Complex regulation of mitochondrial signaling by human adenovirus minor capsid protein VI.

The controlled release of mitochondrial content into the cytosol has emerged as one of the key steps in mitochondrial signaling. In particular, the release of mitochondrial DNA (mtDNA) into the cytosol has been shown to activate interferon beta (IFN-ß) gene expression to execute the innate immune response. In this report, we show that human adenovirus type 5 (HAdV-C5) infection induces the release of mtDNA into the cytosol. The release of mtDNA is mediated by the viral minor capsid protein VI (pVI), which localizes to mitochondria. The presence of the mitochondrial membrane proteins Bak and Bax are needed for the mtDNA release, whereas the viral E1B-19K protein blocked pVI-mediated mtDNA release. Surprisingly, the pVI-mediated mtDNA release did not increase but inhibited the IFN-ß gene expression. Notably, the pVI expression caused mitochondrial leakage of the HSP60 protein. The latter prevented specific phosphorylation of the interferon regulatory factor 3 (IRF3) needed for IFN-ß gene expression. Overall, we assign a new mitochondria and IFN-ß signaling-modulating function to the HAdV-C5 minor capsid protein VI. IMPORTANCE: Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, including conjunctivitis and the common cold. HAdVs need to interfere with multiple cellular signaling pathways during the infection to gain control over the host cell. In this study, we identified human adenovirus type 5 (HAdV-C5) minor capsid protein VI as a factor modulating mitochondrial membrane integrity and mitochondrial signaling. We show that pVI-altered mitochondrial signaling impedes the cell's innate immune response, which may benefit HAdV growth. Overall, our study provides new detailed insights into the HAdV-mitochondria interactions and signaling. This knowledge is helpful when developing new anti-viral treatments against pathogenic HAdV infections and improving HAdV-based therapeutics.

The RNA-binding protein ZC3H11A interacts with the nuclear poly(A)-binding protein PABPN1 and alters polyadenylation of viral transcripts.

Nuclear mRNA metabolism is regulated by multiple proteins, which either directly bind to RNA or form multiprotein complexes. The RNA-binding protein ZC3H11A is involved in nuclear mRNA export, NF-κB signaling, and is essential during mouse embryo development. Furthermore, previous studies have shown that ZC3H11A is important for nuclear-replicating viruses. However, detailed biochemical characterization of the ZC3H11A protein has been lacking. In this study, we established the ZC3H11A protein interactome in human and mouse cells. We demonstrate that the nuclear poly(A)-binding protein PABPN1 interacts specifically with the ZC3H11A protein and controls ZC3H11A localization into nuclear speckles. We report that ZC3H11A specifically interacts with the human adenovirus type 5 (HAdV-5) capsid mRNA in a PABPN1-dependent manner. Notably, ZC3H11A uses the same zinc finger motifs to interact with PABPN1 and viral mRNA. Further, we demonstrate that the lack of ZC3H11A alters the polyadenylation of HAdV-5 capsid mRNA. Taken together, our results suggest that the ZC3H11A protein may act as a novel regulator of polyadenylation of nuclear mRNA.

Fragile X-Related Protein FXR1 Controls Human Adenovirus Capsid mRNA Metabolism.

Human adenoviruses (HAdVs) are widespread pathogens causing a variety of diseases. A well-controlled expression of virus capsid mRNAs originating from the major late transcription unit (MLTU) is essential for forming the infectious virus progeny. However, regulation of the MLTU mRNA metabolism has mainly remained enigmatic. In this study, we show that the cellular RNA-binding protein FXR1 controls the stability of the HAdV-5 MLTU mRNAs, as depletion of FXR1 resulted in increased steady-state levels of MLTU mRNAs. Surprisingly, the lack of FXR1 reduced viral capsid protein accumulation and formation of the infectious virus progeny, indicating an opposing function of FXR1 in HAdV-5 infection. Further, the long FXR1 isoform interfered with MLTU mRNA translation, suggesting FXR1 isoform-specific functions in virus-infected cells. We also show that the FXR1 protein interacts with N6-methyladenosine (m6A)-modified MLTU mRNAs, thereby acting as a novel m6A reader protein in HAdV-5 infected cells. Collectively, our study identifies FXR1 as a regulator of MLTU mRNA metabolism in the lytic HAdV-5 life cycle. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing various self-limiting diseases, such as the common cold and conjunctivitis. Even though adenoviruses have been studied for more than 6 decades, there are still gaps in understanding how the virus interferes with the host cell to achieve efficient growth. In this study, we identified the cellular RNA-binding protein FXR1 as a factor manipulating the HAdV life cycle. We show that the FXR1 protein specifically interferes with mRNAs encoding essential viral capsid proteins. Since the lack of the FXR1 protein reduces virus growth, we propose that FXR1 can be considered a novel cellular proviral factor needed for efficient HAdV growth. Collectively, our study provides new detailed insights about the HAdV-host interactions, which might be helpful when developing countermeasures against pathogenic adenovirus infections and for improving adenovirus-based therapies.

