The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis. Ribonuclease L (RNase L) initiates rapid and widespread degradation of cellular mRNAs in response to double-stranded RNA or viral infection. How the mass degradation of mRNAs affects cellular functions is unknown. Herein, we show that RNase L-mediated mRNA decay is predominately localized to the cytoplasm. Consequently, RNA-binding proteins re-localize from the cytoplasm to the nucleus upon RNase L activation. Coinciding with the influx of RNA-binding proteins into the nucleus, we also observe perturbation of nuclear processing of mRNAs, and these processing defects down-regulate key antiviral cytokines, such as type I interferons. Importantly, we observe these processes occurring during dengue virus and SARS-CoV-2 infection, revealing previously unknown aspects of antiviral gene regulation during pathogenic viral infections. Combined, these findings uncover novel mechanisms by which RNase L regulates host gene expression and expand the potential means by which RNase L blocks protein synthesis. Ribonuclease L (RNase L) is an endoribonuclease that limits the replication of diverse viruses, including influenza virus, chikungunya virus, SARS-CoV-2, and vaccinia virus in mammals –4. RNaseinterferon mRNAs) escape RNase L-mediated mRNA decay. Second, it inhibits the assembly of stress granules [We, and others, recently demonstrated that RNase L activation results in widespread degradation of host mRNAs ,14. Thisgranules , cytoplagranules –18. Thirgranules . Fourth,granules ,20. Lastgranules .Similar to RNase L-mediated mRNA decay, cellular mRNAs are reduced by Kaposi’s sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68) via the SOX and muSOX proteins, respectively, which results in the translocation of host RNA-binding proteins, including PABP, to the nucleus –25. MoreIFN mRNAs. These are general responses since we observe these alterations in RNA processing occur during exposure to exogenous dsRNA, dengue virus infection, or SARS-CoV-2 infection. These findings show that RNase L-mediated mRNA decay alters the balance of RNA-binding protein subcellular localization, host RNA processing events, and antiviral gene expression.Herein, we demonstrate that RNase L-mediated degradation of host mRNAs primarily occurs in the cytoplasm, which leads to the re-localization of many RBPs to the nucleus in a manner dependent on intact nuclear RNA. This demonstrates the principle that the distribution of RBPs between subcellular compartments is dependent on the availability of their binding sites. The influx of RBPs into the nucleus is concurrent with global RNase L-dependent RNA processing alterations including alternative splicing, intron retention and transcription termination defect leading to downstream of gene (DoG) transcript production, which correlate with inhibition of nuclear export of Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, and cellular stresses are known to modulate the location of several RBPs ,28. For RNase L degrades RNA and inhibits stress granules . ConcurrWe transfected parental (WT) or RNase L-KO (RL-KO) A549 cells with or without poly(I:C), a double-stranded viral RNA mimic that initiates the OAS/RNase L, RLR-MAVS, and PKR innate immune response pathways. We first performed immunofluorescence assays for PABPC1, which we previously determined translocates to the nucleus in an RNase L-dependent manner . ConsistWe next analyzed HuR, which primarily localizes to the nucleus in unstressed cells . In WT cOne possibility is that the inhibition of SGs results in RBP re-localization to the nucleus during stress. To examine this, we performed IF on additional RBPs that typically localize to SGs in WT and RL-KO cells transfected with or without poly(I:C). A striking result was that most RBPs we examined translocated to the nucleus in a RNase L-dependent manner in response to poly(I:C), including PABPC4, FAM120A, TIA-1, Caprin-1, and G3BP1 Figs  and S1. One mechanism by which RNase L could alter the localization of RBPs between the nucleus and cytoplasm is differential RNA degradation between the cytoplasm and nucleus, whereby higher RNA decay in the cytoplasm relative to the nucleus would lead to disassociation of RBPs from cytoplasmic RNA more rapidly than from nuclear RNA. To assess this, we quantified poly(A)+ RNA in the cytoplasm and nucleus of WT and RL-KO cells lipofected with or without post-poly(I:C) . We alsoGAPDH mRNA accumulates in the nucleus while being degraded in the cytoplasm when RNase L is activated [Importantly, we did not observe a significant reduction of poly(A)+ RNA staining in the nucleus of WT cells that activated RNase L (RLB-positive) , whereasctivated . These dWhile previous studies have shown that RNase L can localize to both the cytoplasm and nucleus ,33, we oIn principle, RNase L activation could alter RBP localization in two manners. First, RBPs may be subject to post-translational modifications, indirectly promoted by RNase L activation, that increase their nuclear accumulation. Alternatively, free RBPs may shuttle between the nucleus and cytosol faster than RBPs bound to RNA, and therefore the degradation of bulk cytoplasmic RNA would allow RBPs to shuttle to the nucleus, where binding to nuclear RNA would retain the RBPs in the nucleus. A prediction of this latter model is that the accumulation of nuclear RBPs will be dependent on the presence of a pool of nuclear RNA to bind the RBPs and increase their dwell time in the nucleus .To test whether nuclear RNA is required for RBP accumulation in the nucleus, we assayed RBP localization when nuclear RNAs are degraded concurrently with cytoplasmic RNAs in response to RNase L activation . To do tStrikingly, RNase L-mediated degradation of both nuclear and cytoplasmic RNA reduced the levels of PABPC1, PABPC4, HuR, and TIA1 in the nucleus in response to poly(I:C) . SpecifiThus, RNase L activation increases the nuclear localization of several RBPs by increasing the relative number of RBP-binding sites in the nucleus relative to the cytoplasm because of degradation of cytoplasmic but not nuclear mRNAs.The above data imply that RBPs translocated to the nucleus upon RNase L activation associate with RNA. This influx of RBPs would be predicted to compete for RNA-binding with RBPs involved in mRNA processing and thereby alter nuclear RNA processing. To examine if RNase L activation alters RNA processing, we analyzed high-throughput RNA sequencing (RNA-seq) of WT and RL-KO cells following six hours of either mock treatment or poly(I:C) lipofection .To examine if there were changes to alternative splicing, we utilized MAJIQ see  which idThis analysis identified 140 splicing events, across 136 genes, that showed differential splicing either due to poly(I:C) treatment in the WT cells, or were different between the WT and RL-KO cell lines post-poly(I:C). These changes are calculated as the difference in PSI between two conditions, the ΔPSI. The changes in splicing in both cases were generally correlated, which indicates that the majority of changes in alternative splicing observed are due to activation of RNase L . MoreoveTo validate the analysis of RNA-Seq data, we prepared RNA from mock and poly(I:C) treated WT and RL-KO cells and examined a subset of splicing events by low cycle radiolabeled RT-PCR. In all four cases examined , we observed a change in exon inclusion with poly(I:C) treatment that was dependent on RNase L . This deAnother alteration of splicing can be overall decreased splicing rates and the increased retention of introns (intron retention (IR)). To determine if intron retention is affected by RNase L activation, we calculated an intron/exon ratio  per RNA for WT and RL-KO cells with or without poly(I:C) transfection. An increase in intron retention correlates with an increase in the intron/exon ratio.A striking result was that the density plots of all analyzed RNAs showed a shift to higher intron/exon ratios upon poly(I:C) treatment in WT cells, highlighting an RNase L-dependent increase in intron retention . The sam1-5000/exon ratio, where DoG1-5000 corresponds to TPM normalized counts over the first 5000 nt after the annotated gene end  transcriptional read-through, which are observed during viral infection and various other stresses and are consistent with defects in transcription termination –39. To dend Figs  and S4 , we observed DoG transcripts, which were notably absent in poly(I:C)-transfected RL-KO cells . For exaWe considered the possibility that the RNase L-dependent effects on splicing and DoG transcription identified via our