OPENSUBJECT AREAS:
RNA SPLICINGSMALL RNASGENE EXPRESSION
TRNAS

Received
27 February 2013
Accepted
10 July 2013
Published
31 July 2013
Correspondence andrequests for materials
should be addressed to
Y.S. (ysekine@rikkyo.
ac.jp)* These authors
contributed equally tothis work.
1 Current address:
Department of
Horticulture, Chiba
University, Matsudo,
Chiba 271-8510,
Japan.
{ Current address:
Donnelly Centre for
Cellular andBiomolecular
Research, University ofToronto, Toronto, ON
M5S3E1, Canada.
{ Current address:
Samuel Lunenfeld
Research Institute, Mt.
Sinai Hospital,
Toronto, ON
M5G1X5, Canada.

Identification of highly-disrupted tRNA
genes in nuclear genome of the red alga,
Cyanidioschyzon merolae 10D
Akiko Soma1*1, Junichi Sugahara2*, Akinori Onodera1, Nozomu Yachie2{{, Akio Kanai2,
Satoru Watanabe3, Hirofumi Yoshikawa3, Mio Ohnuma4, Haruko Kuroiwa4, Tsuneyoshi Kuroiwa4& Yasuhiko Sekine1
1

Department of Life Science, College of Science, Rikkyo University, Toshima, Tokyo 171-8501, Japan, 2Institute for Advanced
Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan, 3Department of Bioscience, Tokyo University of Agriculture,
1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan, 4Kuroiwa Initiative Research Unit, College of Science, Rikkyo University,
Toshima, Tokyo 171-8501, Japan.

The limited locations of tRNA introns are crucial for eukaryal tRNA-splicing endonuclease recognition.
However, our analysis of the nuclear genome of an early-diverged red alga, Cyanidioschyzon merolae,demonstrated the first evidence of nuclear-encoded tRNA genes that contain ectopic and/or multiple
introns. Some genes exhibited both intronic and permuted structures in which the 39-half of the tRNA
coding sequence lies upstream of the 59-half, and an intron is inserted into either half. These highly
disrupted tRNA genes, which account for 63% of all nuclear tRNA genes, are expressed via the orderly andsequential processing of bulge-helix-bulge (BHB) motifs at intron-exon junctions and termini of permutedtRNA precursors, probably by a C. merolae tRNA-splicing endonuclease with an unidentified subunit
architecture. The results revealed a considerable diversity in eukaryal tRNA intron properties andendonuclease architectures, which will help to elucidate the acquisition mechanism of the BHB-mediateddisrupted tRNA genes.

C

yanidioschyzon merolae 10D is an ultrasmall unicellular red alga that inhabits an extreme environment(pH 1–3, 40–50uC), and represents one of the most ancestral forms of eukaryotes1–3. A primary search of
the complete 16.5 Mbp nuclear genome sequence predicted only 30 tRNA genes, which are insufficient todecode 61 codons2. This paucity of tRNA genes was partly solved by our discovery of a distinctive type of
disrupted tRNA gene; we identified 11 circularly permuted tRNA genes, in which the 39-half of the tRNA coding
sequence lies upstream of the 59-half in the genome4. Biochemical analyses defined a processing pathway in which
the termini of the tRNA precursor (pre-tRNAs) are ligated to form a characteristic circular RNA intermediate,
which is then processed at the acceptor stem to generate the functional termini4. Notably, the sequences adjacentto the termini in these pre-tRNAs potentially form a bulge-helix-bulge (BHB) motif, which was originally foundaround the intron-exon junctions of nuclear and archaeal tRNAs5. To date, permuted tRNAs have only beenfound in some unicellular algae and archaea6,7. These observations suggest that BHB motifs, and the tRNA intron
splicing system by which they are processed, must have been a prerequisite for the development of such disrupted
tRNA genes in early-rooted eukaryotes and archaea.
Here, we report the identification of an alternative type of BHB-mediated disrupted tRNA gene that harbors
atypical introns. Introns found in nuclear and archaeal tRNAs are cleaved by the proteinaceous tRNA-splicing
endonuclease8–11. Nuclear tRNA introns are generally short, comprise a relaxed BHB motif, and are locatedexclusively between positions 37 and 38 (37/38), which is 39 adjacent to the anticodon (the canonical position)5,12.
This limited location of the intron is considered crucial for the precise recognition of pre-tRNA by the eukaryal
tRNA-splicing endonucleases8–11,13–18. However, we found that a number of C. merolae nuclear-encoded tRNA
genes contain single or multiple ectopic introns, which has not been reported in eukaryotes. Some genes contained both intronic and permuted gene structures. Further analysis revealed that the BHB motifs in pre-tRNAs
are processed in an order that correlates with the theoretical free energy and the position of each motif in thecloverleaf structure. It also indicated that the C. merolae tRNA-splicing endonuclease may show a non-canonicalsubunit composition. Our findings provide a new insight into the tRNA processing system in eukaryotes and a

SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

1www.nature.com/scientificreports

G

A
A
Phe
G
U
Leu
C
A
G
Leu
U
C
A
G Ile
U
iMet
C eMet
A
G
U
C

Val

AGA (1)GAA (2)
UAA (1)
CAA (1)
AAG (0)Pro
CAG (0)GAU (1)
UAU (3)
CAU (0)CAU (2)
AAC (0)UAC (0)CAC (0)Tyr

Ser
CGA (1)
AGG (2)

Thr

Ala

C

U
GUA (2)

stop
Trp

stop

His

GUG (0)UGG (1)
CGG (1)Gln

CUG (0)AGU (0)Asn

UGU (1)
CGU (0)Lys

AGC (1)Asp

UGC (0)CGC (2)GluCys

GUU (1)Arg
Ser

UUU (0)CUU (0)Arg

GUC (2)
UUC (2)Gly

GCA (0)A

CCA (1)
ACG (2)
UCG (2)
CCG (3)GGCU (1)U
UCU (3)
CCU (1)
GCC (0) C
UCC (0)*CCC (1)Figure 1 | Anticodons in C. merolae. Newly identified and revised tRNA
genes are indicated in red and blue, respectively. All anticodons arepresented in the 59 to 39 direction. Intron numbers are shown inparentheses. Anticodons of tRNAs encoded by a permuted gene are boxed.
Two distinct genes that produce identical mature tRNA sequences encodetRNAGly(CCC) (indicated by an asterisk). iMet and eMet mean initiatorand elongator tRNAMet(CAU), respectively.

deeper understanding of the evolutionary aspects that governed theformation of BHB-mediated disrupted tRNA genes.

