Takahiro Nakamura1,2,3,*, Yusuke Yagi1,2 and Keiko Kobayashi1,2
Review
Mechanistic Insight into Pentatricopeptide Repeat Proteins
as Sequence-Specific RNA-Binding Proteins for Organellar
RNAs in Plants
1
Department of Research Superstar Program, Institute of Advanced Study, Kyushu University, Fukuoka, 812-8581 Japan
Faculty of Agriculture, Kyushu University, Fukuoka, 812-8581 Japan
3
Biotron Application Center, Kyushu University, Fukuoka, 812-8581 Japan
*Corresponding author: E-mail, [email protected]; Fax,+81 92 642 3370.
(Received December 12, 2011; Accepted April 26, 2012)
2
The pentatricopeptide repeat (PPR) protein family is highly
expanded in terrestrial plants. Arabidopsis contains 450 PPR
genes, which represents 2% of the total protein-coding
genes. PPR proteins are eukaryote-specific RNA-binding proteins implicated in multiple aspects of RNA metabolism of
organellar genes. Most PPR proteins affect a single or small
subset of gene(s), acting in a gene-specific manner. Studies
over the last 10 years have revealed the significance of this
protein family in coordinated gene expression in different
compartments: the nucleus, chloroplast and mitochondrion.
Here, we summarize recent studies addressing the mechanistic aspect of PPR proteins.
Keywords: Chloroplast Mitochondria Pentatricopeptide
repeat protein PPR RNA-binding protein RNA
processing.
Abbreviations: CMS, cytoplasmic male sterility; crr, chlororespiratory reduction; LRR, leucine-rich repeat; MORF, multiple organellar RNA-editing factor; NBS, nucleotide-binding
site; PPR, pentatricopeptide repeat; Rf, restorer-of-fertility;
SMR, small mutS-related; TPR, tetratricopeptide repeat.
Organelle Gene Expression and
Pentatricopeptide Repeat Proteins
Mitochondria and chloroplasts originated from endosymbiotic
processes billions of years ago (Kurland and Andersson 2000,
Bhattacharya et al. 2007). The vast majority of the endosymbionts’ genes have been transferred to the nucleus during
evolution. Currently, organelles contain limited genetic information for their specific function, i.e. photosynthesis or respiration, and for the core components of gene expression.
Therefore, numerous nuclear-encoded factors are imported
into the organelles to maintain organelle biogenesis. For example, approximately 2,000 gene products are estimated to
be in the mitochondrial proteome; however, only approximately 40 proteins are encoded by the mitochondrial
genome (Millar et al. 2005). On the other hand, organelles
transmit their functional and developmental state to the nucleus, which is known as retrograde signaling (Pogson et al.
2008). Thus, the biochemical and genetic features of plant
organelles arose in the context of coordinated co-evolution
between the organellar and nuclear genomes.
The expression of an organelle gene, i.e. the final quantity of
the translated product, is predominantly determined by the
regulation and/or consequence of extensive, multiple posttranscriptional processes such as RNA cleavage, splicing, RNA
editing, RNA stabilization and translation. In the last two
decades, numerous nuclear-encoded proteins involved in the
metabolism of organellar RNA have been identified (del Campo
2009, Jacobs and Kuck 2011, Ruwe et al. 2011). Importantly,
they have been shown to mediate specific RNA processing
events of a single or small subset of gene(s). Recent analyses
have shown that a plant-specific family of pentatricopeptide
repeat (PPR) motif-containing proteins is implicated in many
crucial RNA processing events of organellar genes.
The PPR motifs and proteins were first recognized by in silico
analysis during the sequencing of the Arabidopsis thaliana
genome (Aubourg et al. 2000, Small and Peeters 2000). The
PPR motif-containing proteins are characterized by the presence of tandem arrays of a degenerate 35 amino acid repeat, the
PPR motif, repeated in tandem 2–26 times. A striking feature of
this protein is its high expansion in terrestrial plants. The
Arabidopsis genome encodes 450 PPR proteins, although the
PPR genes are limited to <30 in other eukaryotes (Lurin et al.
