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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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Localization of YFP fusion to OZ1 N-terminal sequence at punctuate loci in chloroplasts.Leaf protoplasts from Arabidopsis Ler were transfected with a construct encoding the first 100 amino acids of OZ1 fused with YFP under the control of a tandem CaMV 35S promoter. Protoplasts were examined for fluorescence 3 days after transfection. (A) Chlorophyll autofluorescence is marked as blue. (B) YFP is yellow. (C) Merge of chlorophyll and YFP signals. (A-C) OZ1 is targeted to chloroplasts.
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pgen.1005028.g012: Localization of YFP fusion to OZ1 N-terminal sequence at punctuate loci in chloroplasts.Leaf protoplasts from Arabidopsis Ler were transfected with a construct encoding the first 100 amino acids of OZ1 fused with YFP under the control of a tandem CaMV 35S promoter. Protoplasts were examined for fluorescence 3 days after transfection. (A) Chlorophyll autofluorescence is marked as blue. (B) YFP is yellow. (C) Merge of chlorophyll and YFP signals. (A-C) OZ1 is targeted to chloroplasts.

Mentions: In order to investigate the subcellular location of OZ1, we fused the N-terminal sequences encoding 100 amino acids to yellow fluorescent protein (YFP) and transfected protoplasts. Confocal microscopic imaging revealed that OZ1-YFP is located in the chloroplasts at punctate loci (Fig. 12). Previously, the entire coding region of OZ1 (VAR3) was fused to GFP and observed to be located within chloroplasts at punctate loci [28]. OZ1 was also detected in chloroplast nucleoid preparations by mass spectrometry (ppdb.tc.cornell.edu). In addition, Target P predicts all of the other OZ1 family members to be organelle targeted [30], one in mitochondria and two in plastids (S2 Table). Proteomics studies have also found OZ4 (At1g48570) in both chloroplast nucleoids and stroma (ppdb.ts.cornell.edu).


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)

Localization of YFP fusion to OZ1 N-terminal sequence at punctuate loci in chloroplasts.Leaf protoplasts from Arabidopsis Ler were transfected with a construct encoding the first 100 amino acids of OZ1 fused with YFP under the control of a tandem CaMV 35S promoter. Protoplasts were examined for fluorescence 3 days after transfection. (A) Chlorophyll autofluorescence is marked as blue. (B) YFP is yellow. (C) Merge of chlorophyll and YFP signals. (A-C) OZ1 is targeted to chloroplasts.
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Related In: Results  -  Collection

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

pgen.1005028.g012: Localization of YFP fusion to OZ1 N-terminal sequence at punctuate loci in chloroplasts.Leaf protoplasts from Arabidopsis Ler were transfected with a construct encoding the first 100 amino acids of OZ1 fused with YFP under the control of a tandem CaMV 35S promoter. Protoplasts were examined for fluorescence 3 days after transfection. (A) Chlorophyll autofluorescence is marked as blue. (B) YFP is yellow. (C) Merge of chlorophyll and YFP signals. (A-C) OZ1 is targeted to chloroplasts.
Mentions: In order to investigate the subcellular location of OZ1, we fused the N-terminal sequences encoding 100 amino acids to yellow fluorescent protein (YFP) and transfected protoplasts. Confocal microscopic imaging revealed that OZ1-YFP is located in the chloroplasts at punctate loci (Fig. 12). Previously, the entire coding region of OZ1 (VAR3) was fused to GFP and observed to be located within chloroplasts at punctate loci [28]. OZ1 was also detected in chloroplast nucleoid preparations by mass spectrometry (ppdb.tc.cornell.edu). In addition, Target P predicts all of the other OZ1 family members to be organelle targeted [30], one in mitochondria and two in plastids (S2 Table). Proteomics studies have also found OZ4 (At1g48570) in both chloroplast nucleoids and stroma (ppdb.ts.cornell.edu).

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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