Results
Approximately 63% of tRNA genes in the C. merolae nuclear
genome are disrupted by introns. A genome-wide search fortRNA genes was performed using the SPLITS and SPLITSX
programs to detect cis-spliced tRNAs (intron-containing tRNAs)
and trans-spliced tRNAs (split tRNAs)19–22. A BLAST search was
performed to detect conserved tRNA sequences. This identified 27
intronic tRNA genes, including nine newly identified and 12 revised
genes (Fig. 1). Among them, 12 appeared to be the first examples ofnuclear-encoded tRNA genes containing multiple introns: nine
genes with two introns, three with three introns and 15 with asingle intron. Four genes encoding tRNALeu(UAA), tRNAArg(CCU),
and two copies of tRNAGly(CCC) had both intronic and permuted
structures, meaning that their pre-tRNAs required removal of an
intron as well as swapping of the 59- and 39-halves. Our search for
BHB-mediated disrupted tRNA genes led to the identification of a
sufficient repertoire of anticodons. C. merolae contains disrupted
tRNA genes that exhibit intronic (23/43), permuted (7/43), or both
(4/43) structures that account for 79.1% (34/43) of all nuclear tRNA
genes. The proportion of intronic tRNA genes in C. merolae (62.8%;
27/43) is much higher than that found in other eukaryotes12,
including Saccharomyces cerevisiae (21.5%, 59/275), Arabidopsis
thaliana (13.2%, 83/630), Drosophila melanogaster (5.2%, 15/290)
and Homo sapiens (4.3%, 28/648). However, it is comparable to thelevels found in several archaea (up to 87%), which contain the highest
number of introns in any organism7,22.
The genomic intron sequences varied from 11–69 bp;
tRNASer(AGA) and tRNAAsn(GTT) contained the shortest introns,
11 bp, in the anticodon-loop, while tRNAArg(TCT) contained thelongest introns, 69 bp, in the D-loop (Fig. 2).
Eukaryal tRNA genes are generally transcribed by RNA polymerase III via an intragenic bipartite promoter consisting of A and B
boxes, which are conserved sequences in the D-arm and TYC-arm,
respectively23. Transcription is initiated by the binding of a multisubunit transcription factor (TFIIIC) to the promoter sequence24.
However, the A and B boxes of atypical intronic tRNA genes in C.
merolae are not recognized uniformly by the transcription
machinery because of the interruption of either box by the intron
SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

and the variable intron sequences between the boxes, making thepositional relation of the A and B boxes inadequate for binding of
TFIIIC. This is also true for the permuted tRNA genes, in which the A
and B boxes are not recognized by TFIIIC because they are inverted4.
Additionally, no homologs of the TFIIIC components that bind to
the A and B boxes have been identified in C. merolae. Instead, aTATA-like sequence and a T-stretch, which are probably the promoter and termination signal, respectively24,25, are found near mosttRNA genes (see Supplementary Fig. S1 online). No promoter or
termination signal is found between the exon segments of the intronic tRNA genes, suggesting that a set of putative tRNA segments istranscribed as a linear RNA. The above observations indicate that C.
merolae employs the upstream TATA-like sequence and a noncanonical transcription system independent of TFIIIC, which would
allow transcription of various types of disrupted (intronic, permuted,
and both) and non-disrupted tRNA genes.
C. merolae tRNA introns form various types of BHB motifs andare scattered throughout the cloverleaf structure. In contrast to the
known nuclear tRNA genes whose introns are located exclusively at
the canonical 37/38 position12, 42 introns in 27 tRNA genes of C.
merolae were randomly distributed along the cloverleaf structure(Fig. 2). Although some (17 introns) were found at the 37/38
position, others were observed at novel positions for nuclear
tRNA: the D-stem (five introns), the D-loop (14 introns), thevariable-arm (one intron), the TYC-stem (one intron) and the
TYC-loop (four introns). The finding that C. merolae nuclear
tRNA genes contain introns at various positions is inconsistent
with a requirement for introns to be located at particular positions
such that they are recognized by known tRNA-splicing endonucleases in eukaryotes9,10.
The intron-exon junctions of nuclear pre-tRNAs generally form a
relatively relaxed BHB motif. This is denoted as the hBH or BHL(BHB-like) motif, and consists of a single 3-nt bulge and an internalloop separated by a 4-nt helix5,12. In C. merolae, the hBH motif isoften located at position 37/38 (Fig. 2), as represented bytRNAAsn(GUU) (Fig. 3A). Our analysis revealed other types of
BHB motifs at position 37/38, such as a strict hBHBh9 motif intRNAIle(UAU) (Figs. 2 and 3C) and an unstructured BHB motif withno central 4-bp helix H in tRNAPhe(GAA) (Figs. 2 and 3B). Various
types of BHB motifs were also identified at positions outside 37/38,
including hBHBh9, BHL (hBH and HBh9) and a motif without H (noH) (Fig. 2).
These features of tRNA genes in C. merolae – the localization of
introns at positions other than 37/38 and identification of various
types of BHB motifs – have never before been observed in a nuclear
genome, although they do occur in some archaeal phyla, such as
Crenarchaeota and Nanoarchaeota5,12,26–28.
Processing of the pre-tRNAs derived from disrupted genes. We
performed northern blotting analysis using C. merolae total RNA toverify whether the highly disrupted tRNA genes are indeed
transcribed in the cell. Northern blots using probes complementary to the 39-halves of tRNAAsn(GUU), tRNAPhe(GAA) andtRNAIle(UAU), which harbor one, two and three introns, respectively, detected bands of the appropriate size (Supplementary Fig.
S2 online), demonstrating that C. merolae disrupted tRNA genes are
expressed. Their different mobilities in Fig. S2D, despite their almost
identical predicted size, have been observed in PAGE and Northernblot analysis of tRNAs4,29–31, and it may be attributed to differences intheir overall secondary structures, which may be unfolded even in the
denaturing gel, and to different post-transcriptional modifications.
No bands for pre-tRNAs harboring unspliced introns were detected,
suggesting that intron processing occurs rapidly in the cells, making
the processing intermediates so scarce that they are nearly impossible
to detect, even by northern blotting. In fact, it was reported that theover-expression of tRNA genes or knockdown of the processing
2

www.nature.com/scientificreportsA

Tyr(GTA) 24 1/2 HBh’
Arg(CCG) 40 1/3 no hAla(CGC) 15 1/2 HBh’Ile(TAT) 30 1/3 hBHBh’

