Mitochondrial Double-Stranded RNA in Exosome Promotes Interleukin-17 Production Through Toll-Like Receptor 3 in Alcohol-associated Liver Injury.

BACKGROUND AND AIMS: Mitochondrial double-stranded RNA (mtdsRNA) and its innate immune responses have been reported previously; however, mtdsRNA generation and its effects on alcohol-associated liver disease (ALD) remain unclear. Here, we report that hepatic mtdsRNA stimulates toll-like receptor 3 (TLR3) in Kupffer cells through the exosome (Exo) to enhance interleukin (IL)-17A (IL-17A) production in ALD. APPROACH AND RESULTS: Following binge ethanol (EtOH) drinking, IL-17A production primarily increased in γδ T cells of wild-type (WT) mice, whereas the production of IL-17A was mainly facilitated by CD4+ T cells in acute-on-chronic EtOH consumption. These were not observed in TLR3 knockout (KO) or Kupffer cell-depleted WT mice. The expression of polynucleotide phosphorylase, an mtdsRNA-restricting enzyme, was significantly decreased in EtOH-exposed livers and hepatocytes of WT mice. Immunostaining revealed that mtdsRNA colocalized with the mitochondria in EtOH-treated hepatocytes from WT mice and healthy humans. Bioanalyzer analysis revealed that small-sized RNAs were enriched in EtOH-treated Exos (EtOH-Exos) rather than EtOH-treated microvesicles in hepatocytes of WT mice and humans. Quantitative real-time PCR and RNA sequencing analyses indicated that mRNA expression of mitochondrial genes encoded by heavy and light strands was robustly increased in EtOH-Exos from mice and humans. After direct treatment with EtOH-Exos, IL-1ß expression was significantly increased in WT Kupffer cells but not in TLR3 KO Kupffer cells, augmenting IL-17A production of γδ T cells in mice and humans. CONCLUSIONS: EtOH-mediated generation of mtdsRNA contributes to TLR3 activation in Kupffer cells through exosomal delivery. Consequently, increased IL-1ß expression in Kupffer cells triggers IL-17A production in γδ T cells at the early stage that may accelerate IL-17A expression in CD4+ T cells in the later stage of ALD. Therefore, mtdsRNA and TLR3 may function as therapeutic targets in ALD.

Mitochondrial mRNA translation initiation contributes to oxidative metabolism in the myocardia of aged, obese mice.

Being both advanced in age and obese each contribute to cardiac hypertrophy in a unique manner. Electron transport complexes I and IV are implicated in deficient electron transport during cardiomyopathies and contain the majority of protein subunits that are transcribed and translated by machinery localized within the mitochondria. PURPOSE: To assess myocardial mt-mRNA translation factors in relation to mitochondrial content and mtDNA-encoded protein using a mouse model of aged obesity and to test the relationship of mt-mRNA translation initiation factor 2 (mtIF2) to oxidative capacity and the cellular oxidation-reduction (redox) state in cardiomyocytes. METHODS: Male C56BL/6 J mice fed lean or high fat diet were aged to either ~3 months or ~22 months, the heart was excised and analyzed using immunoblot and qPCR to assess differences in mitochondrial mRNA translation machinery. Using H9c2 cardiomyocytes, mtIF2 was knocked-down and oxidative metabolic characteristics assessed including oxidation/reduction state, bioenergetic flux, and hypoxic resistance was tested. RESULTS: Aged, obese mouse hearts were ~40% larger than young, lean controls and contained ~50% less mtIF2 protein alongside ~25-50% lower content of Cytb, a protein encoded by mtDNA. Reducing the level of mtIF2 by shRNA is associated with ~15-20% lower content of OXPHOS complex I and IV, ~30% lower optical redox ratio, ~40% oxygen reserve capacity, and ~20% less cell survival following hypoxia. CONCLUSION: We present evidence of altered mt-mRNA translation during cardiac hypertrophy in aged obesity. We build on these results by demonstrating the necessity of mtIF2 in maintaining oxidative characteristics of cardiac muscle cells.

Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication.

Mammalian mitochondria operate multiple mechanisms of DNA replication. In many cells and tissues a strand-asynchronous mechanism predominates over coupled leading and lagging-strand DNA synthesis. However, little is known of the factors that control or influence the different mechanisms of replication, and the idea that strand-asynchronous replication entails transient incorporation of transcripts (aka bootlaces) is controversial. A firm prediction of the bootlace model is that it depends on mitochondrial transcripts. Here, we show that elevated expression of Twinkle DNA helicase in human mitochondria induces bidirectional, coupled leading and lagging-strand DNA synthesis, at the expense of strand-asynchronous replication; and this switch is accompanied by decreases in the steady-state level of some mitochondrial transcripts. However, in the so-called minor arc of mitochondrial DNA where transcript levels remain high, the strand-asynchronous replication mechanism is instated. Hence, replication switches to a strand-coupled mechanism only where transcripts are scarce, thereby establishing a direct correlation between transcript availability and the mechanism of replication. Thus, these findings support a critical role of mitochondrial transcripts in the strand-asynchronous mechanism of mitochondrial DNA replication; and, as a corollary, mitochondrial RNA availability and RNA/DNA hybrid formation offer means of regulating the mechanisms of DNA replication in the organelle.

PKR Senses Nuclear and Mitochondrial Signals by Interacting with Endogenous Double-Stranded RNAs.

Protein kinase RNA-activated (PKR) induces immune response by sensing viral double-stranded RNAs (dsRNAs). However, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. Here, we capture these dsRNAs by formaldehyde-mediated crosslinking and immunoprecipitation sequencing and find that various noncoding RNAs interact with PKR. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). MtRNAs can form intermolecular dsRNAs owing to bidirectional transcription of the mitochondrial genome and regulate PKR and eIF2α phosphorylation to control cell signaling and translation. Moreover, PKR activation by mtRNAs is counteracted by PKR phosphatases, disruption of which causes apoptosis from PKR overactivation even in uninfected cells. Our work unveils dynamic regulation of PKR even without infection and establishes PKR as a sensor for nuclear and mitochondrial signaling cues in regulating cellular metabolism.