Multiple PPR protein interactions are involved in the
RNA editing system in Arabidopsis mitochondria
and plastids
Nuria Andrés-Colása, Qiang Zhua,1, Mizuki Takenakab, Bert De Rybelc,d,e, Dolf Weijersc,
and Dominique Van Der Straetena,2
a
Laboratory of Functional Plant Biology, Department of Biology, Ghent University, B-9000 Gent, Belgium; bMolekulare Botanik, Universitaet Ulm, D-89069
Ulm, Germany; cLaboratory of Biochemistry, Wageningen University and Research, 6708 WE Wageningen, The Netherlands; dDepartment of Plant
Biotechnology and Bioinformatics, Ghent University, 9052 Ghent, Belgium; and eCenter for Plant Systems Biology, Vlaams Instituut voor Biotechnologie,
9052 Ghent, Belgium
Edited by Robert Haselkorn, University of Chicago, Chicago, IL, and approved June 29, 2017 (received for review April 7, 2017)
SLO2
| DYW2 | NUWA | PPR | RNA editing
Downloaded by guest on June 4, 2020
T
he PPR family is classified into the P and PLS subfamilies,
depending on the PPR signature domains. The P subfamily
was described to participate in RNA stabilization, cleavage, splicing, and translation (1). Their members can bind specific RNA
sequences to protect them from endonucleases or modify their
secondary structure to recruit other RNA maturation factors (2).
The P-type PPR protein PPME was the first one described with a
role in RNA editing via RNA binding (3). The PLS subfamily is
further divided into PLS, E, E+, and DYW subgroups, based on
the C-terminal extensions and plays a major role in C-to-U RNA
editing in plant organelles. Among them, E and E+-type PPR
proteins do not have the DYW domain, which is considered to be a
part of the catalytic domain due to the similarity to cytidine deaminase (2), suggesting the requirement of forming protein complexes with a DYW protein for a complete editing event. Indeed,
the E-type PPR protein CRR4 forms a complex with DYW1,
which consists of only a DYW domain, to edit an ndhD site in
chloroplasts (4). However, such transassociated DYW-type PPR
protein in an editing complex was reported only at this site in
chloroplasts. It is still unclear whether this scenario is universal for
numerous RNA editing sites in plant mitochondria. Not only the
composition but the stoichiometry of the proteins in the editosome
complexes remains unsolved (5). Some PPR proteins can homodimerize to bind RNA, although dimerization can also occur in the
absence of the RNA target (6).
Most of the PPR proteins are located in mitochondria (65%) or
chloroplasts (17%) (7). Systematic localization experiments and
data integration showed that PPR dual targeting occurs more
frequently than expected (7, 8). Interestingly, some heat shock
proteins (HSPs) have also been associated with editing processes,
translocating the editing factors into the required organelle (9).
www.pnas.org/cgi/doi/10.1073/pnas.1705815114
Previously, we characterized the mitochondrial E+-type PPR
protein SLO2, which participates in the editing of mttB-144, mttB145, nad4L-110, nad7-739, mttB-666, nad1-2, and nad1-40 sites,
affecting four proteins of complex I in the mitochondrial electron
transport chain (10). However, SLO2 lacks a catalytic domain responsible for the editing task. In this report, we aimed to elucidate
which proteins participate in the working mechanism of SLO2 by
identifying its interacting partners.
Results
Immunoprecipitation with SLO2 Reveals PPR- and HSP-Type Candidate
Interactors. As a first approach to identify interacting partners of
the E+-type PPR protein SLO2, we performed an immunoprecipitation and mass spectrometry (IP–MS) assay. Total protein
extracts from Arabidopsis seedlings expressing a green fluorescent
protein (GFP)-tagged SLO2 protein (SLO2-GFP), either under
control of the SLO2 or the CaMV 35S promoter, were taken as
starting material. Among the top 25 interactors, after selection of
significant data (P value <0.05) and sorting based on the ratio of
pSLO2::SLO2 to the Col-0 wild-type control (Fig. S1), three types
of proteins were chosen: PPR proteins with a DYW domain
(DYW2-At2g15690 and MEF57-At5g44230), P-type PPR proteins
Significance
RNA editing is a posttranscriptional regulation process essential
for organellar function. The PPR protein family plays a major role
in RNA editing, but the precise function of each member and its
interactions in editosome complexes remain unclear. This research
demonstrates that three PPR proteins (SLO2, NUWA, and DYW2)
interact in mitochondria and provides evidence for the following:
(i) P-type PPR protein NUWA assists in the interaction between
SLO2 and DYW2, and (ii) DYW2 participates in E+ editosomes. We
hypothesize that this mechanism is common in both mitochondria
and chloroplasts, where the E+-type PPR proteins, such as SLO2,
target the specific RNA sites, with DYW2 suggested to provide the
editing catalytic domain and NUWA assisting the interaction between the former ones.
Author contributions: N.A.-C., Q.Z., and D.V.D.S. designed research; N.A.-C., Q.Z., M.T.,
and B.D.R. performed research; N.A.-C., Q.Z., M.T., B.D.R., D.W., and D.V.D.S. analyzed
data; Q.Z., M.T., B.D.R., and D.W. revised and commented on the draft manuscript;
D.V.D.S. coordinated the project; and N.A.-C. and D.V.D.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
Present address: Basic Forestry and Proteomics Center, Fujian Provincial Key Laboratory
of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology,
Fujian Agriculture and Forestry University, 350002, Fujian, China.