RNA-seq analysis could be a technical artifact of cytoplasmic-specific RNA decay, which we postulated could increase nuclear RNA processing intermediates relative to mature, cytoplasmic RNAs. However, three observations indicate that this does not solely account for the increased intron and DoG RNA reads.First, we did not observe RNase L-dependent RNA processing alterations in all genes, indicating specificity to RNase L-dependent RNA processing effects. For example, although both GAPDH and ACTB mRNAs are substantially reduced in the cytoplasm but not in the nucleus in an RNase L-dependent manner , we did Third, we confirmed RNase L-dependent DoG formation in the long non-coding RNA, NORAD, via smFISH. We stained cells with smFISH probes targeting either the NORAD RNA region or the region of the NORAD gene downstream of the transcription termination/polyadenylation site (PAS) twelve hours post-poly(I:C) lipofection . ConsistThese observations indicate that RNase L activation results in widespread nuclear RNA processing alterations, including changes in alternative splicing, intron retention and reduced transcription termination.IFNB1 and IFNL1 in WT cells in comparison to RL-KO cells  to allow for more time for cells to induce interferon genes and increase the potential RNase L-mediated effects on interferon mRNAs.A key aspect of the cellular response to dsRNA is the induction of antiviral gene expression. Given this, we examined how RNase L activity affected the processing of mRNAs transcriptionally induced by dsRNA, including type I and type III interferons (IFNs). We observed that RNase L altered the RNA processing of the pre-mRNAs for both types of interferons. For example, we observed increased RNA-seq reads mapping downstream of KO cells , indicatetention . To valiIFNL1 and IFNB1 genes. First, in a portion of WT cells that induced IFNB1 or IFNL1 expression in response to poly(I:C), as determined by co-smFISH for the CDS regions of these genes   . We note49 cells .IFNB1 and IFNL1 DoG-1 RNA staining was significantly less abundant in RL-KO cells in comparison to WT cells , and this was observed in both WT and RNase L-KO cells  lipofection.We have previously documented that RNase L activation can lead to nuclear retention of host and viral mRNAs . The obsIFNB1-DoG RNAs are almost exclusively localized in the nucleus, even in cells that have exported IFNB1 mRNAs that only stain for the CDS region to the cytosol . However, we cannot rule out the formal possibility that all the nuclear retained IFNB1 RNAs could contain DoGs, but with some being too short to hybridize to the DoG probes. However, we did not observe a gradual reduction in RNA-seq reads in the DoG-1 region of IFNB1 mRNAs  was much higher than the number of both DoG-1 and intron foci in the nucleus , both of which activate RNase L [IFNB1 of IFNL1 DoGs or introns.One limitation of the above experiments is that we have used poly(I:C) as a dsRNA mimic of viral infection. To verify that these alterations were relevant to viral infection, we examined whether RNase L-dependent re-localization of RBPs and nuclear RNA processing alterations occurred during viral infection. We analyzed PABPC1 localization via immunofluorescence in WT and RL-KO A549 cells following infection with either dengue virus serotype 2 (DENV2) or SARS-CoV-2  transfection or viral infection triggers the accumulation of RBPs in the nucleus and affects nuclear RNA processing of antiviral mRNAs, with a stronger effect on transcriptional termination leading to the production of DoGs for these mRNAs.Taken together, the analysis of IFNB1 RNA export and processing. This is based on earlier work that the accumulation of PABPC1 in the nucleus inhibits mRNA export, promotes hyperadenylation of mRNAs, and inhibits transcription [IFNB1 mRNA and DoG transcriptional read-through of the IFNB1 gene.An unresolved issue is the specific RBPs that accumulate in the nucleus and which transcripts are affected by one or more specific RBPs. One hypothesis is that PABPC1 accumulation in the nucleus could alter cription ,25,26. TIFNB1 mRNA retention by measuring the immunofluorescence intensity of PABPC1 in the nucleus of cells that induced IFNB1 mRNA, which we measured by smFISH. These analyses revealed that IFNB1 mRNA could be primarily localized to the cytoplasm in cells that contained nuclear PABPC1 accumulation , similar to previous studies [IFNB1 mRNA. Importantly, in cells that contained nuclear mRuby2-PABPC1-NRS, IFNB1 mRNA was abundant in the cytoplasm (IFNB1 mRNA export. studies . Six houytoplasm  and equiytoplasm . These dXRN1 transcript (siXRN1). Western blot analysis demonstrated siXRN1-specific reduction of XRN1 protein + RNA or northern blot of reporter RNAs ,22. MoreIFNB1 DoG RNAs in WT cells than RL-KO cells, indicating that DoG RNA generation is a consequence of RNase L activation in response to SARS-CoV-2 infection. Whether this is beneficial for SARS-CoV-2 since it reduces host antiviral gene expression is unclear. However, since RNase L reduces SARS-CoV-2 replication in cultured cells by only 3–4 fold, presumably via decay of the viral mRNA [in vivo.An unexpected observation is that SARS-CoV-2 infection, which results in rapid degradation of host mRNAs even in the absence of RNase L through the action of the SARS-CoV-2 Nsp1 protein , does noral mRNA , perhapsIFNB1 transcripts that contained DoG RNA disseminated throughout the nucleus , may prove to be new antiviral mechanisms that perturb viral gene expression or promote the host immune response. Moreover, since RBPs can directly modulate transcription ,46, this2 and 37 degrees Celsius in Dulbecco’s modified eagle’ medium (DMEM) supplemented with fetal bovine serum  and penicillin/streptomycin (1% v/v). Routine testing for mycoplasma contamination was performed by the cell culture core facility. African green monkey kidney cells  were maintained at 5% CO2 and 37 degrees Celsius in DMEM supplemented with FBS (10% v/v), 2 mM non-essential amino acids, 2 mM l-glutamine, and 25 mM HEPES buffer.Parental and RNase L knockout A549 cell lines are described in . Cells wFor transfections, high-molecular weight poly(I:C) (InvivoGen: tlrl-pic) and 3-μl of lipofectamine 2000 (Thermo Fisher Scientific) per 1-ug or poly(I:C) was used per manufacturer’s instructions. Cells were transfected with 500-ng of poly(I:C) per volume of media (1-ug in 6-well format).Viral infections are described in ,20. BrieThe generation of the pLenti-ef1-blast–mRuby-2-PABPC1 lentiviral plasmid is described in . Brieflyhttps://github.com/Dowell-Lab/RNAseq-Flow) as follows. Reads were trimmed using BBduk version 38.05 with the following flags ktrim = r qtrim = r, trimq = 10, k = 23 mink = 11 hdist = 1 maq = 10 minlen = 25 ). Trimmed reads were mapped to the human reference genome (GRCh38/hg38) using HISAT2 version 2.1.0 with the very-sensitive, pen-noncansplice 14, mp 1,0, and sp 3,1 flags [https://tools.thermofisher.com/content/sfs/manuals/ERCC92.zip), counted using featureCounts and the ERCC Spike-Ins size factors derived by DESeq2. Significant differentially upregulated or downregulated RNAs were selected by baseMean >5, padj < 0.05 and a log2fold change of 1. Nonsignificant genes were selected by padj > 0.05 and log2fold change of -0.5 to 0.5.Our previously published RNA-seq data  were re-,1 flags . SAMtool,1 flags . TDV fil,1 flags . Normali,1 flags . Exon or,1 flags . GTFtool,1 flags  and to gIntron retention analysis was performed on all RNAs with the Deseq2 baseMean cutoff > 30, to remove RNAs with too low counts. Intron/exon ratios were derived by diving the TPM normalized intron counts by TPM normalized exon counts. Intron/exon ratios > 8.5 were a result from “towers” in intronic regions, counts in non-annotated regions, noise due to low counts or read-through from an upstream gene. Therefore, all intron/exon ratios >8.5 were removed from analysis.We note that for upregulated genes, a shift towards higher intron/exon ratio is visible in wild-type cells compared to RL-KO cells, consistent with an increase in intron retention. However, an additional shift is also visible in both unstressed conditions . This is1-5000 counts were divided by the TPM normalized exon counts to derive and a DoG/exon ratio. For the same reasons as describe for intron/exon ratios, DoG/exon ratios > 5 were removed from analysis. Moreover, small RNAs (exon size < 300bp) were removed from analysis.DoG formation was estimated similarly, by generating TPM normalized counts over the first 5000 pb following the exon annotation. These TPM normalized DoGIn our ratio analysis, genes were