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Cite this article: Omotezako T, Onuma TA,
Nishida H. 2015 DNA interference: DNAinduced gene silencing in the appendicularian
Oikopleura dioica. Proc. R. Soc. B 282:
20150435.
http://dx.doi.org/10.1098/rspb.2015.0435
Received: 24 February 2015
Accepted: 27 March 2015
Subject Areas:
developmental biology, molecular biology
Keywords:
DNA interference, transcriptional gene
silencing, post-transcriptional gene silencing,
chordate, Oikopleura dioica, Brachyury
Author for correspondence:
Tatsuya Omotezako
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2015.0435 or
via http://rspb.royalsocietypublishing.org.
DNA interference: DNA-induced gene
silencing in the appendicularian
Oikopleura dioica
Tatsuya Omotezako, Takeshi A. Onuma and Hiroki Nishida
Department of Biological Sciences, Graduate School of Science, Osaka University, 1 –1 Machikaneyama-cho,
Toyonaka, Osaka 560-0043, Japan
RNA interference is widely employed as a gene-silencing system in eukaryotes
for host defence against invading nucleic acids. In response to invading
double-stranded RNA (dsRNA), mRNA is degraded in sequence-specific
manner. So far, however, DNA interference (DNAi) has been reported only
in plants, ciliates and archaea, and has not been explored in Metazoa. Here,
we demonstrate that linear double-stranded DNA promotes both sequencespecific transcription blocking and mRNA degradation in developing embryos
of the appendicularian Oikopleura dioica. Introduced polymerase chain reaction
(PCR) products or linearized plasmids encoding Brachyury induced tail malformation and mRNA degradation. This malformation was also promoted by
DNA fragments of the putative 50 -flanking region and intron without the
coding region. PCR products encoding Zic-like1 and acetylcholine esterase
also induced loss of sensory organ and muscle acetylcholinesterase activity,
respectively. Co-injection of mRNA encoding EGFP and mCherry, and PCR
products encoding these fluorescent proteins, induced sequence-specific
decrease in the green or red fluorescence, respectively. These results suggest
that O. dioica possesses a defence system against exogenous DNA and RNA,
and that DNA fragment-induced gene silencing would be mediated through
transcription blocking as well as mRNA degradation. This is the first report
of DNAi in Metazoa.
1. Introduction
Most organisms have gene-silencing systems for protection of cells against
invading nucleic acids, such as viruses and transposons. A host defence
system was first reported in the petunia flower [1], where overexpression of
mRNAs involved in floral pigmentation unexpectedly induced a reduction of
such pigmentation. The gene-silencing mechanism known as RNA interference
(RNAi) has been clarified in Caenorhabditis elegans [2], where double-stranded
RNA (dsRNA) induces sequence-specific mRNA degradation. In RNAi, Argonaute protein binds to dsRNA to form a protein –nucleic acid complex, which
recognizes and cleaves the target mRNA [3]. It is now known that RNAi operates in a broad range of organisms from fungi to vertebrates [4,5]. On the other
hand, DNA interference (DNAi) has also been reported in a few species of
plants, ciliates and archaeans. In tobacco and Adiantum, introduction of DNA
fragments without a promoter has been shown to induce sequence-specific
gene silencing. These phenomena were termed DNAi [6–8]. In Paramecium,
a ciliate, plasmid DNA carrying a protein-encoding region induces posttranscriptional gene silencing of the corresponding endogenous gene [9].
Furthermore, a recent study of the archaean Thermus thermophilus has suggested
that it may have a system for DNA-induced DNAi [10]. Argonaute protein in
T. thermophilus has shown to cleave exogenously supplied DNA strands in a
sequence-specific manner [10]. While RNAi is a highly conserved genesilencing system in a wide range of eukaryotes, DNAi has not been explored
in any multicellular animals studied to date. In addition to DNAi, the phenomenon of co-suppression by transgenic DNA has been reported in many
& 2015 The Author(s) Published by the Royal Society. All rights reserved.
