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超越 PPR-RNA 识别码:许多因素影响 RNA 编辑因子 PPR56 的多靶向特性。

Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56.

机构信息

IZMB-Institut für Zelluläre und Molekulare Botanik, Abteilung Molekulare Evolution, Universität Bonn, Bonn, Germany.

出版信息

PLoS Genet. 2023 Aug 21;19(8):e1010733. doi: 10.1371/journal.pgen.1010733. eCollection 2023 Aug.

Abstract

The mitochondrial C-to-U RNA editing factor PPR56 of the moss Physcomitrium patens is an RNA-binding pentatricopeptide repeat protein equipped with a terminal DYW-type cytidine deaminase domain. Transferred into Escherichia coli, PPR56 works faithfully on its two native RNA editing targets, nad3eU230SL and nad4eU272SL, and also converts cytidines into uridines at over 100 off-targets in the bacterial transcriptome. Accordingly, PPR56 is attractive for detailed mechanistic studies in the heterologous bacterial setup, allowing for scoring differential RNA editing activities of many target and protein variants in reasonable time. Here, we report (i) on the effects of numerous individual and combined PPR56 protein and target modifications, (ii) on the spectrum of off-target C-to-U editing in the bacterial background transcriptome for PPR56 and two variants engineered for target re-direction and (iii) on combinations of targets in tandem or separately at the 5'- and 3'-ends of large mRNAs. The latter experimentation finds enhancement of RNA editing at weak targets in many cases, including cox3eU290SF as a new candidate mitogenome target. We conclude that C-to-U RNA editing can be much enhanced by transcript features also outside the region ultimately targeted by PPRs of a plant editing factor, possibly facilitated by its enrichment or scanning along transcripts.

摘要

苔藓植物Physcomitrium patens 的线粒体 C-to-U RNA 编辑因子 PPR56 是一种 RNA 结合五肽重复蛋白,带有末端 DYW 型胞嘧啶脱氨酶结构域。将 PPR56 转入大肠杆菌后,它能忠实作用于其两个天然的 RNA 编辑靶标 nad3eU230SL 和 nad4eU272SL,还能在细菌转录组中的 100 多个非靶标上把胞嘧啶转化为尿嘧啶。因此,PPR56 非常适合在异源细菌体系中进行详细的机制研究,使人们能够在合理的时间内对许多靶标和蛋白变体的差异 RNA 编辑活性进行评分。在这里,我们报告了(i)大量单个和组合的 PPR56 蛋白和靶标修饰的影响,(ii)在细菌背景转录组中 PPR56 和两个针对靶标重新定向设计的变体的脱靶 C-to-U 编辑谱,以及(iii)在大 mRNA 的 5'和 3'末端串联或分别靶向的靶标组合。后一种实验发现,在许多情况下,包括 cox3eU290SF 作为新的候选线粒体基因组靶标,弱靶标上的 RNA 编辑得到了增强。我们得出结论,除了植物编辑因子的 PPR 最终靶向的区域之外,转录本特征也能极大地增强 C-to-U RNA 编辑,这可能是通过 PPR 沿转录本的富集或扫描而实现的。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/766e/10482289/a6943280c938/pgen.1010733.g001.jpg

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