RNA Editing in Plants and Its Evolution

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RNA Editing in Plants
and Its Evolution
Mizuki Takenaka, Anja Zehrmann, Daniil Verbitskiy,
Barbara Härtel, and Axel Brennicke
Molekulare Botanik, Universität Ulm, 89069 Ulm, Germany;
email: mizuki.takenaka@uni-ulm.de, anja.zehrmann@uni-ulm.de,
daniil.verbitskiy@uni-ulm.de, barbara.haertel@uni-ulm.de, mo.bo@uni-ulm.de
Annu. Rev. Genet. 2013. 47:335–52
The Annual Review of Genetics is online at
genet.annualreviews.org
This article’s doi:
10.1146/annurev-genet-111212-133519
c 2013 by Annual Reviews.
Copyright All rights reserved
Keywords
mitochondria, plastids, PPR proteins, MORF proteins, cytidine
deamination, RNA-protein interaction
Abstract
RNA editing alters the identity of nucleotides in RNA molecules such
that the information for a protein in the mRNA differs from the prediction of the genomic DNA. In chloroplasts and mitochondria of flowering plants, RNA editing changes C nucleotides to U nucleotides; in
ferns and mosses, it also changes U to C. The approximately 500 editing
sites in mitochondria and 40 editing sites in plastids of flowering plants
are individually addressed by specific proteins, genes for which are amplified in plant species with organellar RNA editing. These proteins
contain repeat elements that bind to cognate RNA sequence motifs just
5 to the edited nucleotide. In flowering plants, the site-specific proteins
interact selectively with individual members of a different, smaller family of proteins. These latter proteins may be connectors between the
site-specific proteins and the as yet unknown deaminating enzymatic
activity.
335
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INTRODUCTION
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RNA editing:
posttranscriptional
modification of RNA
that changes the
information content
The term RNA editing describes processes
that alter the identity of nucleotides in RNA
molecules or that add or delete nucleotides so
that the information in the mature RNA differs from that defined in the genome. Diverse
processes of RNA editing are found in viruses,
primitive eukaryotes, vertebrates, fungi, and
plants. RNA-editing processes are used as control checkpoints, can restore the function of the
encoded protein, and can create different proteins. Excellent reviews of the various processes
in different organisms have recently appeared
and describe their mechanistic and functional
aspects and their origins (1, 23, 40, 56, 64, 88).
Here, we focus on the RNA-editing process in
land plants in which this is an essential step of
RNA maturation in the two organelles with resident genomes: mitochondria and plastids (14,
21, 37, 70).
RNA EDITING IN PLANTS
In flowering plants (angiosperms), RNA editing
was first recognized in mitochondria in 1989 as
sequence differences between DNA and RNA
(16, 27, 32). These differences of U nucleotides
in the RNA in positions of C nucleotides in
the DNA were found to be caused by substitutional C-to-U changes in the RNA (Figure 1a).
The amino acid codons specified after editing
are more similar to those present at the respective positions of orthologous proteins in
other organisms. Three years later, the same
type of RNA editing involving C-to-U changes
was also documented in plastids (33). Editing
in both organelles was subsequently reported
in all land plants, including all major plant lineages from the bryophytes to gymnosperms and
in all angiosperms (73, 74, 78, 91). The notable
exceptions are some species of liverworts in the
branch of the Marchantiales, in which the messenger RNAs (mRNAs) remain as specified by
the genomes in plastids as well as in mitochondria (Figure 1b) (66). At present, no RNA editing has been observed in cytoplasmic RNAs of
plants. The process seems to be confined to the
two organelles.
336
Takenaka et al.
Another species-specific feature is the distribution of the U-to-C reverse reaction RNA
editing, which occurs only in ferns, mosses,
and Lycopodiaceae in addition to many C-toU changes (Figure 1b) (24, 42). In flowering plants, posttranscriptional mRNA editing
is performed generally as C-to-U alterations of
nucleotide identity (22).
Most of the RNA-editing events occur at
the first or second positions of codons and thus
usually alter the codon given in the genome
and transcribed into the precursor mRNA (premRNA) (Figure 1a). Consequently, the mature RNA specifies a different amino acid than
that encoded by the genomic DNA. To predict the final protein sequence synthesized from
a given gene, it is therefore not sufficient to
determine the genomic sequence because only
the mature mRNA sequence contains this information. The C-to-U RNA editing can alter
any codon containing a C nucleotide, including
the generation of initiation codons by changing
ACG to AUG and the introduction of translation termination signals by changing CGA to
UGA or CAA to UAA. Conversely, the U-toC RNA editing found in mosses and ferns can
convert translational stop codons, such as UAA
termination signals, to a CAA triplet coding for
the amino acid glycine. The final open reading
frames can thus not only become different from
those originally encoded by the genome but can
also be extended or shortened.
RNA editing in plant organelles is a posttranscriptional process. The competence of
lysates from mitochondria or plastids to faithfully edit an in vitro synthesized and added RNA
molecule shows that this editing does not depend on a close link to the transcription machinery (7, 30, 55, 63, 76, 84, 85). However,
only a few sites can be edited in vitro, suggesting that often the editing activity may require
a complex machinery of several proteins that is
not readily assembled on in vitro–added RNA
molecules.
The C-to-U and the U-to-C types of RNA
editing occur in plastids and in plant mitochondria in not only mRNAs but also in
transfer RNAs (tRNAs), introns, and 5 - and
GE47CH15-Takenaka
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29 October 2013
Primary
transcript
14:23
Ala Thr Arg Gln
GCU ACG CGC CAG
RNA editing
RN
Mature RNA
GCU AUG UGC UAG
Ala Met Cys STOP
b
0
Chara (stonewort)
0
Chaetosphaeridium
ext.