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(a)
ATG
1
–1970
2815
exon 248 bp
intron
intron-2
exon-50 bp
exon-20 bp
pBS-Bra
UTR
5¢Bra-3
(b) PCR-EGFP
5¢Bra-2
5¢Bra-1
(c) PCR-248 bp
0/91
(i)
33/119
PCR-full
ORF
intron
UTR
( f ) PCR-exon
(e) PCR-intron
(h) exon-20 bp
133/147
44/49
44/54
87/93
(k) PCR-5¢Bra-2
(l) PCR-5¢Bra-3
(m)
( j) PCR-5¢Bra-1
PCR-UTR
37/45
130/148
5/48
3/88
EGFP (4)
248 bp (3)
full (3)
intron (2)
intron-2 (3)
exon (2)
exon-20 bp (2)
exon-50 bp (4)
UTR (2)
5¢Bra-1 (3)
5¢Bra-2 (2)
5¢Bra-3 (2)
proportion (%)
(n) 100
50
0
0/42
RNAi
normal
short
shrunken
lethal
Figure 1. Tail malformation induced by PCR products encoding the Brachyury gene or cDNA sequence. (a) Regions targeted by each of the PCR products. Upper box
is a gene model of Brachyury and the regions targeted by each of the PCR products are shown by blue bars underneath. Yellow, grey, blue and white boxes indicate
the ORF, intron, UTR and inter-genic region, respectively. The starting methionine and stop codon are indicated as ATG at positions 1 and *. (b – m) Phenotype of
larvae injected with various PCR products of Brachyury and EGFP as a control. Each PCR product (0.3 mg ml21) was co-injected with H2B-EGFP mRNA
(1.3 mg ml21) and only larvae with EGFP fluorescence localizing into nucleus were scored. exon-20 bp and exon-50 bp DNA were prepared by annealing of
20 and 50 bp of synthesized oligonucleotides, respectively. Total numbers of shrunken tails among those of larvae with EGFP fluorescence are shown at the
bottom. Scale bar, 50 mm. (n) Average proportions of hatched larvae that exhibited each phenotype in two to four independent injections (shown in parentheses).
S.d. is shown by vertical bars. The results obtained in animals injected with PCR-EGFP, PCR-Bra-248 bp, PCR-Bra-full, PCR-Bra-intron, PCR-Bra-intron-2, PCR-Bra-exon,
exon-20 bp, exon-50 bp, PCR-Bra-UTR, PCR-50 Bra-1, PCR-50 Bra-2 and PCR-50 Bra-3 are shown in various colours depicted on the right.
organisms [11,12]. In C. elegans, introduction of transgenic
copies of a gene inhibits expression of the transgene as well
as endogenous homologous genes, partly using cellular components of RNAi [12]. This co-suppression is distinct from
DNAi, because co-suppression depends on the presence of
a promoter region of transgene and probably transcription
from transgenes [11,12].
In this study, we found a DNAi phenomenon in the chordate Oikopleura dioica, and demonstrate the existence of a
new gene-silencing system in Metazoa. An RNAi-mediated
gene knockdown method has already been established in
this animal [13]. We found that microinjection of doublestranded DNA fragments of the Brachyury gene induced tail
malformation reminiscent of the phenotypes induced by
RNAi. DNA fragments of the putative 50 -flanking region
and intron without the coding region also induced the
same phenotype. This malformation was also promoted by
linearized plasmid DNAs encoding partial Brachyury
cDNA sequences. Quantitative RT-PCR analyses showed
that this DNA fragment-induced effect is sequence-specific
and reduces the amount of the corresponding transcript.
DNA fragments of Zic-like1 and Acetylcholinesterase (AChE)
induced the expected loss-of-function phenotypes (i.e. loss
of sensory organ and muscle AChE activity, respectively).
These results suggest that O. dioica possesses a defence
system against exogenous DNA and RNA, and that DNA
fragment-induced gene silencing would be mediated through
transcription blocking as well as mRNA degradation.
2. Results and discussion
(a) Double-stranded DNA induces sequence-specific
gene knockdown phenotypes
To investigate the response of O. dioica to invading DNA, we
injected O. dioica with a polymerase chain reaction (PCR) product of 248 bp encoding a partial Brachyury cDNA sequence
(PCR-Bra-248 bp) (figure 1a). For introduction of DNA into
oocytes, the PCR product and mRNA encoding a fluorescent
fusion protein, H2B-EGFP, were co-injected into the ovary of
pre-spawning adults [13]. We selected larvae after hatching
with EGFP fluorescence in the nuclei (22% + 10% s.d. of a
single cohort on average, n ¼ 11) that were considered to
have incorporated PCR product, and these larvae were used
for further analyses. More than 90% of larvae that incorporated
PCR-Bra-248 bp showed tail malformation (figure 1c,n), and
the phenotype closely resembled the result of dsRNAmediated knockdown of Brachyury mRNA (figure 1m) [13].