2004). All PPR proteins are nuclear encoded, whereas a substantial portion of them have been thought to target to either the
mitochondria or the chloroplasts.
A number of genetic analyses have shown that the phenotypes of PPR mutants are highly diverse, and their impacts are
often severe. The disruption of a single PPR gene, in many cases,
Plant Cell Physiol. 53(7): 1171–1179 (2012) doi:10.1093/pcp/pcs069, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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has resulted in embryonic lethality, presumably because of the
impairment of mitochondrial function (Cushing et al. 2005,
Ding et al. 2006, Gutierrez-Marcos et al. 2007). However, it is
now generally accepted that PPR proteins are gene-specific
RNA-binding proteins for organelle gene expression involved
in all aspects of RNA processing that are found in plant organelles, such as RNA cleavage, splicing, stabilization, degradation,
editing and translation (Delannoy et al. 2007, SchmitzLinneweber and Small 2008, del Campo 2009, Jacobs and
Kuck 2011, and references cited therein). Several PPR proteins
are also involved in transcription (Ikeda and Gray 1999, Ding
et al. 2006, Pfalz et al. 2006). In this review, we focus on recent
progress in the molecular insight into PPR proteins.
Definition of the PPR Motif as an
RNA-Interacting Unit
The PPR motif was identified as a novel motif, related to the
tetratricopeptide repeat (TPR) motif. Whereas the TPR motif
can be found in both prokaryotes and eukaryotes, the PPR
motif is prevalent in organellar proteins. Recently, the principal
structure of the PPR motif, namely a pair of antiparallel
a-helices, was determined in a structure of a mitochondrial
RNA polymerase containing two PPR motifs (Ringel et al.
2011; Fig. 1A). The consecutive helical hairpins in a PPR tract
are predicted to form a curved structure resembling half a
donut or crescent (Fig. 1B) (Kurland and Andersson 2000,
Delannoy et al. 2007). This structure is similar to those found
in the helical repeat protein family, such as the TPR motif
(34 amino acids), HEAT domain (39 amino acids), Arm motif
(38 amino acids) and Puf domain (36 amino acids) (Groves and
Barford 1999). The HEAT, Arm and TPR motifs are responsible
for protein–protein interactions, and the concave surface of the
crescent is the interaction site (Blatch and Lassle 1999).
However, the Puf domain uses the same surface for RNA binding (Wang et al. 2002, Gupta et al. 2008). A similar
ligand-binding surface can be expected for the PPR protein.
The PPR motif was originally identified as a 35 amino acid
motif and was later subdivided into the P (classical PPR; 35
amino acids), PPR-like S (short; 31 amino acids), PPR-like L1
(long; 35 amino acids) and L2 (36 amino acids) motifs based
on their sequence characteristics (Lurin et al. 2004). However,
the limitation of structural and functional knowledge about
PPR motifs and the mechanism by which they contact RNA
makes it challenging to define the functionality of PPR motifs
accurately, as well as the differences among the PPR subtypes.
The current domain search programs predict the PPR motif
with various definitions of the length and start position.
Recently, we biochemically evaluated the RNA binding characteristics of the PPR motif; we proposed that the Pfam model
better defines the PPR motif as a functional RNA-interacting
unit, which defines the first amino acid of the PPR motif as
the beginning of helix A (http://Pfam.sanger.ac.uk/, PF01535;
Fig. 1D). In addition, we proposed that the 34th amino acid
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in the Pfam model should be re-numbered ‘ii (2)’ because the
position should be designated as two amino acids before the
first amino acid of the next PPR motif. This numbering also
facilitates compensating for the length differences among PPR
subtypes described above (P, S, L1 and L2; Fig. 1D). It may be
noted that we cannot find obvious differences in the RNA
binding characteristics among the PPR subtypes to date
(K. Kobayashi and T. Nakamura, unpublished data).
The five residues [first, fourth, eighth, 12th and ‘ii’(2)
amino acids] exposed on the solvent surface seem to organize
the RNA-binding surface (Ringel et al. 2011, Kobayashi et al.