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Takakia
509
Anthoceros
Isoetes (quillwort)
11
ext.
HORNWORTS
315
Adiantum
35
Oryza
43
Nicotiana
41
Arabidopsis
0
Chloroplast editing sites
222
1,560
LYCOPODS
2,139
Selaginella
104
500
ext.
MOSSES
Physcomitrella
ext.
1,000
0
Marchantia
1
35
ext.
LIVERWORTS
ext.
C to U
U to C
0
Haplomitrium
0
433
0
GREEN ALGAE
ext.
FERNS
481
SEED PLANTS
536
430
0
500
1,000
1,500
2,000
2,500
Mitochondrial editing sites
Figure 1
Plant organellar RNA editing alters nucleotide identities in almost all land plants. (a) The C-to-U alteration can change amino acid
codons and introduce translational start and stop codons. This results in different amino acids being incorporated into the mature RNA
than were predicted from the genomic DNA. Plants shown in the photographs are from left to right: the liverwort Marchantia
polymorpha, a representative fern, and two angiosperms (flowering plants). (b) In all land plant lineages, RNA editing changes C
nucleotide identities to U in mitochondria and plastids. In green algae, no editing has been reported to date. In the branch of the
liverworts that contains the species Marchantia polymorpha, editing has been lost secondarily. Numbers of editing sites are given for
species in which the full complement has been analyzed. In some species, extensive editing has been reported, although the full extent
still needs to be determined.
3 -untranslated sequences (6, 12, 24, 49). In ribosomal RNAs, editing appears to be absent
or very infrequent. The reason for this suppression of editing can only be speculated; it
may be connected to a rapid compartmentalization of the rRNAs by protein coverage. In
introns, editing seems to be required in some instances for efficient splicing. In tRNAs, editing
events can be required for processing of precursor RNA molecules (6). However, in both
organelles, RNA editing is most prevalent in
the coding regions of mRNAs. The amino acids
specified by the codons generated by editing in
the mRNA are generally better conserved in
evolution than the amino acids encoded by the
genomic DNA (27). This observation suggests
that RNA editing in plants restores codons altered by mutation to (again) encode the amino
acids that are optimal or even required for function of the respective protein. RNA editing can
www.annualreviews.org • RNA Editing in Plants
337
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then be considered to act as an indirect repair
mechanism that corrects DNA mutations on
the RNA level.
PPR proteins:
pentatricopeptide
repeat proteins
THE RNA: CIS ELEMENTS
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The nucleotide to be edited must be recognized
and targeted within the multitude of C nucleotides in the population of RNA molecules.
In recent years, in vivo [trans-plastidic (9–11,
47)], in vitro (55, 84, 85), and in organello (7, 19,
36) investigations have identified the crucial cis
elements in the polynucleotide RNA chain for
a number of editing sites. There is no common
denominator distinguishing a C to be edited
from an unedited C, implying that editing sites
are addressed individually through a unique sequence pattern. In all instances, stretches of 20
to 25 nucleotides in the RNA mostly upstream
(5 ) of the editing site provide a specific sequence context that is recognized by the editing
activity (9, 15, 85).
When mutations of these sequence regions
from several editing sites were tested in in vitro
and in organello assays, the crucial nucleotides
were found to reside in a window between 5
and 15 nucleotides upstream of the edited C (9,
15, 19, 36, 55, 84, 85). The gap to the edited C
suggests that the site-specific trans-factors do
not actually discriminate the nucleotides adjacent to the edited nucleotide. Mechanistically,
either the site-specific factors stretch across this
gap or one or more other proteins are recruited
for the deamination reaction. Such spatial constraints may also be responsible in selecting the
nucleotide identity just upstream of the edited
C. At this −1 position, G and A nucleotides are
highly underrepresented and can be found only
at a very few editing sites (22).
In some instances, nucleotide identities farther from this central motif appear to influence
attachment of the respective specificity factors
(85). Nucleotides more than 30 moieties upstream from the edited C or closer to this position and even downstream in the mRNA influence RNA editing in vitro. These very variable
parameters suggest that individual recognition
patterns are used at each site, further support338
Takenaka et al.
ing the inference that different trans-factors are
involved at different editing events. Indeed, several site-specific trans-factors have been identified in recent years that confirm that the unique
RNA sequence motifs upstream of editing sites
are individually recognized by specific proteins
encoded in the nuclear genome.
THE PROTEINS:
TRANS-FACTORS
Pentatricopeptide Repeat Proteins
Are the Specificity Factors
The first such trans-factor was identified in
2005 for an RNA-editing event in plastids by
tracing a rather unspecific mutant phenotype
to the nuclear gene responsible (41). The physiological defect identified as a disturbed function of the plastid NADH dehydrogenase is
caused by a single RNA-editing defect in the
RNA for a specific subunit of this protein
complex. The affected editing event creates an
AUG translational start from the genomic ACG
codon. Consequently, without this editing
event, the NADH dehydrogenase subunit protein is not synthesized and the complex cannot
be functionally assembled in the mutant of the
RNA-editing specificity factor.
