ADAR1-mediated A-to-I RNA editing is essential for erythropoiesis [RNA-seq]

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) RNA editing sites in wild type mice that were not edited or reduced in editing frequency in ADAR1 deficient murine erythroid cells. Secondly, to determine the transcription consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Total RNA from E14.5 fetal liver of embryos with an erythroid restricted deletion of ADAR1 (KO) and littermate controls (WT), in duplicate. cDNA libraries were prepared and RNA sequenced 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 >0.7, p<0.0001 between biological duplicates per genotype. Clusters of hyper-editing were onserved in long, unannotated 3'UTRs of erythroid specific transcripts. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 5 log2FC) in KO fetal liver compared to WT. 11.332 (6,894 novel) A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data. Conclusions: Our study represents the first detailed analysis of erythroid 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 prevent sensing of endogenous transcripts, likely via a RIG-I like receptor mediated axis. Fetal liver mRNA profiles of E14.5 wild type (WT) and ADAR Epor-Cre knock out mice were generated by deep sequencing, in duplicate using Illumina HiSeq 2000.研究目的：ADAR1介导的RNA编辑对造血发育至关重要。本研究的目标分为两点：其一，通过鉴定野生型小鼠中在ADAR1缺陷型小鼠红细胞系细胞内未发生编辑或编辑频率降低的A-to-I（即G）RNA编辑位点，筛选ADAR1特异性RNA编辑位点；其二，阐明ADAR1介导的A-to-I编辑缺失所带来的转录组学后果。 研究方法：本研究采集了携带红细胞系特异性ADAR1敲除（KO）的胚胎及同窝野生型（WT）对照的E14.5胎肝总RNA，设置生物学重复两份。随后构建cDNA文库并利用Illumina HiSeq2000进行RNA测序。对通过质量过滤的测序读段，先通过TopHat进行序列比对，再使用Cufflinks开展转录本水平分析。采用SYBR Green染料法进行实时定量逆转录聚合酶链反应（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）完成序列比对，再进行方差分析（ANOVA）。最终通过Sanger测序确认RNA编辑位点。 研究结果：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及ADAR1 E861A突变型小鼠的胎肝中鉴定出14484个转录本。各基因型的生物学重复之间，RNA-seq数据的拟合优度（R²）大于0.7，p值小于0.0001。在红细胞系特异性转录本的长链未注释3'非翻译区（3'UTR）中，可观察到高度编辑的位点簇。与WT组相比，KO组胎肝中的干扰素刺激基因出现显著上调，最大对数2倍变化（log2FC）可达5。通过分析RNA-seq数据中的碱基错配，共鉴定出11332个A-to-I RNA编辑位点（其中6894个为新发现位点）。 研究结论：本研究首次利用RNA测序技术，结合生物学重复，对红细胞系转录组及A-to-I RNA编辑位点进行了系统性详细分析。A-to-I RNA编辑是ADAR1的核心功能，其可通过激活RIG-I样受体（RIG-I like receptor）通路，避免机体识别内源性转录本，这一过程对正常生理功能至关重要。本研究通过Illumina HiSeq 2000平台，设置两份生物学重复，完成了E14.5胎龄野生型（WT）及ADAR Epor-Cre敲除小鼠的胎肝mRNA表达谱测序。