Arg(CCT) 27 2/2 HBh’
Pro(AGG) 17 1/1 HBh’Arg(TCG) 47 1/2 HBh’
Pro(TGG) 22 1/1 HBh’73
1
Arg(CCT)
Lys(TTT)
Ala(TGC)

Ser(GCT) 26 1/1 HBh’Arg(TCT) 69 1/3 HBh’

1/2 hBH
1/1 hBH
1/1 hBHBh’

’
Y

R
A
15

Glu(TTC) 20 1/2 HBh’
Asp(GTC) 18 1/2 HBh’

G
G

8

R

A

26

48 Y

Y
U

Ala(AGC) 16 1/1 HBh’

B=3

> 2
h’ =

3’
5’
> 2
h=

H=4

Gly(CCC)a 13 2/2 no hGly(CCC)b 13 2/2 no hAla(CGC) 21 2/2 HBh’Thr(TGT) 31 1/1 HBh’eMet(CAT) 30 1/2 HBh’Arg(TCT) 55 2/3 hBHBh’

B

R
54
C
T U

Ile(TAT) 22 3/3 hBHBh’

Arg(CCG) 60 2/3 HBh’
Leu(CAA) 65 1/1 HBh’
Leu(TAA)
1/2 HBh’Phe(GAA) 28 1/2 hBHBh’
Glu(TTC) 40 2/2 HBh’A

B=3iMet(CAT)R 37

1/1 HBh’Arg(ACG) 22 2/2 HBh’

Pro(CGG) 15 1/1 hBH
Ser(AGA) 11 1/1 hBH
eMet(CAT) 19 2/2 no H Arg(CCG) 33 3/3 no H
Tyr(GTA) 15 2/2 hBHBh’ Asp(GTC) 21 2/2 hBH
Leu(TAA) 21 2/2 hBHBh’
Phe(GAA) 34 2/2 no H
Ile(TAT) 20 2/3 hBHBh’
Pro(AGG) 12 1/1 hBH
Trp(CCA) 12 1/1 hBH
Leu(CAG) 1/1 hBHBh’
Arg(ACG) 32 1/2 hBH
Thr(CGT) 1/1 hBH
Ile(GAT) 14 1/1 no H
Thr(AGT) 1/1 hBH
Arg(TCG) 16 2/2 hBH
Gln(CTG) 1/1 hBH
Ser(CGA) 51 1/1 hBHBh’ Gly(CCC)a 1/2 hBH
Asn(GTT) 11 1/1 hBH
Gly(CCC)b 1/2 hBH
Arg(TCT) 13 3/3 hBH

Figure 2 | Distribution of BHB motifs in intronic and/or permuted tRNAs in C. merolae. (A) Arrows indicate the positions of the BHB motifs. The
length of the intron, number and type of BHB motifs is indicated for each tRNA. For example, ‘‘Ile(TAT) 22 3/3 hBHBh9’’ at the position between 53
and 54 means that the BHB is from tRNAIle(UAU), is 22 nt long, and is the third of three BHB motifs. BHB motifs at the termini of the permuted
pre-tRNAs are shown in double squares. The numbering of the tRNA positions follows that outlined in Reference 12. (B) The BHB motif is classified by
one or two 3-nt-bulges (denoted as B 5 3) separated by a central 4-bp-helix (denoted as H 5 4) and flanked by two helices (denoted as h or h9), each withmore than two base-pairings.

enzymes in the cell is required to detect precursors or processing
intermediates of tRNAs and non-coding RNAs by PAGE or
Northern blotting32–34.
The introns in most C. merolae pre-tRNAs can form a BHB motif
independently. These introns are not nested, as occurs in somearchaea, in which the last intron can form a BHB motif only after
the other introns have been processed26,35. To analyze the processing
intermediates and to clarify the processing pathway in C. merolae,
reverse transcription polymerase chain reaction (RT-PCR) was performed on three tRNAs and the resulting products were sequenced(Fig. 3). PCR products indicated by arrows in Fig. 3 were annotatedby gel purification of the bands, cloning and sequencing analysis(Supplementary Table 1 online, Supplementary Figs. 3–5 online).
While the signal representing unprocessed tRNAs was not detectedin the northern blot analysis (Supplementary Fig. S2 online), RTPCR products derived from various processing intermediates weredetected with various band intensities (Fig. 3). Amplification of the
RT-PCR products of tRNA processing intermediates is influenced by
the amount of the target molecule and by the modification of nucleotides, which may inhibit the reverse transcription reaction36–38. Thus,
the intensities of the PCR bands do not always reflect the amount of
SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

the target RNA molecules present in the cell. The bands for intermediates would be detectable because of significant amplification of asmall amount of cDNA by PCR.
tRNAAsn(GUU) contains a single intron composed of an hBH
motif at position 37/38 (Fig. 3A). RT-PCR using N59-primer 1 andN39-primer 1 (indicated by arrows in Fig. 3A) amplified both a pretRNA containing the intron and a processed form (mature form)(Fig. 3D, lane 1).
tRNAPhe(GAA) contains two introns, in the D-stem (intron 1) andat position 37/38 (intron 2), which comprise a hBHBh9 and a no H
motif, respectively (Fig. 3B). RT-PCR with F59-primer 1 and F39primer 1 produced PCR products representing a pre-tRNA with two
introns, a processing intermediate in which intron 1 was removed
but intron 2 was retained, and a processed form (mature form) inwhich both intron 1 and 2 were removed (Fig. 3E, lane 1). No productwas detected for an alternative intermediate in which intron 2 wasremoved but intron 1 was retained, even when using a primer set (59primer 2 and 39-primer 2) specifically designed to amplify such anintermediate (Fig. 3E, lane 2). Although it remains possible that anintermediate harboring only intron 1 was produced but not detected
because of technical difficulties, the above results suggest a pathway3www.nature.com/scientificreports
B