Analysis of other mutants incapacitated in
various plastid functions led to further similar
proteins, all of them uniquely addressing one
or a very few editing sites in plastid mRNAs
(29, 44, 57, 59). The first factor for editing
events in mitochondrial mRNAs was identified
by genomic mapping of ecotype-specific editing
variants and tracing these to the altered genes
(93). More recently, a direct screening approach
has been developed to identify specific RNAediting aberrations in a randomly mutated plant
population and to trace the respective mutation
to an individual plant and therein to the gene
affected (28, 75, 77, 86, 87, 93).
The nuclear-encoded factors required for
editing of one to as many as eight to ten sites in
mitochondria and plastids belong to the family
of the pentatricopeptide repeat (PPR) proteins.
This protein family is specifically amplified in
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plants, whereas the nuclear genomes of fungi,
protists, and animals code for only few members
of this class of proteins (2, 38, 46, 57, 58, 60, 69,
71). Almost all of the PPR proteins are targeted
to mitochondria or plastids (the two organelles
with genomes), where they are involved in various RNA-processing steps, including intron
splicing, endonucleolytic processing, RNA stability, and access to translation.
The RNA-editing factors in plants belong
to an approximately 200-member-strong subgroup of the PPR proteins, which is characterized by a mixture of 35-mer amino acid repeats
(P) and PPR repeats that are slightly longer (L),
with as many as 37 amino acids, or shorter (S),
with 31 to 34 amino acids (46, 69, 71). At their
C termini, following the last PPR motif, these
RNA-editing proteins are extended by an E (extension) domain. The E region displays some
features of PPR elements, suggesting that such
an element was part of the E-domain ancestor and that some properties may have been retained (Figure 2).
About half of the E-type PPR proteins
are further extended by an approximately
100-amino-acid-long DYW region. This element is named after the three highly conserved
C-terminal amino acids: aspartic acid (D), tyrosine (Y), and tryptophan (W) (Figure 2). The
DYW region is essential in some of the DYW
PPR proteins but is not required for functional
activity in others, at least in complementation
assays of mutants (17, 57). One protein in Arabidopsis, DYW1, consists of a well-conserved
DYW domain and of a region with rudimentary similarity to a partial E domain (Figure 2)
(13). The DYW1 protein interacts in plastids
with the first identified RNA-editing specificity
factor CRR4, which lacks a DYW region, to
edit the ACG codon to an AUG. Both proteins
are required here, suggesting that a DYW
domain is essential but may be provided in trans
if not present in cis on the PPR editing factor.
The nuclear genome of Arabidopsis thaliana
codes for 194 E-type PPR proteins. Six additional genes code for PPR proteins that consist
of the PLS repeat elements that are characteristic for this subgroup but do not encompass
an E domain. These approximately 200 proteins are not enough to individually specify the
450–500 editing sites in mitochondria and plastids. Indeed, many of these proteins are found
to target several sites. Although several of the
assigned RNA-editing PPR proteins appear to
address individual sites, some are required for
as many as six or even eight editing events. Surprisingly, the common targets of a given PPR
protein sometimes show very little sequence
similarity in their cis elements upstream of the
edited nucleotides, suggesting that different nucleotide combinations may confer recognition
and binding of the same PPR protein (79).
In some instances, such flexible connections
between the PPR proteins and the RNA sequence lead to overlapping specificities, resulting in two PPR proteins able to target the same
editing site. Evidence for such redundancies is
mostly indirect from those cases in which a
complete knockout of one PPR protein only reduces editing at a given site but does not lead to a
complete loss of the nucleotide conversion (93).
At these sites, another PPR protein presumably
must be able to provide the residual activity, albeit less efficiently. Direct evidence has so far
been reported for the two rather similar PPR
proteins MEF8 and MEF8S (87). The acronym
MEF designates the PPR proteins identified as
mitochondrial RNA-editing factors. The target
sites of MEF8 and MEF8S seem to be identical, and the editing levels at the sites are directly correlated with the expression pattern of
the resident intact PPR protein in a knockout
plant of the respective other protein.
The overlapping specificities and target sequences of different RNA-editing PPR proteins
have consequences for our view of the specificity of the RNA-PPR interaction. There may
actually be a large number of such redundancies and hidden targets, which could imply a
more degenerate and flexible RNA-PPR code
interaction.
E (extension)
domain: the extension
domain evolved from
an ancient PPR
element; only found in
RNA-editing PPR
proteins
DYW domain:
another extension
beyond the E domain
with signature amino
acids of cytidine
deaminases and
containing the three
highly conserved
amino acids D
(aspartic acid),
Y (tyrosine), and
W (tryptophan) at the
C terminus
PLS repeats: PPR
repeats that consist of
P (normal PPR), L
(longer PPR), and S
(shorter PPR) motifs.
