RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering

Abstract

Plant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members of the RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family are essential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified as minor mitochondrial editing factors whose silencing reduces editing efficiency at ;6% of the mitochondrial C targets. Here we report the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls ;44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. The orrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4 mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and a Gly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain is required for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by its interaction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a major mitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals that adequate mitochondrial editing is necessary for normal plant development.

An Organelle RNA Recognition Motif Protein is Required for Photosynthetic Subunit psbF Transcript Editing

“…ORRM1 is known to be a chloroplast RNA editing trans-factor (Sun et al, 2013), while ORRM2, ORRM3, and ORRM4 are known be required for the editing of mitochondrial RNA editing sites (Shi et al, , 2016b.…”

“…Analysis of STS-PCR-seq data was performed as described by Bentolila et al (2013). The statistical analysis to determine which editing site is significantly affected in the orrm6-2 mutant compared with the wild type is very similar to the one performed previously (Shi et al, , 2016b. Briefly, we performed a x 2 test with 1 degree of freedom for each mutant biological replicate and each wild-type replicate to test for a significant difference in editing extent.…”

“…The coding sequences of ORRM6, RIP1/MORF8 (At3g15000), RIP2/MORF2 (At2g33420), RIP9/MORF9 (At1g11430), OZ1 (At5g17790), LPA66 (At5g48910), RARE1 (At5g13270), and ORRM1 (At3g20930) without the stop codon were amplified from full-length cDNAs as described above and in previous studies (Bentolila et al, 2012;Sun et al, 2013Sun et al, , 2015Shi et al, 2015Shi et al, , 2016b, cloned into the pCR8/GW/TOPO TA vector (Thermo Fisher), and sequenced to confirm the absence of errors. The confirmed fragments were then subcloned into XNGW and XCGW vectors (Ohashi-Ito and Bergmann, 2006) by LR recombination reactions.…”

“…The confirmed fragments were then subcloned into XNGW and XCGW vectors (Ohashi-Ito and Bergmann, 2006) by LR recombination reactions. The two vectors have been used previously to demonstrate interactions between different RNA editing factors, such as ORRM4 and RIP1/MORF8 (Shi et al, 2016b). All the primers used are listed in Supplemental Data Set S2.…”

“…ORRM2, ORRM3, and ORRM4 are targeted to the mitochondria, and none of them have RIP/MORF boxes. The orrm2, orrm3, and orrm4 mutants displayed decreased editing extents at 35, 32, and 262 mitochondrial RNA editing sites, respectively (Shi et al, , 2016b.…”