A

C
73 A

73G

N5'-primer1

G1
G
U
A
G
U
A
U

U G
A
A
C
C UC GG 18
G A GC
G
A
G
U U
U
G
C
G
G

tRNAAsn(GUU)CC
A
N3'-primer1
U
C
A
C G A
U
CUCUC
A
G A GA G
C
U U C
U
54
G
A G
A
C
G
C
C A38
a
g a
a
u
g

C
U
G
U
U
A 37
a
a
g
a c

F5'-primer1F5'-primer2
a

c u a

u
u

c g a

c ug a g ga
ugc u c gGA

c c cgU

CG A C UCG A

GGG A
a

23 G C

a

G1
C
C
A
U
U
G

G

U
U
G
C
A

tRNAPhe(GAA)

I5'-primer2F3'-primer1
GC GC C

C U A

C
U
G
A
A
G 37
g
c

U

a 12 CGAUG
UC
c a a cggucg g c u
g
13
cc gcc gu c g a
c u a c c C AGcC

CG CG G
C
U U C
54
U
A
G G
A
A
F3'-primer2
C
G
UA
38 g
gc
a
g
c
u
intron 2gA

intron 1

73 A

N5'-primer
N3'-primer

E1
1F5'-primer 1F3'-primer 1

intron 1

tRNAIle(UAU)37 U

G

I3'-primer2Ag
G C U U54 c g c g u
C G C GA48

c g c a cGug c g u g

a a
g
g u

53

intron 3

A
G
C
U
C
G 38
A
a
gu

C
U
U
A

a
c
u

a
g
c
a
g
a
c

F

2
2

I5'-primer
I3'-primer

1
1

2
1

u

c
g
u
g
g
u

intron 2

3
2

231
194
Pre-tRNAPhe (+intron1, 2)

118

Pre-tRNAPhe (+intron 2)

Pre-tRNAIle (+intron1, 2, 3)

118*Pre-tRNAIle (+intron1, 2)

74

Mature tRNAIle

Pre-tRNAIle (+intron2, 3)
Pre-tRNAIle (+intron2)

118
Mature tRNAPhe

74
(bp)

(bp)

Mature tRNAAsn

74
(bp)

Lane

C

UGU

I5'-primer3 GG18UGCGU A GA
C
G
A
G
U

194

Pre-tRNAAsn (+intron 1)

C G C U C

A

231
194

231

I3'-primer1

U

24nt

D

U
G
A
G
G
G
C

A1
C
U
C

I5'-primer1

G

12

intron 1

C
G
G
U
A
G
C

M 1

Lane M 1

2

Lane

M

1

2

3

Figure 3 | RT-PCR amplification of intronic tRNAs. Inferred pre-tRNA secondary structures for three tRNA genes are shown. (A) tRNAAsn(GUU) witha single intron at the canonical position 37/38. (B) tRNAPhe(GAA) with two introns, one in the D-stem (intron 1) and the other at 37/38 (intron 2). (C)tRNAIle(UAU) with three introns, one in the D-stem (intron 1), one at 37/38 (intron 2), and the third in the TYC-loop (intron 3). 59- and 39-primers for
RT-PCR are indicated as solid and broken arrows, respectively. Arrowheads indicate the positions to be processed. Intron sequences are shown inlower case. The numbering of the tRNA positions follows Reference 12. (D–F) PCR products amplified from the cDNA of processing intermediates andmature forms are indicated. Annotation of each PCR product was based on gel purification of the bands, cloning and sequencing analysis. Predicted sizes
for each PCR product are described in Supplementary Table 1 online. Although I39-primer 2 was designed to hybridize to a region within intron 3,
the I59-primer 3 and I39-primer 2 combination produced a PCR product representing pre-tRNAIle (1intron 2) and mature tRNAIle (lane 3). This is
attributable to the tendency of the I39-primer 2 to hybridize to the TYC-stem of tRNAIle(UAU), because of sequence similarity between the TYC-stemand 39 region of intron 3. The asterisk corresponds to a PCR product of pre-tRNAIle (1intron 2, 3) found in lane 3 (see details in the methods for theretardation of band mobility). Lane M: DNA molecular weight marker (WX174/HaeIII).

in which removal of intron 1 occurs before removal of intron 2. We
then calculated the free energies of the BHB motifs to examine
whether the processing pathway correlates with the stability of the
BHB motifs. The free energies (DGs) of the BHB motifs in introns 1
and 2 from pre-tRNAPhe(GAA) were calculated as 211.1 kcal mol21
and 1.15 kcal mol21, respectively. This result implies that the processing of intron 1, in which the DG of the BHB motif is much lower,
precedes that of intron 2.
Analysis of tRNAIle(UAU), which contains three introns with
hBHBh9 motifs in the D-stem (intron 1), position 37/38 (intron 2)
and the TYC-loop (intron 3) (Fig. 3C), verified the sequences of a
primary transcript containing all three introns (Fig. 3F, lane 2), anintermediate in which only intron 3 was removed (Fig. 3F, lane 2), anintermediate in which only intron 1 was removed (Fig. 3F, lane 3),
an intermediate in which only intron 2 was retained (Fig. 3F, lane 3)
and a fully processed form (mature form) in which all three introns
were removed (Fig. 3F, lanes 1 and 3). No product was produced for
an intermediate in which intron 2 was removed before intron 1 and 3(Fig. 3F, lanes 1–3). These results show that the processing of introns
1 and 3 precedes that of intron 2. Processing intermediates containing introns 1 and 2 or introns 2 and 3 indicate that the removal of
intron 1 or 3 can occur in either order. The DGs of the BHB motifs ofintrons 1, 2, and 3 were calculated as 211.2 kcal mol21, 23.5 kcal
mol21 and 210.9 kcal mol21, respectively. Thus, like tRNAPhe(GAA),SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