This pattern is
generally present in
PPR-type
RNA-editing factors in
plant organelles
The PPR Protein to RNA Code
RNA binding of PPR proteins has been
shown for several RNA-editing proteins but
www.annualreviews.org • RNA Editing in Plants
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Arabidopsis cp
CRR22
cp S
P
L
S
P
L
CLB19
CRR4
cp
L
P
P
S
P
L
S
P
L
S
P
L2
S
E
cp P
L
S
S
P
L
S
P
L2
S
E
S
S
S
S
P
L
S
P
L2
S
E
L
S
cp
DYW1
DYW
DYW
Arabidopsis mt
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MEF11
mt
L
S
MEF21
mt S
MEF19
P
L
S
P
L
S
P
L
S
P
L2
S
E
mt S
P
L
S
S
P
L
S
P
L2
S
E
P
S
P
L
S
P
L
S
P
L2
S
E
L
S
P
L2
S
E
L
S
P
L2
S
L
mt
MEF8
DYW
DYW
Physcomitrella mt
PpPPR_56
mt
L
S
P
L
S
P
L
S
P
E
DYW
Figure 2
Structure of pentatricopeptide repeat (PPR) proteins involved in plant organellar RNA editing. The proteins
that recognize a specific RNA sequence just upstream of an RNA-editing site belong to the superfamily of
PPR proteins. Aligned here are several representative editing factors from plastids (cp; CRR22, CLB19, and
CRR4) and mitochondria (mt; MEF11, MEF21, and MEF19). These examples display the principle of the
structure and the variations in the numbers of the three different types of repeats (S, small; P, medium sized;
L, long repeats). The RNA-editing subfamily of the PPR proteins is characterized by a modular structure
within which each of the PLS repeats presumably contacts one nucleotide. The function of the extension (E)
domain is not yet clear, and the optional C-terminal DYW domain may provide deaminase activity for the
C-to-U nucleotide conversion in the RNA. In both compartments, proteins with a DYW domain and few if
any PPR elements can be found. As examples, DYW1 in plastids and MEF8 in mitochondria are depicted.
All RNA-editing PPR proteins in the moss Physcomitrella patens contain E and DYW domains. On the left of
the respective protein structure, the N-terminal elements labeled cp or mt denote the predicted respective
target sequences. The N-terminal part of the MEF8 E domain shows relatively low similarity to other E
domains (light green).
is most intensively documented for PPR
proteins involved in other RNA-processing
reactions (59, 69, 90). For PPR proteins
protecting against endo- or exonucleases, tight
contact to the RNA is expected and indeed
found to be very sequence specific. However,
RNA-editing PPR proteins are expected to
bind reversibly because the mature RNA
340
Takenaka et al.
needs to be readily accessible to the ribosome
for protein synthesis. Nevertheless, attachment of some PPR proteins to their specific
RNA targets has been observed in several
instances. The main problem encountered
with these assays is the difficulty of obtaining
PPR proteins by expression of their coding
sequences (20) in bacterial cells. In bacteria,
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29 October 2013
14:23
the PPR proteins are synthesized but usually
aggregate or are sequestered into inclusion bodies and are not soluble. This holds true for the
expression of single PPR repeats as well as for
the E and DYW domains without any repeat.
Although no direct structural data of the
PPR proteins could be obtained, predictions
of their 3D structures generally suggest two
helical structures within each repeat unit (20,
39, 53, 69, 71). Starting from the presumption
that each repeat attaches to one nucleotide in
the RNA, amino acid coincidences with nucleotide identities in the target RNA can be calculated. These presumptions seem to be valid,
given that the parameters identified through
such alignments yield reasonably accurate computational correlation tools, which have been
used to correctly predict the target sites for
previously unassigned RNA-editing PPR proteins (3, 29, 39, 53, 80). These assignments
even allow the intelligent manipulation of the
RNA target sites to eventually generate PPR
proteins, which can be designed to bind to
any given RNA sequence. This fact has been
proven in assays of PPR proteins in which
only the crucial nucleotide determinator amino
acid identities were altered. The recoded PPR
proteins bind specifically to the correspondingly altered RNA sequence (3). Such designed
RNA-binding proteins complement the DNAbinding TAL proteins, which likewise use a
34-mer–amino acid repeat structure to define a
specific sequence pattern as a target for binding
in double-stranded DNA nucleotide polymers
(8).
Multiple Site-Specific Proteins:
Multiple Organellar RNA-Editing
Factors
Along with the RNA sequence-specific PPR
proteins, another group of proteins is required
for RNA editing at all sites in flowering plant
mitochondria as well as plastids (Figure 3). In
the genome of Arabidopsis, ten members of this
small family of proteins are encoded; two are
targeted to plastids and five or six are targeted
to mitochondria, with one or two possibly im-
ported into both organelles (5, 81). One protein
contains only half of the only conserved region
and may be a pseudogene. These proteins are
each required for numerous editing sites and
have been designated accordingly as multiple
organellar RNA-editing factors (MORFs) (81).
Mutation of one such MORF results in loss
of RNA editing at several sites and reduction of
editing at many sites. In the plastid, mutation of
either of the two MORFs targeted exclusively
to this compartment affects almost all editing
sites, suggesting that both proteins are involved
in editing at a given site. At some sites, editing
is completely lost when only one of the two factors is absent (81). Both plastid MORF proteins
are therefore predicted to form homomers and
heteromers, implying that they may, in some
instances, be able to provide their function in
editing as homomers. At other sites heteromers
may be required, but at most of the editing
sites either the homomer or heteromer seems
to be functional, although the heteromers appear to be more prevalent. This inference is suggested by the reduced editing observed at most
of the plastid editing sites when either of the
two MORFs is missing (81).
