Project description:Adenosine-to-inosine (A-to-I) RNA editing, which is catalyzed by a family of adenosine deaminase acting on RNA (ADAR) enzymes, is important in the epitranscriptomic regulation of RNA metabolism. However, the role of A-to-I RNA editing in vascular disease is unknown. Here we show that cathepsin S mRNA (CTSS), which encodes a cysteine protease associated with angiogenesis and atherosclerosis, is highly edited in human endothelial cells. The 3â?² untranslated region (3â?² UTR) of the CTSS transcript contains two inverted repeats, the AluJo and AluSx+ regions, which form a long stemâ??loop structure that is recognized by ADAR1 as a substrate for editing. RNA editing enables the recruitment of the stabilizing RNA-binding protein human antigen R (HuR; encoded by ELAVL1) to the 3â?² UTR of the CTSS transcript, thereby controlling CTSS mRNA stability and expression. In endothelial cells, ADAR1 overexpression or treatment of cells with hypoxia or with the inflammatory cytokines interferon-Î³ and tumor-necrosis-factor-Î± induces CTSS RNA editing and consequently increases cathepsin S expression. ADAR1 levels and the extent of CTSS RNA editing are associated with changes in cathepsin S levels in patients with atherosclerotic vascular diseases, including subclinical atherosclerosis, coronary artery disease, aortic aneurysms and advanced carotid atherosclerotic disease. These results reveal a previously unrecognized role of RNA editing in gene expression in human atherosclerotic vascular diseases. 1) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in poly(A) RNA-seq data derived from endothelial cell transcriptome after ADAR1 or ADAR2 knockdown (n=2 biological replicates per condition, total n=8 biological samples). 2) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from peripheral blood mononuclear cells (n=12 total biological samples; n=4 replicates per condition). 3) Evaluation of transcriptome expression and RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total-RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions (n=2 biological replicates per condition, total n=4 biological samples). 4) Evaluation of RNA editing sites (A-to-G and T-to-C nucleotide mismatches) in total RNA-seq data derived from endothelial cell transcriptome under basal and hypoxic conditions after ADAR1 knockdown (n=3 replicates per condition, total n=12 biological samples). 5) HuR iCLIP RNA-sequencing data derived from HUVEC HuR iCLIP after ADAR1 knockdown (scrambled control and siADAR1, n=1 per condition, total n=2 biological samples).

Project description:Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) mismatches in RNA-seq data compared to mm9 reference genome in wild type mice that were not edited or reduced in editing frequency in ADAR1E861A editing deficient mice. Secondly, to determine the transcriptional consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Fetal liver mRNA profiles of embryonic day 12.5 wild-type (WT) and ADAR1 editing-deficient (ADAR1E861A) mice were generated by RNA sequencing, in triplicate (biological replicates), using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.94 between biological triplicates per genotype. Approximately 4.4% of the transcripts showed differential expression between the WT and ADAR1E861A fetal liver, with a LogFC≥1.5 and p value <0.05. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 11 logFC) in ADAR1E861A fetal liver compared to WT. 6,012 A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data of WT and ADAR1E861A fetal liver. Conclusions: Our study represents the first detailed analysis of fetal liver transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to suppress interferon signaling to endogenous RNA. Fetal liver mRNA profiles of E12.5 wild type (WT) and ADAR E861A mutant mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 200.

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Mitochondrial hypoxic stress induces RNA editing by APOBEC3G in lymphocytes

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