Institute of Molecular Biology and Biophysics, ETH Zurich, Schafmattstrasse 20, CH-8093 Zürich, Switzerland.
Biochimie. 2012 Jul;94(7):1499-509. doi: 10.1016/j.biochi.2011.12.017. Epub 2011 Dec 23.
Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.
作用于 RNA 的腺苷脱氨酶（ADAR）催化双链 RNA（dsRNA）底物中的腺苷向肌苷（A-to-I）编辑。肌苷在翻译机制中被读为鸟苷；因此，编码序列中的 A-to-I 编辑事件可能导致遗传信息的重编码。虽然脊椎动物有两种具有催化活性的酶，即 ADAR1 和 ADAR2，但果蝇只有一种与 ADAR2 相关的 ADAR 蛋白（dADAR）。控制 dsRNA 底物中特定腺苷的底物识别和编辑的结构决定因素仅部分了解。在这里，我们报告了 dADAR 的 N 端 dsRNA 结合域（dsRBD）的溶液结构，并使用 NMR 化学位移扰动来鉴定参与 RNA 结合的蛋白质表面。此外，我们表明果蝇 ADAR 编辑哺乳动物 GluR-2 前 mRNA 中的 R/G 位点，该位点自然被 ADAR1 和 ADAR2 修饰。然后，我们构建了一个模型，展示了 dADAR dsRBD1 如何结合 GluR-2 R/G 茎环。该模型表明，与 RNA 糖磷酸骨架相互作用的大多数侧链只需很小的位移即可适应 dsRNA 结合，因此已准备好与它们的 dsRNA 靶标结合。它还预测 dADAR dsRBD1 与 dsRNA 的结合特异性不如 ADAR2 的 dsRBD 强。总之，这项研究为果蝇 ADAR 对 dsRNA 底物的识别提供了新的见解。