The actual function of the MORF proteins
in the hypothetical editosome in plastids and
mitochondria is as yet unknown. Because they
show no similarity to known functional protein domains (specifically, they do not carry
any cytidine deaminase signatures), they may
provide a function as connectors between the
RNA-binding PPR proteins and the actual enzymatic activity (Figure 4). This notion is supported by the observation that MORF proteins
can interact with the PPR proteins involved in
RNA editing. The mitochondrial MORF proteins discriminate between different PPR proteins in yeast two-hybrid assays (81). In some
instances, the MORF protein required for editing a given site indeed interacts with the specific
PPR protein, which is also essential for processing this site. The MORF proteins may be
involved in bridging the distance of four nucleotides between the nucleotides contacted by
the PPR proteins and the actually edited C moiety to guide the enzyme.
www.annualreviews.org • RNA Editing in Plants
Multiple organellar
RNA-editing factors
(MORF) proteins:
individual MORF
proteins are involved
in RNA editing at
numerous sites in
flowering plants
Editosome: the
hypothetical protein
complex that associates
with mitochondrial or
plastid RNA to alter a
C or U nucleotide to
the respective other
identity
341
29 October 2013
14:23
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Figure 3
Multiple organellar RNA-editing factor (MORF) proteins are required for RNA editing in plastids and mitochondria of flowering
plants in addition to the pentatricopeptide repeat (PPR) proteins. The small family of MORF proteins is only found in flowering plants,
suggesting these are a recent addition to the RNA-editing process. In Arabidopsis thaliana, nine genes encode proteins with full-length
conserved central domains; MORF10 contains only part of this region. Two of the nine full-length genes are specific for the plastid
(MORF2 and MORF9; shaded green), MORF8 is dual targeted to both organelles, and the other MORFs are specific to mitochondria.
This unrooted tree visualizes the species-specific variation of genes for MORF proteins between the flowering plants rice (Oryza),
poplar (Populus), grape (Vitis), and thale cress (Arabidopsis). This suggests that similar species-specific MORF proteins are recent
derivates and may at least partially substitute for each other. Different numbers of genes for MORF proteins are encoded in other
flowering plant species. MORFs are not found in nonflowering plants.
342
Takenaka et al.
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MORF
proteins
E domain
Cytidine
deaminase?
DYW domain
PPR domains
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Figure 4
Model of the presently identified composition of the hypothetical editosome in flowering plant organelles. A
pentatricopeptide repeat (PPR) protein binds to a specific combination of nucleotides in the RNA. One or
more multiple organellar RNA-editing factor (MORF) proteins interact with the PPR protein and attract
the enzymatic activity. This most likely deaminase activity may be a DYW domain from a respective
(second) PPR protein or an entirely different moiety. Bullets represent nucleotides in the RNA. Cartridges
in the PPR proteins denote the degenerate repeats of approximately 35 amino acids. The E and DYW
domains of the respective PPR proteins are indicated.
The only discernible feature present in all
nine members of the MORF family is the
so-called MORF box, which is centrally located
in most MORF proteins. The function of this
conserved MORF box domain is unknown; it
may be a point of contact to the PPR proteins
(5, 81).
The Enzyme
The enzyme catalyzing the actual C-to-U
conversion reaction for RNA editing in plant
mitochondria and plastids has not yet been
identified. So far, several conditions have been
characterized that are only partially compatible
with either of the classic deamination or
transamination reactions.
Functional in vitro assays and the absence
of any in vivo intermediates with termini at
editing sites suggest that the sugar-phosphate
backbone remains intact during editing (7, 30,
31, 76). The C nucleotide is not excised and
substituted by a U, but the C is modified in
place in the RNA chain. This conclusion is
supported by in vitro assays that show that an
α-phosphate-labeled cytosine integrated in a
substrate RNA is recovered as an α-labeled
uridine (7, 63). These observations suggest that
the reaction proceeds as a C-to-U deamination
step analogous to the single nucleotide conversion in the pathway of uridine and cytidine
biosynthesis. Here, a classic cytidine deaminase
enzyme is involved, of which seven are encoded
in the Arabidopsis genome (18). However, none
of them appears to be active in organellar
RNA editing. Precedence for the adaptation
of such a mononucleotide deaminase to be
able to act on polynucleotide chains is found
in the mammalian apolipoprotein C-to-U
RNA editing (54). This, as well as the classic
mononucleotide-specific cytidine deaminases,
requires bound zinc atoms for its active center.
Zinc chelators, however, do not reduce or
block the plant mitochondrial activity in
in vitro assays (76). Analogous assays with
plastid extracts did detect an inhibition by the
chelators, leaving a classic cytidine deaminase
activity as a possibility (31).
As a plausible alternative, it has been
suggested that the PPR protein–integral DYW
domains (in which the crucial amino acid patterns of classic cytidine deaminases appear to be
conserved) supply the cytidine deaminase activity (67). This very attractive hypothesis implies
that for editing sites recognized by E-class PPR
proteins without their own DYW domain, an
additional DYW class PPR protein or a protein
consisting of little more than a DYW region
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is recruited. Support comes from the identification that the DYW1 protein is required
together with the E-class PPR protein CRR4
for the editing at one plastid site, from the
in vivo interaction of these two proteins, and
from the correct function of a chimeric fusion
protein of both entities (13). In most instances,
PPR–PPR DYW protein interactions may be
mediated by the MORF proteins, which may
supply the linker between the two PPR-like
proteins (Figure 4). However, a C-to-U
deaminating activity of the DYW domains still
needs to be shown.
Yet another alternative source of the enzyme could be a modified transaminase activity. Transaminases are involved in amino acid
biosynthesis pathways and require an acceptor
molecule for the amino group. Several acceptor molecules known from the amino acid pathways have been tested in vitro, but none were
found to influence the reaction (76). The very
selective in vitro assays suggest that additional
as-yet-unassigned proteins may play a role in
RNA editing in plants. The positive effect of
added ATP on the in vitro assays is on accessory
functions rather than the reaction itself (76).