not filtered for “clean” genes, genes that have counts due to expression and not due to read-through transcription from up-coming gene . HoweverFor the splicing analysis, raw fastq reads were trimmed to remove adapters and low quality reads with bbduk/38.79 , alignedValidation of splicing events by low-cycle radiolabeled PCR was performed as previously described  with theImmunoblot analysis was performed as described in . Mouse ahttps://biosearchassets.blob.core.windows.net/assets/bti_custom_stellaris_immunofluorescence_seq_protocol.pdf). GAPDH smFISH probes labeled with Quasar 570 dye (SMF-2026-1) were purchased from Stellaris. Custom IFNB1, IFNL1, SARS-CoV-2, and DENV2 smFISH probes [http://biosearchtech.com/stellaris-designer. Reverse complement DNA oligos were purchased from IDT  was used at 1:1000. Rabbit polyclonal anti-HuR antibody (Proteintech: 11910-AP) was used at 1:500. Rabbit polyclonal anti-PABPC4 antibody (Thermo Scientific: 14960-1-AP) was used at 1:300. Rabbit anti-FAM120A (Sigma–Aldrich: HPA019734) was used at 1:500. Rabbit polyclonal anti-TIA1 (Abcam: ab40693) was used at 1:500. Rabbit polyclonal anti-Caprin1 (Fisher Scientific: 50–554–357) was used at 1:500. Rabbit polyclonal anti-PUM1 (Thermo Fisher Scientific: PA5-30327) was used at 1:500. Mouse anti-ataxin2 antibody (BD biosciences: 611378) was use at 1:500. Rabbit polyclonal anti-FMRP (Abcam: ab17722) was used at 1:500. Mouse monoclonal anti-G3BP antibody (Abcam: ab56574) was used at 1:1000. Goat anti-mouse IgG FITC (Abcam: ab97022) was used at 1:1000. Goat anti-rabbit IgG Alexa Fluor 647 (Abcam: ab150079) was used at 1:1000.Coverslips were mounted on slides with VECTASHIELD Antifade Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) . Images were obtained using a wide-field DeltaVision Elite microscope with a 100× objective using a PCO Edge sCMOS camera. Between 10 and 15 Z planes at 0.2 μm per section were taken for each image for smFISH analyses. Five Z planes at 0.3 μm per section were taken for each image for immunofluorescence analyses. Deconvoluted images were processed using ImageJ with FIJI plugin. Z planes were stacked, and minimum and maximum display values were set in ImageJ for each channel to properly view fluorescence. Fluorescence intensity was measured in ImageJ. Single cells were outlined by determining the cell boundaries via background fluorescence and mean intensity was measured in the relevant channels. Imaris Image Analysis Software (Bitplane)  was used to quantify smFISH foci. Single cells were isolated for analysis by defining their borders via background fluorescence. Total foci above background threshold intensity were counted. Afterward, the nucleus marked with DAPI was masked, and foci were counted in the cell at the same intensity threshold cutoff, yielding the cytoplasmic foci count. For quantitative analyses, cells were analyzed in at least three fields of view from a single experiment in which all experimental and control samples were prepared simultaneously. Graphs generally display a single experimental replicate. However, several graphs show multiple replicates, which we note in the figure legends.BioRender.com.The model figure was created with https://www.socscistatistics.com/tests/anova/default2.aspx) or student’s t-test (Excel). The specific test used for each figure is specified in each figure legend. * p<0.05, **p>0.01, ***p>0.001 unless otherwise noted in the figure legend.P-values were derived either by one-way ANOVA with Tukey HSD  IF for indicated proteins in WT and RL-KO A549 cells four following transfection with or without poly(I:C). (D) IF for indicated proteins in WT and RL-KO A549 cells four hours post-poly(I:C). (E) Quantification of the nuclear: cytoplasmic ratio of proteins shown in (D).(TIFF)Click here for additional data file.S2 Fig(A) Number of splicing events with probability>0.9 for a |ΔPSI|>10%. (B) Dot plot of showing the ΔPSI (in fraction form) of splicing events in untreated vs poly(I:C)-treated WT and RL-KO cells. (C) Histograms showing the number of splicing events in each comparison binned by the probability that the |ΔPSI| > 10%(TIFF)Click here for additional data file.S3 Fig(A) Distribution of intron/exon ratios in WT and RL-KO cells following mock or poly(I:C) lipofection of RNAs that are unchanged (padj > 0.5) in WT cells following poly(I:C) lipofection. B) Read coverage mapping to an unchanged RNA–TTC32. (C) RNAs that are downregulated . D) Read coverage mapping to a down-regulated RNA–TMEM60. (E) RNAs that are upregulated . F) Read coverage mapping to an up-regulated RNA–NOCT. Blue and red traces correspond to mapped reads on positive and negative strand respectively.(TIFF)Click here for additional data file.S4 Fig(A) Distribution of DoG/exon ratios in WT and RL-KO cells following mock or poly(I:C) lipofection of RNAs that are unchanged (padj > 0.5) in WT cells following poly(I:C) lipofection. B) Read coverage mapping to an unchanged RNA–TTC32. (C) RNAs that are downregulated . D) Read coverage mapping to a down-regulated RNA–TMEM60. IGV trace scale was decreased 2.5x after autoscaling. (E) RNAs that are upregulated . F) Read coverage mapping to an up-regulated RNA–NOCT. Blue and red traces correspond to mapped reads on positive and negative strand respectively.(TIFF)Click here for additional data file.S5 Fig(A) smFISH for GAPDH or ACTB mRNA in WT and RL-KO cells following poly(I:C) transfection. (B) IGV track of GAPDH and ACTB genes in WT and RL-KO cells post-poly(I:C).(TIFF)Click here for additional data file.S6 Fig(A) IGV tracks for NORAD in WT and RL-KO cells. Below, smFISH probes targeting the NORAD RNA or DoG are shown. (B) smFISH for NORAD CDS and DoG RNA in WT or RL-KO cells twelve hours following mock transfection or transfection with poly(I:C).(TIFF)Click here for additional data file.S7 Fig(A) smFISH for IFNB1-CDS and IFNB1-DoG sixteen hours post-lipofection of poly(I:C), as shown in (TIFF)Click here for additional data file.S8 Fig(A) Co-smFISH for IFNB1 CDS and DOG-2 region in A549 WT and RL-KO cells sixteen hours post-lipofection with poly(I:C). (B) similar to (A) but for IFNL1 CDS and DoG-2 region.(TIFF)Click here for additional data file.S9 Fig(A) smFISH for IFNB CDS and DoG-1 in WT and RL-KO cells, as well as RL-KO cells stably expressing either RNase L or RNase L-R667A  twelve hours after transfection with poly(I:C). (B) Quantification of IFNB-DoG smFISH foci as represented in (A).(TIFF)Click here for additional data file.S10 Fig(A) smFISH for IFNB1-CDS and IFNB1-DoG eight hours post-lipofection of poly(I:C). Red arrows indicate cells that contain high levels of both nuclear-retained IFNB1-CDS and IFNB1-DoG foci that mostly co-localize. White arrows indicate cells that do not contain high levels of IFNB1-DoG foci, which remain largely localized the IFNB1 genomic loci, and IFNB1-CDS is primarily localized to the cytoplasm. Yellow arrows demarcate cells that contain high levels of nuclear-localized IFNB1-CDS but not IFNB1-DoG foci. (B) Box plots displaying of the number of IFNB1-DoG foci localized to the nucleus or cytoplasm in WT or RL-KO cells as represented in (A). Scatter plot of the ratio (nucleus/cytoplasm) of IFNB1-CDS foci (x-axis) and nuclear IFNB1-DoG foci in WT and RL-KO cells as represented in (A). (D) Scatter plots of the quantity of nuclear IFNB1-DoG foci (y-axis) and the quantity of nuclear IFNB1-CDS foci (x-axis) in WT and RL-KO cells as represented in (A). (E) smFISH for IFNL1-CDS and IFNL1-DoG in WT or RL-KO eight hours post-poly(I:C) lipofection.(TIFF)Click here for additional data file.S11 Fig(A) smFISH for GAPDH and DENV2 mRNAs in WT A549 cells 48 hours post-infection with DENV2 (MOI = 0.1). (B) similar to (A) but staining for PABP via IF.