2012; Fig. 1A, D). The RNA binding capacity of the PPR motif
could involve complex cooperation among the RNA-interacting residues. It seems that the first, fourth and ‘ii’ amino acids
may be responsible for the specific recognition of RNA bases, as
also proposed by their high rates of diversification in the
restorer-of-fertility (Rf)-like PPR proteins and by constrained
modeling of the PPR–RNA complex (positions 1, 3 and 6 in
Fujii et al. 2011). The positively charged eighth and 12th residues are significant for the PPR–RNA interaction, as suggested
by the general preference of a basic residue for binding to the
phosphate of a nucleic acid (Delannoy et al. 2007, Kobayashi
et al. 2012).
The mechanism for nucleotide discrimination is, however,
still largely unknown. An analysis of the editing factors suggested that the editing PPR proteins generally distinguish pyrimidines from purines, and, at some positions, recognize
specific bases (Hammani et al. 2009). Our biochemical study
also indicated the presence of PPR motifs displaying a preference for adenine or purine, which contained a characteristic
amino acid signature at the first, fourth and ‘ii’ residues
(Kobayashi et al. 2012). Elucidation of the RNA recognition
mechanism of the PPR motif will require further analyses.
Significance of Amino Acids Making up the
PPR Motif
Recent progress has shown the principle framework of the PPR
motif as an RNA-binding unit and the role of the respective
amino acids. The PPR motif consists of 35 amino acids, which
contains a loop region, helix A and helix B (Fig. 1C, D). The
RNA-interacting residues lie on the loop before helix A and
on one side of helix A [first, fourth, eighth, 12th and ‘ii’(2)].
The amino acids required for helix formation are positioned at
the third, sixth, seventh and 10th positions in helix A and at the
19th, 22nd, 23rd and 26th positions in helix B.
Various PPR mutants carrying an amino acid substitution
(by ethyl methanesulfonate treatment) have been isolated so
far. The mutated amino acid position and the putative role of
the amino acids in the PPR motif are summarized in Fig. 1D.
The mutated positions are spread across the entire motif. A
mutation in the amino acids required for helix formation could
result in loss of the helical repeat structure of the PPR protein.
Indeed, mutation of the sixth residue in mef11-1 and pgr3-3 as
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Functional aspects of PPR proteins
Fig. 1 Framework of the PPR motif. (A) Structure of two PPR motifs. Two PPR motifs in the human mitochondrial RNA polymerase (residues
263–330; PDB, 3SPA) are shown as lateral and top views. The positions of RNA-interacting residues (first, fourth, eighth, 12th and ‘ii’) within the
first PPR motif are represented by red-colored sticks. (B) Predicted structure of the PPR array. The structural model for the Arabidopsis CRR4
protein generated by the Phyre program (Kelley and Sternberg 2009). The region containing nine PPR motifs (fourth to 12th) is represented. The
helical repeats are colored alternately in blue and yellow. (C) Cartoon diagram for the position of the amino acids in the PPR structure. The amino
acids involved in the RNA interaction and helix formation are highlighted by red and dashed lines, respectively. (D) PPR amino acid sequences
and their roles. The sequences of PPR motifs and their subtypes (S, L1 and L2) are shown (as in Kobayashi et al. 2012). The helix and loop
structures are schematically represented. The number below the sequence indicates the first digit of the position of the PPR motif. The 34th
amino acid is re-designated as the ‘ii’ amino acid. ‘Role’ indicates the function of the amino acids: helix formation (B) or RNA interaction (R).
‘RNA’ indicates the effect of an amino acid substitution in an in vitro RNA binding assay (Kobayashi et al. 2012); ‘+’ and ‘–’ indicate loss and no
effect, respectively, of the RNA binding capacity. PPR mutant names and the mutated amino acid positions are also shown; ‘+’ indicates that the
mutation results in severe impairment of protein function, ‘±’ indicates partial impairment, ‘’ indicates no effect, and ‘c’ indicates a compensatory effect (see text). Detailed information on the mutants is shown in Table 1.
well as the 19th residues in mef1-1 and crr4-2 resulted in loss of
protein function (see Table 1 for references and detailed
information).