The ATP enhancement implies that a step is involved that requires the energy supply. The reaction step could be traced to the release of the
RNA from an attached protein, such as glutamate dehydrogenase, which is abundant in mitochondria. The protein was found to inhibit
RNA editing in vitro but is specifically blocked
from binding to the RNA by the presence of
ATP (76). Furthermore, the ATP can be largely
substituted by NTP and even dNTP, suggesting that for the RNA-editing step an RNA helicase may be activated, which unwinds and clears
the target RNAs from attached nonediting proteins. All proteins involved must be identified
and analyzed to understand and to eventually
rebuild the plant plastid and mitochondrial editosomes in vitro.
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GE47CH15-Takenaka
EVOLUTION OF RNA EDITING
IN PLANTS
The evidence gathered to date suggests that
RNA editing in plant organelles evolved inde344
Takenaka et al.
pendently from RNA processes in distant evolutionary lineages of animals, fungi, and protozoans when the first plants moved from the
aquatic environment onto the land and developed into the ancestors of the Lycopodiaceae
(21, 24, 38, 65, 73, 74, 83, 91). No editing has
been observed in any alga, whereas it is prevalent in all land plants. The only exceptions are
several species of the Marchantiales, which presumably lost RNA editing secondarily (25, 26,
66, 89).
Within the liverwort branch, several features other than the loss of editing have changed
during the evolution of the land plants. For example, the frequency of editing events per RNA
unit varies greatly between different lineages. In
flowering plants, 400–600 RNA-editing events
occur in mitochondria, and 30–40 such alterations occur in plastids (Figure 1) (51, 62). In
basal vascular plants, such as Isoetes engelmanii
or Lycopodium, 1,000 to 1,500 nucleotides are
altered in mitochondria (24). On the other end
of the spectrum is the moss Physcomitrella patens,
in which two RNA-editing events occur in plastids and eleven C nucleotides are changed to U
in mitochondria (65).
The number of the genes for RNA editing–
type PPR proteins in a given plant species often
roughly matches the number of editing sites
in the organelles. The 400–500 editing sites in
flowering plants are served by 200 editing-type
PPR proteins; in the moss Physcomitrella, the
13 sites are recognized by 10 editing-type
PPR proteins, and in the liverwort Marchantia,
where no editing is observed, there are no
genes for editing-type PPR proteins found
in the genome. In Lycopodium, the number
of editing PPR proteins is slightly increased
compared with their number in flowering
plants, although the number of editing sites
is two to three times larger. It is possible
that more sites are addressed by a single PPR
protein in Lycopodium than in higher plants,
or maybe the entire editing machinery has
evolved differently. This scenario is a distinct
option, considering that in Lycopodium no
MORF proteins are encoded in the genome.
Many E-type PPR proteins, which require a
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deaminase in trans and an adaptor molecule
analogous to the MORF proteins, are found in
the Lycopodium genome. Similarly, no genes for
MORF proteins are found in Physcomitrella. In
the moss and also in the protist Naegleria, this
may be correlated with the presence of only
DYW-domain-containing PPR proteins, but
the functional significance of the coincidence
will probably become apparent only after the
actual contribution of the MORF proteins to
the editing machinery has been clarified.
Evolution of the U-to-C-Type
RNA Editing
Another difference between the land plant lineages is the occurrence of the reverse RNAediting reaction, which alters a U nucleotide
to a C nucleotide. This type of editing occurs
in hornworts, lycopods, and ferns but is absent
from liverworts, mosses, and flowering plants
(24, 73, 91). This distribution suggests that this
type of editing arose after the split of the mosses
and the hornworts and later was lost or greatly
reduced in the branch that led to the seed plants.
The presence of U-to-C-type RNA editing
requires a specific enzymatic activity. With only
circumstantial information about the enzyme
involved in the C-to-U type of reaction, two
major scenarios are presently feasible. Either
two distinct enzymes perform the two opposing
reactions or one protein molecule catalyzes
both directions of the reaction. In the first
instance, the C-to-U reaction could be accelerated by a deaminase as discussed above and the
amination of the U to a C could be performed
by a specific enzyme adapted, for example,
from the nucleotide metabolism-catabolism
repertoire. The most likely precedence for
the second scenario, a single enzyme for both
reactions, would be a transaminase activity
recruited from one of those involved in amino
acid biosynthesis/degradation pathways. As
outlined above, this would require an acceptor
molecule on which the amino group can be
deposited in the C-to-U reaction and from
where the amino group could be retrieved in
the U-to-C amination of the nucleotide in the
RNA. In addition, the direction of the reaction
needs to be tightly controlled by accessory
proteins, such as distinct types of PPR proteins.
Evolution of the Pentatricopeptide
Repeat Proteins
Similar to the U-to-C editing that arose during
the evolution of the vascular plants and was lost
in the branch that led to the flowering plant
species, individual RNA-editing sites seem to
appear and disappear during evolution (4, 68,
82, 92). The most striking example is the complete loss of all editing sites in the Marchantia
clade of liverworts. But even between closely
related species, such as Arabidopsis and Brassica,
differences in individual editing sites are observed. For example, in the nad3 mRNA, site
nad3-64 is edited in Arabidopsis thaliana but not
in Brassica napus. This rather fast evolution of
appearing and disappearing editing sites suggests that the specificity factors of editing, the
PPR proteins, can mutate rapidly to alter their
target restrictions.
