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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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Yeast two-hybrid assay of interaction of ORRM1 and OZ1.(A) OZ1 interacts with ORRM1 in the yeast two-hybrid assays. (B) N-terminus of ORRM1 mediates the interaction with OZ1. AD-Empty, pGADT7 empty vector. BD-Empty, pGBKT7 empty vector. Yeast single transformants were mated to make double transformants in order to test interactions. Yeast were grown in-leucine-tryptophan double-dropout media overnight before they were harvested and diluted into cell density 106/ml and 105/ml. 10μl of each dilution was spotted onto the-leucine-tryptophan—histidine—adenine quadruple dropout plates. Pictures were taken 3 days after inoculation. nORRM1: amino acids 55–274 ORRM1, which contains the RIP-RIP domain. cORRM1: amino acids 275–374 of ORRM1, which contains the RRM domain.
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pgen.1005028.g009: Yeast two-hybrid assay of interaction of ORRM1 and OZ1.(A) OZ1 interacts with ORRM1 in the yeast two-hybrid assays. (B) N-terminus of ORRM1 mediates the interaction with OZ1. AD-Empty, pGADT7 empty vector. BD-Empty, pGBKT7 empty vector. Yeast single transformants were mated to make double transformants in order to test interactions. Yeast were grown in-leucine-tryptophan double-dropout media overnight before they were harvested and diluted into cell density 106/ml and 105/ml. 10μl of each dilution was spotted onto the-leucine-tryptophan—histidine—adenine quadruple dropout plates. Pictures were taken 3 days after inoculation. nORRM1: amino acids 55–274 ORRM1, which contains the RIP-RIP domain. cORRM1: amino acids 275–374 of ORRM1, which contains the RRM domain.

Mentions: A yeast two-hybrid (Y2H) assay was employed to examine the interaction between OZ1 and ORRM1. Both OZ1 and ORRM1 are plastid-targeted proteins, so the predicted transit peptide sequences were removed from each before cloning them into AD/BD fusion constructs. As shown in Fig. 9A, OZ1 interacts with ORRM1 in yeast. The interaction is not affected by the position of the fusion protein since both AD-OZ1/BD-ORRM1 and its reciprocal pair AD-ORRM1/BD-OZ1 showed interaction, implicating a genuine interaction between these two proteins. ORRM1 was further divided into nORRM1 and cORRM1, encompassing the RIP-RIP and the RRM domain respectively. nORRM1 but not cORRM1 interacts with OZ1, indicating the RIP-RIP domain actually mediates the interaction with OZ1 (Fig. 9B).


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)

Yeast two-hybrid assay of interaction of ORRM1 and OZ1.(A) OZ1 interacts with ORRM1 in the yeast two-hybrid assays. (B) N-terminus of ORRM1 mediates the interaction with OZ1. AD-Empty, pGADT7 empty vector. BD-Empty, pGBKT7 empty vector. Yeast single transformants were mated to make double transformants in order to test interactions. Yeast were grown in-leucine-tryptophan double-dropout media overnight before they were harvested and diluted into cell density 106/ml and 105/ml. 10μl of each dilution was spotted onto the-leucine-tryptophan—histidine—adenine quadruple dropout plates. Pictures were taken 3 days after inoculation. nORRM1: amino acids 55–274 ORRM1, which contains the RIP-RIP domain. cORRM1: amino acids 275–374 of ORRM1, which contains the RRM domain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4359148&req=5

pgen.1005028.g009: Yeast two-hybrid assay of interaction of ORRM1 and OZ1.(A) OZ1 interacts with ORRM1 in the yeast two-hybrid assays. (B) N-terminus of ORRM1 mediates the interaction with OZ1. AD-Empty, pGADT7 empty vector. BD-Empty, pGBKT7 empty vector. Yeast single transformants were mated to make double transformants in order to test interactions. Yeast were grown in-leucine-tryptophan double-dropout media overnight before they were harvested and diluted into cell density 106/ml and 105/ml. 10μl of each dilution was spotted onto the-leucine-tryptophan—histidine—adenine quadruple dropout plates. Pictures were taken 3 days after inoculation. nORRM1: amino acids 55–274 ORRM1, which contains the RIP-RIP domain. cORRM1: amino acids 275–374 of ORRM1, which contains the RRM domain.
Mentions: A yeast two-hybrid (Y2H) assay was employed to examine the interaction between OZ1 and ORRM1. Both OZ1 and ORRM1 are plastid-targeted proteins, so the predicted transit peptide sequences were removed from each before cloning them into AD/BD fusion constructs. As shown in Fig. 9A, OZ1 interacts with ORRM1 in yeast. The interaction is not affected by the position of the fusion protein since both AD-OZ1/BD-ORRM1 and its reciprocal pair AD-ORRM1/BD-OZ1 showed interaction, implicating a genuine interaction between these two proteins. ORRM1 was further divided into nORRM1 and cORRM1, encompassing the RIP-RIP and the RRM domain respectively. nORRM1 but not cORRM1 interacts with OZ1, indicating the RIP-RIP domain actually mediates the interaction with OZ1 (Fig. 9B).

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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