2
To whom correspondence should be addressed. Email: dominique.vanderstraeten@
ugent.be.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1705815114/-/DCSupplemental.
PNAS | August 15, 2017 | vol. 114 | no. 33 | 8883–8888
PLANT BIOLOGY
Recent identification of several different types of RNA editing
factors in plant organelles suggests complex RNA editosomes within
which each factor has a different task. However, the precise protein
interactions between the different editing factors are still poorly
understood. In this paper, we show that the E+-type pentatricopeptide repeat (PPR) protein SLO2, which lacks a C-terminal cytidine
deaminase-like DYW domain, interacts in vivo with the DYW-type
PPR protein DYW2 and the P-type PPR protein NUWA in mitochondria, and that the latter enhances the interaction of the former ones.
These results may reflect a protein scaffold or complex stabilization
role of NUWA between E+-type PPR and DYW2 proteins. Interestingly, DYW2 and NUWA also interact in chloroplasts, and DYW2-GFP
overexpressing lines show broad editing defects in both organelles,
with predominant specificity for sites edited by E+-type PPR proteins.
The latter suggests a coordinated regulation of organellar multiple
site editing through DYW2, which probably provides the deaminase
activity to E+ editosomes.
E+
DYW
P type
At2g13600
SLO2
At5g44230
MEF57
L1
P
S S P L1 S P L2 S
At2g15690
DYW2
L1 S P L2 S
At3g60980
P486
P P
At3g60960
P487
P
At3g49240
NUWA
HSP60
L1 S S P L1 S P L1 S S P L1 S P L2 S
P
P
P
P S P S
P P
S
P
P
E+
E
S
P
E
P
E
1,974 bp
657 aa
DYW
DYW
2,094 bp
697 aa
E+
1,740 bp
579 aa
1,239 bp
412 aa
1,212 bp
403 aa
P P P S
P
1,889 bp
629 aa
3,476 bp
585 aa
At2g33210
HSP60.2
At3g13860
HSP60.3A
At3g23990
HSP60.3B
3,563 bp
572 aa
3,266 bp
577 aa
Fig. 1. Scheme of the primary structure of SLO2 and its interacting partners. The names/ID and locus codes and the type of PPR protein are indicated on the left,
and the sizes in base pairs (bp) and amino acids (aa) on the right side of each scheme. The coding/exon fragments are indicated with gray boxes. The different PPR
protein motifs (13, 37) are indicated with colors and capital letters (L1, brown; L2, red; S, yellow; P, orange; E, light green; E+, dark green; and DYW, blue).
A
DYW
MEF57 DYW2
P type
P486
P487
NUWA APEM9
PEX6
Strong Strong Strong Strong Strong Strong
No
n.c.
HSP60 HSP60.3A Strong Strong Strong Strong Strong Strong
No
n.c.
HSP60.3B Strong Strong Strong Strong Strong Strong
No
n.c.
n.c.
Strong
INPUT
HSP60.2
HSP60.2
No
DYW2
SLO2
HSP60.3B
HSP60.3B
NUWA
HSP60.3B
HSP60.3B-HA
NUWA-GFP
NUWA
DYW2
No
HSP60.3B-HA
No
HSP60.3B-HA
DYW2-GFP
HSP60.2
No
SLO2-HA
HSP60.3B-GFP
SLO2
B
No
SLO2-HA
No
HSP60.2-HA
NUWA-GFP
APEM9
HSP60.2-HA
HSP60.2
E+
SLO2
HSP60.2-HA
DYW2-GFP
localized in mitochondria (10), its interactors are also expected
to show mitochondrial localization. All of the SLO2 interacting
partners were predicted to be localized in mitochondria by the
SUBAcon program (12), except MEF57, predicted to be in plastids. For DYW2 and NUWA, proteomic data and reporter studies
indicated dual targeting to mitochondria and chloroplasts (7, 8).
NUWA mitochondrial localization was confirmed in Arabidopsis
transgenic seedlings under the native promoter (13). In Fig. S2A,
we show that the GFP signal of all GFP-tagged SLO2 candidate
interactors consistently colocalized with the mitochondrial marker
mitotracker/mt-rb in Nicotiana leaves, corroborating their mitochondrial localization, including for MEF57. In addition, a remarkably strong signal of NUWA-GFP was more frequently detected in
chloroplasts (Fig. S2B) than in mitochondria. Likewise, DYW2GFP showed strong chloroplast signals in several cells, in a dotted
pattern (Fig. S2B), supporting dual localization for both proteins.
The background signal at high laser gain, including chloroplast
autofluorescence, is shown in Fig. S3A. HSP60 proteins, apart from
the mitochondrial signal, also exhibited a signal associated with the
cytosol (Fig. S2A).
SLO2-HA
Several SLO2 Interacting Partners Are Uniquely Localized in Mitochondria;
Others Are Dual Localized in Mitochondria and Chloroplasts. As SLO2 is
with the signal of the mitotracker/mt-rb mitochondrial marker
(Fig. S4), proving that the interactions occur in mitochondria.
To further substantiate the interaction of SLO2 and its binding
PPR partners with the HSP60 chaperones, we coimmunoprecipitated human influenza hemagglutinin (HA)-/GFP-tagged
SLO2, DYW2, or NUWA proteins together with GFP-/HAtagged HSP60.2 and HSP60.3B proteins. Fig. 2B shows that
HSP60.2 and HSP60.3B immunoprecipitated together with
SLO2, DYW2, or NUWA, whereas APEM9 did not (Fig. S5A).