(TIFF)Click here for additional data file.S12 FigIFNB1 mRNA smFISH foci in the nucleus of A549 cells expressing either mRuby2-PABPC1 or mRuby2-PABPC1-NRS eight hours post-poly(I:C). Between 17–31 cells were analyzed from two independent experiments. (B) Nucleus to cytoplasm ratio of PABP fluorescence intensity in A549 cells forty-eight hours post-transfection of control siRNA (siNC) or siRNAs targeting XRN1 (siXRN1). Between 14–19 cells were analyzed from two independent experiments. (C) Quantification of total and nuclear IFNB1-CDS or IFNB1-DoG smFISH foci in A549 cells transfected with siNC or siXRN1 siRNAs eight hours post-transfection of poly(I:C). Between 16–28 cells were analyzed from two independent experiments. Statistical significance was determined using student’s ttest. *< 0.05, *(A) Quantification of the percent of (TIFF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Hyperuricemia and gout are two of the most common metabolic disorders worldwide; their incidence is increasing with changes in lifestyle, and they are correlated with many diseases, including renal and cardiovascular diseases. The majority of studies on hyperuricemia and gout have focused on the discovery of the associated genes and their functions and on the roles of monocytes and neutrophils in the development of gout. Virtually no studies investigating the epigenomics of gout disease or exploring the clinical significance of such research have been conducted. In this study, we observed that the expression of enzymes involved in RNA modifications or RNA editing was affected in uric acid (UA)- or monosodium urate (MSU)-treated cell lines. RNA alternative splicing and splicing factors were also affected by UA or MSU treatment. We used transcriptome sequencing to analyze genome-wide RNA splicing and RNA editing and found significant changes in RNA splicing and RNA editing in MSU- or UA-treated THP-1 and HEK293 cells. We further found significant changes of RNA modifications, editing, and splicing in patients with gout. The data indicate that RNA modifications, editing, and splicing play roles in gout. The findings of this study may help to understand the mechanism of RNA splicing and modifications in gout, facilitating the development of new diagnostic and therapeutic strategies. Hyperuricemia and gout are two of the most common metabolic diseases worldwide. Gout is more common in men than in women, and the male to female ratio ranges from 3:1 to 10:1. The global prevalence and incidence of gout are in the ranges of 1–4% and 0.1–0.3%, respectively . The preGout is a chronic disease characterized by the deposition of monosodium urate (MSU) crystals in joints and soft tissues, and its cause is related to an imbalance between urate intake/production and excretion, which leads to urate accumulation and crystallization in tissues . MSU cryAlthough the mechanism by which inflammation is activated in gout has been discovered, the mechanism of epigenomic regulation of gout remains unclear. Technological breakthroughs have led to epigenomics becoming one of the most rapidly expanding fields in biology . Epigene2. Uric acid (UA) was dissolved in 1 N sodium hydroxide and diluted in culture media to 3.5, 7, and 10.5 mg/dL. Media containing UA were adjusted to pH 7.4. MSU was dissolved in phosphate-buffered saline and diluted in culture media to 3.5, 7, and 10.5 mg/dL. Cells were seeded on a culture dish and treated with UA- or MSU-containing media for 48 h.THP-1 cells were grown in lipopolysaccharide-free complete RPMI medium containing 10% fetal bovine serum. HEK293 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. HUVEC cells were grown in Cascade Biologics Medium 200  plus Cascade Biologics Low Serum Growth Supplement  on a gelatin-coated dish. Cells were grown at 37°C in an incubator with 5% CORNA was extracted from UA- or MSU-treated cells or from the buffy coat of patients with gout by using TRIzol Reagent  or the NucleoSpin RNA kit , according to the manufacturer’s instructions. Two micrograms of RNA were subjected to reverse transcription polymerase chain reaction (RT-PCR) using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit , according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed on the Roche LightCycler 480 Real-Time PCR System using Universal ProbeLibrary System . The PCR process involved the following steps: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, 72°C for 1 min, and final extension at 72°C for 7 min. The PCR products were separated by 3% agarose gel electrophoresis. The intensity of the PCR products was analyzed using LabWorks Image Acquisition and Analysis Software . The primers for RT-PCR and real-time PCR are listed in t-tests were conducted to evaluate differences between groups. A probability of less than 0.05 was considered to be statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.0001.Statistical analysis was performed using Microsoft Excel 2010  and GraphPad Prism 5.01 . Student’s Tris-buffered saline with Tween 20 (TBST) for 60 min. The PVDF membranes were incubated with the primary antibody in 1% BSA overnight and then incubated with horseradish peroxidase–conjugated secondary antibody for 60 min. After washing three times with TBST, the PVDF membranes were exposed to the ECL Plus substrate  for 5 min. Chemiluminescence signals were detected, and the expression levels were analyzed using LabWorks Image Analysis Software. The primary antibodies used were anti-PUS1 , anti-PUS7 , anti-NSUN3 , anti-NSUN4 , anti-METTL14 , anti-YTHDC1 , anti-YTHDF3 , anti-ADAR1 , anti-RED1 , anti-SRPK1 , anti-SRPK2 , anti-SF2/ASF , anti-hnRNPA1 (Sigma-Aldrich), anti-Sam68 , anti-histone H1 , anti-ß-actin , and anti-tubulin .Proteins were extracted from UA- or MSU-treated cells or from the buffy coat of patients with gout by using RIPA lysis buffer  and were analyzed through SDS-PAGE: the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes  after electrophoresis. The PVDF membranes were blocked with 5% bovine serum albumin (BSA) solution in Blood samples obtained from 67 patients with gout were approved by the China Medical University and Hospital Research Ethics Committee (CMUH108-REC2-051). Approximately 10 mL of blood was collected in an EDTA tube, and buffy coat was used for RNA and protein extraction.That RNA was of high quality  was confirmed using the Agilent Bioanalyzer 4200 . One microgram of RNA was used for library preparation of the TruSeq Stranded mRNA Library Prep kit , according to the manufacturer’s instructions. Briefly, mRNA was purified using poly-A magnetic beads and fragmented through enzyme treatment. Subsequently, double-strand cDNA synthesis, end-repair, adaptor ligation, and enrichment PCR were performed. Samples were subjected to 2 × 150-bp paired-end sequencing using the Illumina NovaSeq 6000 platform.Base calling and quality scoring were performed with an updated implementation of Real-Time Analysis on the NovaSeq 6000 system. We used bcl2fastq Conversion Software v2.20.0.422) to demultiplex data and convert BCL files to FASTQ files. To facilitate downstream analysis, a quality control (QC) step was performed that involved the use of Trimmomatic tools (v0.39) . Sequenc.0.422 toEpigenomic regulations include DNA and RNA levels, and over 17 and 160 types of modifications have been detected in DNA and RNA, respectively , 29, 30.ADAR1 and ADAR2 expressions in THP-1 and HUVEC cells treated with UA or MSU . In addition to gene expression, we analyzed RNA splicing changes with RNA editing and found significant changes in alternative splicing of CARD8 with RNA editing on the splice site in MSU- or UA-treated THP-1 cells. Alternative splicing of non-coding RNA AC012313.3 with RNA editing on the splice site induced substantial changes in UA-treated HEK293 cells. These findings suggest that RNA editing was affected by MSU and UA treatment in a genome-wide manner.RNA editing is another critical mechanism in gene regulation. RNA editing is different from other RNA modifications because it alters the cellular fate of RNA molecules and their sequence relative to the genome. Adenosine to inosine (A-to-I) RNA editing is the most common type of RNA editing in vertebrates (higher eukaryotes). This editing reaction is catalyzed by the adenosine deaminase acting on RNA (ADAR) protein family . A-to-I A or MSU . The exp with UA , and thewith MSU . We alsowith MSU . Increaswith MSU , whereaswith MSU . No chanwith MSU , but thewith MSU . The quawith MSU ; 6,845 ewith MSU . A totalwith MSU ; 2,612 ewith MSU . Subsequectively . We furtectively . We alsoectively –39. FiveBCL-x, SMAC, and HIPK3 in UA- or MSU-treated cell lines; these genes were involved in apoptosis regulation. We found significant changes of BCL-x splicing in THP-1 cells treated with UA .Original datasets are available in a publicly accessible repository: NCBI Sequence Read Archive (SRA). The original contributions presented in the study are publicly available. This data can be found here: The studies involving human participants were reviewed and approved by China Medical University and Hospital Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.C-MH, P-HH, and J-GC designed the study. Y-CC, I-LL, Y-TL, J-CY, C-LL, and T-YL carried out the experiments. H-DC and S-JT performed the bioinformatic analyses. Y-CC and T-YL wrote the manuscript in consultation with C-MH, P-HH, and J-GC. J-GC supervised the project. All authors contributed to the article and approved the submitted version.


