Department of Biochemistry, McGill University, Montreal, H3G1Y6 Quebec, Canada.
Institute of Parasitology, McGill University, Ste. Anne de Bellevue, H9X 3V9 Quebec, Canada.
RNA. 2020 Jul;26(7):827-835. doi: 10.1261/rna.075598.120. Epub 2020 Apr 10.
The RNA editing core complex (RECC) catalyzes mitochondrial U-insertion/deletion mRNA editing in trypanosomatid flagellates. Some naphthalene-based sulfonated compounds, such as C35 and MrB, competitively inhibit the auto-adenylylation activity of an essential RECC enzyme, kinetoplastid RNA editing ligase 1 (KREL1), required for the final step in editing. Previous studies revealed the ability of these compounds to interfere with the interaction between the editosome and its RNA substrates, consequently affecting all catalytic activities that comprise RNA editing. This observation implicates a critical function for the affected RNA binding proteins in RNA editing. In this study, using the inhibitory compounds, we analyzed the composition and editing activities of functional editosomes and identified the mitochondrial RNA binding proteins 1 and 2 (MRP1/2) as their preferred targets. While the MRP1/2 heterotetramer complex is known to bind guide RNA and promote annealing to its cognate pre-edited mRNA, its role in RNA editing remained enigmatic. We show that the compounds affect the association between the RECC and MRP1/2 heterotetramer. Furthermore, RECC purified post-treatment with these compounds exhibit compromised in vitro RNA editing activity that, remarkably, recovers upon the addition of recombinant MRP1/2 proteins. This work provides experimental evidence that the MRP1/2 heterotetramer is required for in vitro RNA editing activity and substantiates the hypothesized role of these proteins in presenting the RNA duplex to the catalytic complex in the initial steps of RNA editing.
RNA 编辑核心复合物（RECC）催化鞭毛原生动物中线粒体 U-插入/缺失 mRNA 编辑。一些萘基磺化化合物，如 C35 和 MrB，竞争性地抑制必需的 RECC 酶——原生动物 RNA 编辑连接酶 1（KREL1）的自动腺苷酰化活性，该酶是编辑的最后一步所必需的。以前的研究表明，这些化合物能够干扰编辑体与其 RNA 底物之间的相互作用，从而影响构成 RNA 编辑的所有催化活性。这一观察结果表明，受影响的 RNA 结合蛋白在 RNA 编辑中具有关键功能。在这项研究中，我们使用抑制性化合物分析了功能编辑体的组成和编辑活性，并鉴定了线粒体 RNA 结合蛋白 1 和 2（MRP1/2）作为其首选靶标。虽然已知 MRP1/2 异四聚体复合物结合指导 RNA 并促进其与同源预编辑 mRNA 的退火，但它在 RNA 编辑中的作用仍然是一个谜。我们表明，这些化合物影响 RECC 和 MRP1/2 异四聚体之间的关联。此外，用这些化合物处理后纯化的 RECC 表现出体外 RNA 编辑活性受损，但令人惊讶的是，添加重组 MRP1/2 蛋白后可恢复其活性。这项工作提供了实验证据，证明 MRP1/2 异四聚体是体外 RNA 编辑活性所必需的，并证实了这些蛋白质在 RNA 编辑初始步骤中将 RNA 双链体呈递给催化复合物中的假设作用。