This phenotype was categorized into four groups—normal,
short tail, shrunken tail and lethal—on the basis of the previous RNAi experiment [13]. Embryos injected with a PCR
Proc. R. Soc. B 282: 20150435
(g) exon-50 bp
(d)
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full
2
*
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(b) Linear DNA, but not circular DNA, exerts knockdown
effects
To investigate the difference in the response to circular and
linear DNA, we injected O. dioica with four different configurations of a plasmid harbouring a partial Brachyury cDNA
insert of 721 bp (pBS-Bra in figures 1a and 2c). These
comprised a circular plasmid pBS-Bra, linearized plasmids
pBS-Bra/NotI and pBS/BamHI, and only the insert region
pBS-Bra/NotI,BamHI, respectively. Most of the larvae injected
with pBS-Bra and pBS-Bra/NotI showed normal development
(figure 2d,e), whereas those injected with pBS-Bra/BamHI or
pBS-Bra/NotI,BamHI showed tail malformation (figure 2d,e).
These results indicate that a linear DNA configuration is essential for induction of tail malformation phenotypes, or that the
vector sequence connected to the 50 end of the Brachyury
sequence inhibits the capacity to induce malformation.
(c) Linear double-stranded DNA induces a gene-specific
phenotype
Next, to test whether PCR products targeting other genes would
induce specific phenotypes, PCR products of the nerve-specific
gene Zic-like1 or the muscle marker gene Acetylcholinesterase
(AChE) were injected. Zic-like1 is a zinc finger motif protein
expressed zygotically only in neural tissue (corresponding to
zinc finger protein ap-zic [GSOIDT00008831001] in the OikoBase genome browser at http://oikoarrays.biology.uiowa.
edu/Oiko/). In larvae injected with PCR-Zic-like1 (covering
446 bp), loss of the otolith in the brain vesicle was observed at
5.5 h post-fertilization (hpf) (figure 3a). This phenotype was in
accord with the result of RNAi-mediated knockdown of Ziclike1 (figure 3a). To investigate the effect of PCR-AChE (covering
819 bp), the amount of AChE protein was monitored by histochemical staining in 7 hpf larvae. In uninjected controls, two
row of AChE staining were observed in muscle cells on both
sides of the tail (figure 3b). In juveniles injected with PCR-AChE,
staining of muscle cells was not detected, although staining was
detected in small cells on the left side of the tail (figure 3b). These
signals corresponded to the nerve cells in the nerve cord that are
present on the left side and express unknown cholinesterase
activity, having been hidden by strong muscle staining in the
controls. The phenotype induced by each PCR product supported the contention that linear DNAs induced gene-specific
knockdown.
3
Proc. R. Soc. B 282: 20150435
To investigate crucial concentration of the injected DNA,
we diluted PCR-Bra-248 bp DNA (initial concentration was
0.3 mg ml21). As shown in figure 2b, 1/5 concentration
(0.06 mg ml21) was effective. Proportions of the shrunken
tail phenotype at 0.3 and 0.06 mg ml21 were 92% (+1% s.d.)
and 80% (+5% s.d.), respectively. By contrast, 1/25 and 1/
125 concentrations were not effective any more (figure 2b).
Therefore, 0.06 mg ml21 is the minimum concentration.
Next, we examined whether the knockdown phenotype
depends on the length of the target. PCR products that
were 100 bp long (PCR-intron and PCR-exon; figure 1e,f,n;
electronic supplementary material, table S1) worked. By contrast, the 50 bp product (exon-50; figure 1h,n) exerted
moderate effect (22% shrunken tail), and the 20 bp product
(exon-20; figure 1g,n) showed no effect. This is consistent
with the previous result, in which PCR-Bra-intron-2 (74 bp)
also showed weaker effect (51% shrunken tail).
rspb.royalsocietypublishing.org
product encoding EGFP (PCR-EGFP) developed normally
(figure 1b,n). The tail malformation is not due to mutation
caused by homologous recombination between the O. dioica
genome and the PCR products. Sequencing of Brachyury
gene in a PCR-Bra-248 bp injected short-tail larva demonstrated that there are no mutations (n ¼ 2, data not shown).
These results together suggested that the PCR products had
induced sequence-specific gene knockdown phenotypes.
