understand lncRNA’s function due to its large size, conformational flexibility, and low abundance. However, the specific subcellular localization of lncRNA leads to stoichiometrically measurable effects . LncRNA The NEAT1 lncRNA, a dysregulated lncRNA in cancer, is an architectural component of the paraspeckle nuclear body. In the presence of DNA damage response (DDR), p53 protein is activated, which leads to the NEAT1-mediated paraspeckle formation that attenuates oncogene-dependent activation of p53 . MALAT1 Glutamine metabolism is an important source of carbon and nitrogen for several biosynthetic processes. The rate-limiting step of glutaminolysis is the generation of glutamate from glutamine catalyzed by glutaminase-1 (GLS1) . GlutamiPhase separation provides a framework to understand how an individual cell coordinates specific processes at subcellular levels that are simultaneously robust and adaptive. Like the membranous barrier, phase boundary ensures the controlled localization of biomolecules without the complication of transport through a barrier. The formation of biological condensates is a tightly regulated thermodynamic process that depends on the abundance, sequence, and structure of scaffold and client molecules. Various key mechanisms such as environmental conditions, chaperones, presence of a membrane, and posttranslational modifications can work in concert to affect the physio-chemical property, function, and homeostasis of condensates.LncRNAs are key molecules in a cell, whether they are part of the phase condensate or not, as they perform various functions in the cell such as gene regulation, including genetic and epigenetic modifications, swimming to different locations within the cell, and having the potential to interact with several biopolymers such as DNA, RNA, proteins, and lipids. The knowledge of LLPS and of lncRNAs being part of phase condensates unfolds whole new possibilities. This efflorescing concept gives a better understanding of how lncRNAs can get concentrated within a particular cell site while interacting with various biopolymers and performing specialized tasks. Further exploration of phase condensation may reveal secrets about the additional functions of known lncRNAs. LLPS can, indeed, be an educator, answering various unsolved questions and providing space to lncRNAs to execute their superpowers. However, a few questions remain to be clarified, like how a few condensates of a particular lncRNA can define its activity at thousands or more locations? Are various effector sites part of a single condensate? While the dynamic nature of phase condensates is well-appreciated, a better understanding of whether this mobility can help simultaneously reach the enormous number of effector sites is needed.Though phase separation is important for normal cell functioning, some phase-separated condensates are only present in cancer cells, such as FUS-CHOP-mediated phase separation is present in sarcoma . Some coThe studies on lncRNA-associated liquid–liquid phase separation in cancer are limited. Further investigation is required to improve the understanding of the role of lncRNA for LLPS formation in cancer to improve the therapeutic strategies. In addition, phase condensates may increase the drug efficacy or drug resistance in cancer cells depending on their physicochemical properties to form phase condensates . It can


Nup98, an essential component of the nuclear pore that also participates in annulate lamella pore structures localized in the cytosol, is involved in hepatitis C virus (HCV) assembly. Here, we combined confocal microscopy and biochemical assays to study the interplay between Nup98, core , and viral genomes. Our results show that in HCV-infected cells, core protein is necessary and sufficient to induce relocalization of Nup98 from annulate lamellae to lipid droplet-apposed areas in which core/NS5A and HCV genomic RNA [(+)RNA] are clustered to promote viral assembly. Furthermore, we found that Nup98 interacts with HCV RNA and that upon Nup98 downregulation, the viral (+)RNA genome was specifically excluded from areas that contain active translating ribosomes and the core and NS5A proteins. Altogether, these results indicate that Nup98 is recruited by HCV core from annulate lamellae to viral assembly sites to locally increase the concentration of (+)RNA genome, which may favor its encapsidation into nascent virions. Flaviviridae family and is a major cause of liver disease worldwide, with an estimated 71 million infected people (Hepatitis C virus (HCV) is a positive-strand RNA virus of the d people  have emed people .The HCV genome encodes a single polyprotein that is processed in 10 mature viral proteins consisting of (i) an assembly module (core-E1-E2-p7-NS2) encompassing the capsid protein (core) as well as the E1 and E2 surface glycoproteins that are incorporated in viral particles, as well as the p7 and NS2 proteins that support virion assembly, and (ii) a replication module encompassing the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B, which are sufficient to support viral RNA replication but which also contribute to virion production.The assembly of HCV particles occurs at endoplasmic reticulum (ER)-derived membranes in close proximity to lipid droplets (LD) and viral replication complexes . In addiNumerous viral pathogens are believed to use or hijack nuclear pore complex (NPC) components to promote their replication, irrespective of a requirement of nucleus functions in their life cycles: particularly, positive-strand RNA viruses such as picornaviruses, rhabdoviruses, retroviruses, and hepaciviruses. Among them, HCV was shown to interfere with localization of some NPC components and to induce their redistribution in the cytoplasm of infected cells, in