Mitochondrial double-stranded RNA: Discovery, regulation, and function

Abstract

Pattern recognition receptors (PRRs) recognize pathogen molecules such as virus double-stranded RNAs (dsRNAs), leading to the induction of innate immune responses. Protein kinase R-activated (PKR) is the first identified PRR as an interferon-induced kinase in response to dsRNAs produced by infected viruses. Recently, growing evidence suggests that PKR can also be activated by endogenously expressed dsRNAs. To examine the identity of endogenous dsRNAs that activate PKR, these dsRNAs are captured with an anti-PKR antibody and analyzed by formaldehyde-mediated crosslinking and immunoprecipitation sequencing. Surprisingly, the majority of the PKR-interacting RNA repertoire is occupied by mitochondrial RNAs (mtRNAs). My diversified analysis shows the presence of mt-dsRNAs through intermolecular interaction of heavy and light strand mtRNAs owing to bidirectional transcription of the mitochondrial genome. Moreover, mt-dsRNAs activate PKR and its downstream proteins to modulate subsequent cell signaling even in uninfected cells. Considering the overactivation of PKR and mitochondrial dysfunction commonly observed in osteoarthritis (OA), a representative degenerative disease, the role of mt-dsRNAs during OA development is analyzed. Indeed, under OA-eliciting conditions, the cytosolic efflux of mt-dsRNAs is facilitated in chondrocytes, leading to the activation of innate immune signaling. In addition to PKR, mt-dsRNAs released to the extracellular space activate TLR3, another dsRNA-sensing PRR, expressed in neighboring chondrocytes. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage obtained from OA patients and surgery-induced OA mice, respectively, further support my in cellulo data. To further find key regulatory factors of mt-dsRNAs that act as antigens for innate immune responses, CRISPR screening on RNA-binding proteins residing in the mitochondrial matrix is employed. Strikingly, 5-methylcytosine ($m^5C$) rRNA methyltransferase NSUN4 is a crucial regulator of mt-dsRNAs. Methylation by NSUN4 readily occurs on mRNAs and non-coding RNAs (ncRNAs) from both heavy and light strand RNAs transcribed from the mitochondrial genome. Cells depleted of NSUN4 show decreased methylated RNA but increased overall mt-dsRNA levels. Analysis of previously published databases reveals that recognized by complement C1q binding protein (C1QBP) recognizes methylated mtRNAs. Modified mtRNAs recognized by C1QBP interact with and recruit polynucleotide phosphorylase (PNPase), a well-known mitochondrial 3′-to-5′ exoribonuclease, resulting in RNA degradation. Taken together, my work discovers the presence of mt-dsRNAs as a novel class of endogenous dsRNAs that activate various dsRNA-sensing PRRs, suggesting nuclear and mitochondrial signaling cues in regulating cellular metabolism. A comprehensive understanding of innate immune activation by mt-dsRNAs during the development of OA provides a potential target for degenerative diseases associated with mitochondria. Lastly, my study provides the post-transcriptional regulatory mechanism of mt-dsRNAs and establishes RNA m5C modification as a molecular marker for mtRNA decay.