To test whether the DNA-induced malformation was
sequence-dependent, we tested 10 PCR products targeting
distinct regions of the Brachyury gene: PCR-Bra-full, PCR-Braintron, PCR-Bra-intron-2, PCR-Bra-exon, exon-20 bp, exon-50 bp,
PCR-Bra-UTR, PCR-50 Bra-1, PCR-50 Bra-2 and PCR-50 Bra-3
(figure 1a). More than 80% of larvae injected with PCR-Brafull, PCR-Bra-intron, PCR-Bra-exon, PCR-Bra-UTR or PCR50 -Bra-1 similarly showed tail malformation (figure 1d–f,i,j,n).
In addition, PCR-Bra-intron-2 showed a weaker effect (51%
shrunken tail), probably because length of the fragment is
short (only 74 bp; electronic supplementary material, table
S1), and it is not effective enough, as mentioned later. Therefore, PCR products targeting not only Brachyury-encoding
regions but also the introns and one of the 50 -flanking regions
(PCR-50 -Bra-1, covering 2217 to 2393 bp upstream from the
starting methionine) are able to efficiently exert knockdown
effects.
In general, O. dioica mRNAs have tens to a few hundred
bases of 50 UTR [14]. In O. dioica, Brachyury cDNA is reported
to have 50 UTR of 216 bp in length, and does not have the
spliced reader sequence [15]. We confirmed that PCR-50 Bra-1 does not correspond to 50 UTR of Brachyury mRNA
in three ways. First, our 50 -RACE analysis showed that
Brachyury cDNA does not have longer UTR in consistent
with the previous report. Second, we also carried out RTPCR to test the possibility that the PCR-50 -Bra-1 corresponds
to the 50 UTR of Brachyury mRNA. PCR primers covering the
reported 50 UTR and coding region amplified an appropriate
band. On the other hand, forward primer covering the
30 region of PCR-50 Bra-1 (figure 1a) could not amplify the Brachyury sequence (data not shown). Third, RNAi
targeting the sequence covered by PCR-50 -Bra-1 did not
show any knockdown effect (0% shrunken tail, n ¼ 3).
These results indicate that PCR-50 Bra-1 indeed corresponds
to the 50 -flanking region, but not 50 -UTR, of the Brachyury
gene. By contrast, use of PCR-50 Bra-2 (2617 to 2793 bp)
and PCR-50 Bra-3 (21268 to 21444 bp) resulted in gradual
loss of the capacity to induce tail malformation, which
occurred in only 9% (+4% s.d.) and 4% (+2% s.d.) of the
larvae, respectively (figure 1k,l,n). Thus, the efficiency of
the knockdown effect seems to decline with increasing
distance of the target from the Brachyury coding region.
Nonetheless, PCR-50 Bra-2 (2617 to 2793 bp) and PCR50 Bra-3 still exert a weak effect. The effect of PCR products
targeting the intron and the 50 -upstream region of the Brachyury gene suggests that the introduced DNA might interfere
with the transcriptional process.
We examined the possibility that injected DNAs affect
mRNA maturation. RT-PCR of Brachyury cDNA covering the
PCR-Bra-intron target region was carried out. If PCR-Bra-intron
prevents intron excision, the 506 bp band would be detected in
addition to the normally spliced 294 bp band. Injection of PCRBra-intron did not give any additional band (figure 2a), and
reduction of the 294 bp band was just observed, suggesting
mRNA maturation was not affected by PCR-Bra-intron.
