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RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release

ABSTRACT: Mitochondria are essential regulators of innate immunity. They generate long double-stranded RNAs (mt-dsRNAs) and release them to the cytosol to trigger immune response under pathological stress conditions. Yet, the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on RNA-binding proteins residing in mitochondria and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mitochondrial RNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mitochondrial RNAs and establishes a molecular mark for mitochondrial RNA decay and cytosolic release.

ORGANISM(S): Homo sapiens

PROVIDER: GSE246026 | GEO | 2024/08/16

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute 2 protein to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA-mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’. To assess whether nuclear-encoded miRNA are detectable in human mitochondria, we performed the following four steps approach. First, cultured HeLa cells were allowed to reach 80-100% confluence and subjected to fractionation in order to isolate the cytosolic fraction. From the same HeLa cells, mitochondria were isolated by immunomagnetic Anti-TOM22 MicroBeads from the Mitochondria Isolation Kit (Miltenyi Biotec). In total, six mitochondria preparations were perfromed, three of these were additionally treated with RNase A. Second, total RNA was extracted from the mitochondrial and cytosolic fractions. Third, mitochondrial and cytosolic RNA were respectively profiled by microRNA microarray analysis. Last, data were analyzed and normalized.Three independent assays were performed.

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RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release

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