Tailoring of the optimized emulsomal surface formulation was performed using either cationization or PEGylation approaches to boost blood–brain barrier penetration. The pharmacokinetic assessment in rats showed significantly improved bioavailability of VNP emulsomal formulations compared to the oral market product. Additionally, surface-tailored emulsomes exhibited significantly higher brain levels compared to the optimized emulsomes. Based on these findings, the proposed surface-tailored emulsomes could be considered as a promising platform for achieving high brain levels of VNP following intranasal administration.Vinpocetine (VNP), a semisynthetic active pharmaceutical ingredient, is used for oral management of cerebrovascular diseases because of its ability to enhance the blood flow to the brain. However, despite that, the therapeutic application of VNP is restricted due to its reduced bioavailability and diminished brain levels that could be attributed to its low aqueous solubility, short half-life, and presystemic metabolism exposure. Accordingly, the goal of this work was to explore the ability of surface-tailored intranasal emulsomes to boost brain delivery of the drug. A 3 The blood–brain barrier (BBB) permeability and specificity represent crucial challenges for drug delivery to the brain in safe and adequate manner  disorders represent a major public health challenge  core enclosed by a phospholipid (PL) multilayer sheath. It combines advantageous characteristics of both nanoemulsions and liposomes -14,15-eburnamine) is a synthetic derivative of vincamine alkaloid used for the management of several CNS disorders including cerebrovascular ischemia, Alzheimer’s disease, and other different types of dementia , tristeararin (TS), tripalmitin (TP), stearylamine (SA), and cellophane membrane (MWCO 12000 − 14000 Da) were obtained from Sigma-Aldrich . Lipoid S 100® (L-α-Phosphatidylcholine from soya) and  (MPEG-DSPE) were a gift from Lipoid GMBH . All the remaining chemicals/materials were of analytical grade and obtained from Thermo Fisher Scientific Inc.  unless specified otherwise.2.2.The previously reported modified thin film hydration method of Paliwal et al.  was empl2.3.n = 6). Emulsomes were diluted appropriately prior to measurement.Zetasizer Nano S  was employed to measure the mean vesicle size (VS) and zeta potential (ZP) of VNP emulsomes  of VNP loaded emulsomes was determined indirectly. Emulsomal dispersions were subjected to ultracentrifugaiton at 100,000 rpm for 60 min at 4 °C for separation of unentrapped VNP . PBS at pH 6.8 was used for residue washing; the residue was subjected to ultracentrifugation again for 60 min. PBS at pH 6.8 was used to appropriately dilute the mingled supernatant prior to quantification of drug using a UV spectrophotometer  at 2.4.1.in vitro release using modified USP dissolution tester   for X1,  for X2, while X3 was explored at two levels (TS and TP). Eighteen runs were generated as per the design (E1-E18), while six runs were prepared for the optimized with surface modification (E19-E24) (3E19-E24) .1), ZP , EE , and RE at 24 h  were investigated as responses and Design-Expert software  was used for statistical data analysis. The model with the best-fit value of each parameter was chosen for each dependent variable. Analysis of variance (ANOVA) was employed to assess the significance of the independent variables on the responses and the interaction between them at 95% significance level. Further, the optimized VNP emulsomes were selected based on the desirability function. The criteria set for optimized emulsomes were minimized VS and RE24h, and maximized absolute ZP value and EE%.VS  along with drug and emulsomal constituents. The preparation was completed by using the previously mentioned method described in subsection 2.2 . The emulsomal formulation was applied after dilution to a carbon coated grid and allowed for adsorption for 2 min. Staining of the emulsomal dispersion was then performed by using phosphotungsitc acid. The grid was allowed for air-drying after excess stain removal. The samples were then visualized with magnification power of 22500X  were provided by the animal facility, King Abdulaziz University . The study was approved by the Research Ethics Committee, Faculty of Pharmacy (Ref. # PH-109-41). The random distribution of rats among 4 groups was performed; Groups I, II, and III were composed by animals in which the new formulations were tested, while the animals of Group IV received the standard control. The rats involved in the study received VNP in a dose of 10 mg/kg . Both plasma and brain homogenates were subjected to storage at −80 °C until analysis  was used . Statistical analysis was performed by using GraphPad Prism software, version 8.3.0 . ANOVA followed by a post hoc test were used for multiple comparisons. Specifically, Two-way ANOVA followed by Sidak’s multiple comparisons were applied for statistical analysis of plasma concentrations. Cmax and AUC were statistically analyzed using One-way ANOVA followed by Tukey’s multiple comparisons, while Tmax was statistically analyzed using nonparametric Kruskal–Wallis test. The statistical significance was set at p-value ≤ .05.Data were expressed as mean ± standard deviation (3.3.1.R2. The VS and RE data fitted the two-factor interaction (2FI) model, while the ZP and EE data fitted the linear model. The predicted R2 for VS, ZP, EE, and RE were 0.9569, 0.8463, 0.9178, and 0.8976, respectively, while the adjusted R2 for the studied variables were 0.9910, 0.9032, 0.9483, and 0.9785, respectively. The lowest predicted residual sum of squares (PRESS) confirms the validity of the selected models . Additionally, the interaction terms X1X2 and X2X3, corresponding to the interaction between PL:CH and either PL:SL or SL type (X2X3) were also significant . The three-dimensional surface plots presented in VS is one of the influential criteria that can exert an impact on drug entrapment, release, and cellular uptake or penetration via BBB  and PL:CH molar ratio (X2) exhibited significant impact on ZP (p < .001).ZP describes the surface charge of the vesicles and indicates their physical stability against aggregation. The vesicular dispersion is regarded as stable system for absolute ZP values greater than 30 mV owing to electrostatic repulsion that hinders vesicles clumping . This result is in agreement with the work carried out by Nayak et al. reporting no marked differences among various SL types in lipid nanoparticulate formulations . Nevertheless, SL type was not significant (p = .694). The entrapment of an active molecule in lipidic vesicular systems could enhance its biological availability as well as provide a controlled release profile . Furthermore, the interaction terms X1X3 and X2X3, corresponding to the interaction between SL type and either PL:SL ratio or PL:CH, were significant at the 95% level of significance.All the formulations exhibited slow drug release during the first 2 h with no observed burst release (data not shown). This finding might be due to the lipophilic nature of the drug that leads to its encapsulation within the lipophilic part of the emulsomes bilayer. Subsequent release of VNP from the proposed formulations was gradual over a period of 24 h, in accordance with previous studies , PL:CH (2:1), and SL type (TS) (E12). The combination of the optimized levels is predicted to fulfill the set goals with an overall desirability of 0.718. Thereof, E12, which is prepared at the combination of the optimized levels, was selected for further investigations. The measured responses of the optimized formulation are listed in Selection of the optimized formulation was performed on the basis of the desirability function. The criteria set for the optimization process was minimizing VS and RE3.7.Tailoring of the selected optimal VNP emulsomes surface was performed via either cationization or PEGylation using SA or MPEG-DSPE, respectively, with the aim of enhancing penetration via BBB. The development of cationic emulsomes by using SA allows for electrostatic attraction between the surface positive charge and the negative charges on the BBB resulting