Mutation of putative RNA-interacting residues also resulted
in loss of protein function of LPA66 (lpa66-1, fourth residue),
MEF14 (mef14-1, ‘ii’ residue) and DMR1 (dmr1-1, ‘ii’ residue). A
mutation of the eighth basic residue of PET309 (pet309-1)
caused a partial impairment of protein function, i.e. reduced
growth of yeast. Coincidently, the basic residues at the eighth
and 12th positions could be involved in the RNA-binding ability
of the PPR motif, but were less significant than other
RNA-interacting residues (first, fourth and ‘ii’) in vitro
(Kobayashi et al. 2012).
Other amino acids in helix A seem to be involved in RNA
interactions. For example, substitution of the second and 14th
residues resulted in loss of RNA-binding capacity in vitro.
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Table 1 PPR mutants with single amino acid substitutions
Name
Species
crr2-2
AT
a
Amino acid substitution
Phenotype
G309R (24th, eighth motif)
Defect in NDH activity
Hashimoto et al. (2003)
crr4-1
AT
G425R (16th, 11th motif )
Defect in NDH activity
Kotera et al. (2005)
crr4-2
AT
A397V (19th, 10th motif)
Defect in NDH activity
Kotera et al. (2005)
crr4-4
AT
E462K (18th, 12th motif)
Partial defect in NDH activity
Kotera et al. (2005)
dmr1-1
Yeast
D785V (ii, 11th motif)
No respiratory growth at 36.5 C, reduced growth at 30 C
Lipinski et al. (2011)
dmr1-2
Yeast
T351A (31st, fourth motif)
No respiratory growth at 36.5 C
Lipinski et al. (2011)
dmr1-2
Yeast
D785V (ii, 11th motif)
Wild-type-like at 30 C
Lipinski et al. (2011)
ecb2-2
AT
T500I (second, 14th motif )
Delayed greening
Cao et al. (2011)
lpa66-1
AT
G415R (fourth, 11th motif)
Impaired PSII activity
Cai et al. (2009)
mef1-1
AT
G337E (19th, 10th motif)
Defect in RNA editing(rps4-956, nad7-963, nad2-1160)
Zehrmann et al. (2009)
mef1-2
AT
S559F (DYW)
Defect in RNA editing (rps4-956, nad7-963, nad2-1160)
Zehrmann et al. (2009)
mef7-1
AT
G238R (32nd, sixth motif )
Defect in RNA editing (ccb206-28, cod-325, nad2-1433, nad4L-41)
Zehrmann et al. (2012)
mef14-1
AT
D455N (ii, 12th motif)
Reduction in RNA editing to 30% (matR-1895)
Verbitskiy et al. (2011)
mef11-1
AT
L48F (sixth, second motif )
Defect in RNA editing (cox-422, ccb203-378, nad4-124)
Verbitskiy et al. (2010)
mef11-3
AT
V125T (17th, fourth motif )
No effect
Verbitskiy et al. (2010)
pet309-1
Yeast
K424A (eighth, third motif)
Reduced growth
Tavares-Carreon (2008)
pet309-2
Yeast
R427A (11th, third motif )
Reduced growth
Tavares-Carreon (2008)
Yamazaki et al. (2004)
b
Reference
pgr3-1
AT
T610I (second, 15th motif )
Defect in Cyt b6/f complex and NDH activity
pgr3-2
AT
T505I (second, 12th motif )
Defect in Cyt b6/f complex
Yamazaki et al. (2004)
pgr3-3
AT
L1108F (sixth, 27th motif)
Defect in NDH activity
Yamazaki et al. (2004)
a
AT indicates Arabidopsis thaliana.
