Xrn1 Exoribonuclease-An Intrinsic Marker of Yeast Population Growth.

BACKGROUND: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown. METHODS: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δxrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy. RESULTS: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy. CONCLUSIONS: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.

Evidence that Xrn1 is in complex with Gcn1, and is required for full levels of eIF2α phosphorylation.

The protein kinase Gcn2 and its effector protein Gcn1 are part of the general amino acid control signalling (GAAC) pathway best known in yeast for its function in maintaining amino acid homeostasis. Under amino acid limitation, Gcn2 becomes activated, subsequently increasing the levels of phosphorylated eIF2α (eIF2α-P). This leads to the increased translation of transcriptional regulators, such as Gcn4 in yeast and ATF4 in mammals, and subsequent re-programming of the cell's gene transcription profile, thereby allowing cells to cope with starvation. Xrn1 is involved in RNA decay, quality control and processing. We found that Xrn1 co-precipitates Gcn1 and Gcn2, suggesting that these three proteins are in the same complex. Growth under starvation conditions was dependent on Xrn1 but not on Xrn1-ribosome association, and this correlated with reduced eIF2α-P levels. Constitutively active Gcn2 leads to a growth defect due to eIF2α-hyperphosphorylation, and we found that this phenotype was independent of Xrn1, suggesting that xrn1 deletion does not enhance eIF2α de-phosphorylation. Our study provides evidence that Xrn1 is required for efficient Gcn2 activation, directly or indirectly. Thus, we have uncovered a potential new link between RNA metabolism and the GAAC.

Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

Structured 3' UTRs destabilize mRNAs in plants.

BACKGROUND: RNA secondary structure (RSS) can influence the regulation of transcription, RNA processing, and protein synthesis, among other processes. 3' untranslated regions (3' UTRs) of mRNA also hold the key for many aspects of gene regulation. However, there are often contradictory results regarding the roles of RSS in 3' UTRs in gene expression in different organisms and/or contexts. RESULTS: Here, we incidentally observe that the primary substrate of miR159a (pri-miR159a), when embedded in a 3' UTR, could promote mRNA accumulation. The enhanced expression is attributed to the earlier polyadenylation of the transcript within the hybrid pri-miR159a-3' UTR and, resultantly, a poorly structured 3' UTR. RNA decay assays indicate that poorly structured 3' UTRs could promote mRNA stability, whereas highly structured 3' UTRs destabilize mRNA in vivo. Genome-wide DMS-MaPseq also reveals the prevailing inverse relationship between 3' UTRs' RSS and transcript accumulation in the transcriptomes of Arabidopsis, rice, and even human. Mechanistically, transcripts with highly structured 3' UTRs are preferentially degraded by 3'-5' exoribonuclease SOV and 5'-3' exoribonuclease XRN4, leading to decreased expression in Arabidopsis. Finally, we engineer different structured 3' UTRs to an endogenous FT gene and alter the FT-regulated flowering time in Arabidopsis. CONCLUSIONS: We conclude that highly structured 3' UTRs typically cause reduced accumulation of the harbored transcripts in Arabidopsis. This pattern extends to rice and even mammals. Furthermore, our study provides a new strategy of engineering the 3' UTRs' RSS to modify plant traits in agricultural production and mRNA stability in biotechnology.

Hypoxia-driven deSUMOylation of EXOSC10 promotes adaptive changes in the transcriptome profile.

Reduced oxygen availability (hypoxia) triggers adaptive cellular responses via hypoxia-inducible factor (HIF)-dependent transcriptional activation. Adaptation to hypoxia also involves transcription-independent processes like post-translational modifications; however, these mechanisms are poorly characterized. Investigating the involvement of protein SUMOylation in response to hypoxia, we discovered that hypoxia strongly decreases the SUMOylation of Exosome subunit 10 (EXOSC10), the catalytic subunit of the RNA exosome, in an HIF-independent manner. EXOSC10 is a multifunctional exoribonuclease enriched in the nucleolus that mediates the processing and degradation of various RNA species. We demonstrate that the ubiquitin-specific protease 36 (USP36) SUMOylates EXOSC10 and we reveal SUMO1/sentrin-specific peptidase 3 (SENP3) as the enzyme-mediating deSUMOylation of EXOSC10. Under hypoxia, EXOSC10 dissociates from USP36 and translocates from the nucleolus to the nucleoplasm concomitant with its deSUMOylation. Loss of EXOSC10 SUMOylation does not detectably affect rRNA maturation but affects the mRNA transcriptome by modulating the expression levels of hypoxia-related genes. Our data suggest that dynamic modulation of EXOSC10 SUMOylation and localization under hypoxia regulates the RNA degradation machinery to facilitate cellular adaptation to low oxygen conditions.