proximity to LDs, where they partially colocalize with HCV core protein , 6. InteNup98 is a major constituent of the NPC, a huge protein complex of around 100 to 125 MDa that, by spanning the nuclear envelope, regulates the exchange of proteins and RNA between the nucleus and the cytoplasm. The NPC is composed of multiple copies of about 30 different nucleoproteins (Nups) that are organized in different subcomplexes: the cytoplasmic filament, Y-complex, inner ring complex, transmembrane Nups, central FG Nups, and the nuclear basket RNAs] in subcellular regions promoting replication, assembly, and translation. We also highlight that Nup98 interacts with the viral genome, and we detected a strong apposition of viral genome with Nup98 within infected cells. Finally, we show that the core retrieves Nup98 protein from annulate lamellae, hence allowing an enrichment of Nup98 with viral (+)RNAs in the core region and thus promoting HCV assembly.First, we sought to reproduce results from previous studies , 6 in or10.1128/mbio.02923-21.1FIG S1in situ hybridization and analyzed by confocal microscopy. The number of (+)RNA dots per cell was quantified with ImageJ. Download FIG S1, PDF file, 0.1 MB.Nup98 is a proviral factor for HCV particle assembly. (A and B) Huh7.5 cells transduced with lentiviral vectors expressing different shRNAs against Nup98 or with an empty vector (shCtrl) were infected with Jc1 virus (MOI of 0.2) and were harvested at 72 h postinfection. (A) Expression of Nup98 and β-actin proteins was analyzed by Western blotting. Quantification of Nup98 signals was normalized against internal control β-actin signals and expressed as a percentage of Nup98 expression relative to the shCtrl condition. (B) The levels of intracellular and extracellular infectivity were determined from cell lysates or supernatants of infected cells, respectively. (C) Huh7.5 cells transduced with sh5, the most efficient shRNA against Nup98, or with an empty vector (shCtrl) were infected with Jc1 or JFH1 viruses (MOI of 0.2) and were harvested at 72 h postinfection, and the intracellular genomes were quantified by qPCR. The most efficient shRNA against Nup98 (sh5) is referred to as shNup98 in all subsequent figures. (D) Subgenomic replicon (SGR) cells were transduced with an empty vector (shCtrl) or with sh5 and fixed at 72 h posttransduction. HCV (+)RNA was then stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To further confirm the specific effect of Nup98 on HCV assembly, we expressed in Nup98-downregulated cells an shRNA-resistant version of Nup98 fused to an amino-terminal Flag tag (FlagNup98). While this allowed us to restore the level of Nup98 close to that in cells expressing a control shRNA  and B, wNext, we sought to understand how Nup98 could promote the early steps of assembly of HCV particles. Specifically, we investigated the formation of E2/core/NS5A clustered structures, which gradually emerge at early time points postinfection in the cytoplasm of infected cells and mark the functional connection of replication complexes to viral particle assembly sites . InteresAltogether, these results suggested that Nup98 plays a role in virion production at a step prior to assembly of viral particles.in situ hybridization (FISH) and immunostaining analyses, we found that HCV (+)RNAs RNAs and (−)RNAs, respectively, and their colocalization with HCV assembly components in Nup98-downregulated cells. Through combined fluorescent  (+)RNAs  to D and (+)RNAs , we foun (+)RNAs  to D. Mo (+)RNAs  to D.10.1128/mbio.02923-21.2FIG S2in situ hybridization. Colocalization of Nup98 (green channel) with core (red channel) and HCV (−)RNA (blue channel) was analyzed by confocal microscopy. (B) The percentage of core area without (−)RNA, (C) the total area of core analyzed, and (D) the number of RNA dots per infected cell were quantified with ImageJ. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. (E) Pipeline of the segmentation analysis used in FIG S2, PDF file, 0.4 MB.The viral genome is excluded from translation/replication/assembly sites upon Nup98 downregulation. (A to D) Huh7.5 cells transduced with lentiviral vectors expressing an empty vector (shCtrl) or an shRNA against Nup98 (shNup98) were infected with Jc1 or JFH1 viruses (MOI of 0.2) and were fixed at 72 h postinfection. (A) Following staining of Nup98 and HCV core, HCV (−)RNAs were stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02923-21.3FIG S3in situ hybridization. Colocalization of NS5A (blue channel) with core (green channel) and HCV (+)RNA (red channel) (A) or NS5A (green channel) with core (red channel) and HCV (−)RNA (blue channel) (E) was analyzed by confocal microscopy. For clarity, the enlarged areas are pseudocolored red for (+)RNA and (−)RNA and green for NS5A. The percentage of NS5A area without RNA species (B and F), the total area of NS5A analyzed (C and G), and the number of RNA dot species (D and H) per infected cell were quantified with ImageJ. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. Download FIG S3, PDF file, 0.5 MB.Nup98 maintains the viral genome near replication/assembly sites. Huh7.5 cells transduced with lentiviral vectors expressing an empty vector (shCtrl) or an shRNA against Nup98 (shNup98) were infected with Jc1 or JFH1 viruses (MOI of 0.2) and were fixed at 72 h postinfection. Following staining of HCV core and NS5A, HCV (+)RNA (A to D) and (−)RNA (E to H) species were stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We then sought to address the specificity of this (+)RNA partitioning. First, by assessing HCV (−)RNA that were unfrequently associated with core or NS5A, we did not observe an alteration of their localization upon Nup98 downregulation  to H. Se10.1128/mbio.02923-21.4FIG S4in situ hybridization. (A) Colocalization of core (blue channel) with the nLuc RNA (green channel) and HCV (+)RNA (red channel) was analyzed by confocal microscopy. (B) The percentage of core area without nLuc RNA and (C) the number of nLuc RNA dots per infected cell were quantified with ImageJ. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. (D and E) Huh7.5 cells transduced with lentiviral vectors expressing an empty vector (shCtrl) or an shRNA against Nup98 (shNup98) were infected with Jc1 virus (MOI of 0.2) and were fixed at 24, 48, or 72 h postinfection. Following staining of HCV core and NS5A, HCV (+)RNA was stained by fluorescent in situ hybridization, and the colocalization of (+)RNA with core or NS5A was analyzed by confocal microscopy. (D) The percentage of core area without (+)RNA or (E) the percentage of NS5A area without (+)RNA was quantified with ImageJ. Download FIG S4, PDF file, 1.7 MB.The RNA partitioning upon Nup98 downregulation is specific to HCV (+)RNA and is a progressive event. Huh7.5 cells transduced with lentiviral vectors expressing an empty vector (shCtrl) or an shRNA against Nup98 (shNup98) were transduced with a lentiviral vector expressing the nLuc reporter gene, infected or not with Jc1 or JFH1 viruses, and fixed at 72 h postinfection. Following immunostaining of core, the nLuc RNA and HCV (+)RNA were stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, we addressed the kinetics of the HCV (+)RNA aggregation and segregation in and from core/NS5A areas, respectively. We found that the proportion of core-positive areas devoid of (+)RNA progressively decreased over time , resulting in ca. 5% of core regions lacking (+)RNA at late time points postinfection . In contTyr, which is incorporated into nascent polypeptide chains upon its entry in the ribosome A site. Intracellular PMY staining of HCV-infected cells revealed cells exhibiting either a high translational activity and no infection or, alternatively, poor translational activity and expression of HCV infection markers, as judged by expression only in the latter cells of HCV core protein and (+)RNAs RNAs are partitioned in HCV-infected cells upon Nup98 silencing, we analyzed their intracellular localization with respect to active translation sites, using a subcellular ribopuromycylation assay . This asRNAs see , which aRNAs see , active RNAs see . InteresRNAs see  to D, PMRNAs see  to H. Th10.1128/mbio.02923-21.5FIG S5in situ hybridization. Colocalization of puromycin (PMY) (green channel) with core (blue channel) and HCV (+)RNA (red channel) was analyzed by confocal microscopy. Individual channels were pseudocolored white, and infected cells were labeled with yellow stars. Scale bars of merged panels represent 30 μm. Download FIG S5, PDF file, 2.3 MB.HCV infection decreases the overall cellular translation. Huh7.5 cells transduced with lentiviral vectors expressing an empty vector (shCtrl) or an shRNA against Nup98 (shNup98) were infected with Jc1 or JFH1 viruses (MOI of 0.2). At 72 h postinfection, active translating ribosomes were labeled with puromycin followed by digitonin extraction, and cells were fixed. Following staining of puromycin and HCV core, HCV (+)RNAs were stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Altogether, our results suggested that Nup98 helps to maintain HCV (+)RNA at intracellular regions of translation, replication, and assembly. Thus, by concentrating these viral RNAs within these areas, Nup98 may favor the generation of new replication organelles and the assembly of new progeny virions.Since Nup98 harbors an RNA-binding domain , we wondNext, we wondered if Nup98 could be detected in proximity to viral RNA at the single-cell level. We distinguished Nup98 located at the nuclear pore versus in the cytosol, which appeared as dots throughout the cytoplasm in both Jc1 and JFH1 cell culture-grown HCV (HCVcc)-infected cells . Specifi10.1128/mbio.02923-21.6FIG S6in situ hybridization. (A) Colocalization of Nup98 (blue channel) with core (green channel) and HCV (+)RNA (red channel) (top row) or Nup98 (green channel) with core (red channel) and HCV (−)RNA (blue channel) (bottom row) was analyzed by confocal microscopy. For clarity, the enlarged areas showing apposition (right panels) are pseudocolored red for (+)RNA and (−)RNA and green for Nup98. (B) The percentage of cytoplasmic Nup98 with apposed RNA and the number of RNA dots of each RNA species per infected cells were quantified with ImageJ. (C and D) Huh7.5 cells infected with Jc1 virus (MOI of 0.2) or mock infected were fixed at 72 h postinfection. Following immunostaining of Nup98, HCV (+)RNA and actin or GAPDH RNA was stained by fluorescent in situ hybridization. (C) Colocalization of Nup98 (blue channel) with HCV (+)RNA (red channel) and actin or GAPDH RNA (green channel) was analyzed by confocal microscopy. (D) The percentage of cytoplasmic Nup98 with apposed RNA and the number of RNA dots of each RNA species per cell were quantified with ImageJ. (E and F) Huh7.5 cells were transduced with nLuc vector, infected, or not, with JFH1 virus, and fixed at 72 h postinfection. Following immunostaining of Nup98, the nLuc RNA and HCV (+)RNA were stained by fluorescent in situ hybridization. (E) Colocalization of Nup98 (blue channel) with the nLuc RNA (green channel) and HCV (+)RNA (red channel) was analyzed by confocal microscopy. (F) The percentage of cytoplasmic Nup98 with apposed RNA and the number of RNA dots of each RNA species per cell were quantified with ImageJ. