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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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Transient silencing of OZ1 in Arabidopsis results in chloroplast editing defects.Two replicates for each treatment were assayed by PPE. Not inoculated, untreated plants. GFP silenced, inoculated with Agrobacteria harboring a GFP silencing construct. OZ1-silenced, plants that were inoculated with Agrobacteria harboring a GFP and OZ1 co-silencing construct. Average for each group is displayed in a third bar. E: edited band, U: unedited band, O: oligonucleotide. Significance ** (P<0.01), *** (P<0.001)
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pgen.1005028.g005: Transient silencing of OZ1 in Arabidopsis results in chloroplast editing defects.Two replicates for each treatment were assayed by PPE. Not inoculated, untreated plants. GFP silenced, inoculated with Agrobacteria harboring a GFP silencing construct. OZ1-silenced, plants that were inoculated with Agrobacteria harboring a GFP and OZ1 co-silencing construct. Average for each group is displayed in a third bar. E: edited band, U: unedited band, O: oligonucleotide. Significance ** (P<0.01), *** (P<0.001)

Mentions: Since a second T-DNA mutant in the coding region of OZ1 was not available, we performed Virus Induced Gene Silencing (VIGS) to transiently silence OZ1 expression in young Arabidopsis seedlings. To monitor the silencing efficiency, a GFP co-silencing marker harbored in the VIGS construct was used [29]. Agrobacteria carrying either the OZ1/GFP co-silencing construct or the GFP silencing construct alone were inoculated into 2 week-old 35S::GFP expressing Arabidopsis seedlings. After growth in long days for 5 more weeks, the editing extents in RNA from GFP-silenced leaves and from uninoculated plants were analyzed by PPE. There were no differences between leaves of GFP-silenced plants and untreated plants (Fig. 5). ndhB C836 editing extent decreased from 97% in the untreated control to 47% in OZ1 silenced plants (P<0.01). rpoA C200 editing extent dropped from 74% in untreated control to 29% in OZ1 silenced plants (P<0.001). These results agree with the data from oz1–1 mutants, in which editing is abolished at both sites. The residual editing in the silenced plants is probably caused by incomplete depletion of OZ1 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)

Transient silencing of OZ1 in Arabidopsis results in chloroplast editing defects.Two replicates for each treatment were assayed by PPE. Not inoculated, untreated plants. GFP silenced, inoculated with Agrobacteria harboring a GFP silencing construct. OZ1-silenced, plants that were inoculated with Agrobacteria harboring a GFP and OZ1 co-silencing construct. Average for each group is displayed in a third bar. E: edited band, U: unedited band, O: oligonucleotide. Significance ** (P<0.01), *** (P<0.001)
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005028.g005: Transient silencing of OZ1 in Arabidopsis results in chloroplast editing defects.Two replicates for each treatment were assayed by PPE. Not inoculated, untreated plants. GFP silenced, inoculated with Agrobacteria harboring a GFP silencing construct. OZ1-silenced, plants that were inoculated with Agrobacteria harboring a GFP and OZ1 co-silencing construct. Average for each group is displayed in a third bar. E: edited band, U: unedited band, O: oligonucleotide. Significance ** (P<0.01), *** (P<0.001)
Mentions: Since a second T-DNA mutant in the coding region of OZ1 was not available, we performed Virus Induced Gene Silencing (VIGS) to transiently silence OZ1 expression in young Arabidopsis seedlings. To monitor the silencing efficiency, a GFP co-silencing marker harbored in the VIGS construct was used [29]. Agrobacteria carrying either the OZ1/GFP co-silencing construct or the GFP silencing construct alone were inoculated into 2 week-old 35S::GFP expressing Arabidopsis seedlings. After growth in long days for 5 more weeks, the editing extents in RNA from GFP-silenced leaves and from uninoculated plants were analyzed by PPE. There were no differences between leaves of GFP-silenced plants and untreated plants (Fig. 5). ndhB C836 editing extent decreased from 97% in the untreated control to 47% in OZ1 silenced plants (P<0.01). rpoA C200 editing extent dropped from 74% in untreated control to 29% in OZ1 silenced plants (P<0.001). These results agree with the data from oz1–1 mutants, in which editing is abolished at both sites. The residual editing in the silenced plants is probably caused by incomplete depletion of OZ1 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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