Natural Variation in Arabidopsis Leads to the Identification of REME1, a Pentatricopeptide Repeat-DYW Protein Controlling the Editing of Mitochondrial Transcripts

Abstract

In vascular plants, organelle RNAs are edited by C-to-U base modification. Hundreds of mitochondrial C residues are targeted for editing in flowering plants. In this study, we exploited naturally occurring variation in editing extent to identify Required for Efficiency of Mitochondrial Editing1 (REME1), an Arabidopsis (Arabidopsis thaliana) pentatricopeptide repeat protein-encoding gene belonging to the DYW subclass that promotes editing of at least two C residues on different mitochondrial transcripts. Positional cloning identified REME1 unambiguously as the gene controlling editing of nad2-558. Virus-induced gene silencing of REME1 confirmed its role in editing of nad2-558 and allowed us to identify orfX-552 as a second C whose editing is positively controlled by REME1. An unexpected outcome of REME1 silencing was the finding of a number of mitochondrial C targets whose editing extent exhibits a significant and reproducible increase in silenced tissues. That increase was shown to be partly due to the virus inoculation and partly to REME1-specific silencing. Analysis of an insertional T-DNA mutant within the REME1 coding sequence confirmed the findings of the virus-induced gene silencing experiments: decrease in editing extent of nad2-558 and orfX-552 and increase in editing extent of two sites, matR-1771 and rpl5-92. Transgenic complementation of the low-edited accession (Landsberg erecta) restored the editing of nad2-558 and orfX-552 to high-edited accession (Columbia)-type levels or to even higher levels than Columbia. There was no effect of the transgene on editing extent of matR-1771 and rpl5-92. The strategy and tools used in this report can be applied to identify additional genes that affect editing extent in plant mitochondria.

RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing

“…In addition to RIP1, mutations in two PPR proteinencoding genes have been shown to increase editing extent. Mutation of REME1, which encodes a PPR-DYW protein, negatively affects editing of both nad2-558 and orfX-552 but also increases editing extent in at least two sites, matR-1771 and rpl5-92 (23). A null mutant of PPR596, which encodes a PPR-P protein, showed an increase of editing extent in several sites in the rpS4 transcript (45).…”

“…PPE analysis on mitochondrial sites cob-118, cob-325, nad6-26, and nad6-161 was conducted as in ref. 23 with primers cob-118, cob325, nad6-26, and nad6-161 (SI Appendix, Table S3). …”

“…Among the 13 Arabidopsis members of the PLS subfamily reported to be mitochondrial editing factors (22)(23)(24)(25)(26)(27)(28)(29), only 5 members have been observed to control the editing extent at more than one site. MEF1 and MEF11 control the editing of three sites (22,27), whereas REME1, SLO1, and OTP87 control two sites each (23,24,29). Whether 100 PPR proteins are sufficient to recognize the over 500 C targets of editing in Arabidopsis mitochondria is presently unknown.…”

“…The PLS subfamily, which is specific to the plant kingdom, can be further separated into smaller subclasses based on two C-terminal motifs, the E and DYW motifs (14). All of the well-characterized organelle editing factors that are required for editing at specific sites are members of the PLS subfamily of PPR proteins (11,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

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

“…RNA extraction and RT-PCR methods were as previously described (4) and chloroplast isolation as described in Hayes and Hanson (55). Primers to amplify the mitochondrial and plastid transcripts have been previously described (4,29,56). Analysis of RNA editing by PPE was done as in ref.…”