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.

Fig. 1.

A rip1 mutant exhibits dwarf phenotype and increases in RIP1 transcript. (A) Map of At3g15000 (RIP1) with exons shown as black rectangles, T-DNA insertions shown as triangles, the region used for VIGS indicated, and the location of primers used for quantitative RT-PCR shown as facing arrows. (B–D) WT, heterozygous, and homozygous progeny of a heterozygous plant carrying the FLAG_150D11 insertion. Plants are 32 d old. (Scale bars: B and C, 10 mm; D, 1 mm.) (E) The expression of RIP1 is increased four- to sixfold in the T-DNA mutant compared with WT. Quantitative RT-PCR measured the level of RIP1 transcript in two homozygous mutants (M1 and M2) and two homozygous WT siblings (W1 and W2). Quantitative RT-PCR assays were replicated three times for each plant. The expression level was arbitrarily set at 100 for W1. SDs are indicated (n = 3).

Fig. 2.

Mutation in RIP1 affects the editing extent of plastid sites. (A) Acrylamide gels separate the PPE products obtained from sibling plants: two homozygous WT (+/+), two heterozygous (−/+) mutants, and two homozygous mutants (−/−). E, edited; P, primer; U, unedited. The name of the site assayed is given above each gel. (B) The quantification of editing extent derived from the measure of the band’s intensity is represented by a bar below each lane of the acrylamide gels. The average is given for each genotypic class with SD. The sites petL-5, ndhD-2, and accD-794 show a significant decrease of the editing extent in the mutant, representative of the majority of the plastid sites showing an effect of the RIP1 mutation. The site rps12-(i1)-58 shows a significant increase of editing extent in the mutant compared with the WT and heterozygous plants as observed only in two other plastid editing sites.

Fig. 3.

RIP1 is dual-targeted to mitochondria and chloroplasts. Protoplasts prepared from leaves of Arabidopsis accession Col-0 were transfected with a construct encoding an RIP1-GFP fusion protein under the control of a 35S promoter. Protoplasts were examined for fluorescence 16 h after incubation with the construct. (A and D) GFP signal is green. (B and E) Mitochondria (red) were labeled with Mitotracker Orange. (C) Merge of GFP and mitochondrial signal is yellow. (F) Chlorophyll autofluorescence is shown in blue. (G) Merge of D–F gives turquoise signals where GFP and chlorophyll overlap and yellow images where GFP and Mitotracker overlap.

Fig. 4.

Editing extent is not uniformly affected along mitochondrial transcripts in rip1 mutants. (A) Portions of electrophoretograms from RT-PCR bulk sequencing of nad2 are shown for the homozygous T-DNA mutant (−/−) and homozygous WT (+/+). Below the electrophoretograms are given the position of the editing site in the nad2 transcript with the amino acid change on editing between parenthesis and the number of sites in nad2 sharing the same molecular phenotype. The editing phenotype of the mutant was classified in one of five categories above the electrophoretograms from C = 0 (no effect of the mutation on the editing extent) to T = 0 (total loss of editing in the mutant). The C target of editing is highlighted by a black shade for T and a gray shade for C, and it is shown according to its position in the codon. (B) Distribution of the effect of the RIP1 mutation on the editing extent of mitochondrial sites on nad2 and nad6 transcripts. Each site is represented by a block with background color that indicates the strength of the rip1 mutation’s effect on the editing extent as detected by bulk sequencing. (C) PPE assays confirm the reduction of editing extent of mitochondrial sites in cob and nad6 transcripts previously detected by bulk sequencing (Region of nad6-161 gel lacking signal removed for space considerations). The PPE products run on acrylamide gels are shown on top, with the name and position of the site being assayed above the gel. E, edited; P, primer; U, unedited. Below the gels are shown the electrophoretograms of the editing site.

Fig. 5.

RIP1 silencing recapitulates the effect of rip1 mutation on editing extent of organelle sites. (A) PPE assays on plastid sites with quantification of the editing extent represented by a bar below each lane. The average is given with SDs on the right of each group of plants: RIP1-silenced and two sets of controls (GFP-silenced and uninoculated plants). petL-5 and rps12-intron C targets were chosen for assay, because they exhibit reduction and increase, respectively, in the rip1 T-DNA mutants. (B) PPE assays on mitochondrial sites cob-325 and nad6-161 in RIP1-silenced plants compared with the two sets of controls. cob-325 and nad6-161 are C targets that also show a very strong reduction of editing extent in rip1 mutants. (C) RIP1 silencing does not induce any change in the editing extent of cob-118 and nad6-26, two sites with editing extent that was also not affected in the RIP1 mutant. E, edited; P, primer; U, unedited. (The unedited band is not detectable on the cob-118 PPE gel).

Fig. 6.

Transfection of rip1 mutant with a WT version of RIP1 partially complements the editing defect in both organelles. (A) Transfection increases editing extent of mitochondrial nad6-161 and cob-325. (B) Transfection increases editing extent of plastid accD-794. (Upper) The PPE products obtained from plants either transfected or not with a construct containing a functional copy of RIP1 under the control of a 35S promoter. (Lower) Graphs depicting the quantification of editing extent for each lane; on the right of each group, the average is shown with SD. E, edited; P, primer; U, unedited.

Fig. P1.

The nuclear-encoded protein RIP1 (light blue circles) plays a role in editing of chloroplast and mitochondrial transcripts. The diagram describes the involvement of RIP1 and other unidentified nuclear-encoded proteins (gray circles with question marks) in RNA editing. PPR proteins (various colors) are also nuclear-encoded and translated in the cytoplasm (ribosomes shown in red). Different PPR protein editing factors, such as RARE1 (light green circle), are directed to either chloroplasts or mitochondria and are thought to bind to RNA sequences upstream of their C targets. RIP1 uniquely enters chloroplasts and mitochondria. We propose a model where RIP1 is present in each organelle in multiple editing complexes that contain different PPR proteins.