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(a) 120 bp
212 bp
174 bp
(b)
(c)
4
×1/5 (3)
(2)
proportion (%)
(1)
×1/25 (4)
×1/125 (4)
pBS-Brachyury
50
BamHI
NotI
721 bp
0
(1) control (2) PCR-Bra-intron
100
proportion (%)
(d)
al
rm
no
ort
sh
circle (3)
/NotI (4)
/BamHI (3)
/NotI,BamHI (2)
n
ke
n
hru
s
(e)
l
tha
lea
circle
/NotI
2/84
50
/BamHI
0
normal
short
shrunken
leathal
0/90
/NotI,BamHI
39/45
103/110
Figure 2. Tail malformation induced by linear double-stranded DNA. (a) RT-PCR of Brachyury cDNA covering PCR-Bra-intron target region (red bar). Yellow and grey
boxes show exon and intron, respectively. Blue arrows show PCR primers, and the PCR-Bra-intron target region is shown by a red bar. In the bottom picture, cDNAs
from PCR-EGFP and PCR-Bra-intron injected larvae were used as PCR templates, respectively. If PCR-Bra-intron prevents intron excision, the 506 bp band would be
detected in addition to the normally spliced 294 bp band. (b) Average proportions of hatched larvae that exhibited each phenotype derived from animals injected
with different concentration of PCR-Bra-248 bp. 1/5, 1/25 and 1/125 represent the injected concentration, 0.0600 mg ml21, 0.0120 mg ml21 and
0.0024 mg ml21 of PCR-Bra-248 bp, respectively. (c) A partial Brachyury cDNA sequence (blue arrow) was cloned into pBluescript. Direction of arrow indicates
that of the inserted gene. Restriction enzyme recognition sites (BamHI and NotI) are shown by red lines. (d) Proportions of each of the phenotypes in hatched
larvae. Each construct (0.2 mg ml21) was injected into the gonad. Blue, red, green and yellow bars show the results for injection of circular pBS-Bra, pBS/NotI, pBS/
BamHI and pBS/NotI,BamHI, respectively. (e) Representative phenotype resulting from each injection. Ratios of larvae with shrunken tails are shown at the bottom.
Scale bar, 50 mm.
(d) Introduced double-stranded DNA reduces
the amount of mRNA
Next, we examined which processes, from the gene to the
protein level, were involved in the effect of introduced DNAs.
In order to investigate the effect of introduced DNAs on the
amounts of mRNA, we measured the mRNAs of Brachyury
and Thrombospondin, a target gene of Brachyury [16], using
quantitative real-time PCR. PCR-Bra-248 bp reduced the
amount of Brachyury and Thrombospondin mRNA to 21% and
9% of that in the control group, respectively (figure 4a). On the
other hand, PCR-thrombospondin did not reduce the amount
of Brachyury mRNA, but reduced that of Thrombospondin
mRNA to 25% (figure 4b). This result demonstrated that the
introduced DNA specifically reduced the mRNA of the targeted
genes and those downstream from them.
(e) Introduced double-stranded DNA reduces the
amounts of protein synthesis from exogenous
mRNAs
We then examined how introduced DNA reduces the amount
of mRNA, either by blocking its transcription or degrading it,
as in the case of RNAi. PCR products targeting the intron or
50 -upstream regions of Brachyury induced tail malformation,
as mentioned above, thus indicating that introduced DNAs
reduce the amount of mRNA through transcriptional inhibition. To investigate whether PCR products also degrade
mRNA, O. dioica was co-injected with H2B-mCherry mRNA,
H2B-EGFP mRNA, and PCR-EGFP or PCR-mCherry (covering
128 bp of the EGFP sequence and 126 bp of the mCherry
sequence). In embryos injected with PCR-EGFP, only the
green fluorescence of EGFP was reduced (figure 4c, middle
row). Quantitative analysis using the method of Omotezako
et al. [13] showed that the ratio of fluorescence obtained by
dividing the EGFP green signal by the mCherry red signal
(EGFP/mCherry) was reduced to 42% (+17% s.d.) of the
control (figure 4d). Likewise, in embryos injected with PCRmCherry, the red fluorescence of mCherry was reduced to
76% (+14% s.d.) of the control (mCherry/EGFP) (figure 4c,
bottom row; figure 4e). These results show that the introduced
DNA exerts its effect by reduction of the amount of mRNA
through mRNA degradation, at least in part.
Our present results have revealed a new gene-silencing
phenomenon induced by linear double-stranded DNA. In
O. dioica, exogenously supplied double-stranded DNA induced
sequence-specific blocking of transcription and degradation of
Proc. R. Soc. B 282: 20150435
500 bp
300 bp
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100
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(a)
control
5
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30/30
(b)
RNAi
PCR-Zic-like1
4/21
control
PCR-AChE
4/16
Figure 3. Phenotype induced by PCR-Zic-like1 and PCR-AChE. (a) Phenotype of larvae injected with PCR-Zic-like1 (0.1 mg ml21) and uninjected control at 5.5 hpf.
Ratios of larvae in which an otolith was observed are shown at the bottom. (b) Histochemical staining for acetylcholinesterase was performed in juveniles injected
with PCR-AChE (0.2 mg ml21) and uninjected controls at 7 hpf. Ratios of juveniles that showed AChE staining in muscle cells are shown at the bottom. Scale bar,
50 mm.
