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-touridine (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. Virusinduced 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.nucleoid | RNA editosome | dual targeting P osttranscriptional C-to-U RNA editing occurs in plastid and plant mitochondrial transcripts. In a typical vascular plant, ∼30 C targets in chloroplasts and over 500 C targets in mitochondria are targeted for editing (1, 2). The majority of the editing events results in encoding of a different amino acid than the one predicted from the genomic sequence. The editing-encoded amino acid is usually more conserved relative to residues present in homologous proteins in other organisms than the genomically encoded amino acid. Because there is presently no known case in which useful genetic variation results from partial editing of a transcript population, the current concept is that editing is a correction mechanism for thymidine-to-cytidine (T-to-C) mutations that have arisen in plant organelle genomes (1,3,4).Little is known about the molecular apparatus that is responsible for recognizing the correct C target for editing and converting it to U, although plant mitochondrial RNA editing was discovered over 20 y ago (5-7). cis-Elements for recognition of editing sites have been identified proximal and 5′ to the nucleotide to be modified (8-10). As few as 22 nt in sequence surrounding the C target is sufficient to specify RNA editing (9). In 2005, a pentatricopeptide repeat (PPR) motif-containing protein termed CRR4 was discovered to ...

An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize

“…4C). We examined the organelle transcriptome of the mutant for editing defects because other proteins carrying RIP domains have been shown to be editing factors (19,20). We analyzed the plastid RNA editing extent with a new methodology based on RNA-seq.…”

“…A blastp search for homologs to the RIP1 protein returned the 10 previously reported members of the RIP family (19,20) as well as a unique RIP family member, encoded by the gene At3g20930 (Fig. 1A).…”

“…1A). This protein was not previously described as either a RIP or MORF protein (19,20). We used the MEME software (22) to identify four highly significant motifs in the RIP family (Fig.…”

“…We identified Arabidopsis dual-targeted protein RIP1, an essential plant-editing factor that is required for the editing of numerous Cs both in plastids and mitochondria (19). A rip1 mutant plant exhibited reduced editing efficiency at 266 mitochondrial C targets, with a major loss of editing for 108.…”

“…Other members of the RIP family have also been shown to be required for organelle editing (20). RIP proteins are able to interact selectively with PPR trans-actors and also with each other (19,20); however, their function in the plant editosome remains unclear.…”