in adsorptive-mediated endocytosis and enhanced permeation via BBB  and their corresponding surface-tailored formulations. The sharp bright contour in 3.9.3.9.1.p = .001). Tukey’s HSD test, performed to point out the source of variation, showed a significant variation between the optimized emulsomes (E12) and each of the SA surface-tailored formulations . Nevertheless, no significant difference was found when using different concentrations of SA. The marked increase observed in VS of SA surface-tailored emulsomes compared to the corresponding optimized formulation could be attributed to the characteristics of the SA solution. Being viscous in nature, SA solution might influence the emulsomal dispersibility; further and mostly important, it might attract the surface polar heads of the PL bilayer owing to its positive charge. Pushing the surface polar heads of the PLs outwards might result in increased spacing. This observation is supported by the results reported by Narayan et al. (p = .002). As per the results of Tukey’s HSD test, the optimized VNP emulsomes differ significantly from the surface-tailored emulsomes prepared at various concentrations of MPEG-DSPE ; however, the effect of MPEG-DSPE concentration did not significantly influence the size. The marked reduction in the VS upon PEGylation might imply the penetration of the PEGylated polymer into emulsomal bilayer, pressing them together, and thus, enhancing PL packaging within the emulsomes bilayer . Tukey’s HSD test showed that E12 differs significantly from the cationic SA surface-tailored emulsomes . However, the difference between different concentrations of SA was not significant. The observed shift in the ZP is attributed to the key role played by the positive charge induction property of SA. Similar positive shift in the ZP upon addition of SA during pramipexole liposomal formulation was reported by Ghule & Bhoyar . Tukey’s HSD test showed that the ZP of each PEGylated emulsomal formulation differs significantly from the corresponding optimized non-PEGylated emulsomes (E12) . Additionally, a statistically significant difference (p = .023) between E22 and E24, prepared at 0.1 and 0.3 M MPEG-DSPE, respectively, was found. The impact of MPEG-DSPE on ZP could be explained on the basis of the charge-shielding influence of polyethylene glycol moiety . The observed non-significant increase in the EE% might be due to the increased spacing in the PL bilayer, caused by the electrostatic interaction between positively charged SA and negatively charged polar head groups, with consequent increase in the entrapping volume within the lipid bilayer. However, it was observed that the rise in SA concentration above 0.5 M caused decreased drug entrapment. This could be attributed to the increased repulsion between PL layers at higher SA concentrations, resulting in distortion of emulsomes structure and formation of unstable vesicles. These findings are in agreement with previous research studies reporting similar effects for both SA addition and SA variation concentration on EE vesicular systems . Tukey’s HSD test revealed that E12 differs significantly from each of the surface-modified emulsomes prepared at 0.2 and 0.3 M of the PEGylated polymer . Furthermore, a statistically significant difference was found between E22, prepared at 0.1 M, and E24, prepared at 0.3 M (p = .019). The increased drug entrapment of MPEG-DSPE surface-tailored emulsomes in comparison to the non-PEGylated optimized formulation might be due to the existence of PEG moieties on the emulsomal external surface that leads to vesicular stabilization and reduced VNP leakage. Additionally, incorporation of the PEGylated polymer could result in higher lipid amount available for better entrapment of the lipophilic drug. This result is in line with those reported by Sufhakar et al. that observed the enhanced entrapment of ritonavir within PEGylated liposomes . With the aim to point out the difference source Tukey’s HSD test was performed revealing that E12 does not differ significantly from any of its corresponding cationic emulsomes. Nevertheless, a significant difference was observed between E19, prepared at 0.25 M, and E21, prepared at 0.75 M (p = .032). The observed drug release delay upon addition of 0.25 M of SA could be explained based on the electrostatic attraction between the anionic drug and the cationic emulsomes. This interaction could result in confinement of the drug within the emulsomes with consequent reduced release. However, the observed faster release at higher SA concentrations could be explained on the basis of the previously described emulsomal structure disruption and reduced drug entrapment. Delayed release from positively charged vesicles compared to the neutral and anionic forms has been previously reported in different studies . Tukey’s HSD test showed that RE24h of each of PEGylated emulsome formulations differs significantly from that of the optimized formulation . Conversely, no significant effect was revealed for the concentration of MPEG-DSPE. The reported delay of VNP release upon addition of MPEG-DSPE might be due to the increased total amount of lipids available, thus, enhancing the encapsulation as well as the confinement of the lipophilic VNP within the formulation, also controlling its release. Similar pattern for drugs’ release from PEGylated vesicular systems have been described by Sudhakar et al.  characterized by highest EE% and ZP magnitude, and lowest VS and RE3.10.Figure 7.Intranasal instillation was performed as a single shot of 0.375 mL in each nostril using polyethylene tubes. Mean VNP concentrations in plasma  and braiCmax) and the time needed to reach it (Tmax), in addition to the computed area under the plasma-concentration time curve (AUC0-12 and AUC0-∞) are reported in The determined pharmacokinetic parameters, namely maximum plasma concentration (3.10.1.Cmax and AUC) . Tukey’s HSD test revealed that the aforementioned parameters were significantly higher for intranasal emulsomes in comparison to the oral market product (p < .0001). This could be attributed to the bypass of first pass metabolism (hepatic) as a consequence of the intranasal administration. However, both SA and MPEG-DSPE surface-tailored emulsomes showed significantly lower Cmax, AUC0-12, and AUC0-∞ compared to the optimized surface nontailored emulsomes at 95% level of significance, suggesting the possible ability of the surface modification to boost drug availability at brain rather than plasma level. Regarding Tmax, all the emulsomal formulations reached the highest plasma concentration after 0.5 h compared to 1 h needed for the market product. However, no statistical difference was found among Tmax of the formulations as per Kruskal–Wallis test results (p = .467). The lower Tmax of the emulsomes could be attributed to the rich vasculature of the nasal cavity, that allows for faster absorption compared to the oral route  . ANOVA s3.10.2.Cmax, AUC0-12, and AUC0-∞ for the intranasal emulsomal formulations compared to the market product. All formulations reached the maximum brain concentration 1 h after administration. As per ANOVA results, a statistical significant difference in the Cmax, AUC0-12, and AUC0-∞ among the tested formulations was observed (p < .0001). Tukey’s HSD test revealed that the aforementioned parameters were significantly higher following intranasal administration of emulsomes compared with oral administration of the market product, (p ≤ .05). Additionally, the multiple comparison revealed significantly higher parameters for each of the surface-tailored emulsomal formulations compared to the corresponding surface non-tailored emulsomes (p < .001).The higher brain levels of VNP achieved by the nasal emulsomes could be attributed to the uptake of VNP emulsomes via either the systemic or olfactory pathways. Unlike the oral route, the nasal one could achieve direct absorption of VNP into the systemic circulation bypassing the pre-systemic metabolism, thus more drug is available to reach the brain. Additionally, the olfactory pathway allows for drug uptake directly from the nasal cavity into cerebrospinal fluid and brain tissues .


