Yingying Yang, Kira Ritzenhofen, Jessica Otrzonsek, Jingchan Xie, Mareike Schallenberg-Rüdinger, Volker Knoop
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 recent years have seen much progress towards understanding the molecular machinery behind cytidine-to-uridine RNA editing in plant chloroplasts and mitochondria 1–4]. Among other insights, very early functional studies on plant RNA editing based on in organello, in vitro or transplastomic studies had already demonstrated that the specificity for identifying cytidine targets largely resides in their immediate sequence environment, mainly within circa 20 upstream nucleotides [5–14]. The molecular characterization of the trans-acting specificity factors, however, ultimately relied on reverse genetic approaches leading to the identification of CRR4 as a first identified chloroplast and MEF1 as the first mitochondrial RNA editing factor in Arabidopsis thaliana [15,16]. Such site-specific editing factors feature sequence-specific RNA-binding pentatricopeptide repeats (PPRs) of the so-called PLS-type followed by “extension” domains E1 and E2 plus a C-terminal DYW cytidine deaminase, which may alternatively be supplied in trans.
Materials and method
Cloning for expression of Physcomitrium patens PPR56 variants and targets in Escherichia coli was based on vector pET41Kmod as outlined earlier . PPR56 coding sequences (lacking the N-terminus with signal peptide and including only 14 amino acids upstream of the first clearly identified PPR) are cloned via gateway cloning downstream of an N-terminal His6 tag and the maltose-binding protein (MBP) for improved protein solubility  behind a T7 promoter controlled by the lac operator. RNA editing target sequences were cloned behind the protein sequence upstream of a T7 terminator. Here, we also created a new vector variant pET41Kmod2 (S2 Fig) with further restriction sites allowing for cloning targets also upstream of the respective coding region. To that end, we made use of a former XbaI site to create a NotI-EcoRI-PacI-PstI multiple cloning site (MCS) upstream of the ribosome binding site (RBS) in pET41Kmod. Target sequences including flanking restriction sites were generated with synthesized oligonucleotides for both DNA strands (Integrated DNA technologies Europe, BVBA, Leuven, Belgium) and ligated into dephosphorylated vectors after hybridization and phosphorylation.
PPR56, mutant nomenclature and the vector assay systems
PPR56 is a plant C-to-U RNA editing factor equipped with a highly conserved carboxyterminal DYW-type cytidine deaminase domain linked to an upstream PLS-type PPR array via the E1 and E2 extension motifs (Fig 1A). For clarity, we here introduce nomenclature standards to label mutations on the protein or on the target side, respectively, that have been introduced for studying RNA editing functionality. For mutations on the protein side, we use a protein domain label behind a pipe symbol, followed by a colon and the position and amino acid identities in single-letter annotation before and after changes, e.g. PPR56|DYW:G3A for the mutation converting the glycine of the conserved PG box (Figs 1B and S1) into alanine. As a shorthand notation for mutations targeting the crucial positions ‘5’ and ‘L’ of a given PPR, we simply indicate the introduced identities without numbering, e.g. PPR56|P-6ND>TD for the mutation converting the native ND combination in PPR P-6 for a conceptually better match to the guanidine that is naturally present in position -9 upstream of the nad4eU272SL editing site (Fig 1A).
Mutating the DYW domain: different effects on two native targets
All of our experimentation showed that the nad4 target of PPR56 is more resilient towards changes both on the target side and on the protein side than the nad3 editing target site, which proved to be much more sensitive. Notably, the higher sensitivity of the nad3 target towards changes also extended to mutations in the DYW domain of PPR56 (Fig 1B). The carboxy-terminal DYW domain of plant RNA editing factors has long been suspected, and is meanwhile well confirmed, as the catalytic cytidine deaminase domain [36,46,48,55–57]. Many of the highly conserved amino acid residues in the DYW domain are essential for functionality as here again confirmed with a set of mutations in the DYW domain of PPR56. However, while six mutants with single amino acid exchanges in the DYW domain of PPR56 lost RNA editing activity on both targets, seven others affected RNA editing at the nad3eU230SL target more strongly than at the nad4eU272SL site (Fig 1B).
We gratefully acknowledge the computer resources and support provided by the Paderborn Center for Parallel Computing (PC2). We wish to thank Bastian Oldenkott, Philipp Gerke and Simon Zumkeller in our group for the establishment and help in further development of bioinformatic pipelines and Sarah Brenner for technical assistance. We especially like to thank Elena Lesch for establishing the program to generate construct-specific DNA references. We also like to thank Bastian Oldenkott for designing the initial PPR model for PPR protein figures. We thank Mark Hermann Vegas and Grazia Margherita Willerscheidt for cloning constructs and performing initial E. coli experiments as part of their experimental Bachelor theses work.
Citation: Yang Y, Ritzenhofen K, Otrzonsek J, Xie J, Schallenberg-Rüdinger M, Knoop V (2023) Beyond a PPR-RNA recognition code: Many aspects matter for the multi-targeting properties of RNA editing factor PPR56. PLoS Genet 19(8): e1010733. https://doi.org/10.1371/journal.pgen.1010733
Editor: Bao-Cai Tan, Shandong University, CHINA
Received: April 4, 2023; Accepted: July 30, 2023; Published: August 21, 2023
Copyright: © 2023 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The RNAseq data have been deposited in the SRA archive as BioProject PRJNA984633. All other relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.