Project description:We expressed different isoforms of ADAR proteins in human cells without A-to-I RNA editing activity to test which isoform(s) was able to rescue the interferon induction phenotypes caused by activation of MDA5 in the absense of double-stranded RNA editing. Using the rescue cells, we sequenced the RNA and profiled RNA editing in order to identify the key RNA editing substrates for MDA5 sensing.
Project description:Endogenous double-stranded RNA (dsRNA) triggers pro-inflammatory signaling and has been shown to accumulate in Alzheimer’s disease (AD), but the origins of this dsRNA are incompletely understood. Transposable elements (TEs), which are non-coding DNA sequences capable of forming dsRNA, are a potential endogenous source of dsRNA in AD. We generated dsRNA immunoprecipitation (J2 dsRNA antibody) data on astrocytes treated with or without a siRNA to knock down the dsRNA editing enzyme ADAR1. We analyzed TE transcripts enriched in dsRNA pools when ADAR1 was knocked down, and we identified putative dsRNA-prone TEs, which were also present in secondary analyses of datasets profiling RNAs bound to PKR, MDA5, and the p110 isoform of ADAR1.
Project description: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.
Project description:Although recent evidence suggests that overlapping sense/antisense transcription is a common feature in higher eukaryotes, the possibility that overlapping transcripts could interact to each other and bear a specific biological function has not been explored. Here we show that a plethora of sense/antisense transcript pairs are co-expressed from 8q24.21 within the same cell and acquire a stable double-stranded RNA conformation. Interestingly, these molecules display predominantly nuclear localization and establish specific interactions with nuclear components. A detailed characterization of a particular sense/antisense pair (ndsRNA-2a) revealed that this molecule displays differential localization throughout the cell cycle, interacts with RCC1 and RAN and through the latter with the mitotic RANGAP1-SUMO1/RANBP2 complex. Notably, an increased number of bi/multi-nucleated cells and chromatin bridges were observed upon ndsRNA-2a overexpression, whereas strand-specific ndsRNA-2a knockdown leads to mitotic catastrophe and cell death. This suggests a functional role of ndsRNA-2a in cell cycle progression that critically requires its double stranded nature. Finally, the identification of hundreds of sense/antisense transcripts pairs harboring ndsRNA profile signatures and their regulation by cellular cues suggests that ndsRNAs constitute a novel class of regulatory molecules that are likely to be involved in a plethora of biological processes. PLB985 long (3x datasets) and small (3x datasets) strand specific RNA-Seq for captured RNAs. Global PLB985 for long (2x datasets) and small RNAs (2x datasets). Global libraries for EtOH (vehicle) treated (1x dataset) or retinoic acid induced differentiated PLB985 cells (1x dataset).