Abstract
In plant organelles, RNA editing is a post-transcriptional mechanism that converts specific cytidines to uridines in RNA of both mitochondria and plastids, altering the information encoded by the gene. The cytidine to be edited is determined by a cis-element surrounding the editing site that is specifically recognized and bound by a trans-acting factor. All the trans-acting editing factors identified so far in plant organelles are members of a large protein family, the pentatricopeptide repeat (PPR) proteins. We have identified the Organelle Transcript Processing 87 (OTP87) gene, which is required for RNA editing of the nad7-C24 and atp1-C1178 sites in Arabidopsis mitochondria. OTP87 encodes an E-subclass PPR protein with an unusually short E-domain. The recombinant protein expressed in Escherichia coli specifically binds to RNAs comprising 30 nucleotides upstream and 10 nucleotides downstream of the nad7-C24 and atp1-C1178 editing sites. The loss-of-function of OTP87 results in small plants with growth and developmental delays. In the otp87 mutant, the amount of assembled respiratory complex V (ATP synthase) is highly reduced compared with the wild type suggesting that the amino acid alteration in ATP1 caused by loss of editing at the atp1-C1178 site affects complex V assembly in mitochondria.
Highlights
In flowering plants, RNA editing comprises conversion of specific cytidine residues to uridine in both mitochondrial and
Phenotypic Analysis of Arabidopsis Organelle Transcript Processing 87 Mutant—To identify genes that are involved in the editing of organellar transcripts in Arabidopsis, we initiated a reverse genetic screen of T-DNA insertion lines in which genes encoding pentatricopeptide repeat (PPR) proteins of the E and DYW subgroups are disturbed in Arabidopsis (A. thaliana) ecotype Columbia (Col-0) plants
Chloroplast Biogenesis Is Not Affected in otp87—PPR proteins are involved in a wide range of post-transcriptional processes in plant organelles and we postulated that Organelle Transcript Processing 87 (OTP87) is involved in one of these processes
Summary
All primers used in this study are listed in supplemental Table S1. Plant Material—A. thaliana ecotype Columbia (Col-0) was used in this study. Analysis of Targeting via GFP Fusions—The first 300 bp of the coding sequences of the PPR genes were amplified using Phusion DNA polymerase (Finnzymes) with primers listed in supplemental Table S1 containing the attB sites for Gateway cloning according to the manufacturer’s instructions (Invitrogen). This fragment was cloned in-frame with the GFP coding sequence. Genetic Complementation—The 3099 bp fragment containing the coding sequence of OTP87 and the 5Ј-intergenic region was amplified by PCR on total cellular DNA. Immunoblot Analysis—Chloroplasts were isolated from the leaves of 4-week-old plants as previously described (6), and samples were normalized by measuring chlorophyll concentration. Bioinformatic Analysis—Consensus for editing sites recognized by OTP87 was calculated by hand and searched against the Arabidopsis mitochondria or chloroplast genomes sequence using fuzznuc from the EMBOSS package (41)
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