Concomitantly with the loss of an editing
site by its conversion to a genomic T, the
respective specificity conferring PPR protein is
free to mutate or even to become lost from the
nuclear genome. The remaining constraints
on this PPR protein depend on its requirement
for editing at other target sites. These sites
may be contacted through a different set of
the repeats in this PPR protein that would
consequently need to be maintained. This
idea may be difficult to analyze experimentally
because along with the sites for which a given
PPR protein is essential, there are usually other
targets requiring this factor. As detailed above,
these may be hidden by also being targets
of other PPR proteins and therefore cannot
be recognized as such in mutants of a single
gene. For example, the PPR protein MEF10 is
assigned to one target site in Arabidopsis, but its
homolog in grape, which may or may not be an
ortholog, is not required for this site because
this nucleotide is already encoded as a T in the
grape mitochondrial genome (28). Although
this grape PPR protein is the most similar to
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MEF10 in Arabidopsis, it has accumulated so
many amino acid differences in evolution that
its actual function is likely to have shifted to a
different editing site. At present, only one true
pair of orthologous proteins has been identified, the PPR2263 protein in maize and MEF29
in Arabidopsis, which are similar in sequence and
structure and target the same editing sites (72).
The above example also demonstrates the
rule rather than the exception in editing: RNAediting sites are necessary for optimal mitochondrial and plastid (protein) function and
survival of the plant. Generally, the amino acid
encoded by the edited codon is much better
conserved with the respective proteins from
other species. Furthermore, as in MEF10, if
an editing event is lost, usually the genomic
sequence has preempted the requirement for
this reaction by already encoding the T at the
respective position. This finding suggests that
editing is important and, with the sum of its
many sites, required for the plant. Direct evidence for the requirement of editing at individual sites is seen in mitochondria when homozygous knockout plants of respective PPR
proteins are not viable and is seen in plastids
when such mutants can grow only on sugarsupplementing media.
Although in many instances the knockout
mutation of a given PPR protein and the consequential loss of this editing event have no
detectable phenotype in the greenhouse, their
true positive value may only become apparent
in the competitive native environment. Many
of these editing events without any overt effect
in the pampered conditions of the greenhouse
may be crucial under certain environmental
challenges. In particular, the loss of editing reactions at several sites in mitochondria produce pleiotropic phenotypes that are often connected to altered responses and even to survival
under various stress conditions, such as drought
or salt challenges (29, 44, 72, 94). Whether this
is a secondary effect or whether RNA editing
can be regulated to adapt the physiological response of the plant still needs to be investigated
(35, 43, 50, 52).
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GE47CH15-Takenaka
346
Takenaka et al.
Selection and Maintenance of
RNA-Editing Sites
The general if small advantage of individual
editing events also becomes apparent by the inferred selection pressure on the persistence of
these sites. Such a positive selective force can
be postulated from the observation that most
editing events occur in coding regions and in
positions where RNA editing changes codon
identities. In addition, editing events in silent
codon positions tend to be edited in only some
molecules in the steady-state organellar mRNA
population. The partial conversion of such presumably neutral sites suggests that these may
be side effects of PPR proteins primarily acting at other sites. This fact and the nonrandom
distribution of edited nucleotides suggest that
a selective pressure acts on nonsilent sites to
maintain their editing.
Similar positive selection seems to apply to
RNA-editing events in introns that most frequently occur in domains V and VI of the conserved group II intron structures. These are the
best-conserved regions and are essential for the
splicing reaction. Their secondary and tertiary
structures can only fold properly after editing in
some instances, and when inserted into yeast introns, only the edited intron version promotes
splicing (12). In tRNAs, editing events are required for proper folding of the tRNA 3D structure, and processing of the tRNA from its respective precursor RNA is compromised before
editing has occurred at some sites. However,
RNA editing is not necessarily the first processing step. Intron splicing, mRNA end-trimming,
and RNA editing can occur in various orders;
some sites near exon borders are edited
only after splicing of the nearby intron (6,
49).
The examples of editing required for
other processing steps prompt speculation
about a potential regulatory function of RNA
editing in controlling the available pool
of mature functional RNA molecules. In
mRNAs, this could be achieved most economically through the control of an AUG translation initiation codon from an ACG triplet. This
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change is observed in a single plastid mRNA
and in a single mitochondrial mRNA. The affected plastid mRNA codes for the NdhD subunit of the NADH dehydrogenase, a protein
complex that improves the efficiency of photosynthesis but is not essential for survival of the
plant. In mitochondria, an AUG translational
start codon is generated in the nad1 mRNA
coding for a subunit of the respiratory chain
NADH dehydrogenase.
Editing events that create AUG codons, that
help to fold introns and tRNAs, and that remove
translational stops in ferns and Lycopodium may
be employed in regulatory functions. At other
sites, partial or slow RNA editing may create
protein variants for optimal functions in different physiological requirements (45, 61). Considering the most likely evolutionary order of
events, these regulatory effects are likely to have
been secondarily recruited.
ORIGIN OF EDITING:
SPECULATIONS
The potential sense of RNA editing in terms of
a payoff for the plant has been debated without
a clear outcome. Any such interpretation and
justification requires the framework of the
origin of this editing process and the questions
of why it was started and how it was initially
established. Most likely, this process started
by mutation of an enzyme able to perform the
deamination or transamination reaction (34,
48). With this ability established, thymidine
nucleotides in the genome could be substituted
by cytidines with the information content being
corrected in the RNA. The number of editing
sites increased up to the present-day numbers
with the amplification of the site-specific PPR
proteins. This amplification is possibly limited
by the burden of eventually carrying an excess
of genes for the PPR, the MORF, and other
RNA-editing proteins.