Taken together, these results support the protein interaction of
SLO2, DYW2, and NUWA with HSP60.2 and HSP60.3B in
mitochondria.
SLO2-HA
HSP60.2-GFP
(NUWA-At3g49240, P486-At3g60980, and P487-At3g60960), and
mtHSP60 chaperones (HSP60.2-At2g33210, HSP60.3A-At3g13860,
and HSP60.3B-At3g23990), considered to be part of the import
apparatus specific to the mitochondrial matrix (11) (Fig. 1). The
limited number of PPR motifs in the DYW2 protein (Fig. 1), is likely
to be insufficient for specific recognition of target RNAs and is
reminiscent of the DYW1 protein in chloroplast (4).
HA-Ab
GFP-Ab
Downloaded by guest on June 4, 2020
SLO2 and Its Binding Partners Interact in Vivo with HSP60 Import
Factors in Mitochondria. The suggested interaction between
SLO2 and the mtHSP60 chaperones (Fig. S1), motivated us not
only to confirm that these interactions occur in vivo in mitochondria, but also to check a possible interaction between the
SLO2 binding PPR proteins and mtHSP60 import factors. All
interactions were tested by bimolecular fluorescence complementation (BiFC). Both potential protein partners were tagged
with the respective halves of the split-yellow fluorescent protein (YFP) protein and coinfiltrated pairwise in Nicotiana
leaves. As summarized in Fig. 2A, the YFP signal was recovered
when combining SLO2 or the selected PPR proteins with each
of the mtHSP60 proteins, corroborating their interaction. No
signal was recovered when combining them with ABERRANT
PEROXISOME MORPHOLOGY 9 (APEM9) protein (14)
(Fig. 2A and Fig. S3). The recovered YFP signal colocalized
8884 | www.pnas.org/cgi/doi/10.1073/pnas.1705815114
GFP-IP
HA-Ab
GFP-Ab
Fig. 2. Protein interaction of SLO2 and its partners MEF57, DYW2, P486, P487,
and NUWA with mtHSP60 import factors. (A) Summary of the protein interactions between SLO2 or its partners and the HSP60 import factors in mitochondria, detected by BiFC assay in Figs. S3 B–D and S4. “Strong” and “weak” indicate
the strength of the interactions according to the signal intensity under similar
conditions. No, no interaction; n.c., not checked. (B) Total protein extracts (input)
and GFP-immunoprecipitated proteins (GFP-IP) from N. benthamiana leaves
infiltrated with SLO2-HA or HSP60.2-/HSP60.3B-HA together with HSP60.2-/
HSP60.3B-GFP or DYW2-/NUWA-GFP constructs, 3 d after infiltration, analyzed by Western blot with anti-HA and anti-GFP antibodies (HA-Ab and GFPAb). N. benthamiana leaves infiltrated with the corresponding HA-tag constructs
alone were taken as negative controls of the immunoprecipitation. Representative blots of at least two independent experiments for each combination are
shown. Blots were cropped to the bands of interest (full-length blots in Fig. S5B).
The contrast/brightness was adjusted for good visualization.
Andrés-Colás et al.
+ NUWA
A
DYW2
P486
P487 NUWA MEF57 DYW2 APEM9 PEX6
Weak
Weak
Weak
Weak Strong Weak Strong
No
n.c.
n.c.
DYW2
-
-
Weak
Weak Strong
-
-
No
MEF57
-
-
Weak
Weak Strong
-
-
No
n.c.
APEM9
No
No
No
No
No
n.c.
Strong
SLO2
DYW2
SLO2-HA
DYW2-GFP
+NUWA-His
NUWA
SLO2-HA
DYW2-GFP
DYW2
NUWA
SLO2-HA
+NUWA-His
NUWA
NUWA-HA
DYW2-GFP
SLO2
NUWA-HA
SLO2
DYW2
No
NUWA-HA
SLO2-GFP
MEF57
No
NUWA-HA
SLO2
B
INPUT
DYW
MEF57
SLO2-HA
DYW2-GFP
DYW
SLO2
P type
SLO2-HA
E+
DYW
SLO2-HA
MEF57-GFP
Next we verified by BiFC whether the interaction of SLO2 with the
P- and DYW-type PPR proteins detected by immunoprecipitation
occurs in mitochondria. As presented in Fig. 3A, the recovery of
the YFP signal when combining SLO2 with each PPR protein,
confirmed the physical interaction of SLO2 with all of the tested
PPR proteins. The colocalization of the recovered YFP signal with
the mitotracker/mt-rb mitochondrial marker (Fig. S6A) certified
that the interaction takes place in mitochondria. However, the
recovery of the YFP signal was observed with a low frequency (less
than 10% of the cells) and low intensity, except when combining
SLO2 with NUWA (Fig. 3A and Fig. S6A). Because BiFC analyses
tend to also detect rather weak and transient interactions, to corroborate the stable interaction between SLO2 and the DYW proteins or the P-type PPR protein NUWA, we coimmunoprecipitated
tagged DYW proteins (MEF57-GFP and DYW2-GFP) and P-type
PPR protein (NUWA-HA), together with tagged SLO2 (SLO2-HA
or SLO2-GFP). As shown in Fig. 3B and Fig. S5A, SLO2 was
immunoprecipitated together with all of the candidates tested, except with MEF57 and APEM9 control. These results endorse the
specific interaction of SLO2 with all of the candidates tested, except
for MEF57.
