Transcript The Pentatricopeptide Repeat Protein OTP87 Is Essential for RNA

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, 67084 Strasbourg Cedex, France, ;
¶Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany, and ; ‖Department of Botany, Graduate
School of Science, Kyoto University, Kyoto 606-8502, 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
From Lurin et al. 2004
Based on those whose functions are known,
they mediate aspects of RNA processing,
especially RNA editing.
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.
OTP87 is Dualtargeted to
Mitochondria
and Chloroplasts
C. RFP fused to the presequence of cytochrome
oxidase IV (mito. marker)
A,B. Amino
acids 1-100 of
OTP87 fused to
GFP.
D. RFP fused to the rbcS protein (plastid marker)
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;286:21361-21371
©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;286:21361-21371
©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.