0.8
(c)
bright field
H2B-mCherry
0.6
0.4
0
(b)
control
PCR-THS
0.8
0.6
PCR-mCherry
relative amount
1.0
Brachyury THS
PCR-EGFP
0.2
0.4
0.2
0
Brachyury THS
H2B-EGFP
(d)
fluorescent intensity
EGFP/mCherry
control
PCR-Bra
control
relative amount
1.0
1.0
control
PCR-EGFP
0.8
0.6
*
0.4
0.2
0
(e)
1.0
fluorescent intensity
mCheery/EGFP
(a)
control
PCR-mCh
*
0.8
0.6
0.4
0.2
0
Figure 4. PCR products eliciting mRNA reduction. (a,b) Results of quantitative real-time PCR. Ratios of the amounts of Brachyury and Thrombospondin mRNA in 3 hpf
larvae injected with (a) PCR-Bra-248 bp (0.3 mg ml21) and with (b) PCR-thrombospondin (0.7 mg ml21) to that in larvae injected with control PCR-EGFP
(0.1 mg ml21) are shown. Three separate experiments per subject were performed, and the average values are shown. (c) Fluorescence in embryos at 2 hpf.
Top panels show mCherry and EGFP fluorescence in embryos co-injected with H2B-EGFP and H2B-mCherry mRNAs (1.7 mg ml21). Middle and bottom panels represent
embryos that were co-injected with an mRNA mixture and PCR-EGFP or PCR-mCherry (0.1 mg ml21), respectively. Scale bar, 50 mm. (d,e) Quantitative representation
for (c). *p , 0.05 (Wilcoxon– Mann – Whitney test) compared with the control groups. Bars indicate standard deviations.
mRNA, resulting in gene-specific knockdown phenotypes. We
term this phenomenon ‘DNAi’ in O. dioica for the following
reasons. This gene-silencing phenomenon is different from
co-suppression with transgenic DNA in fly and nematodes,
which requires promoter region [11,12]. Microinjection of promoterless DNA fragments encoding exon, intron or UTRs
were sufficient to induce gene silencing in O. dioica. This
characteristic is consistent with the DNAi in plants [7,8]. However, the DNAi in O. dioica differs from DNAi in plants in
several aspects. In O. dioica, PCR products targeting the intergenic region and intron also induced sequence-specific gene
silencing. In plants, however, only promoterless-cDNA matching to exons could induce gene silencing. Another difference is
conformation of DNA. In O. dioica, DNAi is specifically
induced by linear DNA, whereas both linear and circular
DNA induced DNAi in plants. Overall, this study represents
the first example of DNAi in multicellular animals.
The molecular pathway that mediates DNAi in O. dioica
will be the focus of future investigations. As a host defence
against invading DNA, it has recently been reported that
Argonaute protein of T. thermophilus (TtAgo) mediates
small single-stranded DNA-induced DNAi [10]. Similarly to
TtAgo, Argonaute of O. dioica may mediate DNAi, inhibiting
transcription and degrading mRNA. In a BLAST search of the
O. dioica genome database (OikoBase) [17], we found nine
Argonaute family protein homologues bearing both the
Proc. R. Soc. B 282: 20150435
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and BamHI-BraR primers using Brachyury cDNA inserted into
the pCR-TOPO vector, and this was inserted between the NotI
and BamHI digestion sites of the pBluescript vector.
(c) Microinjection into the ovary
(d) Preparation of PCR products
PCR products to be injected were amplified using KOD plus
(TOYOBO) and gene-specific primers or universal primers (see
primer list in electronic supplementary material, table S1). We
ordinarily get a single band. In one case where non-specific
bands were detected, we purified an appropriate band from gel.
exon-20 bp and exon-50 bp were generated by annealing of synthesized oligonucleotides. Sense and antisense oligonucleotides
were heated at 958C for 8 min and gradually cooled into 258C
using thermal cycler. They were purified by phenol–chloroform,
chloroform and ethanol precipitation, and dissolved in water for
microinjection.
(e) Acetylcholinesterase histochemistry
3. Material and methods
(a) Laboratory culture of Oikopleura dioica
Wild animals were collected at Sakoshi Bay and Tossaki Port in
Hyogo prefecture, Japan, to start a culture. The animals have
been cultured over generations for more than a year in the
laboratory, as described previously [13].
