DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites

Abstract

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5 0 sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the À1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells

“…Although the large sets of identified off-targets argue for freely diffusing RNA editing factors in the cytosol, our experimental setup may generally favor the delivered native targets located on the same transcript behind their respective coding regions. First evidence in that direction comes from a very recent study indeed finding somewhat lower RNA editing efficiencies when modifying the previously established bacterial assay system ( 28 ) by providing the RNA editing targets on a separate second vector ( 66 ).…”

DYW cytidine deaminase domains have a long‐range impact on RNA recognition by the PPR array of chimeric plant C‐to‐U RNA editing factors and strongly affect target selection

“…The structure of the DYW cytidine deaminase domain of OTP86, a chloroplast RNA editing factor in A. thaliana (Hammani et al., 2009), has been obtained by X‐ray crystallography (Takenaka et al., 2021). Moreover, a chimera of PPR56 in fusion with the OTP86 DYW domain proved to be functional in the E. coli assay system (Maeda et al., 2022; Takenaka et al., 2021). We independently created PPR56/OTP86 chimeras with a fusion point at the end of the E2 motif and, again, replaced only the gating domain of PPR56 with the one of OTP86 in an independent chimera (Figure 3).…”

“…In contrast, however, many other recombinant protein chimeras created similarly could not be shown to be functional (Chateigner‐Boutin et al., 2013; Ichinose & Sugita, 2018; Maeda et al., 2022; Okuda et al., 2014) suggesting that there is no simple modular concept. The respective DYW domain and/or the respective E1 and E2 motifs may exert further selectivity for target recognition, especially in the immediate environment of the RNA editing target site and several available data indeed support this conclusion (Ichinose & Sugita, 2018; Maeda et al., 2022; Okuda et al., 2014; Ruwe et al., 2019).…”

“…Indeed, the DYW domain of Arabidopsis chloroplast RNA editing factor OTP86, for which a crystal structure has recently been obtained, could replace the one of Physcomitrium mitochondrial editing factor PPR56 for functional analyses in Escherichia coli (Takenaka et al., 2021). However, this functional chimera appeared to be more of an exception than the rule since testing other RNA editing factor chimeras had only moderate success both in planta (Ichinose & Sugita, 2018) or in the heterologous bacterial system (Maeda et al., 2022).…”

Exploring the RNA Editing Events and Their Potential Regulatory Roles in Tea Plant (Camellia sinensis L.)

“…RNA editing of C-to-U is a deamination reaction [ 22 ]. A number of RNA editing factors were found to be involved in this process [ 3 ], including PLS-type pentatricopeptide repeat (PPR) family proteins [ 23 ], multiple organellar RNA editing factor (MORF, also named RNA editing factor interacting protein (RIP)) family proteins [ 24 , 25 ], organelle RNA recognition motif-containing (ORRM) family proteins [ 26 ], protoporphyrinogen IX oxidase 1 (PPO1) [ 27 ], Chlororespiratory reduction 4 (CRR4) [ 28 ] and organelle zinc finger 1 (OZ1) [ 29 ].…”