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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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Immunoprecipitation of 3FS-ORRM1 using anti-FLAG resins.Two independent IP experiments were performed for each group. (A) Immunoblot of immunoprecipitate (IP), unbound flowthrough (UB) and total input (IN) for both Col and transgenic RecA-3FS-mORRM1. 10 μg total protein loaded for IN and UB. 1% of the IP was loaded. Anti-FLAG-HRP was used to detect FLAG-tagged protein. (B) 10% of the IP was separated by 10% Tris-Glycine SDS-PAGE and silver stained. Arrow indicates location of tagged ORRM1.
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pgen.1005028.g002: Immunoprecipitation of 3FS-ORRM1 using anti-FLAG resins.Two independent IP experiments were performed for each group. (A) Immunoblot of immunoprecipitate (IP), unbound flowthrough (UB) and total input (IN) for both Col and transgenic RecA-3FS-mORRM1. 10 μg total protein loaded for IN and UB. 1% of the IP was loaded. Anti-FLAG-HRP was used to detect FLAG-tagged protein. (B) 10% of the IP was separated by 10% Tris-Glycine SDS-PAGE and silver stained. Arrow indicates location of tagged ORRM1.

Mentions: As is shown in Fig. 2, the anti-FLAG antibody recognizes one band from the transgenic plant samples, but none in the wild-type sample. The unique band’s electrophoretic mobility is slightly slower than that expected for the predicted 42 kD size of the tagged ORRM1, possibly due to post-translational modifications. Anti-FLAG resins retained almost all tagged ORRM1 protein from the extract (Fig. 2A). The elutions from both ORRM1 and negative control were separated by a SDS-PAGE gel and silver stained. The bait, 3FS-mORRM1, is clearly seen in the transgenic plant IP but missing in the Col negative control (Fig. 2B). The immunoprecipitates were subjected to MS/MS mass spectrometry in order to identify ORRM1-binding proteins. The protein encoded by At5g17790 was selected for further investigation because after the ORRM1 peptides, it had the largest number of matches in MS/MS spectra and was not detected in the negative controls. S1 Table describes the peptides detected that resulted in the identification of the At5g17790 as a candidate ORRM1-interacting protein.


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)

Immunoprecipitation of 3FS-ORRM1 using anti-FLAG resins.Two independent IP experiments were performed for each group. (A) Immunoblot of immunoprecipitate (IP), unbound flowthrough (UB) and total input (IN) for both Col and transgenic RecA-3FS-mORRM1. 10 μg total protein loaded for IN and UB. 1% of the IP was loaded. Anti-FLAG-HRP was used to detect FLAG-tagged protein. (B) 10% of the IP was separated by 10% Tris-Glycine SDS-PAGE and silver stained. Arrow indicates location of tagged ORRM1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359148&req=5

pgen.1005028.g002: Immunoprecipitation of 3FS-ORRM1 using anti-FLAG resins.Two independent IP experiments were performed for each group. (A) Immunoblot of immunoprecipitate (IP), unbound flowthrough (UB) and total input (IN) for both Col and transgenic RecA-3FS-mORRM1. 10 μg total protein loaded for IN and UB. 1% of the IP was loaded. Anti-FLAG-HRP was used to detect FLAG-tagged protein. (B) 10% of the IP was separated by 10% Tris-Glycine SDS-PAGE and silver stained. Arrow indicates location of tagged ORRM1.
Mentions: As is shown in Fig. 2, the anti-FLAG antibody recognizes one band from the transgenic plant samples, but none in the wild-type sample. The unique band’s electrophoretic mobility is slightly slower than that expected for the predicted 42 kD size of the tagged ORRM1, possibly due to post-translational modifications. Anti-FLAG resins retained almost all tagged ORRM1 protein from the extract (Fig. 2A). The elutions from both ORRM1 and negative control were separated by a SDS-PAGE gel and silver stained. The bait, 3FS-mORRM1, is clearly seen in the transgenic plant IP but missing in the Col negative control (Fig. 2B). The immunoprecipitates were subjected to MS/MS mass spectrometry in order to identify ORRM1-binding proteins. The protein encoded by At5g17790 was selected for further investigation because after the ORRM1 peptides, it had the largest number of matches in MS/MS spectra and was not detected in the negative controls. S1 Table describes the peptides detected that resulted in the identification of the At5g17790 as a candidate ORRM1-interacting protein.

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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