Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403 USA.
Nucleic Acids Res. 2021 Jun 4;49(10):5985-5997. doi: 10.1093/nar/gkab390.
Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5' end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5' UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPR proteins can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.
五肽重复（PPR）蛋白是螺旋重复蛋白，以模块化的方式结合 RNA，其序列特异性可以通过氨基酸密码来操纵。因此，PPR 重复是设计用于合成生物学应用的 RNA 结合蛋白的有前途的支架。然而，由共识 PPR 基序构建的人工 PPR 蛋白的体内功能才刚刚开始被探索。在这里，我们报告了一种人工 PPR 蛋白 dPPRrbcL 的体内功能，该蛋白由设计用于结合拟南芥叶绿体 rbcL 转录本 5'端附近序列的共识 PPR 基序组成。我们使用功能互补测定法证明，该蛋白在体内特异性地结合其预期的 RNA 靶标，并且通过稳定加工的 rbcL mRNA 替代天然 PPR 蛋白。我们将第二个类似设计的蛋白质靶向 petL 5'UTR，在那里它替代了天然稳定 PPR 蛋白 PGR3，但效率不高。这些结果表明，可以对人工 PPR 蛋白进行工程设计，以模拟作为抗核酸外切酶物理屏障的天然 PPR 蛋白类。