the processing of pre-tRNAIle(UAU) begins with removal of introns 1
or 3, in which the DG of the BHB motif is lower, before intron 2 isremoved. The band indicated by an asterisk (Fig. 3 F, lane 3), which
was estimated to be more than 118 bp, was identified to be derived
from pre-tRNAIle (1intron 2, 3) and was 116 bp in length. Such aninconsistency between the gel mobility and the actual size of PCR
products was probably observed because of irregular structures of in
the DNA. Annealing DNA strands with complementary repeatedsequences sometimes results in DNA species such as cruciform orslipped DNA, which migrate slower in a polyacrylamide gel39,40;
however, such an event in plasmid DNAs is rare41. In our analysis,
PCR products consisting of tRNA sequence with stem-loop structures might have caused unknown structural abnormalities, leading
to inconsistent mobility in gel electrophoresis.
The potential folding of BHB motifs is also found at the junction of
the processing sites in permuted pre-tRNAs. tRNAGly(CCC), possessing both intronic and permuted structures (Fig. 4A–C,
Supplementary Table 1 online, Supplementary Fig. 6 online), wasanalyzed to determine which BHB motif is processed first. RTPCR amplified a product derived from a circular RNA intermediatein which the leader and trailer sequences at the RNA termini were
processed, while the intervening sequence was retained (Fig. 4D).
The circular intermediate lacked intron 1 in the TYC-loop(Fig. 4E), suggesting that the BHB motif of intron 1 is processed4www.nature.com/scientificreports
before that of the termini in pre-tRNAGly(CCC). The DG of the BHB
motif of intron 1 was calculated as 24.8 kcal mol21, which is slightly
lower than that of the BHB motif at the termini (24.43 kcal mol21).
Thus, the processing of the BHB motif of permuted pre-tRNA termini may also follow the order suggested for intronic tRNAs in Fig. 3.
The above results implied a correlation between the processingorder of introns and the theoretical free energy of BHB motifs inintrons. This may explain why the removal of multiple BHB motifs
from pre-tRNAs occurs in an order, even though each BHB motif canfold independently. Alternatively, it may depend on the position of
the BHB motif, because the BHB motif in the anticodon arm is always
the final substrate observed in tRNAPhe(GAA), tRNAIle(UAU), andtRNAGly(CCC). In the latter case, the BHB motif at position 37/38
can be recognized by the C. merolae endonuclease only after the BHB
motifs at the other positions have been processed. These possibilities
have to be further addressed by in vitro splicing analysis.
Unique subunit composition of C. merolae tRNA-splicing endonuclease. The tRNA-splicing endonuclease of the yeast S. cerevisiae
is one of the best-studied eukaryal tRNA-processing enzymes. It
possesses a heterotetrameric structure (abde) comprising two
catalytic subunits (Sen2 and Sen34) and two accessory subunits(Sen15 and Sen54)9–11,16. Sen2 and Sen34 are homologs of thecatalytic subunits of archaeal endonucleases. The interactions
between Sen2 and Sen54, and between Sen15 and Sen34 wereclarified by a yeast two-hybrid (YTH) experiment16. These four
subunits function cooperatively to recognize cleavage sites via ‘‘aruler mechanism’’, in which the endonuclease measures a specified
distance to where the cuts should be made in a pre-tRNA9,10,15–17. In
addition to the localized structural fold of the BHB motif, thismechanism requires the specific recognition of the mature domain
in pre-tRNAs. Thus, the yeast endonuclease would have coevolved
with the introns exclusively localized at position 37/38. A search of
the C. merolae genome identified three homologs of the yeast
endonuclease subunits, cmSen2 (CMN231C), cmSen34 (CMH233C)
and cmSen54 (CMK254C), comprising 246, 435, and 348 aminoacids, respectively (see Supplementary Fig. S7 online). According
to their isolated cDNA sequences, these candidates are predicted to
be functionally active2. However, no apparent homolog of Sen15 was
identified, which would appear to conflict with the notion that all
four subunits are essential for the function of the endonuclease. In
yeast, Sen15 interacts with Sen34 to aid the proper positioning of the
39-splice site8,9,15–17.
To find a functional homolog of Sen15 or another potential subunit of the C. merolae endonuclease, a YTH screening was carriedout. The full-length cDNA cmSen2, cmSen34, or cmSen54 wascloned into a bait protein plasmid, and positive clones were screened
from a C. merolae genomic cDNA library. Screening using cmSen2(CMN231C) as a bait protein allowed the isolation of the cmSen54(CMK254C) fragments from two independent prey clones (24 to 348
or 43 to 348 amino acids). However, no other interactions wereobtained in this analysis. To confirm their specific interaction, wecloned the full-length cDNA of the three cmSen candidates andtested them using a YTH matrix experiment. The reciprocal interaction between cmSen2 and cmSen54 was clearly detected (Fig. 5).
However, no pairwise interaction was detected for cmSen34, implying that the C. merolae endonuclease has a noncanonical subunit
composition, which could not be clarified by YTH analysis. The C.
merolae endonuclease may contain an unidentified subunit with low
homology to known Sen15 proteins. It is also possible that the C.
merolae endonuclease comprises a novel heterotrimeric complex.
Given that Sen15 (129 amino acids) functions cooperatively with
Sen34 in yeast and that cmSen34 (435 amino acids) is larger thanyeast Sen34 (275 amino acids), cmSen34 may fulfill the structuraland functional roles of both Sen34 and Sen15; however, there is nosequence similarity between yeast Sen15 and C. merolae Sen34.
SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

Alternatively, the C. merolae endonuclease may function as a dimer
comprising catalytic subunits depending on the substrate to cleave.
We are currently analyzing this process in vitro using two or threecomponents of the recombinant endonuclease. In addition, we areplanning to perform co-immunoprecipitation analysis to identify theactive form of the endonuclease. Further analysis is required tounderstand the C. merolae tRNA-splicing endonuclease and its processing mechanism, which would be essential for the maturation of
intronic and/or permuted pre-tRNAs.