Candida auris stands out for commonly being highly resistant to antifungal drugs, and some isolates are pan-resistant. The rate of mortality linked to C. auris infections varies from 28% to 78%. In this study, we characterized C. auris extracellular vesicles (EVs) in the presence of caspofungin, an echinocandin, which is the recommended first line antifungal for the treatment of infections due to this emerging pathogen. Furthermore, we also analyzed the protein and RNA content of EVs generated by C. auris cultivated with or without treatment with caspofungin. We observed that caspofungin led to the increased production of EVs, and treatment also altered the type and quantity of RNA molecules and proteins enclosed in the EVs. There were distinct classes of RNAs in the EVs with ncRNAs being the most identified molecules, and tRNA-fragments (tRFs) were abundant in each of the strains studied. We also identified anti-sense RNAs, varying from 21 to 55 nt in length. The differentially abundant mRNAs detected in EVs isolated from yeast subjected to caspofungin treatment were related to translation, nucleosome core and cell wall. The differentially regulated proteins identified in the EVs produced during caspofungin treatment were consistent with the results observed with the RNAs, with the enriched terms being related to translation and cell wall. Our study adds new information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade treatment and persist in the host. The ability of C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents.Antifungal resistance has become more frequent, either due to the emergence of naturally resistant species or the development of mechanisms that lead to resistance in previously susceptible species. Among these fungal species of global threat,  Candida auris is an emerging species characterized in 2009 when it was isolated from the ear canal of a patient in Japan . In. InC. aual sizes . Howeveral sizes . In ordeal sizes , indicatCaspofungin treatment resulted in a change in the composition of RNA in EVs. Distinct classes of RNA molecules were identified as full-length mRNAs and most of the RNAs identified are ncRNAs smaller than 200 nucleotides that included anti-sense RNAs, tRNAs, other ncRNAs and rRNA, as described in other fungi species .In order to identify full-length mRNAs, we filtered transcripts with a 5× of minimum reads coverage A. In totThe ncRNAs represented the bulk of RNA molecules in the EVs. When comparing the three strains, 78 identified ncRNAs were common among them. A total of 80 ncRNAs were identified in at least two strains A. For thIn addition, we also detected in our RNA-seq mRNA fragments that presented low coverage, with reads aligning in specific regions and, interestingly, in the complementary strand of the mRNA (anti-sense RNA-asRNA) A. The leIn addition to RNA composition, we further investigated if caspofungin treatment would change the composition of EV proteins. We observed a difference in the EV protein abundance amongst strains and also in response to caspofungin.C. auris reference Uniprot sequence (UP000230249), considering proteins identified in at least two replicates, 505 proteins were identified. To obtain the proteins differentially expressed in the caspofungin treatment, control data from the different strains were pooled, and these results were used for comparison. Fold Change of 2 and p-value 0.05 was used as cutoff. Under these terms, 67 proteins were considered differentially expressed  can act in all the steps of the RNA cycle, including transcription, translation, and RNA turnover . The asRC. auris EVs upon caspofungin treatment and related these findings to that of the RNA analysis. Most peptides identified were present in the three isolates studies and associated with ribosome pathways and biogenesis and stress response, with many HSPs. Although it is indeed interesting to find a high number of ribosome-related proteins enriched after caspofungin treatment, these results are not isolated, as other studies have found an increased ribosomal protein content after yeast cells were treated with antifungals [Histoplasma capsulatum EVs [Candida glabrata, Candida tropicalis and Candida parapsilosis EVs [Paracoccidioides brasiliensis and C. albicans [C. auris EVs are described as immunogenic in other Candida species, and their presence in the EVs might induce an altered immune response in the host cell [We further studied the protein content of ifungals ,19,20. Tatum EVs  and in Cosis EVs . HSP 82 albicans ,57. Manyost cell ,59,60,61ost cell ,63. The C. auris to efficiently alter the composition of EVs may represent a mechanism for the fungus to mitigate the effects of antifungal agents. This information expands our understanding of fungal EV biology and drug resistance, a serious and emerging global threat.In summary, these results provide information on how an echinocandin can affect the EV pathway, which is associated with the yeast cell being able to evade the treatment and persist in the host. The ability of


Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid—plasma or urine—that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear.Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer.Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs. Prostate cancer (PC) is the second most common cancer in males affecting more than 1.27 million men per year worldwide . It is aLiquid biopsies are samples of biofluids that are used for the analysis of cancer cells or cancer tissue-derived molecules . Liquid p < 0.05), present in the pre-operation EVs (at least 10 raw reads in one of the samples) and decreased in the post-operation EVs  in each RNA biotype.The term “EV” refers to all types of membrane-bound vesicles released from cells in the extracellular space . The maiA total of 30 patients with newly diagnosed resectable PC were enrolled in this study between October 2018 and January 2020 at Riga East University Hospital and were followed-up until September 2021. All patients had elevated levels of PSA (2.5–50 ng/mL) at the time of diagnosis. Patient exclusion criteria included: blood transfusion in the last 6 months, another oncological disease, urinary tract infection and use of long-term urinary catheter. Clinical characteristics of the study population are provided in Sixty ml of the first morning urine were collected, centrifuged at 2000 g for 15 min at room temperature, aliquoted and stored at −80°C. Blood samples were collected in EDTA-coated tubes and processed at room temperature within 2 h. Plasma samples were centrifuged twice at 3000 g for 10 min, aliquoted and stored at −80°C. Samples were collected at two different time points: before radical prostatectomy (PreOp) and 3 months after the surgery (PostOp).Tumor and normal prostate tissue samples were macroscopically dissected immediately after the surgery by an experienced uropathologist. One slice of the tissue specimens was subjected to histological evaluation in order to verify the presence or absence of tumor cells in the tissue specimens and to assess the Gleason score in the given specimen, whereas the other part of the specimen was immediately placed into the RNALater solution  and stored at −20°C until processing.The study was conducted according to the Declaration of Helsinki. The specimens were collected after the patients’ informed written consent was obtained and anonymized. The study protocol was approved by the Latvian Central Medical Ethics Committee (decision No. 01-29.1/488).EVs were isolated from the plasma and urine samples using size exclusion chromatography (SEC) as described before  with somEVs were lysed in RIPA buffer . Protein concentration was measured using PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). LNCaP cells  were used as a positive control. Equal fraction (one fifth) of the EV proteins and 10 µg of cellular proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes and blocked using 10% (w/v) fat-free milk. Membranes were incubated with primary antibodies against TSG101 , Calnexin , CD63  and PDCD6IP/ALIX . Membranes were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, F(ab’)2-HRP:  or goat anti-mouse m-IgGκ BP-HRP  in 1:2000 dilution. Immunoreactive bands were visualized using Western Blotting Detection Reagent kit  and pictures were taking using a Nikon d610 dSLR body (Nikon) with Sigma 35 mm f/1.4 DG HSM Art lens (Sigma).Approximately 20 mg of tissues were homogenized using QIAzol Lysis Reagent  and Lysing Matrix A tubes in a FastPrep-24 homogenizer . RNA was isolated using miRNeasy Micro Kit (Qiagen) following the small RNA enrichment protocol provided by the manufacturer, thus obtaining both, the long and small RNA fraction of each sample. From EVs, RNA was extracted using miRNeasy Micro Kit. On column DNAse treatment was performed according to the manufacturer’s instructions. RNA quantity and quality was assessed using Agilent pico RNA kit and Agilent 2100 Bioanalyzer .