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SUMMARYRIG-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 a long, highly structured, polyA-bearing, non-coding 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 preventing mtRNA-triggered RIG-I-mediated autoinflammation.

Nucleic acid accumulation in repeat expansion disease poses multiple challenges to cellular integrity. Myotonic dystrophy type 2 (DM2) results from large CCTG repeats in the CNBP gene leading to myopathy and an increased prevalence of autoimmunity. Here, we observed that DM2 patients exhibited a type-I interferon signature in blood and cultured fibroblasts. RNA repeat accumulation was prevalent in the cytosol of DM2 patient fibroblasts, facilitating repeat-associated non-AUG translation. The ensuing chronic endoplasmic reticulum (ER) stress response led to an ATF6-controlled induction of type-I IFN dependent on the cGAS/STING pathway. Recapitulating chronic ER stress in the monocytic THP-1 cell line revealed its dependence on mitochondrial DNA (mtDNA). Correspondingly, mitochondrial stress and cytosolic leakage of mtDNA was observed in DM2 patient fibroblasts. Altogether, our study demonstrates a novel mechanism by which large repeat expansions cause chronic ER and mitochondrial stress and induce a type-I interferon response that predisposes to autoimmunity. Keywords: ER stress, ATF6, Autoimmunity, type I IFN, repeat expansions

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., Translation efficiency can be affected by mRNA stability and secondary RNA structures. Here the authors reveal that loss of DHX36 helicase activity leads to an accumulation of translationally inactive target mRNAs with G-rich structures in untranslated regions.

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.