One selective factor for RNA editing might
have been protection against the increased exposure to UV light when plants moved from
water to land. This connection is attractive
considering the establishment of RNA editing
only in the first land plants and its absence in
the alga. However, the potential connection is
presently not discernible because the most exposed nucleotide connections of TT dimers in
the organellar DNA are not statistically more
highly represented in the RNA-editing target
sites than at unedited positions in the genomes.
Furthermore, RNA editing is very frequent in
Isoetes, but these plants have moved back to life
underwater, where they are better protected
from UV irradiation.
Plastids and mitochondria possibly benefit
from RNA editing, which could protect their
genes and eventually their entire genomes from
being transferred into the nucleus. This raison
d’être could be on the level of the divergent
genetic code being used, for example, in mammalian mitochondria. Any direct translocation
of a gene encoded in the mitochondrial genome
into the nuclear genome could not be functionally translated. The different nuclear decoding
system in mammalia and the absence of RNA
editing in plants will result in different, less well
adapted proteins.
The Cost of RNA Editing
Energetically, RNA editing seems a waste of
resources and is outrageously expensive. Also,
the sheer number of nuclear genes being devoted to this process in plastids and mitochondria are extremely costly in terms of genomic
space. Furthermore, considerable energy must
be spent on first synthesizing and incorporating
C ribonucleotides and then converting these to
U ribonucleotides in the RNA. RNA editing
is certainly not essential for land plants, as the
loss of editing in Marchantia proves. So why is
RNA editing still rampant in plants, and why is
it maintained in spite of being so expensive? Is
RNA editing in plant organelles used for regulation of organellar processes? Even if such
a use was secondarily acquired, the control of
the two energy generating organelles by the nucleus may justify the expenses. Answers to these
questions still require an evolutionary rationale.
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CONCLUSION
Recent progress, mostly via genetic studies,
revealed two types of key players in RNA
editing in plant organelles: PPR proteins and
MORF proteins. However, how these proteins
cooperate is still unclear. Identification of the
deaminase enzyme activity remains one of the
main open questions in RNA editing in plants.
The biological significance of RNA editing
in plant organelles, beyond just being toler-
ated through, for example, a regulatory function of organellar genes, needs to be clarified. At present, there is little evidence to support such a function. The modular nature of
sequence-specific RNA binding by the PPR
proteins opens a new field of RNA biotechnology in which proteins analogous to the TALEN
DNA-binding technology are built. The PPR
proteins identified in RNA editing in plant organelles open applications up to interference
with RNA virus infections.
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SUMMARY POINTS
1. Individual RNA-editing sites are recognized by specific PPR proteins.
2. In flowering plants, approximately 200 of the 400 PPR proteins are involved in RNA
editing, and the rest promote various other RNA-processing steps.
3. PPR proteins consist of approximately 35-mer amino acid–repeat units, each of which
can contact a nucleotide in the RNA.
4. The PPR protein–RNA code has been solved to rely on two combinatorial amino acids.
5. In flowering plants, another group of proteins, the MORFs, are essential components of
the RNA editosome and interact with PPR proteins.
FUTURE ISSUES
1. The actual editing enzyme needs to be identified.
2. The functions of the MORF proteins need to be investigated.
3. The connection between PPR specificity factors and MORF proteins should be analyzed.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We thank Dagmar Pruchner, Bianca Wolf, and Angelika Müller for excellent experimental
help. This work was supported by grants from the Deutsche Forschungsgemeinschaft to Mizuki
Takenaka and Axel Brennicke. Mizuki Takenaka is a Heisenberg Fellow.
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Annual Review of
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Causes of Genome Instability
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Radiation Effects on Human Heredity
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Dissecting Social Cell Biology and Tumors Using Drosophila Genetics
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Estimation and Partition of Heritability in Human Populations Using
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Detecting Natural Selection in Genomic Data
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Adaptive Translation as a Mechanism of Stress Response
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Organizing Principles of Mammalian Nonsense-Mediated
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Control of Nuclear Activities by Substrate-Selective
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Genomic Imprinting: Insights From Plants
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Regulation of Bacterial Metabolism by Small RNAs
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Bacteria and the Aging and Longevity of Caenorhabditis elegans
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SIR Proteins and the Assembly of Silent Chromatin in Budding Yeast
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New Gene Evolution: Little Did We Know
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RNA Editing in Plants and Its Evolution
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and Axel Brennicke p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 335
Expanding Horizons: Ciliary Proteins Reach Beyond Cilia
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The Digestive Tract of Drosophila melanogaster
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RNase III: Genetics and Function; Structure and Mechanism
Donald L. Court, Jianhua Gan, Yu-He Liang, Gary X. Shaw, Joseph E. Tropea,
Nina Costantino, David S. Waugh, and Xinhua Ji p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 405
Modernizing the Nonhomologous End-Joining Repertoire:
Alternative and Classical NHEJ Share the Stage
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Enterococcal Sex Pheromones: Signaling, Social Behavior,
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Gary M. Dunny p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 457
Control of Transcriptional Elongation
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The Genomic and Cellular Foundations of Animal Origins
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Genetic Techniques for the Archaea
Joel A. Farkas, Jonathan W. Picking, and Thomas J. Santangelo p p p p p p p p p p p p p p p p p p p p p p p 539
Initation of Meiotic Recombination: How and Where? Conservation
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Bernard de Massy p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 563
Biology and Genetics of Prions Causing Neurodegeneration
Stanley B. Prusiner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 601
Bacterial Mg2+ Homeostasis, Transport, and Virulence
Eduardo A. Groisman, Kerry Hollands, Michelle A. Kriner, Eun-Jin Lee,
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Errata
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Contents