The numbers in parentheses indicate the position of the substituted residue in the PPR motif and the PPR motif number within the protein, respectively.
b
Mutation of corresponding residues in pgr3-1, pgr3-2 and
ecb2-2 resulted in the loss of protein function. PGR3 contains
the highest number of PPR motifs (i.e. 26) in Arabidopsis. This
protein seems to be involved in versatile steps, including stabilization of the petL operon RNA and in the translation of petL
and ndhA. PetL operon mRNA is destabilized in pgr3-1 and
pgr3-2, which carry mutations in the 15th and 12th PPR
motifs, respectively. However, another mutant, pgr3-3, which
has a mutation at the sixth residue in the 26th PPR motif, is only
deficient in the translation of petL and ndhA, suggesting that
the defects caused by a single amino acid substitution could be
involved in specific functions of the protein (Cai et al. 2011).
Based on the mutated positions, the first half of the 26 PPR
motifs could be responsible for the stabilization of petL RNA,
while the second half could play a role in the translation of petL
and ndhA. Mutation of the 11th methionine residue did not
affect RNA binding capacity in vitro, whereas mutation of the
11th arginine residue in yeast pet309 resulted in decreased
growth (pet309-2), suggesting that a basic residue in the 11th
position might be able to compensate for the role of the 12th
position.
Mutation of amino acids in the loop region resulted in diverse effects, presumably affecting the packaging of the helices.
A mutation at the 16th and 32nd residue in crr4-1 and mef7-1,
respectively, resulted in impairment of protein function.
However, mutation of the 17th residue had no effect in
mef11-3. Mutation of the 31st residue (T351A; fourth PPR
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motif of DMR1) partially alleviated the hypomorphic phenotype (i.e. respiratory growth of yeast) of the ‘ii’ mutation in the
other PPR motif (D785V; 11th PPR motif of DMR1) in dmr1-2
at the permissive temperature. Mutations in other residues
have also resulted in loss of protein function, such as the
18th (helix B; crr4-1) and 24th residue (helix B; crr2-2). These
residues are found in the convex region of the PPR structure,
separate from the RNA-interacting surface, which might
suggest an unknown function of the convex region, such as
protein–protein interactions observed in a Pumilio protein
(see below).
It should be mentioned that the substitutions described
above could result in various outcomes such as loss of RNA
binding capacity or absence of the protein itself because of
misfolding. The translational status has not been determined
in many cases, presumably owing to low accumulation of the
proteins. However, the above observations suggest that numerous positions are involved in PPR protein function, in spite of
high amino acid degeneration in the PPR motif.
Significance of Individual PPR Motifs
in a Protein
Although various positions of the amino acids could be
involved in PPR functions, recent analyses have suggested
that not all PPR motifs contribute to protein function. The
PPR protein has been hypothesized to interact with RNA in a
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Functional aspects of PPR proteins
sequence-specific manner via one-motif to one-nucleotide correspondence. For PPR proteins involved in RNA editing, the PPR
tract could recognize the cis-element, which is approximately a
20-mer region located just 50 upstream of the editable cytidine
residue (Okuda et al. 2006, Tasaki et al. 2010). Several PPR
proteins are known to be involved in multiple sites of RNA
editing. However, the cis-elements for these editing sites are
not significantly conserved, and identical nucleotides are
rarely found (Hammani et al. 2009, Zehrmann et al. 2009,
Tang et al. 2010).
A recent biochemical study revealed that OTP82 and CRR22
proteins specifically bind to their respective target elements,
although their nucleotides were partially or barely conserved,
respectively (Okuda and Shikanai 2012). The high-affinity binding of the proteins to unrelated target elements suggests that
only certain specific nucleotides in an RNA sequence are sufficient for high-affinity binding of a PPR protein. These data
imply that only a few PPR motifs could be required for target
discrimination. This possibility is also supported by the fact that
several truncated proteins containing only two PPR motifs
derived from HCF152 displayed low or no RNA binding capacity
(Kobayashi et al. 2012).