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. Download FIG S6, PDF file, 2.6 MB.Nup98 is apposed to (+)RNA. (A and B) Huh7.5 cells infected with JFH1 virus (MOI of 0.2) were fixed at 72 h postinfection. Following immunostaining of Nup98 and HCV core, HCV (+)RNA and (−)RNA species were stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the While Nup98 interacts with actin and GAPDH RNAs , we founAltogether, these results suggested that Nup98 can interact with HCV (+)RNA, as well as with non-HCV RNAs, but can be readily detected in close proximity to HCV (+)RNA, hence suggesting a viral mechanism allowing Nup98 and HCV (+)RNA apposition.Since Nup98 interacts with HCV (+)RNAs  and is rFirst, to address this possibility, we investigated the intracellular sites of colocalization of Nup98 with core and NS5A . Cytopla10.1128/mbio.02923-21.7FIG S7FIG S7, PDF file, 2.8 MB.Nup98 colocalizes with HCV proteins. Huh7.5 cells infected with Jc1 or JFH1 viruses (MOI of 0.2) were fixed at 72 h postinfection. (A) Nup98 and HCV core and NS5A proteins were immunostained, and the colocalization of Nup98 (green channel) with core (red channel) and NS5A (blue channel) was analyzed by confocal microscopy. For clarity, the enlarged areas showing single channels were pseudocolored in grayscale. (B) Nup98, lipid droplets and HCV core protein were immunostained, and the colocalization of lipid droplets (LD) (green channel) with core (red channel) and Nup98 (blue channel) was analyzed by confocal microscopy. For clarity, the enlarged areas showing single channels were pseudocolored in grayscale. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. Download Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Next, since the core protein does not accumulate around LDs in Jc1 HCVcc-infected cells, in contrast to JFH1 HCVcc-infected cells , 17, 18,Then, we investigated the role of core on apposition of Nup98 with HCV (+)RNA. Whereas in SGR cells apposition of Nup98 with viral (+)RNAs was comparable to that of non-viral RNA , we foun10.1128/mbio.02923-21.8FIG S8in situ hybridization. Colocalization of NS5A (blue channel) with core (green channel) and HCV (+)RNA (red channel) was analyzed by confocal microscopy. Scale bars of panels and enlargements from the square area represent 10 μm and 2 μm, respectively. Download FIG S8, PDF file, 2.5 MB.Nup98 coprecipitates HCV RNA in SGR cells. (A and B) Subgenomic replicon (SGR) cells transduced with core to NS2 (C-NS2) lentiviral vectors from the J6 strain were subjected to immunoprecipitation against endogenous Nup98 with anti-Nup98 antibody or control immunoglobulins (rat IgG). (A) The efficiency of the immunoprecipitation was analyzed by Western blotting. (B) HCV RNA and cellular GAPDH RNA coimmunoprecipitated with Nup98 were quantified by qPCR. The specific capture of RNA is expressed as the fold enrichment of RNA bound to Nup98 immunoprecipitated with the Nup98 antibody against control immunoglobulins (rat IgG). (C) SGR cells expressing shRNA against Nup98 (shNup98) or empty vector (shCtrl) were transduced with lentiviral vectors expressing core or C-NS2 from the J6 HCV strain and were fixed at 72 h posttransduction. Following staining of HCV core and NS5A, HCV (+)RNA was stained by fluorescent Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Altogether, these results indicated that individual core expression can regulate Nup98 localization in the cytosol and that core protein colocalizes with Nup98, hence suggesting that core may recruit Nup98 and induce its proximity with HCV (+)RNAs.We then sought to determine if HCV core retrieves Nup98 alone or in combination with other proteins from the nuclear pore complex (NPC) or, alternatively, from annulate lamella (AL) pore complex (ALPC). The latter structures are discrete cytosolic double-membrane structures containing most components of the NPC, with the exception of some Nups such as TPR , a nuclear basket protein , and werFirst, we found that TPR, which readily colocalized with Nup98 at the nuclear membrane , was notSecond, whereas Nup98 colocalized in Huh7.5 cells with RanBP2, we found that cytoplasmic Nup98 was readily disassociated from cytoplasmic RanBP2 in Jc1 and JFH1 HCVcc-infected cells, without any change in its nuclear fraction  and D, a10.1128/mbio.02923-21.9FIG S9FIG S9, PDF file, 1.7 MB.HCV core expression is sufficient to dissociate Nup98 from RanBP2. (A and B) Huh7.5 cells were transduced with lentiviral vectors expressing core or core to NS2 (C-NS2) from J6 or JFH1 strains and were fixed at 72 h posttransduction. (A) Nup98, HCV core, and TPR proteins were immunostained, and the colocalization of Nup98 (green channel) with core (blue channel) and TPR (red channel) was analyzed by confocal microscopy. (B) The degree of overlap between nuclear or cytoplasmic Nup98 and TPR was assessed by quantifying the Manders’ M1 overlap coefficients, representing the overlap of Nup98 signal with TPR signal in the different fractions, with the JACoP plugin of ImageJ. Scale bars of panels and enlargements from the boxed area represent 10 μm and 2 μm, respectively. (C) Huh7.5 cells were infected with Jc1 or JFH1 viruses (MOI of 0.2) and were fixed at 24, 48, or 72 h postinfection. HCV core, Nup98, and RanBP2 were immunostained, and the colocalization of Nup98 with RanBP2 was analyzed by confocal microscopy. The degree of overlap between nuclear or cytoplasmic Nup98 and RanBP2 was assessed by quantifying the Manders’ M1 overlap coefficients, representing the overlap of Nup98 signal with RanBP2 signal in the different fractions, with the JACoP plugin of ImageJ. Download Copyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Finally, since the core protein contains several nuclear localization signals (NLSs) and nuclear export signals (NESs) that may allow the binding of core to karyopherins  and sinc10.1128/mbio.02923-21.10FIG S10FIG S10, PDF file, 2.2 MB.Effect of peptides targeting karyopherins on RanBP2-Nup98 dissociation induced by HCV infection. Huh7.5 cells were infected with Jc1 virus (MOI of 0.2) for 6 h and then incubated with 100 μM each synthetic peptides