SLO2-HA
SLO2 Interacts in Vivo with DYW2 and NUWA Proteins in Mitochondria.
HA-Ab
GFP-Ab
HA-Ab
GFP-IP
GFP-Ab
C
SLO2
SLO2
DYW
NUWA
DYW
or protein stability (15–17), we investigated whether the three
SLO2 associated HSP60 proteins, interacting also with both DYW
proteins (Fig. 2A), could stabilize the DYW proteins or the SLO2/
DYW complexes, directly or through binding their target RNAs.
Such stabilization could result in an increased interaction signal between SLO2 and the DYW proteins. To this end, we tested the interaction by BiFC in the presence and absence of the HSP60 partners
(HSP60-HA), in parallel experiments, in different halves of the same
Nicotiana leaf. The addition of HSP60 chaperones did not result in
an increased interaction signal (Fig. S6A).
P-type PPR proteins also have been described to possess RNA
or complex stabilization properties (2). Therefore, we checked
whether the addition of the P-type PPR proteins detected as
SLO2-interacting partners could improve the interaction between
SLO2 and the DYW proteins. Interestingly, a clear increase in
the SLO2 interaction with DYW2, appreciated by a high frequency
(in around 90% of the cells) and intensity of the recovered YFP
signal in parallel experiments, was detected in mitochondria in the
presence of the P-type PPR protein NUWA (NUWA-HA) (Fig. 3
A and C and Fig. S6A), whereas coexpression of the other two
P-type PPR proteins did not enhance the interaction (Fig. S6A).
The increase in the interaction of SLO2 with DYW2 in the presence of NUWA (NUWA-His) was corroborated by coimmunoprecipitation (Fig. 3B), as reflected by a more intense SLO2-HA
signal in the GFP immunoprecipitate (GFP-IP) compared with the
input signal. The addition of NUWA, on the other hand, did not
result in an enhanced signal for the SLO2/MEF57 interaction (Fig.
3 A and C and Fig. S6A).
Downloaded by guest on June 4, 2020
DYW2 and NUWA Proteins Interact in Mitochondria and Chloroplasts.
To elucidate whether the specific enhancement of the SLO2/
DYW2 interaction by NUWA was conferred through their direct
protein interactions, we compared affinities between each P-type
PPR protein and the two DYW-type PPR proteins by BiFC assay. Although all three P-type PPR proteins could interact with
both DYW-type PPR proteins in mitochondria, NUWA showed
significantly stronger BiFC signal than the other two P-type PPR
proteins (Fig. 3A and Fig. S6B). Interestingly, the interaction
between NUWA and DYW2 took place mainly in mitochondria
but, in some cells, the signal was detected in chloroplasts
(Fig. S6B). This result is in agreement with the identification of
NUWA and DYW2 as interactors of the chloroplastic E+-type
PPR CLB19 (18). Furthermore, the interaction between NUWA
Andrés-Colás et al.
SLO2-nYFP
DYW2-cYFP
SLO2-nYFP
DYW2-cYFP
+NUWA-HA
SLO2-nYFP
MEF57-cYFP
SLO2-nYFP
MEF57-cYFP
+NUWA-HA
Fig. 3. Protein interaction between SLO2 and its PPR interacting partners
MEF57, DYW2, P486, P487, and NUWA. (A) Summary of the protein interactions between SLO2, its DYW- and P-type PPR partners in mitochondria,
detected by BiFC assay in Figs. S3 B–D and S6, as indicated in Fig. 2A. (B) Total
protein extracts (input) and GFP-immunoprecipitated proteins (GFP-IP) from
N. benthamiana leaves infiltrated with SLO2-HA together with MEF57-/
DYW2-GFP, or NUWA-HA together with SLO2-/DYW2-GFP, or infiltrated
with SLO2-HA together with DYW2-GFP or NUWA-His, or the combination of
the three constructs. Proteins were extracted 3 d after infiltration and analyzed by Western blot as indicated in Fig. 2B (full-length blots in Fig. S5B). (C)
N. benthamiana leaves infiltrated with SLO2-nYFP together with MEF57-/
DYW2-cYFP, in the absence or presence of NUWA-HA construct, analyzed by
confocal microscopy 3–6 d after infiltration. Green fluorescence is indicative
of the localization of the reconstituted whole YFP protein (protein interaction). Representative individual cells of at least three independent experiments are shown. Single cells were cropped from the original image.
(Scale bar, 50 μm.) Images for combinations without NUWA-HA (Left), with
really low fluorescent signal, were taken at a higher laser gain condition,
whereas the images where NUWA-HA was added (Right) were taken under
lower gain conditions. For a proper comparison between proteins, images for
both DYW2 and MEF57 proteins were taken under the same conditions. Images were extracted from Fig. S6A.
and DYW2 proteins was corroborated by coimmunoprecipitation (Fig. 3B and Fig. S5A).
DYW2 and NUWA Proteins Form Homomers. We further investigated
the capacity of SLO2 and its interacting PPR partners to homomerize by BiFC assay. Only the PPR proteins DYW2 and NUWA
showed the capacity to form homomers (Fig. 4). Moreover,
whereas NUWA homodimers were formed in both mitochondria
PNAS | August 15, 2017 | vol. 114 | no. 33 | 8885
PLANT BIOLOGY
NUWA Enhances the Interaction Between SLO2 and DYW2 in Mitochondria
in Vivo. Because some HSPs have been described to enhance mRNA
and chloroplasts, DYW2 homomerization was restricted to chloroplasts (Fig. 4).