® Small RNA Library Prep Kit . The libraries were cleaned using Blue Pippin DNA Size Selection with 3% gel Blue Pippin Cassette  setting target length to 130–250 bp. All libraries were constructed in duplicates. The libraries were sequenced on Illumina NextSeq500 instrument using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) .EV-RNA libraries were constructed from half of the entire yield of extracted EV RNA without any size separation steps, whereas tissue small RNA libraries were constructed from 10 ng of tissue small RNA fraction using CleanTagTranscriptome libraries were built in duplicates from 100 ng of long tissue RNA fraction using TruSeq Stranded mRNA library Prep  following manufacturer’s instructions. Libraries were size-selected using Blue Pippin system with 2% gel Blue Pippin Cassette  with a range of 200–600 bp and their quality and quantity was assessed using Agilent DNA kit . Libraries were pooled and sequenced using a NextSeq 500/550 High Output Kit v2.5 (300 cycles) .p-value of ≤0.05 was considered to be significant. The RNAseq datasets are available in ArrayExpress, accession No. E-MTAB-11910.The obtained raw data in FASTQ format were analyzed using ad-hoc R script pipeline. For small RNA libraries, it included the trimming of adapters using Cutadapt , read maA half of the entire yield of extracted EV RNA was reverse-transcribed using miRCURY LNA RT kit (Qiagen) according to the manufacturer’s protocol. A total of 20 µL of PCR reaction containing 1:2 diluted cDNA, 10 µL of 2xEvaGreen Supermix (Bio-Rad) and either 1 µL of miRCURY LNA primer mix (Qiagen) or 2 µL of QuantiNova LNA primer mix (Qiagen)  was loadp value ≤0.05 was considered significant.Statistical analyses were performed using GraphPad Prism 6.2 . Comparison between PreOp vs. PostOp data was assessed using Wilcoxon matched-paired signed rank test. Kruskal–Wallis test with multiple comparisons corrected by Dunn’s test was performed to determine differences among RNA biotypes. 7 to 1.5 × 1010 particles/mL while in plasma it ranged from 5.68 ×109 to 7.10 ×1011 particles per ml (The workflow of this study is shown in s per ml . The EV s per ml  and no ss per ml .S) and normal prostate tissue (NS). Moreover, we reasoned that only fragments of degraded mRNAs and lncRNAs are present in small RNA libraries constructed from tissue specimens. Therefore, in order to obtain unbiased long RNA expression profiles, we also built full transcriptome libraries from tumor (TL) and normal prostate (NL) tissues.To characterize the composition of RNA cargo in plasma and urinary EVs, we performed deep sequencing of RNA libraries constructed from the following bio-specimens from 10 PC patients: pre-operation plasma (PreOpP), 3 months post-operation plasma (PostOpP), pre-operation urine (PreOpU), 3 months post-operation urine (PostOpU), and small RNA fractions of histologically verified PC tissue (TWe have shown before that more than 50% of the EV-associated RNA is attached to the surface of EVs . In the p < 0.05); (2) present in the PreOp EVs (at least 10 raw reads in one of the samples) and (3) decreased in the PostOp EVs as compared to PreOp EVs . RNAs matching these criteria were found in four RNA biotypes: miRNA, piRNA, mRNA and lncRNA  overexpressed in tumor tissues as compared to normal prostate tissues  were designed using GeneGlobe (Qiagen) platform. As sequencing data suggested that the EV-enclosed mRNAs and lncRNAs are fragmented, we selected target regions that were present in the majority of the EV samples. Using this approached we failed to identify suitable target regions for all three lncRNAs and several mRNAs because the fragments were too short or had unacceptably high GC content. Nevertheless, seven RT-ddPCR assays  were successfully designed  and theip = 0.0003), piR-28004  and adenosylmethionine decarboxylase 1 (AMD1)  were significantly decreased in the PostOp as compared to the PreOp samples , present in PreOpU EVs and decreased after the surgery, yet the difference did not reach statistical significance after multiple testing correction.lncRNA Linc00662 has been shown to promote tumorigenesis in PC , CHASERRAmong the biomarker candidates was also piR-28004, an uncharacterized piRNA, whereas the levels of another 8 and 1 piRNA were decreased in the PostOp U and PostOpP EVs, respectively and another 10 were significantly overexpressed in PC tissues. piRNAs are 26–32 nt long RNA molecules that interact with PIWI proteins and act as mediators of various processes including transposon silencing, genome rearrangement, epigenetic regulation of gene expression etc. Initially, piRNAs were thought to function mainly in male germ cells, however it is now clear that they are aberrantly expressed in multiple types of cancer and play essential roles in the progression of cancer and metastasis by transcriptional and post-transcriptional gene silencing . RecentlThe most straight forward way to translate these findings into clinically applicable tools, would be to develop PCR-based assays, however, this is hampered by two major issues: fragmentation of long RNAs and the lack of normalization methods for urinary EV analysis. Long RNAs such as mRNAs and lncRNAs appear to be an attractive type of biomarkers due to higher fold changes, known functional significance in the cancer and considerably higher number of biomarker candidates as to compare with miRNAs or other small non-coding RNAs. However, the design of PCR assays for long RNAs is challenging due to their fragmented nature in EVs . CurrentTo validate RNA sequencing data that are normalized for sequencing depth and RNA composition, PCR normalization method that is based on a set of invariable reference genes is needed. Some potential reference genes have been identified for blood EVs , but notTaken together, these data suggest that the fraction of PC-derived EVs in urine is sufficiently large to allow the detection and tracking of PC-derived RNAs. Hence, urine appears to be a superior source of EV-RNAs for the diagnosis and active surveillance of PC, however, it is unlikely to be suitable for post-operative monitoring of PC progression. For this, blood assays are needed. However, our data suggest that the PC-derived EV fraction in blood plasma at least in patients with localized PC is too low to enable tracking of PC-derived RNAs. Moreover, the composition of plasma EVs is much more complex than urinary EVs as EVs are sampled from many more different cell types, hence EV isolation methods that would allow enrichment with cancer-derived EVs are required for the development of EV-based blood tests for the detection and monitoring of PC.


Circular RNAs (circRNAs) are endogenously expressed covalently circularized RNA structures formed via non-canonical backsplicing of the primary transcript where the downstream donor site is ligated to the upstream acceptor site . DiscoveInterestingly, the altered expression of circRNAs during development, disease progression, the presence in biofluids, and their extraordinary stability make them promising biomarkers for disease diagnosis and hence provide novel approaches for therapeutic intervention . UnderstWang et al., provides a comprehensive summary of the biogenesis and functions of circRNAs, with a major emphasis on the potential molecular mechanisms and their therapeutic applications in urological cancers. This review article is instrumental in developing novel therapeutic strategies for urological cancers in the near future.While it is crucial to elucidate circRNA function in normal cell physiology and biological processes, there is a rising interest in exploring the role of circRNA dysregulation in cancer, diabetes, neurological disorders, and other diseases. The review by Nfix (circNfix), which was found to interact with miR-204-5p miRNA, known to suppress myocyte enhancer factor 2c (MEF2C) expression, thereby promoting muscle cell differentiation, was reported by Das et al.Recently thousands of circRNAs were identified in mouse and human skeletal muscle tissue. Moreover, a subset of highly abundant circRNAs was found in mouse skeletal muscle based on experimental validation by RNase R treatment, RT-qPCR analysis, and sequencing of the backsplice junctions. Examining plausible circRNA-miRNA-mRNA networks led to the discovery of conserved circular RNA Naarmann-de Vries et al., which can detect nearly one hundred circRNAs and their linear counterparts in a single experiment as shown in total human heart RNA samples. Lexo-circSeq can be used to detect any specific circRNA in a large cohort of clinical samples, as demonstrated in hiPSC-derived cardiomyocytes, biopsies from patients diagnosed with hypertrophic cardiomyopathy and dilated cardiomyopathy. Thus, Lexo-circSeq is a cost-effective technique that can monitor circRNA expression patterns across large patient cohorts in terms of circular-to-linear ratios starting with a small input.Major hurdles in circRNA research are their relatively low cellular abundance and the dependence of detection methods on input size and lacuna in downstream processing using conventional RNA-seq and computational methodologies. In this Research Topic, a targeted RNA sequencing approach known as Lexo-circSeq has been proposed by Guria et al., demonstrated a novel template-dependent multiple displacement amplification (tdMDA)-NGS method. The improved-tdMDA-NGS method involves the typical rRNA depletion coupled with cDNA library preparation using H minus reverse transcriptase and RNase H treatment followed by tdMDA using Phi29 DNA polymerase. This cost-effective hybrid NGS approach improves the detection threshold to find even the marginally expressed circRNAs at lower NGS depths. Reportedly, there was a significant rise of nearly 11-fold in the total number of circular RNAs identified in rice plant and HeLa cell line using this improved-tdMDA-NGS method compared to the typical RNA-seq method of circRNA sequencing. Thus, the improved-tdMDA-NGS method can identify and characterize circRNAs in diverse biological samples.Another interesting article by This Research Topic covers the most recent findings concerning the roles of circRNA in health and diseases, as well as articles discussing improved state-of-the-art techniques to aid in accurately detecting circRNAs in clinically challenging scenarios. We believe that the articles highlighted in this Research Topic will be useful for carrying out a study to advance this emerging field of research.