Structural Insights into Human Adenovirus Type 4 Virus-Associated RNA I.

RNA molecules can adopt specific RNA triplex structures to execute critical biological functions. Human adenoviruses (HAdVs) are abundant pathogens encoding the essential, noncoding virus-associated RNA I (VA RNAI). Here, we employ a triplex-specific probing assay, based on the intercalating and cleaving agent benzoquinoquinoxaline 1, 10-phenanthroline (BQQ-OP), to unravel a potential RNA triplex formation in VA RNAI. The BQQ-OP cleavage of the pathogenic HAdV type 4 (HAdV-4) VA RNAI indicates that a potential triplex is formed involving the highly conserved stem 4 of the central domain and side stem 7. Further, the integrity of the HAdV-4 VA RNAI side stem 7 contributes to a potential triplex formation in vitro and virus growth in vivo. Collectively, we propose that the HAdV-4 VA RNAI can potentially form a biologically relevant triplex structure.

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA.

Human adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased, and it accumulated similarly to the pVII protein in the nuclei of virus-infected cells. The lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knockout cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular antiviral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.IMPORTANCE Human adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of antiviral proteins and noncoding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular antiviral protein ZNF622, which may impede lytic HAdV growth.

Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections.IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and MKRN1 proteins may prime MKRN1 for proteasomal degradation, because the MKRN1 protein is efficiently degraded during the late phase of HAdV-C5 infection. Since MKRN1 protein accumulation is also reduced in measles virus- and vesicular stomatitis virus-infected cells, our results signify the general strategy of viruses to target MKRN1.

The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing.

Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection.

An efficient adenovirus infection results in high-level accumulation of viral DNA and mRNAs in the infected cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not necessarily reflect the same abundance in individual cells. Here, we describe a novel padlock probe-based rolling-circle amplification technique that enables simultaneous detection and analysis of human adenovirus type 5 (HAdV-5) genomic DNA and virus-encoded mRNAs in individual infected cells. We demonstrate that the method is applicable for detection and quantification of HAdV-5 DNA and mRNAs in short-term infections in human epithelial cells and in long-term infections in human B lymphocytes. Single-cell evaluation of these infections revealed high heterogeneity and unique cell subpopulations defined by differential viral DNA content and mRNA expression. Further, our single-cell analysis shows that the specific expression pattern of viral E1A 13S and 12S mRNA splice variants is linked to HAdV-5 DNA content in the individual cells. Furthermore, we show that expression of a mature form of the HAdV-5 histone-like protein VII affects virus genome detection in HAdV-5-infected cells. Collectively, padlock probes combined with rolling-circle amplification should be a welcome addition to the method repertoire for the characterization of the molecular details of the HAdV life cycle in individual infected cells.IMPORTANCE Human adenoviruses (HAdVs) have been extensively used as model systems to study various aspects of eukaryotic gene expression and genome organization. The vast majority of the HAdV studies are based on standard experimental procedures carried out using heterogeneous cell populations, where data averaging often masks biological differences. As every cell is unique, characteristics and efficiency of an HAdV infection can vary from cell to cell. Therefore, the analysis of HAdV gene expression and genome organization would benefit from a method that permits analysis of individual infected cells in the heterogeneous cell population. Here, we show that the padlock probe-based rolling-circle amplification method can be used to study concurrent viral DNA accumulation and mRNA expression patterns in individual HAdV-5-infected cells. Hence, this versatile method can be applied to detect the extent of infection and virus gene expression changes in different HAdV-5 infections.

Clamping of RNA with PNA enables targeting of microRNA.

To be able to target microRNAs also at stages where these are in a double stranded or hairpin form we have studied BisPNA designed to clamp the target and give sufficient affinity to allow for strand invasion. We show that BisPNA complexes are more stable with RNA than with DNA. In addition, 24-mer BisPNA (AntimiR) constructs form complexes with a hairpin RNA that is a model of the microRNA miR-376b, suggesting that PNA-clamping may be an effective way of targeting microRNAs.

The adenovirus VA RNA-derived miRNAs are not essential for lytic virus growth in tissue culture cells.

