RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.
RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10.
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DOI：
10.1261/rna.059568.116
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发表时间：
2017-04
期刊：
影响因子：
--
通讯作者：
Barkan A
中科院分区：
文献类型：
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作者：
Miranda RG;Rojas M;Montgomery MP;Gribbin KP;Barkan A
五肽重复 (PPR) 蛋白包含一大家族螺旋重复蛋白，可影响线粒体和叶绿体中的基因表达。 PPR 束可以通过模块化的单重复-单核苷酸机制结合 RNA，其中核苷酸由每个重复中的几个氨基酸的身份指定。这种识别模式，即所谓的 PPR 代码，为预测天然 PPR 结合位点和设计结合特定 RNA 的蛋白质提供了机会。然而，要实现这些目标，必须深入了解决定 PPR-RNA 相互作用的亲和力和特异性的参数。我们报告了对 PPR10 序列特异性的全面分析，PPR10 是一种与玉米叶绿体 atpH 和 psaJ 基因附近约 18 个核苷酸 (nt) 的相似 RNA 序列结合的蛋白质。我们通过分析每个位置单核苷酸变化的影响，评估了 atpH 结合位点中每个核苷酸对 PPR10 体外亲和力的贡献。在一种补充方法中，通过深度测序分析来自部分随机化 RNA 库的 PPR10 结合的 RNA。结果揭示了三个补丁，其中核苷酸同一性对结合亲和力有重大影响。其中包括在 PPR10-RNA 晶体结构中未观察到蛋白质接触的 5 nt，以及当前 PPR 代码观点无法解释的 4 nt。这些发现强调了 PPR-RNA 相互作用的各个方面，这些方面给结合位点预测和设计带来了挑战。
Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat–one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR–RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10–RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR–RNA interactions that pose challenges for binding site prediction and design.