Discussion
In C. merolae, disrupted tRNA genes that exhibit intronic (23/43),
permuted (7/43) or both (4/43) structures account for 79.1% (34/43)
of all nuclear tRNA genes. Some introns of nuclear-encoded tRNAs
are required for post-transcriptional modification9 or regulation of
the cell cycle42, while some are dispensable43. It has also been suggested that fragmented tRNA genes may prevent the integration ofmobile elements44,45. Thus, the conservation of various types of disrupted tRNA genes in the streamlined genome of C. merolae, which
require more extensive processing, may suggest that the fragmentation of tRNA genes or the BHB motif itself are significant for cellfunction. Alternatively, disrupted tRNA genes might have developedby neutral evolution, or may be a remnant of the process for decreasing the number of tRNA genes. Even if such tRNA genes wereacquired in eukaryotes, most would not have been retained becauseof the failure of transcription or subsequent RNA processing.
However, in C. merolae, these genes could have persisted in the
genome owing to the upstream promoter-dependent transcription
system and, especially, the capacity to process non-canonical pretRNAs, including intronic and permuted pre-tRNAs, into the canonical cloverleaf structure.
BHB motifs in intronic pre-tRNAs and their corresponding splicing machinery are commonly found in eukaryotes and archaea,
although their modes of splicing endonuclease recognition differ.
Archaeal endonucleases exhibit symmetrical subunit architecturescomprising catalytic subunits (homologs of Sen2 and Sen34) andrecognize introns in pre-tRNAs in a manner that is basically dependent only on the BHB motifs11,18,27,28. Genome-wide analyses have
expanded the number of archaeal tRNA genes that contain introns
comprising various types of BHB motifs at various positions.
Accordingly, four different types of endonuclease were identified
in archaea, which indicated that the subunit compositions of archaeal
endonucleases correlate with their substrate specificity toward the
BHB motifs22,28,29,46,47. In eukaryotes, the processing mechanisms oftRNA-splicing endonucleases, other than those of yeast, are not fully
understood. The present study showed that C. merolae contains
atypical introns and an endonuclease that is substantially eukaryotic-like, but differs in subunit composition. Homology searching and
YTH analysis could not identify an accessory subunit (Sen15) that isessential for functional multimerization of yeast endonuclease, indicating that the C. merolae endonuclease may contain an unidentified
subunit, or is possibly comprised of a novel heterotrimeric complex.
Alternatively, the C. merolae endonuclease may adopt a different
type of subunit architecture. The yeast Sen54 is indicated to interact
with the D-arm and the acceptor stem in the L-shaped tertiary structure of tRNA17,18; therefore, the C. merolae endonuclease containing
the cmSen54 subunit is not likely to interact with pre-tRNAs harboring ectopic introns, especially in the D-arm. Thus, the C. merolae
endonuclease may act on such pre-tRNAs as a dimer composed ofonly catalytic subunits (cmSen2 and cmSen34) to recognize atypicalintrons. It is tempting to speculate that the C. merolae endonuclease
comprises a different subunit combination; for example, cmSen2cmSen54-cmSen34 or cmSen2-cmSen34, depending on the positions
or types of the BHB motifs in the substrates. If so, BHB motifs at
non-canonical positions will be removed by cmSen2-cmSen34, making the BHB motif at canonical position (37/38) accessible to5www.nature.com/scientificreports

A

B

Leader sequence

Intervening sequence

TATA

3'-halfTATA3'-half
Intron sequence(13 bp)

U C
G
C
C
U
U
A 73
G1 C
G
C
G
C
C
G
A
U
G3'-primer1 G C
A
G
C
UU G
G GC C CU
C 56
intron 1
G A
a
G
U
C
cg
UG U G
u a a
c cc
C
G
C
AC A UGG G U au g u
C
G
G
G
U
U A
U
G
C
55
A
U
A
U
C
G
G5'-primer1 A U38U
C
AU
U
A
U
G
C
G
C
U
C
A
A 37
U
A
A
UA
C
G
U
U
U
U
C
G
G
C

tRNAGly(CCC)a

5'

3'

D

G5'-primer
G3'-primer

Trailer sequence5'-half
Intron sequence(21 or 27 bp)

T-stretch5'-half

T-stretch

C

U C
G
C
C
U
U
A 73
Gly
tRNA (CCC)b
G1 C
G
C
C
G
G
C
A
U
G3'-primer1 G C
A
G
C
UU G
G GC C CU
C 56
intron 1
G A
a
G
U
C
cg
UG U G
u ac
c cc
C
G
C
AC A U
ua u
G
G
G
C
G
U c
G
U G
U A
U
C
G
55
A
U
A
U
G
C
UU
A
G5'-primer1
38 C
AU
U
A
U
G
C
G
C
C
U
A
A 37
U
A
A
UA
C
G
U
U
U
U
C
G
G
C

5'

3'

E

1
1

U C
G
C
C
U
U
A 73
G1 C
G
C
G
C
G3'-primer1 C G
A
U
C
G
U
G
C
U
A
G GCCC
U
G A
G
G
U
C
C
G
G
G
U
U GU GCC
U 56
GC
A CA U
G
G
U G
U A
G
C
A
U
A
U
C
G5'-primer1 G
A
U
38 U
U

tRNAGly(CCC)a, b
231
194
118
74
(bp)

CI-tRNAGly (Δintron 1)

U

Lane

M 1

37A

C C C

Figure 4 | RT-PCR amplification of intronic and permuted pre-tRNAGly(CCC). (A) Schematic representation of a permuted tRNA gene harboring an
intron; tRNALeu(UAA) and tRNAArg(CCU) in the upper panel, tRNAsGly(CCC) in the lower panel. (B, C) The inferred secondary structures of pretRNAGly(CCC)a and pre-tRNAGly(CCC)b are shown. The sequences of the 59-half (blue) and the 39-half (red) of mature tRNA, and the intervening
sequence (green) of pre-tRNA are shown. Arrowheads indicate the processing positions. Intron sequences are shown in lower case. The numbering of thetRNA positions follows Reference 12. (D) The PCR product of cDNA generated from reverse transcription around (E), a circular intermediate oftRNAGly(CCC) without the intron, is indicated as CI-tRNAGly (Dintron 1). 59- and 39-primers are indicated as solid and broken arrows, respectively. Lane
M: DNA molecular weight marker (WX174/HaeIII).

cmSen2-cmSen54-cmSen34, which interacts with the maturedomain of the pre-tRNA, as it does in yeast. RT-PCR analysis(Figs. 3 and 4) showing that the BHB motif at canonical position
37/38 is always the final substrate may support this hypothesis.
An increasing number of reports on BHB-mediated disrupted
tRNA genes support the hypothesis that the BHB motif, and its
SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

processing system, played a central role in the development andmaintenance of such tRNA genes. An evolutionary relationshiphas been suggested to exist between intronic tRNAs and split
tRNAs in archaea7,19,26,44,45,47, while permuted tRNAs might have been
generated by gene duplication, probably of an intronic tRNA
through recombination to form a tandem repeat, followed by the
6