At late times during a lytic infection human adenovirus type 5 produces ∼10(8) copies per cell of virus-associated RNA I (VA RNAI). This short highly structured RNA polymerase III transcript has previously been shown to be essential for lytic virus growth. A fraction of VA RNAI is processed by Dicer into small RNAs, so-called mivaRNAIs, which are efficiently incorporated into the RNA-induced silencing complex. Here, we constructed recombinant adenoviruses with mutations in the seed sequence of both the 5'- and the 3'-strand of the mivaRNAI duplex. The results showed that late viral protein synthesis, as well as new virus progeny formation, was essentially unaffected by the seed sequence mutations under lytic replicative conditions in HeLa or HEK293 cells. Collectively, our results suggest that either strand of the mivaRNAI duplex does not have target mRNA interactions that are critical for the establishment of virus growth under lytic conditions. Further, by depletion of protein kinase R (PKR) in HEK293 cells, we show that the suppressive effect of VA RNAI on the interferon-induced PKR pathway is most critical for late gene expression.

Activation of the Ca2+/NFAT Pathway by Assembly of Hepatitis C Virus Core Protein into Nucleocapsid-like Particles.

Hepatitis C virus (HCV) is the primary pathogen responsible for liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been linked to several aspects of HCV pathology, including oncogenesis, immune evasion and stress responses. We and others have previously shown that C expression in various cell lines activates Ca2+ signaling and alters Ca2+ homeostasis. In this study, we identified two distinct C protein regions that are required for the activation of Ca2+/NFAT signaling. In the basic N-terminal domain, which has been implicated in self-association of C, amino acids 1-68 were critical for NFAT activation. Sedimentation analysis of four mutants in this domain revealed that association of the C protein into nucleocapsid-like particles correlated with NFAT-activated transcription. The internal, lipid droplet-targeting domain was not required for NFAT-activated transcription. Finally, the C-terminal ER-targeting domain was required in extenso for the C protein to function. Our results indicate that targeting of HCV C to the ER is necessary but not sufficient for inducing Ca2+/NFAT signaling. Taken together, our data are consistent with a model whereby proteolytic intermediates of C with an intact transmembrane ER-anchor assemble into pore-like structures in the ER membrane.

Next-Generation Sequencing Analysis of CpG Methylation of a Tumor Suppressor Gene SHP-1 Promoter in Stable Cell Lines and HCV-Positive Patients.

Hepatitis C virus (HCV) is the major causative pathogen associated with hepatocellular carcinoma and liver cirrhosis. The main virion component, the Core (C) protein, is involved in multiple aspects of HCV pathology including oncogenesis and immune evasion. In this study, we established a next-generation bisulfite sequencing (NGS-BS) protocol to analyze the CpG methylation profile at the tumor suppressor gene SHP-1 P2 promoter as a model system. Our data show that HCV C protein expression in the immortalized T cells correlated with a specific CpG methylation profile at the SHP-1 P2. The NGS-BS on HCV-positive (HCV+) patient-derived PBMCs revealed a considerably different CpG methylation profile compared to the HCV C protein immortalized T cells. Notably, the CpG methylation profile was very similar in healthy and HCV+ PBMCs, suggesting that the SHP-1 P2 CpG methylation profile is not altered in the HCV+ individuals. Collectively, the NGS-BS is a highly sensitive method that can be used to quantitatively characterize the CpG methylation status at the level of individual CpG position and also allows the characterization of cis-acting effects on epigenetic regulation.

ZC3H11A loss of function enhances NF-κB signaling through defective IκBα protein expression.

ZC3H11A is a cellular protein associated with the transcription export (TREX) complex that is induced during heat-shock. Several nuclear-replicating viruses exploit the mRNA export mechanism of ZC3H11A protein for their efficient replication. Here we show that ZC3H11A protein plays a role in regulation of NF-κB signal transduction. Depletion of ZC3H11A resulted in enhanced NF-κB mediated signaling, with upregulation of numerous innate immune related mRNAs, including IL-6 and a large group of interferon-stimulated genes. IL-6 upregulation in the absence of the ZC3H11A protein correlated with an increased NF-κB transcription factor binding to the IL-6 promoter and decreased IL-6 mRNA decay. The enhanced NF-κB signaling pathway in ZC3H11A deficient cells correlated with a defect in IκBα inhibitory mRNA and protein accumulation. Upon ZC3H11A depletion The IκBα mRNA was retained in the cell nucleus resulting in failure to maintain normal levels of the cytoplasmic IκBα mRNA and protein that is essential for its inhibitory feedback loop on NF-κB activity. These findings indicate towards a previously unknown mechanism of ZC3H11A in regulating the NF-κB pathway at the level of IkBα mRNA export.