PPR protein genes have evolved and been expanded dramatically during land plant evolution (Fujii and Small 2011). At least
some PPR genes undergo a ‘birth and death’ process (Geddy
and Brown 2007). The appearance of editing site(s) and corresponding recognition factors also varies greatly, even between
closely related taxa (Sasaki et al. 2003, Fiebig et al. 2004, Tillich
et al. 2005). These observations suggest that proteins of this
family, or several motifs within, might not have been fully optimized to the target RNA during evolution.
Mode of Action of PPR Proteins
in RNA Editing
Plant RNA editing in the chloroplasts and mitochondria is the
conversion of C to U, or rarely, U to C, in transcripts (Gray 1996,
Maier et al. 1996). Arabidopsis contains 34 and 488 editing sites
in the chloroplast and mitochondrion genomes, respectively
(Shikanai 2006). In mammals, C to U editing to create a premature stop codon has been observed for apolipoprotein B
mRNA that requires two components: target RNA recognition
and C to U deamination, as provided by APOBEC-1 (apoB
editing catalytic subunit-1) and ACF (APOBEC-1 complementation factor), respectively (Teng et al. 1993, Mehta et al.
2000). However, a different mechanism operates in plant organelle RNA editing. The first site recognition factor identified
in plants was CHLORORESPIRATORY REDUCTION 4 (CRR4)
that is a PPR protein belonging to the PLS subfamily (Lurin
et al. 2004, Kotera et al. 2005). After the discovery of CRR4,
all other genetic analyses of site recognition for RNA editing
have identified PLS subfamily PPR proteins (Castandet and
Araya 2011).
The PLS subfamily contains additional non-PPR motifs
(E and DYW motifs). The DYW motif contains invariant
residues that match the active site of cytidine deaminases,
and the phylogenetic distribution is strictly correlated with
RNA editing, suggesting that the DYW motif is a good candidate for the catalytic domain for RNA editing (Salone et al.
2007). However, DYW motifs in several PPR proteins such as
CRR2 (involved in RNA cleavage between rps7 and ndhB) have
RNA cleavage activity in vitro (Hashimoto et al. 2003,
Nakamura and Sugita 2008, Okuda et al. 2009). In addition,
the DYW motif is often missing from PPR proteins involved
in RNA editing and has been shown to be dispensable for the
in vivo function of CRR22 and CRR28 (Okuda et al. 2009,
Okuda et al. 2010). Further, it has been proposed that the E
domain is required to recruit a still undefined catalytic enzyme,
rather than for RNA recognition (Okuda et al. 2007).
Very recently, participation of an additional, unexpected
class of protein, multiple organellar RNA-editing factor
(MORF), has been identified for RNA editing machinery in
plant organelles (Takenaka et al. 2012). Proteins in this family
require efficient RNA editing of multiple, or even dozens, of
sites via physical interaction with PPR proteins. These data
indicate that the process and machinery of RNA editing
seem to be more complex than previously thought in plant
organelles.
Mode of Action of Classical PPR proteins
The P-type PPR proteins can be found in various eukaryotes.
This classical class of PPR proteins makes up roughly half of the
PPR family in a plant. Most consist of only the PPR tract. Barkan
and her group have proposed that these PPRs act as a ‘cap’ for
processed transcripts (Pfalz et al. 2009, Barkan 2011, Prikryl
et al. 2011). Genes in plant organelles are often transcribed
as a polycistronic transcript containing several open reading
frames. The transcript must be cleaved and/or trimmed into
a particular transcript length. PPR10, which is a P-type PPR
protein containing 16 or 17 PPR motifs, interacts with the processed RNA termini of the intercistronic regions of atpI–atpH
and psaJ–rpl33 (Pfalz et al. 2009). Subsequent analysis has
shown that the association of PPR10 with the processed transcripts protects them from RNase attack and has site-specific
remodeling activity to enhance translation (Prikryl et al. 2011).
This model is also supported by the observation that several
other P-type PPR proteins such as CRP1, PpPPR_38, MRL1 and
HCF152 are likely to bind processed RNA termini and are
required for the accumulation of their processed transcripts
in chloroplasts (Barkan et al. 1994, Meierhoff et al. 2003,
Hattori et al. 2007, Johnson et al. 2010). Moreover, numerous
small RNAs (16–28 nucleotides) have been discovered in the
Arabidopsis chloroplast (Ruwe and Schmitz-Linneweber 2012).
