The Pentatricopeptide Repeat Protein OTP87 Is Essential for RNA Editing of nad7 and atp1 Transcripts in Arabidopsis Mitochondria Kamel Hammani‡§, Catherine Colas des Francs-Small‡, Mizuki Takenaka¶, Sandra K. Tanz‡, Kenji Okuda ‖, Toshiharu Shikanai ‖, Axel Brennicke¶ and Ian Small‡ From the: ‡ Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia, ; § Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, Strasbourg Cedex, France, ; ¶ Molekulare Botanik, Universität Ulm, Ulm, Germany, and ; ‖ Department of Botany, Graduate School of Science, Kyoto University, Kyoto , Japan Ian Small, U. of Western Australia

Background: Arabidopsis and PPR proteins Arabidopsis thaliana – most favored model for plant biology because of its genetics  A small dicot in the mustard family  Short life cycle (seed-to-seed in 6 wks)  Well-developed genetics  Smallest genome of angiosperms (low in repetitive DNA)  Genome has been completely sequenced (transcriptomics and proteomics also well developed) Elliot Meyerowitz, Calif. Institute of Technology

Pentatricopeptide Repeat (PPR) Proteins Contain tandem arrays of pentatricopeptide repeats (PPRs) Found in many eukaryotes, but so many more in angiosperms!  e.g., > 400 in Arabidopsis Most are targeted to organelles (M & C) There are several subgroups Based on those whose functions are known, they mediate aspects of RNA processing, especially RNA editing. From Lurin et al. 2004

Molecular characterization and phenotypic analysis of the Arabidopsis otp87 mutant. Has a T-DNA insertion that knock-outs the PPR protein, OTP87. Mutant can be complemented with wild-type OTP87 gene.

C. RFP fused to the presequence of cytochrome oxidase IV (mito. marker) D. RFP fused to the rbcS protein (plastid marker) A,B. Amino acids of OTP87 fused to GFP. OTP87 is Dual- targeted to Mitochondria and Chloroplasts

Chloroplasts appear normal in the otp87 null mutant.

Two editing sites in mitochondria are affected in the OTP87 KO mutant; 1 each in the atp1 and nad7 mRNAs. Only the former changes an amino acid, however. ©2011 by American Society for Biochemistry and Molecular Biology

ATP synthase complex assembly is defective in the otp87 KO.

Pollen structure of WT and otp87 plants is normal. Hammani K et al. J. Biol. Chem. 2011;286: ©2011 by American Society for Biochemistry and Molecular Biology

Homology between the cis-elements surrounding the two affected editing sites (nad7-C24 and atp1-C1178). Hammani K et al. J. Biol. Chem. 2011;286: ©2011 by American Society for Biochemistry and Molecular Biology

Electrophoretic mobility shift assays using recombinant OTP87 protein. Conclusion: OTP87 binds specifically to small RNAs with the editing sites for nad7 and atp1.

Conclusions Arabidopsis OTP87 is dual-targeted to chloroplasts and mitochondria, but may function only in the latter. OTP87 is required for 2 editing sites in the mitochondria, one each in nad7 and atp1. The loss of atp1 editing leaves a non-conserved amino acid, which appears to inhibit stable assembly of the atp synthase complex. This could account for the phenotype of the OTP87 insertional mutant. Recombinant OTP87 binds to the 2 editing sites specifically, in an in vitro shift assay. OTP87 may recruit the editing enzyme to those sites.

Assessing Strengths & Weaknesses of a Paper Usually consider the following:  Originality (specifically discussed at end of Introduction & beginning of Discussion sections)  Significance (discussed in Abstract and Discussion sections)  Quality of data?  Are conclusions supported by the data?  Is the paper clear and well written? Methods well described? Is the data presentation clear? How could the paper be improved?

Strengths and weaknesses Strengths  Novelty: new protein, and a rare KO mutant in a PPR protein that gives a clear phenotype  Significance: the first protein from Arabidopsis shown to bind to 2 different editing sites in vitro. Will enable further biochemical studies of how it binds RNA, specifically.  Quality of the data was generally high, and supported the conclusions. Weaknesses  Some of the writing, e.g. the figure legends, was not very clear even if the reader was an expert.  Work still provided no clues about what the editing enzyme(s) are.