Hepatitis C Virus Core Protein Down-Regulates Expression of Src-Homology 2 Domain Containing Protein Tyrosine Phosphatase by Modulating Promoter DNA Methylation.

Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The main virion component, the core (C) protein, has been implicated in several aspects of HCV pathology including oncogenesis and immune subversion. Here we show that expression of the C protein induced specific tyrosine phosphorylation of the TCR-related signaling proteins ZAP-70, LAT and PLC-γ in the T cells. Stable expression of the C protein specifically reduced Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1) mRNA and protein accumulation. Quantitative CpG methylation analysis revealed a distinct CpG methylation pattern at the SHP-1 gene promoter in the C protein expressing cells that included specific hypermethylation of the binding site for Sp1 transcription factor. Collectively, our results suggest that HCV may suppress immune responses and facilitate its own persistence by deregulating phosphotyrosine signaling via repressive epigenetic CpG modification at the SHP-1 promoter in the T cells.

Role of CCCH-Type Zinc Finger Proteins in Human Adenovirus Infections.

The zinc finger proteins make up a significant part of the proteome and perform a huge variety of functions in the cell. The CCCH-type zinc finger proteins have gained attention due to their unusual ability to interact with RNA and thereby control different steps of RNA metabolism. Since virus infections interfere with RNA metabolism, dynamic changes in the CCCH-type zinc finger proteins and virus replication are expected to happen. In the present review, we will discuss how three CCCH-type zinc finger proteins, ZC3H11A, MKRN1, and U2AF1, interfere with human adenovirus replication. We will summarize the functions of these three cellular proteins and focus on their potential pro- or anti-viral activities during a lytic human adenovirus infection.

Synthesis, Structure, and Function of Human Adenovirus Small Non-Coding RNAs.

Human adenoviruses (HAdVs) are common pathogens causing a variety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an advantage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV sncRNAs; VA RNA, mivaRNA and MLP-TSS-sRNA will be discussed in the present review. Even though VA RNA has been extensively characterized during the last 60 years, this multifunctional molecule continues to surprise us as more of its structural secrets unfold. Likely, the recent developments on mivaRNA and MLP-TSS-sRNA synthesis and function highlight the importance of these sncRNA in virus replication. Collectively, we will summarize the old and new knowledge about these three viral sncRNAs with focus on their synthesis, structure and functions.

Circulating miRNAs as Potential Biomarkers in Myasthenia Gravis: Tools for Personalized Medicine.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies which attack receptors at the neuromuscular junction. One of the main difficulties in predicting the clinical course of MG is the heterogeneity of the disease, where disease progression differs greatly depending on the subgroup that the patient is classified into. MG subgroups are classified according to: age of onset [early-onset MG (EOMG; onset ≤ 50 years) versus late-onset MG (LOMG; onset > 50 years]; the presence of a thymoma (thymoma-associated MG); antibody subtype [acetylcholine receptor antibody seropositive (AChR+) and muscle-specific tyrosine kinase antibody seropositive (MuSK+)]; as well as clinical subtypes (ocular versus generalized MG). The diagnostic tests for MG, such as antibody titers, neurophysiological tests, and objective clinical fatigue score, do not necessarily reflect disease progression. Hence, there is a great need for reliable objective biomarkers in MG to follow the disease course as well as the individualized response to therapy toward personalized medicine. In this regard, circulating microRNAs (miRNAs) have emerged as promising potential biomarkers due to their accessibility in body fluids and unique profiles in different diseases, including autoimmune disorders. Several studies on circulating miRNAs in MG subtypes have revealed specific miRNA profiles in patients' sera. In generalized AChR+ EOMG, miR-150-5p and miR-21-5p are the most elevated miRNAs, with lower levels observed upon treatment with immunosuppression and thymectomy. In AChR+ generalized LOMG, the miR-150-5p, miR-21-5p, and miR-30e-5p levels are elevated and decrease in accordance with the clinical response after immunosuppression. In ocular MG, higher levels of miR-30e-5p discriminate patients who will later generalize from those remaining ocular. In contrast, in MuSK+ MG, the levels of the let-7 miRNA family members are elevated. Studies of circulating miRNA profiles in Lrp4 or agrin antibody-seropositive MG are still lacking. This review summarizes the present knowledge of circulating miRNAs in different subgroups of MG.

Adenovirus in the omics era - a multipronged strategy.