A
DYW2-GFP-OE2
WT
B
DYW2-GFP-OE1
DYW2-GFP-OE2
DYW2 Is Involved in the Editing of the E+-Type PPR Edited Sites. Fi-
Discussion
PPR Editing Complexes. To date, the interactions between PPR
proteins were restricted to the chloroplastic PPR protein DYW1,
YFP
merge
light
DYW2
DYW2
DYW2-nYFP
DYW2-cYFP
NUWA
NUWA
NUWA-nYFP
NUWA-cYFP
YFP
mitotracker
merge
D 50
30
E+40
E+
E 30
20
DYW
10
P
0
E
20
10
0
Mitochondria Chloroplasts
Non-affected sites
C40
Down-edited sites
nally, to clarify the biological relevance of the SLO2/DYW2 interaction, because we could not analyze homozygous dyw2
knockout lines due to their embryo lethality (18), we generated
Arabidopsis plants overexpressing the GFP-tagged DYW2 gene
(Fig. S7). Two independent lines, containing at least fourfold higher
levels of DYW2/DYW2-GFP transcripts than the DYW2 transcripts
in wild type (Fig. S7A), were isolated. They showed a slow growth
phenotype (Fig. 5 A and B), reminiscent of other PPR loss-offunction mutants. Editing analysis revealed a high number of affected editing sites, mainly down-edited, and some completely
(100%) affected (Dataset S1). In particular, the E+-type PPR
SLO2 edited sites (10), including one identified in this work (Fig.
S8), were down-edited (Dataset S1). Interestingly, this downediting was also the case for most of the sites described to be
edited by other E+-type PPR proteins, but only for a few ones
edited by DYW- or E-type PPRs, both in mitochondria and
chloroplasts (Fig. 5 C and D). This result suggests that DYW2mediated editing events are basically linked to E+-type PPR
proteins. Interestingly, as shown in Fig. S7B, the NUWA expression levels were higher in these DYW2-GFP overexpressing
(DYW2-GFP-OE) lines compared with the wild-type plants,
indicative of coordinated regulation. The work of Guillaumot
et al. (18) on dyw2 and nuwa loss-of-function mutants (18) also
revealed a clear editing defect in most of the E+-type PPR edited
sites, including the SLO2 sites, corroborating a predominant role
of DYW2 and NUWA proteins in E+-type PPR-associated
editing. Moreover, some sites not described to be regulated by
known PPR proteins were also altered in the DYW2-GFP-OE
lines (Dataset S1). This result points toward a hub function for
DYW2; however, further investigation will be needed to establish a clear connection between DYW2 and non-PPR editing
factors. The knockout line of the other DYW-type PPR partner
of SLO2, MEF57 (Fig. S9), shows RNA editing defect at the
nad9-92 site (Fig. S8), but no alteration at the SLO2 edited sites.
DYW
P
Mitochondria Chloroplasts
Fig. 5. Characterization of DYW2-GFP-OE lines. (A and B) Morphology of
adult plants. Images of adult plants from the homozygous DYW2-GFP-OE2 line
and the WT control, grown in parallel (A), and a detail of the morphology in
two independent heterozygous DYW2-GFP-OE lines (B). Representative images
of at least two independent experiments are shown. (C and D) RNA editing
analysis of DYW2-GFP-OE lines. Editing analysis of rosette leaves from WT and
two independent DYW2-GFP-OE lines are shown. Each independent line was
analyzed once. The media and SD of the differences in editing percentage in
both DYW2-GFP-OE lines with respect to the WT (Δ%) were calculated (Dataset
S1). The number of sites modified by a particular type of PPR protein is represented. Both mitochondrial and chloroplastic sites are shown, split into downedited (C) and nonaffected (D). A value of Δ% = 24% was taken as cutoff. The
consistent data in both OE lines are shown. The list of PPR associated with
editing sites was formed according to the literature (2, 20, 38) and this work.
which can function in trans with the E-type PPR protein CRR4 (4).
In this paper, we present evidence for the interaction between
three types of PPR proteins (E+, DYW, and P type) in mitochondria (SLO2, DYW2, and NUWA) (Figs. 1 and 3). The high
NUWA expression levels in the DYW2-GFP-OE lines (Fig. S7B)
reinforces the idea that NUWA is required by DYW2 to form a
functional editosome with other editing factors. In addition, the
dual localization and interaction of the DYW2 and the NUWA
partner proteins (Figs. S2 and S6) suggest a coordinated regulation
of organellar editing, supported by the identification of NUWA
and DYW2 as interactors of the chloroplastic E+-type PPR protein
CLB19 (18) and the broad effect of DYW2 in E+-type PPR edited
sites (Fig. 5) (18). Taken together, we propose that E+-type PPR
proteins target the specific RNA sites, whereas P-type PPR protein
NUWA assists the interaction between the E+ partner and DYW2.