The 50 and 30 termini of chloroplast RNAs coincide with the
ends of the small RNAs. The presence of these small RNA fragments could result from the protective action of PPR proteins
against exonucleases, called PPR footprints (Zhelyazkova et al.
2012). A significant portion of the P-type PPR proteins may
participate in this process.
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A few P-type PPR proteins contain additional motifs, which
might be involved in the catalytic process. For example, OTP51,
which is required for the splicing of ycf3 intron 2 and influences
the splicing of some group-IIa introns in Arabidopsis, contains
two LAGLIDADG motifs similar to those found in group-I
intron maturases (de Longevialle et al. 2008). The small
mutS-related (SMR) domain is found in the C-termini of
eight PPR proteins, including pTAC2 (plastid transcriptionally
active chromosome 2; Pfalz et al. 2006) and GUN1 (genome
uncoupling 1; Koussevitzky et al. 2007). The SMR domain is
highly related to the MutS2 family, which is involved in the
DNA mismatch repair system (Moreira and Philippe 1999).
GUN1 has both DNA and RNA binding activities
(Koussevitzky et al. 2007). PPR proteins with the SMR
domain appear to be involved in the transcription or repair
of chloroplast DNA, rather than RNA metabolism. PPR proteins
with extra motifs are good candidates to elucidate the mode of
action of PPR proteins in RNA/DNA processing.
On the other hand, it is conceivable that PPR proteins without obvious extra motifs could interact with other proteins. In
fact, the physical association of DELAYED GREENING 1 (DG1)
with SIG6, a subunit of chloroplast RNA polymerase, has been
demonstrated (Chi et al. 2010). The HCF152 protein forms a
homodimer via its C-terminal non-PPR regions (Nakamura
et al. 2003). PPR336 associates with the polysome in plant
mitochondria (Uyttewaal et al. 2008b). The Pumilio protein,
which is akin to the PPR protein with respect to the helical
repeat architecture and RNA binding properties, interacts with
other protein factors with its convex region, which is on the
opposite side to the RNA-interacting surface (Edwards et al.
2001). It is necessary to analyze thoroughly the function of the
PPR motif other than interacting with RNA, as well as any
non-PPR regions.
Recently, 62 PPR proteins were detected in the chloroplast
nucleoid-enriched proteome, including RNA-editing factors
(OTP82, CRR22 and CRR28), splicing factors (PPR5 and
OTP51), factors involved in processing and/or stabilization of
specific mRNAs (HCF152, CRR2, MRL1, CRP1, HCF107, PPR10
and PGR3), a putative transcription regulator (pTAC2) and 48
proteins with unknown functions (Majeran et al. 2012). This
proteome analysis suggests that PPR proteins may be spatially
close to dense areas where transcription of their target RNA
occurs. Further studies will be required to determine whether
post-transcriptional events are coupled with transcription in
the chloroplasts and mitochondria, as observed in other eukaryotes and prokaryotes.
Function of Species-Specific PPR Proteins
A comparison of the PPR sets from Arabidopsis, rice and the
moss Physcomitrella patens showed that the vast majority of
PPR genes in seed plants existed before the divergence of
monocots and dicots and the presence of species-specific PPR
protein groups (O’Toole et al. 2008), which belong to the
P-class PPR proteins. The species-specific PPR proteins are
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homologous to the product of Rf genes found in several species.
The Rf genes act to suppress the mitochondrial genetic defect
that causes cytoplasmic male sterility (CMS) and encode PPR
proteins in many cases (Chase 2007). These Rf-like PPR genes
show a number of characteristic features compared with other
PPR genes, including chromosomal clustering, unique patterns
of evolution and high rates of diversifying selection (Fujii and
Small 2011).