SARS-CoV-2 NSP14 governs mutational instability and assists in making new SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the rapidly evolving RNA virus behind the COVID-19 pandemic, has spawned numerous variants since its 2019 emergence. The multifunctional Nonstructural protein 14 (NSP14) enzyme, possessing exonuclease and messenger RNA (mRNA) capping capabilities, serves as a key player. Notably, single and co-occurring mutations within NSP14 significantly influence replication fidelity and drive variant diversification. This study comprehensively examines 120 co-mutations, 68 unique mutations, and 160 conserved residues across NSP14 homologs, shedding light on their implications for phylogenetic patterns, pathogenicity, and residue interactions. Quantitative physicochemical analysis categorizes 3953 NSP14 variants into three clusters, revealing genetic diversity. This research underscoresthe dynamic nature of SARS-CoV-2 evolution, primarily governed by NSP14 mutations. Understanding these genetic dynamics provides valuable insights for therapeutic and vaccine development.

Intermolecular base-pairing interactions, a unique topology and exoribonuclease-resistant noncoding RNAs drive formation of viral chimeric RNAs in plants.

In plants, exoribonuclease-resistant RNAs (xrRNAs) are produced by many viruses. Whereas xrRNAs contribute to the pathogenicity of these viruses, the role of xrRNAs in the virus infectious cycle remains elusive. Here, we show that xrRNAs produced by a benyvirus (a multipartite RNA virus with four genomic segments) in plants are involved in the formation of monocistronic coat protein (CP)-encoding chimeric RNAs. Naturally occurring chimeric RNAs, we discovered, are composed of 5'-end of RNA 2 and 3'-end of either RNA 3 or RNA 4 bearing conservative exoribonuclease-resistant 'coremin' region. Using computational tools and site-directed mutagenesis, we show that de novo formation of chimeric RNAs requires intermolecular base-pairing interaction between 'coremin' and 3'-proximal part of the CP gene of RNA 2 as well as a stem-loop structure immediately adjacent to the CP gene. Moreover, knockdown of the expression of the XRN4 gene, encoding 5'→3' exoribonuclease, inhibits biogenesis of both xrRNAs and chimeric RNAs. Our findings suggest a novel mechanism involving a unique tropology of the intermolecular base-pairing complex between xrRNAs and RNA2 to promote formation of chimeric RNAs in plants. XrRNAs, essential for chimeric RNA biogenesis, are generated through the action of cytoplasmic Xrn 4 5'→3' exoribonuclease conserved in all plant species.

Exoribonuclease RNase R protects Antarctic Pseudomonas syringae Lz4W from DNA damage and oxidative stress.

IMPORTANCE: Bacterial exoribonucleases play a crucial role in RNA maturation, degradation, quality control, and turnover. In this study, we have uncovered a previously unknown role of 3'-5' exoribonuclease RNase R of Pseudomonas syringae Lz4W in DNA damage and oxidative stress response. Here, we show that neither the exoribonuclease function of RNase R nor its association with the RNA degradosome complex is essential for this function. Interestingly, in P. syringae Lz4W, hydrolytic RNase R exhibits physiological roles similar to phosphorolytic 3'-5' exoribonuclease PNPase of E. coli. Our data suggest that during the course of evolution, mesophilic E. coli and psychrotrophic P. syringae have apparently swapped these exoribonucleases to adapt to their respective environmental growth conditions.

The CCR4-NOT complex suppresses untimely translational activation of maternal mRNAs.

Control of mRNA poly(A) tails is essential for regulation of mRNA metabolism, specifically translation efficiency and mRNA stability. Gene expression in maturing oocytes relies largely on post-transcriptional regulation, as genes are transcriptionally silent during oocyte maturation. The CCR4-NOT complex is a major mammalian deadenylase, which regulates poly(A) tails of maternal mRNAs; however, the function of the CCR4-NOT complex in translational regulation has not been well understood. Here, we show that this complex suppresses translational activity of maternal mRNAs during oocyte maturation. Oocytes lacking all CCR4-NOT deadenylase activity owing to genetic deletion of its catalytic subunits, Cnot7 and Cnot8, showed a large-scale gene expression change caused by increased translational activity during oocyte maturation. Developmental arrest during meiosis I in these oocytes resulted in sterility of oocyte-specific Cnot7 and Cnot8 knockout female mice. We further showed that recruitment of CCR4-NOT to maternal mRNAs is mediated by the 3'UTR element CPE, which suppresses translational activation of maternal mRNAs. We propose that suppression of untimely translational activation of maternal mRNAs via deadenylation by CCR4-NOT is essential for proper oocyte maturation.

