Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, United States of America.
Department of Biochemistry & Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
PLoS One. 2019 Apr 29;14(4):e0211525. doi: 10.1371/journal.pone.0211525. eCollection 2019.
Uridylate insertion/deletion RNA editing in Trypanosoma brucei is a complex system that is not found in humans, so there is interest in targeting this system for drug development. This system uses hundreds of small non-coding guide RNAs (gRNAs) to modify the mitochondrial mRNA transcriptome. This process occurs in holo-editosomes that assemble several macromolecular trans factors around mRNA including the RNA-free RNA editing core complex (RECC) and auxiliary ribonucleoprotein (RNP) complexes. Yet, the regulatory mechanisms of editing remain obscure. The enzymatic accessory RNP complex, termed the REH2C, includes mRNA substrates and products, the multi-domain 240 kDa RNA Editing Helicase 2 (REH2) and an intriguing 8-zinc finger protein termed REH2-Associated Factor 1 (H2F1). Both of these proteins are essential in editing. REH2 is a member of the DExH/RHA subfamily of RNA helicases with a conserved C-terminus that includes a regulatory OB-fold domain. In trypanosomes, H2F1 recruits REH2 to the editing apparatus, and H2F1 downregulation causes REH2 fragmentation. Our systematic mutagenesis dissected determinants in REH2 and H2F1 for the assembly of REH2C, the stability of REH2, and the RNA-mediated association of REH2C with other editing trans factors. We identified functional OB-fold amino acids in eukaryotic DExH/RHA helicases that are conserved in REH2 and that impact the assembly and interactions of REH2C. H2F1 upregulation stabilized REH2 in vivo. Mutation of the core cysteines or basic amino acids in individual zinc fingers affected the stabilizing property of H2F1 but not its interactions with other examined editing components. This result suggests that most, if not all, fingers may contribute to REH2 stabilization. Finally, a recombinant REH2 (240 kDa) established that the full-length protein is a bona fide RNA helicase with ATP-dependent unwinding activity. REH2 is the only DExH/RHA-type helicase in kinetoplastid holo-editosomes.
布氏锥虫的尿苷酸插入/缺失 RNA 编辑是一个复杂的系统，在人类中不存在，因此人们有兴趣针对该系统开发药物。该系统使用数百种小的非编码引导 RNA (gRNA) 来修饰线粒体 mRNA 转录组。这个过程发生在全编辑体中，全编辑体围绕 mRNA 组装几个大分子转录因子，包括无 RNA 的 RNA 编辑核心复合物 (RECC) 和辅助核糖核蛋白 (RNP) 复合物。然而，编辑的调控机制仍然不清楚。酶辅助的 RNP 复合物，称为 REH2C，包括 mRNA 底物和产物、多结构域 240 kDa RNA 编辑解旋酶 2 (REH2) 和一个有趣的 8 锌指蛋白称为 REH2 相关因子 1 (H2F1)。这两种蛋白质在编辑中都是必不可少的。REH2 是 DExH/RHA 亚家族 RNA 解旋酶的成员，具有保守的 C 端，包括一个调节性 OB 折叠结构域。在锥虫中，H2F1 将 REH2 招募到编辑装置中，H2F1 的下调导致 REH2 断裂。我们系统的诱变分析确定了 REH2 和 H2F1 中用于组装 REH2C、REH2 稳定性以及 REH2C 与其他编辑转录因子的 RNA 介导相互作用的决定因素。我们鉴定了真核 DExH/RHA 解旋酶中保守的 REH2 中的功能 OB 折叠氨基酸，这些氨基酸影响 REH2C 的组装和相互作用。H2F1 的上调稳定了体内的 REH2。单个锌指的核心半胱氨酸或碱性氨基酸的突变影响了 H2F1 的稳定特性，但不影响其与其他检查编辑成分的相互作用。这一结果表明，即使不是全部，大多数锌指可能有助于 REH2 的稳定。最后，重组 REH2（240 kDa）证实全长蛋白是一种真正的 RNA 解旋酶，具有 ATP 依赖性解旋活性。REH2 是动质体全编辑体中唯一的 DExH/RHA 型解旋酶。
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