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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 expression of OZ1 under a 35S promoter in oz1 mutant plants complements the editing and phenotypic defects.(A) Three different C targets of editing in several different transgenic plants were assayed by Poisoned Primer Extension (PPE). PPE bands were quantified by ImageQuant software and illustrated in graphs.-/-, homozygous oz1 mutant plants;-/- w/35S:OZ1, transgenic oz1 mutant plants transformed with a construct expressing OZ1 driven by 35S promoter. E, edited band; U, unedited band. (B) independent 9-week-old transgenic mutant plants lacking the yellow phenotype observed in the mutant plant.
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pgen.1005028.g008: Stable expression of OZ1 under a 35S promoter in oz1 mutant plants complements the editing and phenotypic defects.(A) Three different C targets of editing in several different transgenic plants were assayed by Poisoned Primer Extension (PPE). PPE bands were quantified by ImageQuant software and illustrated in graphs.-/-, homozygous oz1 mutant plants;-/- w/35S:OZ1, transgenic oz1 mutant plants transformed with a construct expressing OZ1 driven by 35S promoter. E, edited band; U, unedited band. (B) independent 9-week-old transgenic mutant plants lacking the yellow phenotype observed in the mutant plant.

Mentions: Because of the poor growth of the homozygous oz1 mutant plant (Fig. 3D), we decided to transform the heterozygous plant by floral dipping with a construct expressing OZ1 under the control of a 35S promoter. Genotyping the transgenic plants growing on a selectable plate allowed us to recover several independent plants homozygous mutant for the endogenous oz1 alleles but expressing the OZ1 transgene. The introduction of a functional OZ1 complements the editing defect in all the transgenic plants assayed (Fig. 8A). Positional effects on the transgene are known to affect expression and likely resulted in the range of responses in the transformed plants. For example, in different plants, rpoA C200 editing extent ranged from 13%-65% (Fig. 8A). The restoration of editing extent in some transgenic mutant plants is much more pronounced than with the transient expression in the oz1 mutant protoplasts, e.g. 89% vs. 31% for ndhB C836, and reaches almost the level observed in the wild-type plant. In addition to reverting the editing defects, the introduction of OZ1 in planta also suppress the yellow phenotype observed in the mutant plant (Fig. 8B). The reversion of both editing and phenotypic defects by expression of a functional OZ1 demonstrates the role of this protein in both phenomena.


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 expression of OZ1 under a 35S promoter in oz1 mutant plants complements the editing and phenotypic defects.(A) Three different C targets of editing in several different transgenic plants were assayed by Poisoned Primer Extension (PPE). PPE bands were quantified by ImageQuant software and illustrated in graphs.-/-, homozygous oz1 mutant plants;-/- w/35S:OZ1, transgenic oz1 mutant plants transformed with a construct expressing OZ1 driven by 35S promoter. E, edited band; U, unedited band. (B) independent 9-week-old transgenic mutant plants lacking the yellow phenotype observed in the mutant plant.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005028.g008: Stable expression of OZ1 under a 35S promoter in oz1 mutant plants complements the editing and phenotypic defects.(A) Three different C targets of editing in several different transgenic plants were assayed by Poisoned Primer Extension (PPE). PPE bands were quantified by ImageQuant software and illustrated in graphs.-/-, homozygous oz1 mutant plants;-/- w/35S:OZ1, transgenic oz1 mutant plants transformed with a construct expressing OZ1 driven by 35S promoter. E, edited band; U, unedited band. (B) independent 9-week-old transgenic mutant plants lacking the yellow phenotype observed in the mutant plant.
Mentions: Because of the poor growth of the homozygous oz1 mutant plant (Fig. 3D), we decided to transform the heterozygous plant by floral dipping with a construct expressing OZ1 under the control of a 35S promoter. Genotyping the transgenic plants growing on a selectable plate allowed us to recover several independent plants homozygous mutant for the endogenous oz1 alleles but expressing the OZ1 transgene. The introduction of a functional OZ1 complements the editing defect in all the transgenic plants assayed (Fig. 8A). Positional effects on the transgene are known to affect expression and likely resulted in the range of responses in the transformed plants. For example, in different plants, rpoA C200 editing extent ranged from 13%-65% (Fig. 8A). The restoration of editing extent in some transgenic mutant plants is much more pronounced than with the transient expression in the oz1 mutant protoplasts, e.g. 89% vs. 31% for ndhB C836, and reaches almost the level observed in the wild-type plant. In addition to reverting the editing defects, the introduction of OZ1 in planta also suppress the yellow phenotype observed in the mutant plant (Fig. 8B). The reversion of both editing and phenotypic defects by expression of a functional OZ1 demonstrates the role of this protein in both phenomena.

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