Dynamic "Cap"-abilities of P-bodies and the XRN1-EDC4 axis.

RNA turnover regulates the quality and quantity of cellular gene expression through a coordinated cavalcade of enzymes, factors, and phase transitions. In this issue, Brothers et al reveal the importance of balanced communication between the Xrn1 exonuclease and the EDC4 decapping factor to coordinate P-body dynamics and maintain cellular fitness.

The EDC4-XRN1 interaction controls P-body dynamics to link mRNA decapping with decay.

Deadenylation-dependent mRNA decapping and decay is the major cytoplasmic mRNA turnover pathway in eukaryotes. Many mRNA decapping and decay factors are associated with each other via protein-protein interaction motifs. For example, the decapping enzyme DCP2 and the 5'-3' exonuclease XRN1 interact with the enhancer of mRNA-decapping protein 4 (EDC4), a large scaffold that has been reported to stimulate mRNA decapping. mRNA decapping and decay factors are also found in processing bodies (P-bodies), evolutionarily conserved ribonucleoprotein granules that are often enriched with mRNAs targeted for decay, yet paradoxically are not required for mRNA decay to occur. Here, we show that disrupting the EDC4-XRN1 interaction or altering their stoichiometry inhibits mRNA decapping, with microRNA-targeted mRNAs being stabilized in a translationally repressed state. Importantly, we demonstrate that this concomitantly leads to larger P-bodies that are responsible for preventing mRNA decapping. Finally, we demonstrate that P-bodies support cell viability and prevent stress granule formation when XRN1 is limiting. Taken together, these data demonstrate that the interaction between XRN1 and EDC4 regulates P-body dynamics to properly coordinate mRNA decapping with 5'-3' decay in human cells.

Bacillus subtilis NrnB is expressed during sporulation and acts as a unique 3'-5' exonuclease.

All cells employ a combination of endo- and exoribonucleases to degrade long RNA polymers to fragments 2-5 nucleotides in length. These short RNA fragments are processed to monoribonucleotides by nanoRNases. Genetic depletion of nanoRNases has been shown to increase abundance of short RNAs. This deleteriously affects viability, virulence, and fitness, indicating that short RNAs are a metabolic burden. Previously, we provided evidence that NrnA is the housekeeping nanoRNase for Bacillus subtilis. Herein, we investigate the biological and biochemical functions of the evolutionarily related protein, B. subtilis NrnB (NrnBBs). These experiments show that NrnB is surprisingly different from NrnA. While NrnA acts at the 5' terminus of RNA substrates, NrnB acts at the 3' terminus. Additionally, NrnA is expressed constitutively under standard growth conditions, yet NrnB is selectively expressed during endospore formation. Furthermore, NrnA processes only short RNAs, while NrnB unexpectedly processes both short RNAs and longer RNAs. Indeed, inducible expression of NrnB can even complement the loss of the known global 3'-5' exoribonucleases, indicating that it acts as a general exonuclease. Together, these data demonstrate that NrnB proteins, which are widely found in Firmicutes, Epsilonproteobacteria and Archaea, are fundamentally different than NrnA proteins and may be used for specialized purposes.

The PARN, TOE1, and USB1 RNA deadenylases and their roles in non-coding RNA regulation.

The levels of non-coding RNAs (ncRNAs) are regulated by transcription, RNA processing, and RNA degradation pathways. One mechanism for the degradation of ncRNAs involves the addition of oligo(A) tails by non-canonical poly(A) polymerases, which then recruit processive sequence-independent 3' to 5' exonucleases for RNA degradation. This pathway of decay is also regulated by three 3' to 5' exoribonucleases, USB1, PARN, and TOE1, which remove oligo(A) tails and thereby can protect ncRNAs from decay in a manner analogous to the deubiquitination of proteins. Loss-of-function mutations in these genes lead to premature degradation of some ncRNAs and lead to specific human diseases such as Poikiloderma with Neutropenia (PN) for USB1, Dyskeratosis Congenita (DC) for PARN and Pontocerebellar Hypoplasia type 7 (PCH7) for TOE1. Herein, we review the biochemical properties of USB1, PARN, and TOE1, how they modulate ncRNA levels, and their roles in human diseases.