54
C)
G
AD
n2
,e
S e ( C M mpn3
N2 ty v
4
( C 3 1 C ect
Seorn5 MH ))
4
(C 233
C)
M
K2
AD
54
C)
(p
G
Se
AD
n2
,e
Se (CM mpn3
N2 ty v
4
(C 31C ect
Seorn5 MH ))
4
(C 233
C
M
K2 )
54
C)

C)

K2

33

Se

AD

(p

M

(C

4
n5

(C
Se

Se

n3

4

M

H2

y
pt

31

m

N2

,e

(C

M

AD
G

n2

(p

Se

AD

C)

ve

ct

or

)

www.nature.com/scientificreports

BD (pGBTK, empty vector)
Sen2 (CMN231C)
Sen34 (CMH233C)
Sen54 (CMK254C)

-LW

-LWA

-LWH + 1mM 3AT

Figure 5 | Specific interactions between C. merolae Sen proteins. A specificity test using YTH analysis was carried out on SC plates lacking Leu and Trp(-LW), Leu, Trp and adenine (-LWA), or Leu, Trp and His containing 1 mM 3AT (-LWH 1 1 mM 3AT) with a 4-day incubation. Diploid strains
were constructed by mating PJ69-4Aa cells harboring pGBTK or its derivatives (Baits) with PJ69-4Aa cells harboring pGAD or its derivatives (Preys); each
derivative plasmid contained full-length cmSen2 (CMN231C), cmSen34 (CMH233C), or cmSen54 (CMK254C).

loss of the outer segment of each copy4,6,48,49. The existence of anumber of intronic tRNA genes may reflect an evolutionary background that produced a number of the permuted tRNAs in C. merolae. Even though the detailed mechanisms that govern thedevelopment of each type of disrupted tRNA gene remain unclear,
the discovery of various BHB-mediated disrupted tRNA genes may
imply an evolutionary relationship between them.
Some archaea, including Nanoarchaeum equitans and Caldiviga
maquilingensis, harbor many split tRNAs, but few intronic tRNAs7,22.
Other archaea, including Pyrobaculum and Thermophilum, harbor a
number of intronic tRNAs, although Pyrobaculum have no split or
permuted tRNAs22. Unicellular green algae contain some permuted
tRNAs, but almost no intronic tRNAs6. Hence, C. merolae is uniquein containing a number of both atypical intron-containing and permuted tRNAs. Conversely, split tRNAs have not been found in C.
merolae, despite its potential ability to express them. Hitherto, there
has been no report of an organism in which split tRNAs coexist withpermuted tRNAs. Thus, the individual mechanisms and requisite
elements necessary for the acquisition or maintenance of disrupted
tRNA genes are sometimes substantially different, as previously suggested7. Our findings for C. merolae indicate that the processing
system of eukaryal tRNA introns is more divergent and species-specific than previously thought; atypical introns and several types ofsplicing endonucleases may also be present in other eukaryotes.
Previous studies may support this possibility, as evidenced by thediscovery of ectopic intron-containing tRNA genes in the nucleomorph of the cryptomonad, Guillardia theta50; however, most of
their introns cannot form a BHB motif. The absence of accessory
subunit homologs (Sen15 and Sen54) in the A. thaliana endonuclease51 implies the possibility that plant endonucleases have evolved
variations in their subunit architectures.
Considering that the origin of eukaryotes is related to that ofarchaea, C. merolae may retain vestigial traits derived from thearchaeal lineage. However, no sequence similarity was foundbetween the disrupted tRNA genes of C. merolae and archaea.
Additionally, atypical intron-containing, permuted, and split
tRNAs show a discontinuous and patchy distribution among early
eukaryotes and archaea6,7,22. These observations also imply a nonmonophyletic origin for BHB-mediated disrupted tRNA genes andsuggest the possibility that they could have arisen multiple times,
independently, in each lineage. The next challenge will be to estimate
the formation process of each type of disrupted tRNA gene.
SCIENTIFIC REPORTS | 3 : 2321 | DOI: 10.1038/srep02321

The findings presented here reaffirm the unique nature of C. merolae tRNAs, which display various rearrangement patterns of RNA
gene structures and tRNA processing pathways. Further comparative
analysis of C. merolae tRNA genes and the splicing endonucleasewith those of other organisms will help to clarify the mechanismsthat govern the development of BHB-mediated disrupted tRNA
genes.

Methods
Computational prediction of tRNA genes and their BHB motifs in the C. merolae
nuclear genome. The 10D strain of C. merolae was used in this study1. Genome-wide
prediction of tRNA genes in the C. merolae genome was conducted as described
previously2,4 using tRNAscan-SE52, SPLITS20 and SPLITSX21 software with thefollowing parameters: ‘-X 15 -I -36’, ‘-c -p 0.55 -f 3 -h 3’ and ‘-p 0.55 -f 3 -h 3’,. Initial
predictions of tRNA introns and the secondary structures of splicing motifs weremade by SPLITS/SPLITSX. In addition, a BLAST search was performed using
BLASTN with a threshold E-value , 1. The sequences of candidate genes weremanually inspected.
The free energies of the predicted introns were calculated using RNAeval, implemented within the Vienna-RNA package53, and then matched to the BHB secondary
structure model. The BHB secondary structure model consisted of both intron andexon sequences, and was classified into three types based on the reported archaeal
BHB motifs: strict hBHBh9, relaxed HBh9 or relaxed hBH5. Some secondary structures of the predicted tRNAs harboring BHB motifs were revised by comparing themwith known BHB-harboring tRNA structures.
Northern blotting analysis. Cell culture, RNA purification and northern blotting
analysis followed previously described methods4. Total RNA isolated from C. merolae
was separated on a 10% polyacrylamide gel containing 8 M urea and compared to
RNA molecular weight markers (Dynamarker RNA Low, BioDynamics Laboratory,
Tokyo, Japan). The gel was blotted onto Hybond N1 (GE Healthcare Bio-science AB,
Upssala, Sweden) with TBE buffer for northern blotting analysis. The synthetic DNA
oligonucleotides listed below were 59 32P-labeled and used as probes. Hybridization
was performed at 50uC for 6 h. The membrane was then washed with 1 3 SSC buffer(150 mM NaCl and 15 mM sodium citrate) at room temperature for 40 min. The
predicted size of each mature molecule indicated in Fig. S2 was confirmed bysequencing analysis of the corresponding RT-PCR product (Fig. 3, SupplementaryFigs. 3-5 online). The sequences of synthetic DNA oligonucleotides used as probes arelisted below.
Oligo-DNA probes for northern blot analysis.
N-probe 59- cggtagtagagaggctt -39.
F-probe 59- tgccatcgcgcgggatc -39.
I-probe 59- tactcccggcgaggctc -39.
Reverse-transcription polymerase chain reaction (RT-PCR) and DNA
sequencing. Cell culture, RNA purification and RT-PCR analysis followed previously
described methods4. Total RNA prepared from C. merolae cells was reversetranscribed with ReverTraAce (TOYOBO, Osaka, Japan) and PCR-amplified with
Blend Taq (TOYOBO). Total RNA was denatured for 3 min at 94uC and then7