targeting different karyopherins : PEN wasCopyright © 2022 Boson et al.2022Boson et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Altogether these results suggested that viral (+)RNA interacts with Nup98 in annulate lamellae and that core recruits Nup98 from these annulate lamellae to bring viral (+)RNA to HCV assembly sites.Our results underscore a novel mechanism by which a nucleoporin, Nup98, favors the early steps of assembly of HCV particles. Specifically, we found that (i) Nup98 interacts with viral RNA independently of virion assembly, (ii) core recruits Nup98 from annulate lamellae and increases Nup98-HCV (+)RNA apposition, and (iii) core-associated Nup98 maintains viral RNA in intracellular sites of HCV replication, translation, and assembly, in order to promote HCV assembly. Based on this evidence, we propose that annulate lamella (AL) pore complexes (ALPCs) could be found in the “membranous web” (MW) and allow the retention of viral RNA in this specific microenvironment where HCV replication, translation, and assembly occur. Thus, when the replication to assembly switch occurs after a certain threshold of expression of viral assembly components is reached, core recruits Nup98 from these AL to favor encapsidation of viral ribonucleoprotein in nascent particles RNA-core-Nup98 interaction. First, while it may enrich the concentration of HCV (+)RNAs in MW areas, which are specifically depleted from immune sensors , it may In addition to allowing maintenance of viral (+)RNAs within the MW, our results open the possibility that core hijacks Nup98 to bring the viral genome close to the assembly sites, hence promoting the initiation of its encapsidation in nascent virions. Of note, no specific packaging determinant has been identified in HCV RNA to date , which hSeveral RNA viruses have been shown to hijack NPCs; yet, to the best of our knowledge, none of them seem to use nucleoporins and/or NPC components in a manner similar to that underscored here for HCV. Indeed, our results highlight an original mechanism whereby HCV hijacks a cytosolic nucleoporin to promote its replication and assembly, whereas most other viruses directly act on NPC to facilitate viral replication and/or to inhibit innate immunity. On the one hand, several viruses, such as, e.g., influenza A virus , vesicul37–Flaviviridae members that decrease nucleoporin levels  is the “gold standard” method to firmly identify these particular structures, but due to the thickness of the EM samples, they must be perfectly in plane to be correctly visualized. Besides, fluorescence microscopy is a method of choice to link AL visualization with functional analysis. Indeed, recent studies have shed light on AL composition , 23, 24,Collectively, our results provide new insights into the cellular factors and organelles that promote HCV assembly. Specifically, we show that Nup98 could interact with viral RNA in the annulate lamella pore complex upon replication. When assembly of viral particles takes place, core may recruit Nup98 to allow the initiation of encapsidation of viral RNA. Overall, this highlights how HCV could hijack and subvert some cellular components for its own benefit.Huh7.5 cells  and HEK-293T cells (ATCC CRL-1573) were grown in Dulbecco’s modified minimal essential medium (DMEM)  supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% fetal bovine serum.pFK-JFH1wt_dg, pFK-JFH1/J6/C-846_dg, and pWPI-CNS2 Jc1 plasmids , 53 wereThe pFlagNup98-sh5res plasmid was constructed as follows: The pEGFP-Nup98 plasmid was obtained from Jan Ellenberg  . The N-tThe NanoLuc luciferase gene was amplified from pL-Luc (a kind gift from Eric Bergeron) with the Up nLuc BamHI and Low nLuc XhoI primers and inserted in the pHR′-CMV-W-GFP (a kind gift from Didier Trono) lentiviral construct opened with BamHI and XhoI restriction enzymes to generate the pHR′-CMV-nLuc-WPRE lentiviral construct.pSGR-JFH1-neoR is a kind gift from Franck Chisari. psPAX2 and phCMV-G are kind gifts from Didier Trono and Jane Burns, respectively. Lentiviral vectors encoding shRNA against Nup98 , and SHC001 (pLKO.1 control empty vector) were purchased from Sigma-Aldrich. The puromycin resistance gene and its PGK promoter were partially deleted from SHC001 and TRCN0000296863 plasmids by extracting the BspEI fragment to generate SHC001-puro(−) and TRCN0000296863-puro(−), respectively. These plasmids were then used in ribopuromycylation experiments only.Rat anti-Nup98 (clone 2H10), rabbit anti-TPR, and rabbit anti-RanBP2 were purchased from Abcam. Rabbit anti-Flag, mouse anti-PMY (clone 12D10), and mouse anti-β-actin (clone AC74) were purchased from Sigma-Aldrich. The mouse anti-nanoLuc antibody (MAB10026) was purchased from R&D Systems. Rat anti-E2 clone 3/11, mouse anti-NS5A clone 9E10, and mouse anti-core clone 19D9D6 were kind gifts from Jane McKeating , Charles Rice , and Colette Jolivet , respectively.BODIPY 493/503, ProLong glass antifade mountant, Hoechst 33342, and protein G Dynabeads were purchased from Thermo Fisher. Mowiol 40-88, cycloheximide (CHX), and puromycin were purchased from Sigma-Aldrich.in vitro transcribed with a T7 RiboMAX Express Large Scale RNA Production System kit (Promega), and 10 μg of the in vitro-transcribed RNA was electroporated on Huh7.5 cells. At 48 h after electroporation, the cells were cultured under 0.25 mg/mL of neomycin until a uniform layer of resistant cells appeared. The resistant cell population was further validated for NS5A expression by IF and flow cytometry.XbaI-linearized pSGR-JFH1-neoR DNA was HCVcc production procedures were described previously . Extrace6 HEK293T cells were transfected with 2 μg of phCMV-G, 8 μg of psPAX2, and 8 μg of pWPI-CNS2 Jc1, pWPI-CNS2 JFH1, pWPI-core J6, pWPI-core