title: RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing.
abstract: Transcripts of plant organelle genes are modified by cytidine-to-uridine (C-to-U) RNA editing, often changing the encoded amino acid predicted from the DNA sequence. Members of the PLS subclass of the pentatricopeptide repeat (PPR) motif-containing family are site-specific recognition factors for either chloroplast or mitochondrial C targets of editing. However, other than PPR proteins and the cis-elements on the organelle transcripts, no other components of the editing machinery in either organelle have previously been identified. The Arabidopsis chloroplast PPR protein Required for AccD RNA Editing 1 (RARE1) specifies editing of a C in the accD transcript. RARE1 was detected in a complex of >200 kDa. We immunoprecipitated epitope-tagged RARE1, and tandem MS/MS analysis identified a protein of unknown function lacking PPR motifs; we named it RNA-editing factor interacting protein 1 (RIP1). Yeast two-hybrid analysis confirmed RIP1 interaction with RARE1, and RIP1-GFP fusions were found in both chloroplasts and mitochondria. Editing assays for all 34 known Arabidopsis chloroplast targets in a rip1 mutant revealed altered efficiency of 14 editing events. In mitochondria, 266 editing events were found to have reduced efficiency, with major loss of editing at 108 C targets. Virus-induced gene silencing of RIP1 confirmed the altered editing efficiency. Transient introduction of a WT RIP1 allele into rip1 improved the defective RNA editing. The presence of RIP1 in a protein complex along with chloroplast editing factor RARE1 indicates that RIP1 is an important component of the RNA editing apparatus that acts on many chloroplast and mitochondrial C targets.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Stephane
authors.initials: S
authors.lastname: Bentolila
authors.forename: Wade P
authors.initials: WP
authors.lastname: Heller
authors.forename: Tao
authors.initials: T
authors.lastname: Sun
authors.forename: Arianne M
authors.initials: AM
authors.lastname: Babina
authors.forename: Giulia
authors.initials: G
authors.lastname: Friso
authors.forename: Klaas J
authors.initials: KJ
authors.lastname: van Wijk
authors.forename: Maureen R
authors.initials: MR
authors.lastname: Hanson
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D030821:Plants, Genetically Modified; D011485:Protein Binding; D011523:Protoplasts / Q000378:metabolism; D017393:RNA Editing; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; C542387:RARE1 protein, Arabidopsis; C574455:RIP1 protein, Arabidopsis; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D049452:Green Fluorescent Proteins