ADAR2 reproducibly changes abundance and sequence of mature microRNAs in the mouse brain [gene expression]. Mus musculus

Background: Adenosine deaminases that act on RNA (ADARs) bind to double-stranded and structured RNAs and deaminate adenosines to inosines. This A to I editing is widespread and required for normal life and development. Besides mRNAs and repetitive elements, ADARs can target miRNA precursors. Editing of miRNA precursors can affect processing efficiency and alter target specificity. Interestingly, ADARs can also influence miRNA abundance independent of RNA-editing. In mouse embryos where editing levels are low, ADAR2 was found to be the major ADAR protein that affects miRNA abundance. Here we extend our analysis to adult mouse brains where high editing levels are observed. Results: Using Illumina deep sequencing we compare the abundances of mature miRNAs and editing events within them, between wild-type and ADAR2 knockout mice in the adult mouse brain. Reproducible changes in abundance of specific miRNAs are observed in ADAR2 deficient mice. Most of these quantitative changes seem unrelated to A to I editing events. However, many A to G transitions in cDNAs prepared from mature miRNA sequences, reflecting A to I editing events in the RNA, are observed with frequencies reaching up to 80%. About half of these editing events are primarily caused by ADAR2 while a few miRNAs show increased editing in the absence of ADAR2, suggesting preferential editing by ADAR1. Moreover, novel, previously unknown editing events were identified in several miRNAs. In general 64% of all editing events are located within the seed region of mature miRNAs. In one of these cases retargeting of the edited miRNA could be verified in reporter assays. Also, altered processing efficiency upon editing near a processing site could be experimentally verified. Conclusions: ADAR2 can significantly influence the abundance of certain miRNAs in the brain. Only in a few cases changes in miRNA abundance can be explained by miRNA editing. Thus, ADAR2 binding to miRNA precursors, without editing them, may influence their processing and thereby abundance. ADAR1 and ADAR2 have both overlapping and distinct specificities for editing of miRNA editing sites. Over 60% of editing occurs in the seed region possibly changing target specificities for many edited miRNAs. Overall design: Examination of the effect of ADAR2 on gene expression in mature mouse wildtype and ADAR2 knockout brain using Affymetrix® GeneChip® Whole Transcript (WT) Expression Arrays (Analysis by KFB Regensburg, Germany)

背景：作用于RNA的腺苷脱氨酶（ADARs）可结合双链及结构化RNA，将腺苷脱氨基转化为肌苷。这种A-to-I编辑（A-to-I editing）广泛存在，是正常生命活动与发育所必需的。除信使RNA（mRNA）与重复序列外，ADARs还可靶向miRNA前体（miRNA precursors）。miRNA前体的编辑可影响其加工效率并改变靶标特异性。值得注意的是，ADARs还可不依赖RNA编辑的方式影响miRNA的丰度。在编辑水平较低的小鼠胚胎中，ADAR2被证实是影响miRNA丰度的主要ADAR蛋白。本研究将分析拓展至编辑水平较高的成年小鼠大脑。
结果：本研究利用Illumina高通量测序技术，比较了成年小鼠大脑中野生型与ADAR2敲除（ADAR2 knockout）小鼠的成熟miRNA丰度及其内部的编辑事件。在ADAR2缺陷小鼠中，可观测到特定miRNA丰度的可重复变化。其中绝大多数定量变化与A-to-I编辑事件无关。然而，从成熟miRNA序列制备的互补DNA（cDNA）中，存在大量A到G的转换现象，这反映了RNA层面的A-to-I编辑事件，其发生频率最高可达80%。约半数此类编辑事件主要由ADAR2介导，而少数miRNA在ADAR2缺失时编辑水平升高，提示ADAR1具有偏好性编辑的特性。此外，本研究在多个miRNA中发现了此前未被报道的新型编辑事件。总体而言，64%的编辑事件位于成熟miRNA的种子区（seed region）。在其中一例中，编辑后的miRNA的靶标重定位现象可通过报告基因实验验证。同时，在加工位点附近发生的编辑可导致加工效率改变，这一现象也已通过实验证实。
结论：ADAR2可显著影响大脑中特定miRNA的丰度。仅在少数情况下，miRNA丰度的变化可通过miRNA编辑来解释。由此推测，ADAR2结合miRNA前体但不对其进行编辑的行为，可能会影响其加工过程，进而改变miRNA的丰度。ADAR1与ADAR2在miRNA编辑位点的编辑特异性上既存在重叠，也存在差异。超过60%的编辑事件发生在种子区，这可能会改变众多被编辑miRNA的靶标特异性。
整体实验设计：本研究利用Affymetrix® GeneChip® 全转录组（WT）表达阵列，检测ADAR2对成年野生型与ADAR2敲除小鼠大脑成熟基因表达的影响（分析由德国雷根斯堡的KFB实验室完成）