JFH1, pHR′-CMV-nLuc-WPRE, TRCN0000046913, TRCN0000046914, TRCN0000046916, TRCN0000296791, TRCN0000296863, TRCN0000296863-puro(−), SHC001, or SHC001-puro(−) plasmids using calcium phosphate precipitation. At 16 h posttransfection, the medium was changed and harvested 24 h later, filtered through 0.45-μm-pore filters, and stored aliquoted at −80°C. Lentiviral vectors expressing HCV proteins or nLuc were titrated on Huh7.5 cells. Briefly, 105 Huh7.5 cells were transduced with serial dilutions of the lentiviral vectors, and the expression of the HCV core or nLuc was analyzed 72 h later by flow cytometry.A total of 2.4 × 105 cells were seeded on 6-well plates containing uncoated 14-mm-diameter glass coverslips. Eight hours later, cells were transduced with lentiviral vectors expressing a control shRNA (SHC001) or an shRNA against Nup98 . The sh5 shRNA, which conferred the best downregulation of Nup98, was used in all experiments and referenced as shNup98, with the exception of A total of 1 × 106 Huh7.5 or SGR cells were infected with Jc1 at an MOI of 0.5 or transduced with CNS2 Jc1-expressing lentiviral vectors at an MOI of 5, respectively. For experiments with nLuc vector, 1.2 × 106 Huh7.5 cells were transduced with nLuc-expressing lentiviral vector at an MOI of 10. Six days postinfection or transduction, 4 × 106 cells were washed and lysed at 4°C for 30 min in lysis buffer . Lysates were clarified at 18,000 × g at 4°C for 15 min and incubated overnight with 50 μL of protein G Dynabeads coated with 5 μg of anti-Nup98 or anti-rat IgG. Immune complexes were washed 3 times with washing buffer 1  and twice with washing buffer 2  and eluted with Laemmli buffer for 5 min at 95°C before Western blot analysis or RNA quantification.A total of 1.2 × 10Proteins obtained in total cell lysates or by immunoprecipitation were denatured in Laemmli buffer at 95°C for 5 min, separated by SDS-PAGE, and then transferred to nitrocellulose membrane and revealed with specific primary antibodies, followed by the addition of IRdye secondary antibodies , and analyzed by imaging with an Odyssey infrared imaging system CLx (Li-Cor Biosciences).TCTGCGGAACCGGTGAGTA-3′ and 5′-TCAGGCAGTACCACAAGGC-3′; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-AGGTGAAGGTCGGAGTCAACG-3′ and 5′-TGGAAGATGGTGATGGGATTTC-3′; for transferrin (TF), 5′-CCCTTAACCAATACTTCGGCTAC-3′ and 5′-TTTGCCAAGTTCTCAAATATAGTCG-3′; for actin, 5′-TCCGTGTGGATCGGCGGCTCCA-3′ and 5′-CTGCTTGCTGATCCACATCTG-3′; for CIDEB, 5′-AGCCAAAGCATTGGAGACCCTACT-3′ and 5′-TCTGACCAGACTGCAACACCATCA-3′; and for HIV vector (nLuc), 5′-TGTGTGCCCGTCTGTTGTGT-3′ and 5′-GAGTCCTGCGTCGAGAGAGC-3′. RNA levels were normalized with respect to GAPDH RNA levels. As an internal control of extraction, an exogenous RNA from the linearized Triplescript plasmid pTRI-Xef (Invitrogen) was added into the supernatant prior to extraction and quantified with specific primers (5′-CGACGTTGTCACCGGGCACG and 5′-ACCAGGCATGGTGGTTACCTTTGC).HCV RNAs were extracted , reverse transcribed , and quantified  on an Applied StepOne real-time PCR apparatus. The sequences of the primers used for the reverse transcription-quantitative PCR (RT-qPCR) were as follows: for HCV, 5′-2O). At 72 h postinfection, cells were washed with PBS, fixed with 3% paraformaldehyde (PFA), and further stained for core, Nup98, and RanBP2 proteins to assess the Nup98/RanBP2 colocalization by confocal microscopy.Synthetic peptides were manufactured by GenScript (Netherlands) and have been previously described . PEN was4Cl, and permeabilized with 0.1% Triton X-100 for 7 min. Fixed cells were then incubated for 1 h with primary antibodies in 1% BSA–PBS, washed and stained for 1 h with the corresponding fluorescent Alexa Fluor-conjugated secondary antibody  in 1% BSA–PBS. LDs were stained with 10 μg/mL BODIPY 493/503 according to the manufacturer’s instructions. Cells were washed three times with PBS, stained for nuclei with Hoechst for 5 min when stated, washed and mounted in Mowiol prior to image acquisition with LSM-710 confocal microscope.Huh7.5 cells grown on uncoated 14-mm-diameter glass coverslips were infected at MOI of 0.2. At different times postinfection, cells were washed with PBS, fixed with 3% paraformaldehyde in PBS for 15 min, quenched with 50 mM NHViral proteins were first immunostained as described above. After a postfixation step with 3% paraformaldehyde for 15 min and 3 washes with PBS, HCV RNA plus and minus strands were detected using probe sets that target regions between nucleotide positions 3733 to 4870 and 4904 to 5911, respectively . In the JFH1 genome, the nLuc RNA was detected using a probe that targets the full WPRE sequence contained in the lentiviral genome sequence , and actin and GAPDH RNAs were detected with the control probes VA4-10293 and VA4-10641, respectively (Affymetrix), using the QuantiGene ViewRNA ISH cell assay kit (Panomics/Affymetrix) according to the manufacturer’s instructions, except for the protease digestion step, which was omitted. Staining of nuclei, slide mounting, and acquisition were performed as described above.5 Huh7.5 cells were seeded on 6-well plates containing uncoated 14-mm-diameter glass coverslips. Eight hours postseeding, cells were transduced with lentiviral vectors expressing a control shRNA [SHC001-puro(−)] or an shRNA against Nup98 [TRCN0000296863-puro(−)]. Sixteen hours postransduction, cells were infected with Jc1 or JFH1 viruses at MOI of 0.2. At 72 h postinfection, cells were processed for ribopuromycylation as previously described . The cells were then washed once with wash buffer , fixed with 3% paraformaldehyde for 15 min at room temperature, and processed for IF and FISH.A total of 10Huh7.5 cells were grown on high-precision cover glasses and infected (MOI of 0.2). At 72 h postinfection, cells were fixed with 3% paraformaldehyde and stained as described above. Cells were then mounted in ProLong glass antifade mountant prior to image acquisition with Elyra PS-1 microscope. Images were reconstructed using the Zen 2012 Black software.2, which corresponds to half of the resolution limit of confocal laser scanning microscopy, were recorded. For quantification of Nup98 apposed at the edge of LDs or apposed to HCV (+)RNA, we used an ImageJ macro that we previously described . P values of <0.05 were considered statistically significant, and the following denotations were used: ****, P ≤ 0.0001; ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05; ns, not significant (P > 0.05).Significance values were calculated by applying the two-tailed, unpaired Mann-Whitney test for image analyses and the paired


