Laboratoire Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Unité Mixte de Recherche Centre National de la Recherche Scientifique - Université de Lorraine, Biopôle de l'Université de Lorraine, Vandœuvre-lès-Nancy, France.
PLoS One. 2013 Jul 29;8(7):e70313. doi: 10.1371/journal.pone.0070313. Print 2013.
Multiple RNA-guided pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.
多个 RNA 指导的假尿嘧啶合酶、含有指导 RNA 和四个蛋白质的 H/ACA 核蛋白颗粒 (RNP) 催化底物 RNA 中尿嘧啶的特异性转录后异构化为假尿嘧啶。在古菌颗粒中，指导小 RNA (sRNA) 由假尿嘧啶合酶 aCBF5 和核糖体蛋白 L7Ae 锚定。蛋白 aNOP10 与 aCBF5 和 L7Ae 相互作用。第四个蛋白 aGAR1 与 aCBF5 相互作用并增强催化效率。在这里，我们比较了来自 Pyrococcus abyssi 的两种 H/ACA sRNAs，Pab21 和 Pab91 的特征。我们发现 aCBF5 与 Pab91 的结合力比与 Pab21 的结合力弱得多。令人惊讶的是，Pab91 sRNP 的催化效率比 Pab21 sRNP 高。因此，我们研究了组装和两种酶催化活性的差异效率的分子基础。为此，我们比较了在 sRNP 逐步重建过程中这些 sRNAs 中铅诱导切割程度的图谱，并分析了 aNOP10-L7Ae 相互作用缺失的影响。这些探测实验表明，sRNAs 在 RNP 组装过程中经历了一系列构象变化。这些变化也通过圆二色性 (CD) 光谱直接评估，这是一种非常适合分析 RNA 构象动力学的工具。此外，我们的结果表明，在 H/ACA sRNAs 底部形成的 P1 螺旋的构象在 Pab21 中经过优化，以实现有效的 aCBF5 结合和 RNP 组装。此外，P1 交换可改善 Pab91 sRNP 的组装。然而，有效的 aCBF5 结合可能还依赖于假尿嘧啶化口袋，在 Pab21 的情况下，该口袋未针对高活性进行优化。