Human adenoviruses (HAdVs) are common pathogens associated with a wide variety of respiratory, ocular, and gastrointestinal diseases. To achieve its effective lytic mode of replication, HAdVs have to reprogram host-cell gene expression and fine-tune viral gene expression in a temporal manner. In two decades, omics revolution has advanced our knowledge about the HAdV and host-cell interplay at the RNA and protein levels. This review summarizes the current knowledge from large-scale datasets on how HAdV infections adjust coding and noncoding RNA expression, as well as how they reprogram host-cell proteome during the lytic course of infection.

miR-1933-3p is upregulated in skeletal muscles of MuSK+ EAMG mice and affects Impa1 and Mrpl27.

MuSK antibody seropositive (MuSK+) Myasthenia Gravis (MG) typically affects skeletal muscles of the bulbar area, including the omohyoid muscle, causing focal fatigue, weakness and atrophy. The profile of circulating extracellular microRNA (miRNA) is changed in MuSK + MG, but the intracellular miRNA profile in skeletal muscles of MuSK + MG and MuSK + experimental autoimmune MG (EAMG) remains unknown. This study elucidated the intracellular miRNA profile in the omohyoid muscle of mice with MuSK + EAMG. The levels of eleven mouse miRNAs were elevated and two mouse miRNAs were reduced in muscles of MuSK + EAMG mice. Transient expression of miR-1933-3p and miR-1930-5p in mouse muscle (C2C12) cells revealed several downregulated genes, out of which five had predicted binding sites for miR-1933-3p. The mRNA expression of mitochondrial ribosomal protein L27 (Mrpl27) and Inositol monophosphatase I (Impa1) was reduced in miR-1933-3p transfected C2C12 cells compared to control cells (p = 0.032 versus p = 0.020). Further, transient expression of miR-1933-3p reduced Impa1 protein accumulation in C2C12 cells. These findings provide novel insights of dysregulated miRNAs and their intracellular pathways in muscle tissue afflicted with MuSK + EAMG, providing a possible link to mitochondrial dysfunction and muscle atrophy observed in MuSK + MG.

Estrogen Receptor, Inflammatory, and FOXO Transcription Factors Regulate Expression of Myasthenia Gravis-Associated Circulating microRNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate important intracellular biological processes. In myasthenia gravis (MG), a disease-specific pattern of elevated circulating miRNAs has been found, and proposed as potential biomarkers. These elevated miRNAs include miR-150-5p, miR-21-5p, and miR-30e-5p in acetylcholine receptor antibody seropositive (AChR+) MG and miR-151a-3p, miR-423-5p, let-7a-5p, and let-7f-5p in muscle-specific tyrosine kinase antibody seropositive (MuSK+) MG. In this study, we examined the regulation of each of these miRNAs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the Encyclopedia of DNA Elements (ENCODE) to gain insight into the transcription factor pathways that drive their expression in MG. Our aim was to look at the transcription factors that regulate miRNAs and then validate some of those in vivo with cell lines that have sufficient expression of these transcription factors This analysis revealed several transcription factor families that regulate MG-specific miRNAs including the Forkhead box or the FOXO proteins (FoxA1, FoxA2, FoxM1, FoxP2), AP-1, interferon regulatory factors (IRF1, IRF3, IRF4), and signal transducer and activator of transcription proteins (Stat1, Stat3, Stat5a). We also found binding sites for nuclear factor of activated T-cells (NFATC1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), early growth response factor (EGR1), and the estrogen receptor 1 (ESR1). AChR+ MG miRNAs showed a stronger overall regulation by the FOXO transcription factors, and of this group, miR-21-5p, let-7a, and let 7f were found to possess ESR1 binding sites. Using a murine macrophage cell line, we found activation of NF-κB -mediated inflammation by LPS induced expression of miR-21-5p, miR-30e-5p, miR-423-5p, let-7a, and let-7f. Pre-treatment of cells with the anti-inflammatory drugs prednisone or deflazacort attenuated induction of inflammation-induced miRNAs. Interestingly, the activation of inflammation induced packaging of the AChR+-specific miRNAs miR-21-5p and miR-30e-5p into exosomes, suggesting a possible mechanism for the elevation of these miRNAs in MG patient serum. In conclusion, our study summarizes the regulatory transcription factors that drive expression of AChR+ and MuSK+ MG-associated miRNAs. Our findings of elevated miR-21-5p and miR-30e-5p expression in immune cells upon inflammatory stimulation and the suppressive effect of corticosteroids strengthens the putative role of these miRNAs in the MG autoimmune response.