7 hpf larvae were fixed in 5% formaldehyde in artificial seawater
at room temperature for 15 min. After washing with PBS-Tween,
fixed larvae were stained with reaction solution including
65 mM phosphate buffer (pH 6.0), 10 mM sodium citrate, 3 mM
copper sulfate, 0.5 mM potassium ferricyanide and 0.5 mg ml21
acetylthiocholine iodide at room temperature for 2 h.
Data accessibility. All sequences generated for this study have been depos-
(b) Constructs
To generate Brachyury, Acetylcholinesterase and Zic-like1 constructs, O. dioica genomic DNA for Brachyury and partial
cDNAs for Acetylcholinesterase and Zic-like1 (accession no.
KM047670 and KM047669) were isolated and cloned into the
pCR4-TOPO vector (Invitrogen), except for Acetylcholinesterase
cDNA, which was subcloned into the pGEM-T Easy vector
(Promega). To amplify the Brachyury genome sequence, PCR primers were designed for the upstream and downstream flanking
genes of Brachyury, respectively. Primers used for obtaining
genome and cDNA sequences are shown in the primer list (electronic supplementary material, table S1). To generate pBS-Bra,
partial Brachyury cDNA (721 bp) was amplified with NotI-BraF
ited under GenBank accession numbers KC253939, KM047669 and
KM047670. The datasets supporting this manuscript have been
submitted as part of the electronic supplementary material.
Acknowledgements. M. Suzuki, K. Kanae, M. Hayashi, M. Isobe and
R. Amano of our laboratory provided useful help with the culture
of O. dioica. We also thank S. Konishi in our laboratory for isolating
cDNAs of Zic-like1.
Funding statement. This work was supported by grants-in-aid for
Scientific Research from the JSPS to H.N. (22370078 and 26650079)
and to T.A.O. (24870019), and a grant-in-aid for JSPS Fellows to
T.O. (20131402).
Authors’ contributions. T.O., T.A.O. and H.N. designed the study, and wrote
and revised the manuscript. T.O. conducted the experiment and analysed the data. All authors read and approved the final manuscript.
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Proc. R. Soc. B 282: 20150435
PCR products and constructs were injected into the gonads of
maturing adult animals, as described previously [13]. Because
pro-oocytes are connected to a shared cytoplasm through a
pore known as the ring canal in the ovary [18], injected constructs were spread into part of the gonad with a gradient and
incorporated into oocytes through the ring canal. mRNA encoding H2B-EGFP or H2B-mCherry was co-injected with DNA
fragments as an indicator of incorporation. We categorized
resulting larvae into two groups (larvae with and without
fluorescence in the nuclei), although there was a continuous
distribution of different intensities from strongly fluorescent to
non-fluorescent eggs. Larvae categorized into ones with fluorescence were scored and used for analysis. A single injected
animal spawned approximately 200 eggs, and 22% + 10% s.d.
(n ¼ 11) of hatched larvae showed fluorescence derived from
incorporated mRNA on average.
6
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PAZ and PIWI domains. For host defence, some of these
homologues might mediate RNAi in response to dsRNA,
whereas others might mediate DNAi in response to introduced DNA. Although its primary function would be
protection against exogenous nucleic acids, if the invading
DNA were to carry a homologous sequence of the O. dioica
endogenous gene, this host defence system might inhibit
the endogenous gene to induce malformation.
Another intriguing possibility is that the gene silencing in
O. dioica is mediated by RNA, which is transcribed from
introduced DNA. However, while PCR products targeting
the sequences in the 50 -upstream region, PCR-50 Bra-1,
induced knockdown phenotype efficiently, dsRNA targeting
the same sequence showed no effect, as mentioned above.
This shows that transcription suppression by DNAi is unlikely to be mediated by RNA that is transcribed from
introduced DNA. This observation, however, does not
exclude the possibility that mRNA degradation by DNAi is
mediated by transcribed small RNAs.
Another unresolved issue is whether DNAi is a conserved
biological event in metazoans. We have investigated this
possibility using another chordate species, the ascidian
Halocynthia roretzi. However, when we injected H. roretzi
eggs with PCR-HrBra, these animals developed normally
(data not shown). Further investigations will clarify whether
double-stranded DNA-induced gene silencing occurs in other
multicellular animals.
Preparation of double-stranded DNA by PCR is much
easier, faster and cheaper than preparation of dsRNA. Therefore, DNAi could be a feasible technique for characterization
of gene functions in O. dioica. For practical application, PCR
products longer than 100 bp at a concentration of over
0.1 mg ml21 would be recommended.
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