Based on the current evidence (5), we hypothesize that DYW2 would
provide the editing catalytic domain, both in mitochondria and
chloroplasts. A model for the proposed PPR editosome mechanism
is shown in Fig. 6.
light
Cytosol
DYW2
Fig. 4. Dimerization of DYW2 and NUWA. N. benthamiana leaves infiltrated
with DYW2-/NUWA-nYFP together with DYW2-/NUWA-cYFP constructs and analyzed by confocal microscopy 3 d after infiltration. Green and red fluorescence
are indicative of the localization of the reconstituted whole YFP protein (protein
interaction) and the mitotracker mitochondrial marker, respectively. Representative individual cells of at least two independent experiments are shown, including their merged and light fields. Single cells were cropped from the original
image. (Scale bar, 50 μm.) White arrows point to a mitochondrial signal dot and
yellow arrows to a chloroplast signal. The contrast/brightness was adjusted for
good visualization.
8886 | www.pnas.org/cgi/doi/10.1073/pnas.1705815114
NUWA
HSP60
Downloaded by guest on June 4, 2020
SLO2
U
C
NUWA
E+ PPR
NUWA
Mitochondrion
DYW2
C
U
DYW2 NUWA
E+ PPR
DYW2 NUWA
Chloroplast
Fig. 6. Model of the tripartite PPR protein interaction in editosomes in
mitochondria and chloroplasts.
Andrés-Colás et al.
Dual Localization of PPR Proteins. The PPR proteins DYW2 and
NUWA have dual localization in both mitochondria and chloroplasts (Fig. S2). However, the protein characteristics and mechanisms responsible for this dual localization remain unclear. A
significant number of proteins are dual targeted to both organelles
via ambiguous targeting signals (28). It has been proposed that
cytosolic chaperones may play a role in determining targeting specificity (29). In Arabidopsis, the mitochondrial HSP60 family has three
members, considered to be part of the import apparatus specific to
the mitochondrial matrix (30). We would expect that mutants of the
interacting HSP60 proteins in this work affect RNA editing. However, if the three mtHSP60 proteins have any redundant function, a
triple mutant would be needed to observe such editing defects.
Downloaded by guest on June 4, 2020
PPR mRNA and Protein Complex Stabilization. The increased interaction of SLO2 and DYW2 when adding NUWA (Fig. 3) and
the protein interaction of NUWA with both SLO2 and DYW2
(Fig. 3) suggest a possible scaffold or complex stabilization role of
NUWA for the interaction between SLO2 and DYW2 in the
editosome. In this context, a non-PPR editing factor (MORF8) was
described to assist the interaction between an E+-type PPR protein
(MEF13) and another non-PPR factor (MORF3) (31). The fact
that the P-type PPR protein NUWA assists in the interaction between an E+ (SLO2) and a DYW (DYW2) protein, could reflect a
more general mode of action of PPR proteins, hence representing
the key for the discovery of similar tripartite PPR interactions and
shedding light on the complex PPR editosome formation and
action mechanisms.
Our results suggest that HSP60 proteins do not participate in
the protein complex stabilization (Fig. S6A). They may assist in
1. Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant
Biol 65:415–442.
2. Shikanai T (2015) RNA editing in plants: Machinery and flexibility of site recognition.
Biochim Biophys Acta 1847:779–785.
3. Leu KC, Hsieh MH, Wang HJ, Hsieh HL, Jauh GY (2016) Distinct role of Arabidopsis
mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein,
PPME, in nad1 RNA editing. RNA Biol 13:593–604.
4. Boussardon C, et al. (2012) Two interacting proteins are necessary for the editing of
the NdhD-1 site in Arabidopsis plastids. Plant Cell 24:3684–3694.
Andrés-Colás et al.
mitochondrial import of the partner proteins, as previously mentioned, or support RNA binding of the editing complexes. The
chaperone HSP60 was suggested to have a highly specific nucleic
acid-binding activity, presumably implicated in mtDNA stability
(32). In protozoa, it has been demonstrated that a chaperone
activity in the editosome increases the flexibility of U residues in
the pre-mRNAs to facilitate the binding of gRNAs (33), providing
a rational explanation for the U specificity of the editing reaction.
P-type PPR subfamily members can also bind to specific RNA
sequences (2), protecting the RNA from endonucleases and/or
modifying its secondary structure to recruit general factors involved
in RNA maturation. In this context, it will be interesting to check
RNA binding activity of the HSP60 and P-type PPR proteins detected in this work.
Further investigation will be needed to understand the precise
function of SLO2, DYW2, NUWA, and the HSP60 proteins in
the SLO2 editosome complex and beyond.
Materials and Methods
IP–MS Assay. The IP–MS assay was performed as previously described (34),
without modifications. Three biological replicates were used for each line.
BiFC. The predicted subcellular localization was analyzed by SUBAcon and
SUBA3 programs (12, 35). For experimental subcellular localization and BiFC,
the corresponding binary vectors were introduced into Agrobacterium
tumefaciens strain GV3101. Agrobacterium was used to transiently transform young leaves of Nicotiana benthamiana grown on soil under long day
conditions. For details, see SI Materials and Methods.
CoIP. A modified μMACS Epitope Tag Protein Isolation Kit (Miltenyi) protocol
(34) was followed. For details, see SI Materials and Methods.
T-DNA Insertion, Complemented, and Overexpressing Lines. Arabidopsis thaliana T-DNA insertion mutant line N585176 from the SALK collection was used
and complemented with the 35S::MEF57-GFP construct. The DYW2-GFP-OE
lines were generated transforming Col-0 A. thaliana plants with the 35S::
DYW2-GFP construct.
