A comparative analysis of ADAR mutant mice reveals site-specific regulation of RNA editing
A comparative analysis of ADAR mutant mice reveals site-specific regulation of RNA editing
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DOI：
10.1261/rna.072728.119
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发表时间：
2020-04-01
期刊：
影响因子：
4.5
通讯作者：
Kawahara, Yukio
中科院分区：
文献类型：
--
作者：
Cruz, Pedro Henrique Costa;Kato, Yuki;Kawahara, Yukio
腺苷-肌苷RNA编辑是由腺苷脱氨酶作用于RNA（阿达尔）1和ADAR 2催化的一种重要的转录后修饰。对于编码序列（CDS）和microRNA中的许多位点，编辑是高度保守的，并且具有显著的生物学后果，例如，通过改变氨基酸残基和靶识别。然而，还没有进行全面和定量的研究，以确定特定的ADAR如何有助于保守的网站在体内。在这里，我们分别扩增了每个具有编辑位点的RNA区域，并将其组合用于深度测序。然后，我们比较了野生型小鼠、表达失活ADAR 1的Adar 1（E861 A/E861）/fih（-/-）小鼠（Adar 1 KI）和Adar 2-/Gria 2（R/R）（Adar 2 KO）小鼠大脑皮层和脾脏中CDS和microRNA中所有保守位点的编辑率。我们发现，大多数网站显示出一个阿达尔的偏好。相比之下，一些位点，如miR-3099- 3 p，没有显示出阿达尔偏好性。此外，我们发现几个位点的编辑比例，如DACT 3 R/G，在任一阿达尔突变小鼠品系中上调，而ADAR 1和ADAR 2之间的协调相互作用是有效编辑特定位点所必需的，如5-HT 2cRB位点。我们进一步创建了双突变体Adar 1 KI Adar 2KO小鼠，并观察到完全不存在编辑的存活和可生育的动物，证明ADAR 1和ADAR 2是负责体内所有编辑位点的唯一酶。总的来说，这些发现表明，编辑是由ADAR 1和ADAR 2之间的不同相互作用以位点特异性方式调节的。
Adenosine-to-inosine RNA editing is an essential post-transcriptional modification catalyzed by adenosine deaminase acting on RNA (ADAR)1 and ADAR2 in mammals. For numerous sites in coding sequences (CDS) and microRNAs, editing is highly conserved and has significant biological consequences, for example, by altering amino acid residues and target recognition. However, no comprehensive and quantitative studies have been undertaken to determine how specific ADARs contribute to conserved sites in vivo. Here, we amplified each RNA region with editing site(s) separately and combined these for deep sequencing. Then, we compared the editing ratios of all sites that were conserved in CDS and microRNAs in the cerebral cortex and spleen of wild-type mice, Adar1(E861A/E861)/fih(-/-) mice expressing inactive ADAR1 (Adar1 KI) and Adar2 -/Gria2(R/R)(Adar2 KO) mice. We found that most of the sites showed a preference for one ADAR. In contrast, some sites, such as miR-3099-3p, showed no ADAR preference. In addition, we found that the editing ratio for several sites, such as DACT3 R/G, was up-regulated in either Adar mutant mouse strain, whereas a coordinated interplay between ADAR1 and ADAR2 was required for the efficient editing of specific sites, such as the 5-HT2cRB site. We further created double mutant Adar1 KI Adar2KO mice and observed viable and fertile animals with the complete absence of editing, demonstrating that ADAR1 and ADAR2 are the sole enzymes responsible for all editing sites in vivo. Collectively, these findings indicate that editing is regulated in a site-specific manner by the different interplay between ADAR1 and ADAR2.