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A zinc finger motif-containing protein is essential for chloroplast RNA editing.

Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR - PLoS Genet. (2015)

Bottom Line: Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts.The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria.With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

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Stable integration of 35S::RecA-3FS-mORRM1 into the orrm1 mutant restores normal editing level in plastids.(A) Protein sequence of RecA-3FS-mORRM1. Transit peptide sequence from RecA is underlined. Sequence of epitope tags is italic. 3xFLAG, spacer, StrepII and Glycine-Serine linker are labeled with red, yellow, green and blue, respectively. Sequence of mature ORRM1 without the 54 amino acid transit peptide is bolded. (B) Portion of electrophoretograms from RT-PCR bulk sequencing of matK C640, ndhB C872 and ndhG C50 is shown for the Columbia wild-type, orrm1, and a stable transformant expressing RecA-3xFLAG-strepII-mORRM1 under control of a 35S promoter. The editing sites are indicated with arrows. The complementary strand of the sequenced ndhG is shown, as sequencing was done from a reverse direction.
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pgen.1005028.g001: Stable integration of 35S::RecA-3FS-mORRM1 into the orrm1 mutant restores normal editing level in plastids.(A) Protein sequence of RecA-3FS-mORRM1. Transit peptide sequence from RecA is underlined. Sequence of epitope tags is italic. 3xFLAG, spacer, StrepII and Glycine-Serine linker are labeled with red, yellow, green and blue, respectively. Sequence of mature ORRM1 without the 54 amino acid transit peptide is bolded. (B) Portion of electrophoretograms from RT-PCR bulk sequencing of matK C640, ndhB C872 and ndhG C50 is shown for the Columbia wild-type, orrm1, and a stable transformant expressing RecA-3xFLAG-strepII-mORRM1 under control of a 35S promoter. The editing sites are indicated with arrows. The complementary strand of the sequenced ndhG is shown, as sequencing was done from a reverse direction.

Mentions: We investigated whether the epitope-tagged protein (Fig. 1A) could restore editing in transgenic plants obtained by root transformation of orrm1 mutant plants. Transgenic plants of normal phenotype were obtained and RNA was extracted for use in editing assays. Editing extent of matK C640, ndhB C872 and ndhG C50, which exhibit decreased editing in orrm1, was examined by bulk sequencing (Fig. 1B). Editing of all three sites was restored to wild-type level in the RecA-3FS-mORRM1 transgenic plants.


A zinc finger motif-containing protein is essential for chloroplast RNA editing.

Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR - PLoS Genet. (2015)

Stable integration of 35S::RecA-3FS-mORRM1 into the orrm1 mutant restores normal editing level in plastids.(A) Protein sequence of RecA-3FS-mORRM1. Transit peptide sequence from RecA is underlined. Sequence of epitope tags is italic. 3xFLAG, spacer, StrepII and Glycine-Serine linker are labeled with red, yellow, green and blue, respectively. Sequence of mature ORRM1 without the 54 amino acid transit peptide is bolded. (B) Portion of electrophoretograms from RT-PCR bulk sequencing of matK C640, ndhB C872 and ndhG C50 is shown for the Columbia wild-type, orrm1, and a stable transformant expressing RecA-3xFLAG-strepII-mORRM1 under control of a 35S promoter. The editing sites are indicated with arrows. The complementary strand of the sequenced ndhG is shown, as sequencing was done from a reverse direction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359148&req=5

pgen.1005028.g001: Stable integration of 35S::RecA-3FS-mORRM1 into the orrm1 mutant restores normal editing level in plastids.(A) Protein sequence of RecA-3FS-mORRM1. Transit peptide sequence from RecA is underlined. Sequence of epitope tags is italic. 3xFLAG, spacer, StrepII and Glycine-Serine linker are labeled with red, yellow, green and blue, respectively. Sequence of mature ORRM1 without the 54 amino acid transit peptide is bolded. (B) Portion of electrophoretograms from RT-PCR bulk sequencing of matK C640, ndhB C872 and ndhG C50 is shown for the Columbia wild-type, orrm1, and a stable transformant expressing RecA-3xFLAG-strepII-mORRM1 under control of a 35S promoter. The editing sites are indicated with arrows. The complementary strand of the sequenced ndhG is shown, as sequencing was done from a reverse direction.
Mentions: We investigated whether the epitope-tagged protein (Fig. 1A) could restore editing in transgenic plants obtained by root transformation of orrm1 mutant plants. Transgenic plants of normal phenotype were obtained and RNA was extracted for use in editing assays. Editing extent of matK C640, ndhB C872 and ndhG C50, which exhibit decreased editing in orrm1, was examined by bulk sequencing (Fig. 1B). Editing of all three sites was restored to wild-type level in the RecA-3FS-mORRM1 transgenic plants.

Bottom Line: Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts.The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria.With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.

ABSTRACT
C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

Show MeSH
Related in: MedlinePlus