Rf PPR proteins have been proposed to interact with a mitochondrial RNA containing an aberrant open reading frame
associated with CMS, and to prevent the expression of the
mitochondrial gene in various pathways (Kazama et al. 2008,
Uyttewaal et al. 2008a). Recently, several observations have
been made regarding the function of Rf-like PPR proteins in
normal mitochondrial gene expression. One such factor, RNA
PROCESSING FACTOR2 (RPF2), is responsible for natural 50 end
polymorphism of mitochondrial nad9 and cox3 mRNAs in
Arabidopsis (Jonietz et al. 2010). Subsequently, a second
Rf-like PPR protein, Arabidopsis RPF1, was identified and
demonstrated to be required for the efficient generation of a
50 end of the mitochondrial nad4 mRNA (Holzle et al. 2011).
The small RNAs, which were found as the PPR footprints in
chloroplasts as described above, have not been found for these
mitochondrial RNAs, suggesting an alternative mechanism of
the P-type PPR protein in the formation and stabilization of
mRNA termini. In contrast to RPF1 and RPF2, RPF3 is required
for the accumulation of mature ccmC transcripts in mitochondria in Arabidopsis accession Columbia, but not in C24 and
other accessions (Jonietz et al. 2011).
The loss of RPF1, RPF2 or RPF3 seemingly causes no change
to the macroscopic phenotype or development of a functional
male gametophyte. This result is in contrast to the Rf protein
that is required for the production of functional pollen in the
presence of the mitochondrial CMS gene, although the expression of the CMS gene is not implicated in the physiological
aspect in the vegetative cell (Chase 2007). Consequently, the
Rf and Rf-like PPR genes seem to act in a nuclear and cytoplasmic interaction in response to the species-specific variation of
mitochondrial genome sequences and, in some cases, to compensate for a hybrid barrier, as for the yeast PPR protein AEP2
(Lee et al. 2008).
The evolutionary behavior of Rf-like PPR genes can be compared with those of leucine-rich repeat (LRR) domaincontaining genes (Fujii and Small 2011). The LRR domain is
found in plant resistance (R) proteins of the nucleotide-binding
site (NBS)-LRR family, which are responsible for plant innate
immunity (Glowacki et al. 2011). The genes containing an LRR
domain have increased during plant evolution and have been
subjected to diversifying selection. The essential structural
element of the LRR domain is the tandem repeat of 20–30
amino acids comprising an a-helix and b-sheet. The tertiary
structure of tandem LRR domains is usually a horseshoe-shaped
superhelix (Glowacki et al. 2011).
It is noteworthy that the PPR and LRR domains share similar
molecular architecture, which is a repetition of the functional
Plant Cell Physiol. 53(7): 1171–1179 (2012) doi:10.1093/pcp/pcs069 ! The Author 2012.
Functional aspects of PPR proteins
unit comprising several dozen amino acids. The LRR could be
required for protein–protein interactions for defense against
pathogens, whereas the PPR is required for protein–RNA interactions during organellar gene expression. Although most PPR
genes were established before the divergence of monocots and
dicots, the rapid evolution of Rf-like PPR genes suggests that the
nuclear and endosymbiotic-organellar genomes continue a molecular ‘arms-race’ over billions of years, similar to the LRR
against pathogenic invaders.
Perspective
Since the discovery of the PPR motif in 2000, PPR proteins have
become a major research field for organelle biology. Many questions are still unanswered, such as the mechanism of sequencespecific RNA recognition and a detailed mode of action in RNA
processing. The following fundamental questions remains: why
have plants alone acquired so many PPR genes, and why are
proteins with this motif exclusively involved in symbiotic organelle gene expression across organisms? Further research will
determine the physiological and molecular functions of PPR
proteins to reveal the system of gene-specific control of plant
organelle genes, which are likely to be closely linked to the
physiology and evolution of plants.
Funding
This work was supported by the Bio-oriented Technology
Research Advancement Institution [the Program for Basic and
Applied Research for Innovations in Bio-oriented Industry
(BRAIN)]; the Kyushu University Research Superstar Program;
the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grants-in-Aid 22681028 and 22380008].
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