PDF(Pubmed)
Abstract：
Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2\'s preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes\' functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.
摘要：
腺苷-肌苷(A-to-I)RNA编辑是由腺苷脱氨酶作用于RNA(ADAR)家族酶介导的重要转录后修饰,通过将RNA分子中选定的核苷酸A改变为I来扩展转录组。最近,已经探索了使用治疗性指导寡核苷酸在特定位点指导ADAR编辑来纠正RNA中的致病突变。人类有两个活跃的ADAR，其偏好和特异性尚未得到很好的理解。为了研究它们的底物特异性，我们分别介绍了hADAR1和hADAR2，进入裂殖酵母(S.pombe),缺乏内源性ADAR，并评估了它们在体内的编辑活性。使用在最佳生长温度（30°C）下培养的S.pombe的转录组测序，与未编辑的野生型对照菌株相比,我们为hADAR1鉴定了483个A-to-I高置信度编辑位点,为hADAR2鉴定了404个位点.然而,这些位点在hADAR1和hADAR2表达菌株之间大多是不同的，共享33个常见位点，每个菌株的比例低于9%。它们对底物的差异特异性归因于它们对编辑位点的相邻序列的差异偏好。我们发现在相对于编辑站点的-3位置，HDAR1表现出一种向T的趋势，而hADAR2倾向于A。此外，当改变表达hADAR1-和hADAR2的菌株的生长温度时，我们观察到在20和35°C时它们的编辑位点增加，与它们在30°C下生长相比。然而,我们没有观察到hADAR1和hADAR2在三个温度下对相邻序列的偏好发生显著变化。在较低和较高的温度下，RNA编辑位点的巨大变化也被观察到之前在出芽酵母中的hADAR2，这可能是由于在这些不同温度下RNA折叠的影响，在许多其他因素中。我们注意到编辑位点周围较长长度的dsRNA的例子，这些例子在20或35°C诱导编辑，但在其他两个温度条件下不存在。我们发现基因的功能会受到转录本编辑的极大影响，其中50%以上的HADAR1和HADAR2的RNA编辑位点都在编码序列（CDS）中，其中60%以上导致蛋白质产品的氨基酸变化。这项研究揭示了两种活性人类ADARS之间底物选择性的广泛差异，即，ADAR1和ADAR2，并在使用RNA编辑方法利用两种不同的酶在体内治疗人类遗传疾病时提供了新的见解。