In this paper, we extend the tangle-based model with its minimal prime factorization, useful to analyze patterns that characterize the RNA secondary structure.Due to its key role in various biological processes, RNA secondary structures have always been the focus of in-depth analyses, with great efforts from mathematicians and biologists, to find a suitable abstract representation for modelling its functional and structural properties. One contribution is due to Kauffman and Magarshak, who modelled RNA secondary structures as mathematical objects E. coli tRNA and provide some visual examples of interesting patterns.By leveraging the mapping between RNA and tangles, we prove that the prime factorizations of tangle-based models share some patterns with RNA folding’s features. We analyze the We formulate an open question on the nature of the class of equivalent factorizations and discuss some research directions in this regard. We also propose some practical applications of the tangle-based method to RNA classification and folding prediction as a useful tool for learning algorithms, even though the full factorization is not known. A secondary structure appears when the RNA molecule folds onto itself creating additional weaker bonds, called Watson-Crick pairs  and Wobble pairs (G-U). Figure dot-bracket notation, a string in which a pair of matching brackets correspond to a weak bond in the secondary structure and dots unpaired nucleotides. The dot-bracket string can also be represented by a flattened diagram, that is a set of points displayed horizontally (representing the nucleotides) joined by an arc in the upper half part of the diagram (representing the pairs). Since every arc has to connect two dots, every flattened diagram has N arcs and 2N paired dots.In biological cells, RNA is a molecule that regulates a huge variety of functions. It consists of a long chain of smaller molecules, called nucleotides, bonded sequentially (Adenine (A), Guanine (G), Cytosine (C), and Uracil (U)), known as the hairpins, bulges, stems, interior loops  that can be categorized as see Fig. , and mulsee Fig. .Fig. 3A pseudoknot, where an unbonded nucleotide is bonded with another nucleotide in a different loop of the RNA molecule  into the host cells in order to replicate themselves. Moreover, it is still in the debate that the self-replicating capabilities of RNA may have given the basis for early life on Earth even before DNA appeared .This work proposes a different way to investigate RNA folding with an algebraic structure during the process of optimization, exploiting its decomposition in prime factors.A monoid is an algebraic structure made by a set of elements and an associative binary operator equipped with an identity element.N and a set of 2N dots in tangle is a set of N pairs  of distinct dots, such that no dot occurs in more than one edge. Tangles are represented graphically by drawing two rows of N dots labelled with [N] if they are on the top and labelled with invariant and we will represent it by separating edges by commas and pair of dots by colons ;N nucleotides and N pairs;discard the unpaired nucleotides, there are now 2shape  and nucleotides in the bottom row with numbers in enumerate the nucleotides in the top row with numbers in [As Giegerich et al. pointed out, the study of the shape of an RNA secondary structure lifts the user from the burden of paying attention to changes that do not affect the overall desired structure, which means that we do not lose information because we are doing a static analysis [-tangle:X are transversal);a tangle -tangle:X has a lower hook h of size a tangle -tangle:-tangle with the extra condition of having only primes as factors ;-tangle:big hook because they will always have a lower hook h of size all the other tangles  that this subdivision generates. Each region will correspond to a prime, and if two regions Ernst et al. defined a factorization algorithm that constructs a minimal factor list given an input L-tangle . Their a-tangle is a tangle in the form X. To do this, we will merge the lower hook Recall that a X and adding edges a and b such that if e is a hook or a negative transversal, then e is a positive transversal, then We say that we merge a lower hook primes in its factor list, we would like this merging process to maintain the crossing number constant, in this way we are sure to not include any more primes in the non-optimal factor list we are calculating.Since the number of crossings in a tangle corresponds to the number of Letbe atanglewithcnumber of crossings and with a lower hookLetFor all edgescalculateinter(e) to be the number of intersectionsehas with edges inI. Let be the set of edges that intersect both edges inI, for eachcalculate the number of crossings the tanglewould have if we mergedhwitheand pick the tangle whose number of crossings is equal toc. If more than one edge satisfies this last condition, among them, pick the edge that has the least amount of intersections inX.I, it will happen that some edges in X