RNA Editing Analysis. Specific cDNAs were generated as described previously
(36). The sequence data for each gene-specific RT-PCR product were obtained
commercially and compared for analysis (Macrogen). Ratios between heights
of C and T signals were calculated with the DNADynamo software (BlueTractorSoftware). The Arabidopsis slo2-2, slo2-3, complemented slo2-2, and
complemented slo2-3 lines were taken from our previous work (10).
A detailed description of the plasmid construction and full methods are
described in SI Materials and Methods.
Data Availability. All data generated or analyzed during this study are included in this article (and Supporting Information).
ACKNOWLEDGMENTS. N.A.-C. and D.V.D.S. thank Maria Helena de Souza
Goldman for the N. benthamiana seeds and helpful suggestions regarding
experimental design and data interpretation; Irina Vaseva for critical reading of the first draft of this manuscript; and Yordanka Yordanova for experimental help in genotyping the mef57 mutant line. D.V.D.S. acknowledges
the Research Foundation Flanders for financial support (Project G.0C84.14N).
B.D.R. was funded by a Marie Curie long-term postdoctoral fellowship (FP7PEOPLE-2009-IEF-252503) and by The Netherlands Organization for Scientific
Research (NWO; VIDI-864.13.001). D.W. acknowledges funding by NWO (ERACAPS Project EURO-PEC; 849.13.006). M.T. was supported by Deutsche Forschungsgemeinschaft Grants TA624/4-2, TA624/-1, and TA624/10-1.
5. Sun T, Bentolila S, Hanson MR (2016) The unexpected diversity of plant organelle RNA
editosomes. Trends Plant Sci 21:962–973.
6. Yin P, et al. (2013) Structural basis for the modular recognition of single-stranded RNA
by PPR proteins. Nature 504:168–171.
7. Colcombet J, et al. (2013) Systematic study of subcellular localization of Arabidopsis
PPR proteins confirms a massive targeting to organelles. RNA Biol 10:1557–1575.
8. Hooper CM, Castleden IR, Tanz SK, Aryamanesh N, Millar AH (2017) SUBA4: The interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic
Acids Res 45:D1064–D1074.
PNAS | August 15, 2017 | vol. 114 | no. 33 | 8887
PLANT BIOLOGY
Although the interaction between SLO2 and MEF57 detected
by IP–MS could be confirmed by BiFC, it was not corroborated
by coimmunoprecipitation (CoIP) (Fig. 3). The immunoprecipitation was realized from stable transgenic Arabidopsis plants,
whereas the coimmunoprecipitation and BiFC assays were performed on transiently transformed Nicotiana leaves. These differences suggest the possibility that a factor present in Arabidopsis
but not in Nicotiana could assist in the SLO2/MEF57 interaction.
However, the possibility of absence of MEF57/SLO2 interaction
would also be plausible. Indeed, the RNA editing analysis suggests
that MEF57 protein is not required for the SLO2 editing ability
(Fig. S8).
The observed down-editing effect in the DYW2-GFP-OE lines
(Fig. 5 and Dataset S1) could likely be explained by a dominant
negative effect of an inappropriate stoichiometry of the complex
(19) or through capture of DYW2 interacting factors required by
other editing complexes that target the same sites or by competition with other factors for the same editing sites (20–22).
Furthermore, it was reported that the addition of a C-terminal
tag to DYW proteins could inhibit their editing function (23, 24),
while allowing interaction with other editing partners (4, 25–27).
This might lead to a nonfunctional editosome, explaining the
down-editing effect in these overexpressing lines. The observed
DYW2-GFP-OE phenotype (Fig. 5) could be explained by the
broad editing defect that would affect the organellar functions,
impairing plant growth. This broad defect in the essential RNA
editing process, would justify the lethality of the homozygous
dyw2 loss-of-function mutants (18).
Downloaded by guest on June 4, 2020
9. Law YS, et al. (2015) Phosphorylation and dephosphorylation of the presequence of
precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during import into mitochondria from Arabidopsis. Plant Physiol 169:1344–1355.
10. Zhu Q, et al. (2012) SLO2, a mitochondrial pentatricopeptide repeat protein affecting
several RNA editing sites, is required for energy metabolism. Plant J 71:836–849.
11. Murcha MW, et al. (2014) Protein import into plant mitochondria: Signals, machinery,
processing, and regulation. J Exp Bot 65:6301–6335.
12. Hooper CM, et al. (2014) SUBAcon: A consensus algorithm for unifying the subcellular
localization data of the Arabidopsis proteome. Bioinformatics 30:3356–3364.
13. He S, et al. (2017) A novel imprinted gene NUWA controls mitochondrial function in
early seed development in Arabidopsis. PLoS Genet 13:e1006553.
14. Goto S, Mano S, Nakamori C, Nishimura M (2011) Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes. Plant Cell 23:1573–1587.
15. Bender T, Lewrenz I, Franken S, Baitzel C, Voos W (2011) Mitochondrial enzymes are
protected from stress-induced aggregation by mitochondrial chaperones and the
Pim1/LON protease. Mol Biol Cell 22:541–554.
16. Kim KH, et al. (2012) Rescue of PINK1 protein null-specific mitochondrial complex IV deficits
by ginsenoside Re activation of nitric oxide signaling. J Biol Chem 287:44109–44120.
17. Wang R, et al. (2016) HSP90 regulates temperature-dependent seedling growth in Arabidopsis by stabilizing the auxin co-receptor F-box protein TIR1. Nat Commun 7:10269.