Structures of SARS-CoV-2 N7-methyltransferase with DOT1L and PRMT7 inhibitors provide a platform for new antivirals.

The RNA N7-methyltransferase (MTase) activity of SARS-CoV-2's nsp14 protein is essential for viral replication and is a target for the development of new antivirals. Nsp14 uses S-adenosyl methionine (SAM) as the methyl donor to cap the 5' end of the SARS-CoV-2 mRNA and generates S-adenosyl homocysteine (SAH) as the reaction byproduct. Due to the central role of histone MTases in cancer, many SAM/SAH analogs with properties of cell permeability have recently been developed for the inhibition of these MTases. We have succeeded in identifying two such compounds (SGC0946 and SGC8158) that display significant antiviral activity and bind to the SARS-CoV-2 nsp14 N7-MTase core. Unexpectedly, crystal structures of SGC0946 and SGC8158 with the SARS-CoV-2 nsp14 N7-MTase core identify them as bi-substrate inhibitors of the viral MTase, co-occupying both the SAM and RNA binding sites; positing novel features that can be derivatized for increased potency and selectivity for SARS-CoV-2 nsp14. Taken together, the high-resolution structures and the accompanying biophysical and viral replication data provide a new avenue for developing analogs of SGC0946 and SGC8158 as antivirals.

Analogs of the Catechol Derivative Dynasore Inhibit HIV-1 Ribonuclease H, SARS-CoV-2 nsp14 Exoribonuclease, and Virus Replication.

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3'-to-5' exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases.

Targeting RNA oxidation by ISG20-mediated degradation is a potential therapeutic strategy for acute kidney injury.

Oxidative stress plays a central role in the pathophysiology of acute kidney injury (AKI). Although RNA is one of the most vulnerable cell components to oxidative damage, it is unclear whether RNA oxidation is involved in the pathogenesis of AKI. In this study, we found that the level of RNA oxidation was significantly enhanced in kidneys of patients with acute tubular necrosis (ATN) and in the renal tubular epithelial cells (TECs) of mice with AKI, and oxidized RNA overload resulted in TEC injury. We further identified interferon-stimulated gene 20 (ISG20) as a novel regulator of RNA oxidation in AKI. Tubule-specific deficiency of ISG20 significantly aggravated renal injury and RNA oxidation in the ischemia/reperfusion-induced AKI mouse model and ISG20 restricted RNA oxidation in an exoribonuclease activity-dependent manner. Importantly, overexpression of ISG20 protected against oxidized RNA overproduction and renal ischemia/reperfusion injury in mice and ameliorated subsequent protein aggresome accumulation, endoplasmic reticulum stress, and unfolded protein response. Thus, our findings provide direct evidence that RNA oxidation contributes to the pathogenesis of AKI and that ISG20 importantly participates in the degradation of oxidized RNA, suggesting that targeting ISG20-handled RNA oxidation may be an innovative therapeutic strategy for AKI.

ISG20 stimulates anti-tumor immunity via a double-stranded RNA-induced interferon response in ovarian cancer.

Augmentation of endogenous double-stranded RNA (dsRNA) has become a promising strategy for activating anti-tumor immunity through induction of type I interferon (IFN) in the treatment of ovarian carcinoma. However, the underlying regulatory mechanisms of dsRNA in ovarian carcinoma remain elusive. From The Cancer Genome Atlas (TCGA), we downloaded RNA expression profiles and clinical data of patients with ovarian carcinoma. Using the consensus clustering method, patients can be classified by their expression level of core interferon-stimulated genes (ISGs): IFN signatures high and IFN signatures low. The IFN signatures high group had a good prognosis. Gene set enrichment analysis (GSEA) showed that differentially expressed genes (DEGs) were primarily associated with anti-foreign immune responses. Based on results from protein-protein interaction (PPI) networks and survival analysis, ISG20 was identified as a key gene involved in host anti-tumor immune response. Further, elevated ISG20 expression in ovarian cancer cells led to increased IFN-ß production. The elevated interferon improved the immunogenicity of tumor cells and generated chemokines that attract immune cells to infiltrate the area. Upon overexpression of ISG20, endogenous dsRNA accumulated in the cell and stimulated IFN-ß production through the Retinoic acid-inducible gene I (RIG-I)-mediated dsRNA sense pathway. The accumulation of dsRNA was associated with the ribonuclease activity of ISG20. This study suggests that targeting ISG20 is a potential immune therapeutic approach to treat ovarian cancer.