will share a dot with edges in I. We count them too as intersecting.Note that, for the case of edges in h with N edges in the worst case, the time complexity for this heuristic is Merging two edges takes constant time, but the calculation of the crossing number takes nt}O(N2) , and sin-tangleX by transforming it into a -tangle. The idea is to take one of the lower hooks h with size shrink it until it becomes of size one. To do this we compose X with primes until this condition is met. During the shrinkage process, other edges will inevitably change size. In order to decide where we should shrink h, we use a heuristic that chooses a location where the size of the other edges increases the least. We apply this heuristic to the smallest lower hook of X, in this way there will be no smaller lower hook inside of it.We will extract factors from a Given a-tangleX, letbe the smallest lower hook ofXof sizeLetjbe the index of the shrinkage location where the size of the other edges increases the least. Shrink the lower hookhinto locationjby composingXwithandThis procedure yields a-tanglesuch thatThe notation This heuristic is not optimal, but it can be computed in linear time.delete rules and R11–13 move rules. We can use them to minimize a non optimal factor lists by implementing them in a rewriting logic tool .We will now provide an example of the mapping procedure for deriving, from a RNA secondary structure, a tangle with its prime factors.E. coli tRNA in Fig. We will start from the modified This diagram is reduced to obtain a shape diagram Fig. b that caX is a -tangle because there is a lower hook of size 1 . The algorithm now tries to factorize the tangle returned from the last step  is that every secondary structure without pseudoknots maps to a -tangle. The intuition behind this result is that the number of valid ways we can arrange 2N open and closed parenthesis of a single type is the Catalan numberprime as a factor.The resulting tangle is invariant to ument}BN , 23. ThiLet us show some other properties using the example we provided in the previous section Fig. . In the mentclass2pt{minims of an RNA secondary structure, stem or pseudoknot, we write i and ending in position j (with X and an edge gen(e) to indicate the factors that generate it.We will give a mathematical foundation for these empirical results. Given a section If an RNA secondary structure has a stemswiththen the index of every factor of the corresponding tanglewill always be greater or equal to two. The converse is also true.X be the corresponding tangle, then X. The backward argument is also valid. Assume that an RNA shape has an edge Letsbe a stem of an RNA secondary structure and letpbe a pseudoknot starting inside the hairpin ofsand ending outside of it. Thenedge(s) will crossedge(p).edge(s) to be a 2-dimensional closed curve edge(p) starts inside of edge(p) must cross edge(p) must cross edge(s). We can abstract LetXbe a tangle withand letIfandcross, then there existsfor somei.P that generates their crossing. But since every intersection is generated by a prime, P must be a prime. This implies that a prime generates both Since The existence of equivalent factorizations leads us to reason about an open question:commutative factors and, in general, of equivalent factorizations?What is the biological interpretation of equivalent factorizations up to commutativity (R13)equivalent factorizations up to R11 and R12The reason for this distinction is that R13 does not really impose a challenge during factorization, recall that R13 is defined as:We hypothesise two separate research directions, regarding:p connecting stems We can also discuss another research direction by analyzing different mappings from RNA secondary structures to tangles. For example, in the mapping we discussed in this paper, if there is a pseudoknot her Fig. .We did not explore this alternative mapping, so we leave it as a future research direction.Regarding the factorization algorithm, there are also some improvements that can be done with respect to the time complexity. Our methodology uses heuristics to obtain a non-minimal factorization and then refines it by using rewriting logic. This last step becomes prohibitive for large tangles, therefore a faster approach is necessary. During our research, we did not find an algorithm capable of such performances, but we have the hypothesis that the factorization problem for the Brauer Monoid could be solved in polynomial time.Let’s discuss now some practical applications our methodology could be used for.The factor representation we have discussed in this paper can be useful as an additional classification criterion for RNA secondary structures databases, in which a user could query RNAs that are generated only by a particular set of prime tangles, without the need of specifying the exact shape of the RNA molecule they are interested in. This could also lead to interesting applications in the context of sequence alignment, in which two sequences are compared not by the alignment of their nucleotides, but by their factor list.As we discussed in “We have crossed the bridge that Kauffman and Magarshak have built between RNA secondary structures and the Brauer Monoid to pave the way for a novel prime tangle factorization for RNA secondary structures. Our results show that the presence of pseudoknots influences the type of factors the corresponding tangle has. Moreover, we proved that two interconnected sections of the RNA secondary structure will naturally share some factors. Since the exact interpretation of equivalent factorizaion is not clear, we expect further development in this direction. In any case, the proposed approach may reveal useful for reducing the search space for the optimal folding and for structure comparison and classification.