18. Guillaumot D, et al. (2017) Two interacting PPR proteins are major Arabidopsis editing
factors in plastid and mitochondria. Proc Natl Acad Sci USA 114:8877–8882.
19. Veitia RA (2007) Exploring the molecular etiology of dominant-negative mutations.
Plant Cell 19:3843–3851.
20. Bentolila S, Knight W, Hanson M (2010) Natural variation in Arabidopsis leads to the
identification of REME1, a pentatricopeptide repeat-DYW protein controlling the
editing of mitochondrial transcripts. Plant Physiol 154:1966–1982.
21. Blanc V, et al. (2001) Identification of GRY-RBP as an apolipoprotein B RNA-binding
protein that interacts with both apobec-1 and apobec-1 complementation factor to
modulate C to U editing. J Biol Chem 276:10272–10283.
22. Hayes ML, Giang K, Berhane B, Mulligan RM (2013) Identification of two pentatricopeptide repeat genes required for RNA editing and zinc binding by C-terminal
cytidine deaminase-like domains. J Biol Chem 288:36519–36529.
23. Wagoner JA, Sun T, Lin L, Hanson MR (2015) Cytidine deaminase motifs within the
DYW domain of two pentatricopeptide repeat-containing proteins are required for
site-specific chloroplast RNA editing. J Biol Chem 290:2957–2968.
24. Zehrmann A, Verbitskiy D, Härtel B, Brennicke A, Takenaka M (2010) RNA editing
competence of trans-factor MEF1 is modulated by ecotype-specific differences but
requires the DYW domain. FEBS Lett 584:4181–4186.
25. Bentolila S, et al. (2012) RIP1, a member of an Arabidopsis protein family, interacts with the
protein RARE1 and broadly affects RNA editing. Proc Natl Acad Sci USA 109:E1453–E1461.
26. Sun T, et al. (2013) An RNA recognition motif-containing protein is required for plastid
RNA editing in Arabidopsis and maize. Proc Natl Acad Sci USA 110:E1169–E1178.
8888 | www.pnas.org/cgi/doi/10.1073/pnas.1705815114
27. Takenaka M, et al. (2012) Multiple organellar RNA editing factor (MORF) family
proteins are required for RNA editing in mitochondria and plastids of plants. Proc
Natl Acad Sci USA 109:5104–5109.
28. Carrie C, et al. (2009) Approaches to defining dual-targeted proteins in Arabidopsis.
Plant J 57:1128–1139.
29. Kriechbaumer V, von Löffelholz O, Abell BM (2012) Chaperone receptors: Guiding
proteins to intracellular compartments. Protoplasma 249:21–30.
30. Murcha MW, Narsai R, Devenish J, Kubiszewski-Jakubiak S, Whelan J (2015) MPIC: A
mitochondrial protein import components database for plant and non-plant species.
Plant Cell Physiol 56:e10.
31. Glass F, Härtel B, Zehrmann A, Verbitskiy D, Takenaka M (2015) MEF13 requires MORF3 and
MORF8 for RNA editing at eight targets in mitochondrial mRNAs in Arabidopsis thaliana.
Mol Plant 8:1466–1477.
32. Kaufman BA, et al. (2000) In organello formaldehyde crosslinking of proteins to
mtDNA: Identification of bifunctional proteins. Proc Natl Acad Sci USA 97:7772–7777.
33. Leeder WM, Voigt C, Brecht M, Göringer HU (2016) The RNA chaperone activity of the
Trypanosoma brucei editosome raises the dynamic of bound pre-mRNAs. Sci Rep 6:
19309.
34. Wendrich JR, Boeren S, Möller BK, Weijers D, De Rybel B (2017) In vivo identification
of plant protein complexes using IP-MS/MS. Methods Mol Biol 1497:147–158.
35. Tanz SK, et al. (2013) SUBA3: A database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis. Nucleic Acids Res
41:D1185–D1191.
36. Takenaka M, Brennicke A (2007) RNA editing in plant mitochondria: Assays and biochemical approaches. Methods Enzymol 424:439–458.
37. Lurin C, et al. (2004) Genome-wide analysis of Arabidopsis pentatricopeptide repeat
proteins reveals their essential role in organelle biogenesis. Plant Cell 16:2089–2103.
38. Sun T, et al. (2015) A zinc finger motif-containing protein is essential for chloroplast
RNA editing. PLoS Genet 11:e1005028.
39. Tanaka Y, et al. (2012) Gateway vectors for plant genetic engineering: Overview of
plant vectors, application for bimolecular fluorescence complementation (BiFC) and
multigene construction genetic engineering. Basics, New Applications and Responsibilities (InTech, Croatia), pp 35–68.
40. Nakagawa T, et al. (2007) Improved Gateway binary vectors: High-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol
Biochem 71:2095–2100.
41. Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers
for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136.
42. Mohammadzadeh S, et al. (2016) Co-expression of hepatitis C virus polytope–HBsAg
and p19-silencing suppressor protein in tobacco leaves. Pharm Biol 54:465–473.
43. Clough SJ, Bent AF (1998) Floral dip: A simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J 16:735–743.
44. Sparkes IA, Hawes C, Baker A (2005) AtPEX2 and AtPEX10 are targeted to peroxisomes
independently of known endoplasmic reticulum trafficking routes. Plant Physiol 139:
690–700.
Andrés-Colás et al.