DIS3L2 knockdown impairs key oncogenic properties of colorectal cancer cells via the mTOR signaling pathway.

DIS3L2 degrades different types of RNAs in an exosome-independent manner including mRNAs and several types of non-coding RNAs. DIS3L2-mediated degradation is preceded by the addition of nontemplated uridines at the 3'end of its targets by the terminal uridylyl transferases 4 and 7. Most of the literature that concerns DIS3L2 characterizes its involvement in several RNA degradation pathways, however, there is some evidence that its dysregulated activity may contribute to cancer development. In the present study, we characterize the role of DIS3L2 in human colorectal cancer (CRC). Using the public RNA datasets from The Cancer Genome Atlas (TCGA), we found higher DIS3L2 mRNA levels in CRC tissues versus normal colonic samples as well as worse prognosis in patients with high DIS3L2 expression. In addition, our RNA deep-sequencing data revealed that knockdown (KD) of DIS3L2 induces a strong transcriptomic disturbance in SW480 CRC cells. Moreover, gene ontology (GO) analysis of significant upregulated transcripts displays enrichment in mRNAs encoding proteins involved in cell cycle regulation and cancer-related pathways, which guided us to evaluate which specific hallmarks of cancer are differentially regulated by DIS3L2. To do so, we employed four CRC cell lines (HCT116, SW480, Caco-2 and HT-29) differing in their mutational background and oncogenicity. We demonstrate that depletion of DIS3L2 results in reduced cell viability of highly oncogenic SW480 and HCT116 CRC cells, but had little or no impact in the more differentiated Caco-2 and HT-29 cells. Remarkably, the mTOR signaling pathway, crucial for cell survival and growth, is downregulated after DIS3L2 KD, whereas AZGP1, an mTOR pathway inhibitor, is upregulated. Furthermore, our results indicate that depletion of DIS3L2 disturbs metastasis-associated properties, such as cell migration and invasion, only in highly oncogenic CRC cells. Our work reveals for the first time a role for DIS3L2 in sustaining CRC cell proliferation and provides evidence that this ribonuclease is required to support the viability and invasive behavior of dedifferentiated CRC cells.

Expression of interferon-stimulated gene 20 (ISG20), an antiviral effector protein, in glomerular endothelial cells: possible involvement of ISG20 in lupus nephritis.

BACKGROUND: In addition to regulating the antiviral response, increased expression of Toll-like receptor 3 (TLR3) in resident renal cells plays a role in developing some forms of glomerulonephritis. TLR3 activation leads to type I interferon (IFN) production, which induces the expression of IFN-stimulated genes (ISGs). However, the role of ISG20 expression in resident renal cells remains unclear. METHODS: Cultured normal human glomerular endothelial cells (GECs) were treated with polyinosinic-polycytidylic acid (poly IC), Escherichia coli lipopolysaccharide (LPS), R848, and CpG (TLR3, TLR4, TLR7, and TLR9 agonists, respectively). The mRNA levels of ISG20, CX3CL1/fractalkine, and CXCL10/IP-10 were measured by quantitative reverse transcription-polymerase chain reaction. ISG20 protein expression was assessed by Western blotting. RNA interference was used to knockdown IFN-ß and ISG20 expression. CX3CL1 protein levels were assessed by enzyme-linked immunosorbent assay. We performed immunofluorescence to examine endothelial ISG20 expression in biopsy specimens from patients with lupus nephritis (LN). RESULTS: In GECs, the expression of ISG20 mRNA and protein was increased by polyIC, not by LPS, R848, or CpG treatment. Moreover, ISG20 knockdown prevented poly IC-induced CX3CL1 expression but had no effect on CXCL10 expression. Intense endothelial ISG20 immunoreactivity was observed in biopsy specimens obtained from patients with proliferative LN. CONCLUSION: In GECs, ISG20 was regulated via TLR3 but not via TLR4, TLR7, or TLR9 signaling. Moreover, ISG20 was involved in regulating CX3CL1 production. In addition to regulating antiviral innate immunity, ISG20 may act as a mediator of CX3CL1 production, thereby inducing glomerular inflammation, particularly in patients with LN.