RNA Sequencing Quantitative Analysis and identification of RNA editing sites of Wild Type and ADAR1 editing deficient (ADAR1E861A) murine fetal liver RNA

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 identifying 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.研究目的：ADAR1介导的RNA编辑 (RNA editing) 对造血发育至关重要。本研究的首要目标为：以mm9参考基因组 (mm9 reference genome) 为参照，对比野生型小鼠与ADAR1E861A编辑缺陷型小鼠的RNA测序 (RNA-seq) 数据，通过鉴定A-to-I（即G）错配位点，筛选ADAR1特异性RNA编辑位点，其中野生型小鼠的编辑未发生或编辑频率降低；其次，本研究旨在阐明ADAR1介导的A-to-I编辑缺失所引发的转录组学后果。 实验方法：本研究以胚胎发育第12.5天（E12.5）的野生型 (WT) 与ADAR1编辑缺陷型 (ADAR1E861A) 小鼠胎肝为样本，采用Illumina HiSeq2000测序平台进行RNA测序 (RNA-seq)，设置3次生物学重复 (biological replicates) 以获取胎肝mRNA表达谱。对通过质量过滤的测序读段，采用TopHat后接Cufflinks进行转录本水平的数据分析。采用SYBR Green荧光染料法完成定量实时聚合酶链反应 (qRT-PCR) 验证。A-to-I（即G）RNA编辑位点的鉴定参照Ramaswami G.等人2012年发表于《Nature Methods》的方法，先使用Burrows-Wheeler比对工具 (Burrows-Wheeler Aligner, BWA) 进行序列比对，再通过方差分析 (ANOVA) 进行筛选。最终通过桑格测序 (Sanger sequencing) 确认鉴定得到的RNA编辑位点。 实验结果：本研究采用优化后的数据分析流程，通过BWA将每个样本约3000万条测序读段比对至小鼠基因组（版本mm9），并在WT与ADAR1E861A小鼠胎肝中鉴定得到14484个转录本。不同基因型组内的3次生物学重复间，RNA-seq数据的拟合优度 (R²) 均大于0.94。在WT与ADAR1E861A小鼠胎肝中，约4.4%的转录本呈现显著差异表达，其对数倍数变化 (LogFC) ≥1.5且P值<0.05。与WT组相比，ADAR1E861A组胎肝中的干扰素刺激基因 (interferon stimulated genes) 出现显著上调，部分基因的LogFC可达11。通过对WT与ADAR1E861A小鼠胎肝的RNA-seq数据进行错配位点分析，本研究共鉴定得到6012个A-to-I RNA编辑位点。 研究结论：本研究首次利用RNA测序 (RNA-seq) 技术，结合生物学重复 (biological replicates)，对小鼠胎肝转录组与A-to-I RNA编辑位点开展了系统性详细分析。A-to-I RNA编辑是ADAR1的核心功能，其可通过抑制内源性RNA介导的干扰素信号通路发挥必要作用。本研究同样采用Illumina HiSeq2000测序平台，对E12.5天野生型 (WT) 与ADAR E861A突变型小鼠的胎肝进行深度测序，并设置3次生物学重复 (biological replicates) 以获取mRNA表达谱。