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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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OZ1 interacts with other components of the editing complex.(A) OZ1 dimerizes (B) OZ1 interacts with OTP82, CRR28 and RIP1 but not RIP2 or RIP9. (C) ORRM1 interacts with RIP1 and RIP2. All interactions were tested on—Leu-Trp-His-Ade dropout media. Image taken 3 days after spotting of strains.
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pgen.1005028.g010: OZ1 interacts with other components of the editing complex.(A) OZ1 dimerizes (B) OZ1 interacts with OTP82, CRR28 and RIP1 but not RIP2 or RIP9. (C) ORRM1 interacts with RIP1 and RIP2. All interactions were tested on—Leu-Trp-His-Ade dropout media. Image taken 3 days after spotting of strains.

Mentions: We suspected that OZ1 might also interact with other components of chloroplast editosomes in addition to ORRM1, such as additional PPR site recognition factors and members of the RIP/MORF protein family. In order to determine whether OZ1 can dimerize and/or interact with other components of the editing complex, we performed a series of Y2H assays. OZ1 fused to either AD or BD does not show any auto-activation for HIS and ADE reporters, while yeast with AD-OZ1/BD-OZ1 is able to grow on histidine and adenine deficient media, indicating self-interaction (Fig. 10A). OZ1 also interacts with OTP82 and CRR28, as shown in Fig. 10B, a result expected from the effect of the oz1–1 mutation on C targets controlled by OTP82 and CRR28 (Table 1). OZ1 exhibits a weaker interaction with RIP1; fewer colonies are seen in the RIP1/OZ1 combination (Fig. 10B). However, no interaction was observed between OZ1 and RIP2 or RIP9 (Fig. 10B), even though RIP2 and RIP9 are essential for editing of a large number of chloroplast C targets. We considered the possibility that OZ1 associates with RIP2 and RIP9 via ORRM1. To test this hypothesis, we performed a Y2H assay for ORRM1 and RIP proteins (Fig. 10C). Both RIP1 and RIP2 can interact with ORRM1. RIP9 fused with the GAL4 binding domain strongly autoactivates HIS and ADE reporters, so RIP9 was not tested in this experiment. ORRM1 with the GAL4 activation domain shows no autoactivation (Fig. 9A). Our data is consistent with ORRM1 as a mediator of interaction between OZ1 and RIP2 within the chloroplast editosome.


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)

OZ1 interacts with other components of the editing complex.(A) OZ1 dimerizes (B) OZ1 interacts with OTP82, CRR28 and RIP1 but not RIP2 or RIP9. (C) ORRM1 interacts with RIP1 and RIP2. All interactions were tested on—Leu-Trp-His-Ade dropout media. Image taken 3 days after spotting of strains.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359148&req=5

pgen.1005028.g010: OZ1 interacts with other components of the editing complex.(A) OZ1 dimerizes (B) OZ1 interacts with OTP82, CRR28 and RIP1 but not RIP2 or RIP9. (C) ORRM1 interacts with RIP1 and RIP2. All interactions were tested on—Leu-Trp-His-Ade dropout media. Image taken 3 days after spotting of strains.
Mentions: We suspected that OZ1 might also interact with other components of chloroplast editosomes in addition to ORRM1, such as additional PPR site recognition factors and members of the RIP/MORF protein family. In order to determine whether OZ1 can dimerize and/or interact with other components of the editing complex, we performed a series of Y2H assays. OZ1 fused to either AD or BD does not show any auto-activation for HIS and ADE reporters, while yeast with AD-OZ1/BD-OZ1 is able to grow on histidine and adenine deficient media, indicating self-interaction (Fig. 10A). OZ1 also interacts with OTP82 and CRR28, as shown in Fig. 10B, a result expected from the effect of the oz1–1 mutation on C targets controlled by OTP82 and CRR28 (Table 1). OZ1 exhibits a weaker interaction with RIP1; fewer colonies are seen in the RIP1/OZ1 combination (Fig. 10B). However, no interaction was observed between OZ1 and RIP2 or RIP9 (Fig. 10B), even though RIP2 and RIP9 are essential for editing of a large number of chloroplast C targets. We considered the possibility that OZ1 associates with RIP2 and RIP9 via ORRM1. To test this hypothesis, we performed a Y2H assay for ORRM1 and RIP proteins (Fig. 10C). Both RIP1 and RIP2 can interact with ORRM1. RIP9 fused with the GAL4 binding domain strongly autoactivates HIS and ADE reporters, so RIP9 was not tested in this experiment. ORRM1 with the GAL4 activation domain shows no autoactivation (Fig. 9A). Our data is consistent with ORRM1 as a mediator of interaction between OZ1 and RIP2 within the chloroplast editosome.

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