A Conserved Glutamate Residue in the C-terminal Deaminase Domain of Pentatricopeptide Repeat Proteins Is Required for RNA Editing Activity

Abstract

Background: Pentatricopeptide repeat (PPR) proteins that are required for RNA editing frequently include a C-terminal DYW deaminase domain. Results: Mutagenesis of a glutamate residue in the conserved deaminase HXE motif results in loss of editing activity. Conclusion:The glutamate residue is required for editing. Significance: The DYW deaminase domain of PPR proteins has the molecular characteristics of a deaminase.Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast.RNA editing takes place in most land plant chloroplasts and mitochondria (1, 2). In flowering plants, the transcripts of chloroplasts and mitochondria are modified post-transcriptionally by C-to-U editing with about 35 C-to-U editing events in chloroplasts and hundreds of editing sites in the mitochondria (3).Editing in higher plants and in Physcomitrella patens is known to require nuclear proteins (4 -8).Pentatricopeptide repeat (PPR) 2 genes have been shown to be required for RNA editing (9), and form a large family of protein-coding genes in higher plants with over 400 members in Arabidopsis (10). The known editing factors are members of the PLS subfamily of PPR proteins, which are composed of characteristic P, L (long), and S (short) repeats (11). Amino acid residues located in specific locations within the repeats have been shown to specify the base recognized in the cis-element (12)(13)(14), and the PLS repeat domain interacts with specific nucleotides within the cis-element to provide site specificity for RNA editing (12,15).The PLS subfamily of PPR proteins also includes ...

Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants

“…() (top), and those proposed and used in this study (bottom). ‘PG’ indicates the conserved ‘PG box’ (Hayes et al ., ) while HSE and CxDC indicate the zinc‐binding deaminase signature sequence (Salone et al ., ; Hayes et al ., , ; Boussardon et al ., ; Wagoner et al ., ).…”

A genome-wide identification and analysis of the DYW-deaminase genes in the pentatricopeptide repeat gene family in cotton (Gossypium spp.)

“…Cytidine deaminase is a Zn-dependent enzyme in which the CxxCH motif functions as a binding site [20]. The conserved His and Cys residues in the DYW domain combine with Zn ions, while the conserved Glu is required for deamination [8]. Thus, the DYW domain may be involved in nucleotide deaminations.…”

“…Recently, Hayes et al (2015) suggested that the conserved Glu residue of the HXE motif in the DYW domain is necessary for RNA editing. When the codon for the Glu residue in the DYW domain of OTP84 and CREF7 was mutagenized, the transgenic plants containing the mutated genes could not efficiently edit the cognate editing sites [8]. Wagoner et al (2015) proposed that the DYW domain and the E (or E+) domain are essential for cytidine deaminase activity because truncating either domain resulted in a QED1 protein that completely lacked any editing activity.…”

The pentatricopeptide repeat protein EMP9 is required for mitochondrial ccmB and rps4 transcript editing, mitochondrial complex biogenesis and seed development in maize

“…In phylogenetic distribution, the appearance of DYW domains in PPR proteins coincides with the appearance of RNA editing events. Moreover, mutagenesis of the conserved residues in DYW1, QED1, RARE1, OTP84 and CREF7 leads to loss of editing activity (Boussardon et al ., ; Hayes et al ., ; Wagoner et al ., ). However, the deaminase activity was not found by testing recombinant DYW proteins in vitro (Nakamura & Sugita, ; Okuda et al ., ).…”