ADAR2 affects mRNA coding sequence edits but not gene expression or splicing in vivo

Adenosine deaminases (ADARs) are RNA binding proteins that bind to double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, including AMPA-subtype Glutamate channels such as Gria2 that is edited to introduce a Q to R amino acid change. Adar2 knock out mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on the frontal cortices of Adar2-/- Gria2R/R mice and their Adar2+/+ Gria2R/R littermates, quantifying overall gene expression, splicing, and A to I editing in a transcriptome-wide fashion. We found 56 editing sites whose level of editing was significantly diminished in the Adar2 deficient animals. The majority of Adar2 responsive editing sites were in the coding regions of genes. Interestingly, other than Adar2 expression, there were only two additional statistically significant differentially expressed genes, Flnb and Cdh13. There were also only three exons that showed statistically significant differences in expression levels between the two genotypes. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has only modest effects on gene expression or splicing in vivo. 6 ADAR2 Knock out frontal cortices and 6 litter mate control frontal cortices from male mice腺苷脱氨酶（Adenosine deaminases, ADARs）是一类结合双链RNA并可将腺苷转化为肌苷的RNA结合蛋白。RNA编辑可生成多种神经递质受体亚型，涵盖AMPA亚型谷氨酸受体通道，例如经编辑后会引发谷氨酰胺（Q）到精氨酸（R）氨基酸替换的Gria2。Adar2基因敲除小鼠会在出生后不久因癫痫发作死亡，但如果将Gria2的Q/R编辑位点突变以模拟编辑后的状态，则该小鼠可正常存活。我们对Adar2-/- Gria2R/R小鼠及其Adar2+/+ Gria2R/R同窝仔鼠的前额叶皮层开展了RNA测序（RNA-Seq），在转录组范围内定量分析了整体基因表达、可变剪接以及A-to-I编辑事件。本研究共鉴定出56个在Adar2缺陷型小鼠中编辑水平显著降低的编辑位点。Adar2响应性编辑位点大多分布于基因的编码区。值得注意的是，除Adar2自身的表达差异外，仅存在另外两个具有统计学显著性的差异表达基因：Flnb与Cdh13。两种基因型小鼠间仅存在3个外显子的表达水平具有统计学显著性差异。本研究结果表明，ADAR2在蛋白质编码序列的位点特异性改变中发挥关键作用，但在体内对基因表达或可变剪接仅具有温和的调控效应。本数据集包含6只雄性Adar2缺陷型小鼠的前额叶皮层样本，以及6只同窝雄性对照小鼠的前额叶皮层样本。