www.nature.com/scientificreports
incubated on ice for 5 min. Reverse transcription was performed using a reverse
primer (39-primer) at 55uC for 60 min, and 5 mL aliquots of the cDNA solution wereused for PCR (50 mL total reaction mixtures, using a program comprising 25 cycles)
using 59 and 39-primers. PCR products were gel purified and cloned using a TA
cloning kit (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s
protocol. Five clones were sequenced for each product (Supplementary Figs. 3–6
online). Not all of the PCR products could be cloned. The sequences of synthetic DNA
oligonucleotides used as primers for RT-PCR shown in Fig. 3 and Fig. 4 are listed
below.
Oligo-DNA primers for RT-PCR amplification of tRNAAsn(GUU).
N59-1 59- ggtagtatagctcagtcggttag -39.
N39-1 59- cggtagtagagaggcttg -39.
Oligo-DNA primers for RT-PCR amplification of tRNAPhe(GAA).
F59-1 59- gccattgtagctcagcagggag -39.
F39-1 59- tgccatcgcgcgggatc -39.
F59-2 59- gccattgtagctcagcagggagagga -39.
F39-2 59- ccgcggacctttgcatcttca -39.
Oligo-DNA primers for RT-PCR amplification of tRNAIle(UAU).
I59-1 59- actcctgtagct -39.
I39-1 59- tactcccggcgaggctc -39.
I59-2 59- actcctgtagctatcgg -39.
I39-2 59- tactcccggcgaggctcgaacgcgca -39.
I59-3 59- actcctgtagctcagctggt -39.
Oligo-DNA primers for RT-PCR amplification of tRNAGly(CCC)a andtRNAGly(CCC)b.
G59-1 59- tggaagattcccattcttct -39.
G39-1 59- cactacaccagcagcgc -39.
Yeast two-hybrid screening of the C. merolae tRNA-splicing endonuclease. The
YTH analysis used for library screening was conducted according to a previously
described method54. The full-length cDNA of cmSen2 (CMN231C), cmSen34(CMH233C), or cmSen54 (CMK254C) was PCR-amplified and cloned into pGBTK,a GAL4 DNA-binding domain fusion vector. The C. merolae genomic library used forscreening was constructed using in the pACT2 plasmid (TaKaRa). Plasmids wereintroduced into yeast PJ69-4Aa (for the pGBTK derivatives) and PJ69-4Aa (for the
pACT2 derivatives) haploid strains, using TRP1 and LEU2, respectively, as selective
markers. These yeast strains were co-incubated and became diploids. Positive
protein–protein interactions between bait and prey were detected by the ability ofcells to grow on SC-LWH plates containing 5 mM 3-aminotriazol and SC-LWA. The
pACT2 derivatives were extracted from the diploid yeast and the insert junctions with
the GAL4 activation domain were sequenced and compared with the C. merolae
genome database. The synthetic DNA oligonucleotides used as primers for cloning
are listed below.
Oligo-DNA primers used for cloning the C. merolae Sen2 homolog (cmSen2;
CMN231C).
cmSen2Fwd 59- ggggaattcatgagcgttcaagcgcaact -39.
cmSen2Rev 59- gggggatccctacttcgcggcacgctgtt -39.
Oligo-DNA primers used for cloning the C. merolae Sen34 homolog (cmSen34;
CMH233C).
cmSen34Fwd 59- ccccccggggatgtatccaagtgtccagag -39.
cmSen34Rev 59- gggggggtcgactcagggtagagcctcacacc -39.
Oligo-DNA primers used for cloning the C. merolae Sen54 homolog (cmSen54;
CMK254C).
cmSen54Fwd 59- ggggaattcatgcgaaatcagcggggtgg -39.
cmSen54Rev 59- gggggatcctcacagtccctgctcgtcgt -39.
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Acknowledgments
We thank A. Muto and S. Maruyama for valuable discussions; K. Tanaka, O. Misumi, M.
Matsuzaki, Y. Yoshida, N. Sato, T. Fujiwara and M. Monma for technical advice; and R.
Saito, Y. Ohashi and K. Kurokawa for advice on the computational analysis. This work was
supported by a grant from the Nakajima Foundation (to A.S.), a Grant-in-Aid for Young
Scientists B (23770211 to A.S.), the Frontier project ‘Adaptation and Evolution ofExtremophiles’ (to Y.S.) and the Strategic Research Foundation Grant-aided Project forPrivate Universities (S1201003 to Y.S.) from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.

Author contributions
Y.S., A.K., T.K. and A.S. designed the research. A.S., J.S., A.O., S.W., N.Y. and M.O.
performed the research. A.S., J.S., A.O., N.Y., H.Y. and H.K. analyzed the data. A.S., N.Y.,
S.W., A.K., J.S., A.O. and Y.S. wrote the main manuscript text. A.S. prepared Figures 1–4,
Figures S1–7 and Table S1. J.S. and A.O. prepared Figures 2 and S1. S.W. prepared Figure 5.
All authors reviewed and approved the manuscript.

Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Soma, A. et al. Identification of highly-disrupted tRNA genes innuclear genome of the red algae, Cyanidioschyzon merolae 10D. Sci. Rep. 3, 2321;
DOI:10.1038/srep02321 (2013).
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