A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.
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.
From the Developmental and Cell Biology, University of California, Irvine, California 92697.
Michael L
ML
Hayes
From the Developmental and Cell Biology, University of California, Irvine, California 92697.
Kim N
KN
Dang
From the Developmental and Cell Biology, University of California, Irvine, California 92697.
Michael F
MF
Diaz
From the Developmental and Cell Biology, University of California, Irvine, California 92697 rmmullig@uci.edu.
R Michael
RM
Mulligan
D060054:Agrobacterium / Q000235:genetics; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017124:Conserved Sequence; D003564:Cytidine Deaminase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017393:RNA Editing; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014170:Transformation, Genetic; D019076:Transgenes
D029681:Arabidopsis Proteins; D020033:Protein Isoforms; D018749:RNA, Plant; C538752:pentatricopeptide repeat protein, Arabidopsis; D018698:Glutamic Acid; D003564:Cytidine Deaminase
Chloroplasts; Cytidine Deaminase; DYW Deaminase Domain; Metalloenzyme; RNA Editing; RNA Metabolism; RNA-binding Protein; Zinc