Characterization of mtdsRNA levels in Non-Small Cell Lung Carcinoma (NSCLC)
Mitochondrial double-stranded RNA (mtdsRNA) is exported to the cytoplasm when mitochondrial RNA degradation is impaired, serving as a novel Damage-Associated Molecular Pattern (DAMP) for mitochondrial stress.  The mtdsRNA has been detected in certain cancers, but its prevalence and functional role remain largely unexplored.  Moreover, mtdsRNA is not readily detected in large computational datasets.  Here, we used comprehensive computational characterization of NSCLC cell lines and identified elevated light-strand transcription in a subset of cell lines, suggesting a potential for mtdsRNA formation. Our work allowed us to stratify NSCLC lines into groups with high and low mtdsRNA abundance. Despite high cytoplasmic mtdsRNA levels in some lines, we did not observe consistent activation of Type-I interferon (IFN-l) response or ADAR editing. RT-qPCR analysis revealed that only USP18 transcripts were significantly regulated in a subset of NSCLC lines, indicating a partial or suppressed IFN-I response. Strand-specific RT-qPCR also revealed no bias in mitochondrial gene expression to account for this phenomenon. These results suggest that basal mtdsRNA accumulation does not robustly activate IFN-I signaling and may be tolerated in NSCLC. The monoclonal J2 antibody, which detects mtdsRNA, consistently emerged as a reliable marker of intracellular dsRNA burden and could serve as a functional readout of mitochondrial RNA dysregulation. This approach may help stratify tumors based on its immunogenic potential, independent of genomic mutations. Importantly, we report that mtdsRNA is upregulated in a subset of NSCLC cell lines, representing a potential new marker for mitochondrial dysfunction in cancer. Overall design: Experiment 1 was RIP-Seq of three different NSCLC cell lines (H1437, H23, PC9) in three replicates. Experiment 2 was RIP-seq of three different NSCLC cell lines (H1437, H23, PC9) with and without IMT1 treatment.  Experiment 3 was RIP-seq of the NSCLC cell line H23 with and without ADAR1 knockdown.