ABSTRACT
Myotonic dystrophy type 2 (DM2) is a tetranucleotide CCTG repeat expansion disease associated with an increased prevalence of autoimmunity. Here, we identified an elevated type I interferon (IFN) signature in peripheral blood mononuclear cells and primary fibroblasts of DM2 patients as a trigger of chronic immune stimulation. Although RNA-repeat accumulation was prevalent in the cytosol of DM2-patient fibroblasts, type-I IFN release did not depend on innate RNA immune sensors but rather the DNA sensor cGAS and the prevalence of mitochondrial DNA (mtDNA) in the cytoplasm. Sublethal mtDNA release was promoted by a chronic activation of the ATF6 branch of the unfolded protein response (UPR) in reaction to RNA-repeat accumulation and non-AUG translated tetrapeptide expansion proteins. ATF6-dependent mtDNA release and resulting cGAS/STING activation could also be recapitulated in human THP-1 monocytes exposed to chronic endoplasmic reticulum (ER) stress. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic endoplasmic reticulum stress and associated mtDNA leakage. This mtDNA is, in turn, sensed by the cGAS/STING pathway and induces a type-I IFN response predisposing to autoimmunity. Elucidating this pathway reveals new potential therapeutic targets for autoimmune disorders associated with repeat expansion diseases.
Subject(s)
Autoimmune Diseases , Interferon Type I , Myotonic Dystrophy , Humans , Myotonic Dystrophy/genetics , Myotonic Dystrophy/metabolism , DNA, Mitochondrial/genetics , Autoimmunity/genetics , Leukocytes, Mononuclear/metabolism , RNA , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Endoplasmic Reticulum Stress/geneticsABSTRACT
Ustilago maydis is a biotrophic fungus that causes tumor formation on all aerial parts of maize. U. maydis secretes effector proteins during penetration and colonization to successfully overcome the plant immune response and reprogram host physiology to promote infection. In this study, we functionally characterized the U. maydis effector protein Topless (TPL) interacting protein 6 (Tip6). We found that Tip6 interacts with the N-terminus of RELK2 through its two Ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motifs. We show that the EAR motifs are essential for the virulence function of Tip6 and critical for altering the nuclear distribution pattern of RELK2. We propose that Tip6 mimics the recruitment of RELK2 by plant repressor proteins, thus disrupting host transcriptional regulation. We show that a large group of AP2/ERF B1 subfamily transcription factors are misregulated in the presence of Tip6. Our study suggests a regulatory mechanism where the U. maydis effector Tip6 utilizes repressive domains to recruit the corepressor RELK2 to disrupt the transcriptional networks of the host plant.
Subject(s)
Basidiomycota , Plant Diseases , Ustilago , Plant Diseases/microbiology , Zea mays/microbiology , Ustilago/metabolism , Co-Repressor Proteins/metabolism , Carcinogenesis , Fungal Proteins/metabolismABSTRACT
RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.
Subject(s)
DEAD Box Protein 58 , Isoleucine , Receptors, Immunologic , DEAD Box Protein 58/chemistry , DEAD Box Protein 58/genetics , DEAD Box Protein 58/metabolism , Immune Tolerance , Isoleucine/genetics , RNA, Double-Stranded/genetics , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Humans , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolismABSTRACT
Translation efficiency can be affected by mRNA stability and secondary structures, including G-quadruplex structures (G4s). The highly conserved DEAH-box helicase DHX36/RHAU resolves G4s on DNA and RNA in vitro, however a systems-wide analysis of DHX36 targets and function is lacking. We map globally DHX36 binding to RNA in human cell lines and find it preferentially interacting with G-rich and G4-forming sequences on more than 4500 mRNAs. While DHX36 knockout (KO) results in a significant increase in target mRNA abundance, ribosome occupancy and protein output from these targets decrease, suggesting that they were rendered translationally incompetent. Considering that DHX36 targets, harboring G4s, preferentially localize in stress granules, and that DHX36 KO results in increased SG formation and protein kinase R (PKR/EIF2AK2) phosphorylation, we speculate that DHX36 is involved in resolution of rG4 induced cellular stress.
Subject(s)
DEAD-box RNA Helicases/metabolism , G-Quadruplexes , RNA, Messenger/metabolism , Untranslated Regions , Gene Knockout Techniques , HEK293 Cells , Humans , Phosphorylation , Protein Biosynthesis , Ribosomes/metabolism , Stress, Physiological , eIF-2 Kinase/metabolismABSTRACT
Fungalysins from several phytopathogenic fungi have been shown to be involved in cleavage of plant chitinases. While fungal chitinases are responsible for cell wall remodeling during growth and morphogenesis, plant chitinases are important components of immunity. This study describes a dual function of the Ustilago maydis fungalysin UmFly1 in modulation of both plant and fungal chitinases. Genetic, biochemical and microscopic experiments were performed to elucidate the in vitro and in planta functions of U. maydis UmFly1. U. maydis ∆umfly1 mutants show significantly reduced virulence, which coincides with reduced cleavage of the maize chitinase ZmChiA within its chitin-binding domain. Moreover, deletion of umfly1 affected the cell separation of haploid U. maydis sporidia. This phenotype is associated with posttranslational activation of the endogenous chitinase UmCts1. Genetic complementation of the ∆umfly1 mutant with a homologous gene from closely related, but nonpathogenic, yeast fully rescued the cell separation defect in vitro, but it could not recover the ∆umfly1 defect in virulence and cleavage of the maize chitinase. We report on the dual function of the secreted fungalysin UmFly1. We hypothesize that co-evolution of U. maydis with its host plant extended the endogenous function of UmFly1 towards the modulation of plant chitinase activity to promote infection.