Abstract RNA editing is a posttranscriptional process that changes nucleotide sequences, among which cytosine-to-uracil by a deamination reaction can revert non-neutral codon mutations. Pentatricopeptide repeat (PPR) proteins comprise a family of RNA-binding proteins, with members acting as editing trans-factors that recognize specific RNA cis-elements and perform the deamination reaction. PPR proteins are classified into P and PLS subfamilies. In this work, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform trans-factor specific protein isolation. Soybean cis-elements from these three different RNA probes show differences in respect to other species. Pulldown samples were submitted to mass spectrometry for protein identification. Among detected proteins, five corresponded to PPR proteins. More than one PPR protein, with distinct functional domains, was pulled down with each one of the RNA probes. Comparison of the soybean PPR proteins to Arabidopsis allowed identification of the closest homologous. Differential gene expression analysis demonstrated that the PPR locus Glyma.02G174500 doubled its expression under salt stress, which correlates with the increase of its potential rps14 editing. The present study represents the first identification of RNA editing trans-factors in soybean. Data also indicated that potential multiple trans-factors should interact with RNA cis-elements to perform the RNA editing.

中文翻译：

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与质体 RNA 编辑位点相关的大豆反式因子的鉴定

摘要 RNA编辑是一种改变核苷酸序列的转录后过程，其中通过脱氨基反应将胞嘧啶变为尿嘧啶可以恢复非中性密码子突变。五肽重复 (PPR) 蛋白包含一个 RNA 结合蛋白家族，其成员充当编辑反式因子，识别特定的 RNA 顺式元件并执行脱氨反应。PPR蛋白分为P亚族和PLS亚族。在这项工作中，我们设计了基于大豆质体 RNA 编辑位点的 RNA 生物素化探针，以进行反式因子特异性蛋白质分离。这三种不同 RNA 探针的大豆顺式元件显示出与其他物种的差异。将下拉样品提交至质谱分析以进行蛋白质鉴定。在检测到的蛋白质中，有 5 个对应于 PPR 蛋白质。每一种 RNA 探针都会拉下不止一种具有不同功能域的 PPR 蛋白。将大豆 PPR 蛋白与拟南芥蛋白进行比较，可以鉴定出最接近的同源蛋白。差异基因表达分析表明，PPR 位点 Glyma.02G174500 在盐胁迫下其表达量增加了一倍，这与其潜在的 rps14 编辑的增加相关。本研究首次鉴定了大豆中的 RNA 编辑反式因子。数据还表明，潜在的多个反式因子应与 RNA 顺式元件相互作用以进行 RNA 编辑。