Permuted tRNA Genes Expressed via a Circular
RNA Intermediate in Cyanidioschyzon merolae
Akiko Soma, et al.
Science 318, 450 (2007);
DOI: 10.1126/science.1145718
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CORRECTED 30 NOVEMBER AND 21 DECEMBER 2007; SEE LAST 2 PAGES

Fig. 4. RA treatment results in recruitment of the
UTX/MLL complex, concomitant with decreased levelsof trimethyl H3K27 and increased levels of trimethyl
H3K4. (A to T) UTX occupancy(A to D), SUZ12 occupancy (Eto H), trimethyl H3K27 levels(I to L), trimethyl H3K4 levels(M to P), and ASH2L occupancy (Q to T) at HOXA1, HOXA3,
HOXB1, and HOXB2 genes
were analyzed by a qChIP
assay. Data are presented as
the mean ± SEM (error bars)(n ≥ 3 except in the case of
ASH2L, where n = 2). The
relative occupancy represents the fold change in the percent of input over the control (28). The valuesof percent input set as 1 in (A) to (T) are as follows: (A), 0.0028%; (B), 0.0012%; (C), 0.003%; (D),0.004%; (E), 0.046%; (F), 0.031%; (G), 0.099%; (H), 0.035%; (I), 0.039%; (J), 0.16%; (K), 0.02%;(L), 0.022%; (M), 2.47%; (N), 6.3%; (O), 1.11%; (P), 4.96%; (Q), 0.1%; (R), 0.04%; (S), 0.04%;
and (T), 0.07%.

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28. Materials and methods are available as supporting
material on Science Online.
29. We thank A. Shilatifard and T. Jenuwein for their antibodies;
Kazusa DNA Research Institute Foundation for UTX cDNA;
M. Hart, T. Beer, K. Speicher, and D. Speicher for help withmass spectrometry; G. Harris for figure preparation; andH. Hoff and D. Schultz for protein expression. L.D.C. was
supported by a grant from the Spanish–Ministerio de
Educación y Cultura and R.S. was supported by a grant from

NIH (RO1CA090758) and an intramural fund from Centre
de Regulació Genòmica.

Supporting Online Materialwww.sciencemag.org/cgi/content/full/1149042/DC1
Materials and Methods
Figs. S1 to S9Table S1
References
5 June 2007; accepted 20 August 2007
Published online 30 August 2007;
10.1126/science.1149042
Include this information when citing this paper.

that is insufficient to decode all 61 codons
Permuted tRNA Genes Expressed via a number(2). This prominent paucity of tRNA genes
prompted us to search for undiscovered tRNA
genes that may elucidate the evolution of tRNAs
Circular RNA Intermediate inin early eukaryotes.
To search for C. merolae nuclear tRNA
Cyanidioschyzon merolae
genes, we used SPLITS and SPLITSX, new proAkiko Soma,1 Akinori Onodera,1 Junichi Sugahara,2,3 Akio Kanai,2,3,4 Nozomu Yachie,2,3Masaru Tomita,2,3,4 Fujio Kawamura,1,5 Yasuhiko Sekine1,5*
A computational analysis of the nuclear genome of a red alga, Cyanidioschyzon merolae, identified
11 transfer RNA (tRNA) genes in which the 3′ half of the tRNA lies upstream of the 5′ half inthe genome. We verified that these genes are expressed and produce mature tRNAs that areaminoacylated. Analysis of tRNA-processing intermediates for these genes indicates an unusual
processing pathway in which the termini of the tRNA precursor are ligated, resulting in formationof a characteristic circular RNA intermediate that is then processed at the acceptor stem togenerate the correct termini.
yanidioschyzon merolae is an ultrasmall
unicellular red alga that inhabits an extreme environment (1). The completesequence of the nuclear genome of C. merolae
recently became available (2). Genome-wide
analyses (1, 2) and a molecular phylogenetic

C

450

analysis (3) have demonstrated that this organism
is likely to represent one of the most ancestralforms of eukaryote. A search for tRNA genes
from the C. merolae nuclear genome, using thetRNAscan-SE program (4), predicted only 30
tRNA genes encoding 30 species of anticodon, a

19 OCTOBER 2007

VOL 318SCIENCEgrams that can detect cis-spliced tRNAs containing introns in various positions and trans-spliced
tRNAs that are joined at several positions (5–7).
In addition to this analysis, we performed a
BLAST search of tRNA genes with conserved
sequences in the TYC arm or the anticodon arm,followed by manual inspection of the results.
1
Department of Life Science, College of Science, Rikkyo (St.
Paul’s) University, Toshima, Tokyo 171-8501, Japan. 2Institutefor Advanced Biosciences, Keio University, Tsuruoka, 997-0017,
Japan. 3Systems Biology Program, Graduate School of Mediaand Governance, Keio University, Fujisawa, 252-8520, Japan.
4
Faculty of Environment and Information Studies, Keio University, Fujisawa, 252-8520, Japan. 5Research Information
Center for Extremophile, Rikkyo (St. Paul’s) University, Toshima,
Tokyo 171-8501, Japan.

*To whom correspondence should be addressed. E-mail:ysekine@rikkyo.ac.jp

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REPORTSREPORTS
the intragenic bipartite promoter consisting of an
A box and a B box, which are conserved sequences in the D arm and TYC arm (9), theupstream TATA-like sequence may play a central
role in initiation of transcription in C. merolae.
The genomic sequence encoding the intervening
sequence between the 3′ and 5′ halves varies
from 7 to 74 base pairs (bp). Downstream of the
5′ half, a T stretch that corresponds to a termination signal for RNA polymerase III (pol III)is found (10). These observations suggest that thepair of putative tRNA halves is transcribed as alinear RNA. As shown in Fig. 1B and fig. S2,
permuted tRNA genes can be classified into fourtypes on the basis of the location of the junction
between the 3′ end of the 5′ half and the 5′ end of
the 3′ half in the inferred secondary structures of
the tRNAs. The junctions are located at 20/21(between position 20 and 21) in the D loop (I),37/38 in the anticodon loop (II), 50/51 in the
TYC stem (III), and 59/60 in the TYC loop (IV).
We identified one, six, one, and three candidates

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The most important finding was that 11 genes
have a novel gene organization in which the
3′ half of the tRNA sequence lies upstream of the
5′ half in the genome (Fig. 1A and fig. S1). Sucha gene arrangement is accomplished by circulargene permutation (8), and we therefore termed
these genes permuted tRNA genes. As shown infig. S1, a TATA-like sequence is found upstreamof the 3′ half in most of the genes. This sequenceis also conserved in nonpermuted tRNA genes of
the C. merolae nuclear genome. Thus, instead ofFig. 1. Permuted tRNA genes in C. merolae. (A) Schematic representation of a
permuted tRNA gene. (B) Inferred secondary structures for pre-tRNAs of four typesof permuted tRNA genes. Arrowheads indicate the positions to be processed. The
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SCIENCE

intron sequence is shown in lower case. The numbering of the tRNA positions isaccording to (19). Single-letter abbreviations for the amino acid residues are as
follows: A, Ala; G, Gly; K, Lys; L, Leu; M, Met; Q, Gln; R, Arg; and T, Thr.
VOL 318

19 OCTOBER 2007451

for the type I to IV tRNAs, respectively. Notably,
the sequences adjacent to those junctions intRNA precursors (pre-tRNAs) potentially form
bulge-helix-bulge (BHB) motifs, which wereoriginally found around the intron-exon junctionsof nuclear and archaeal tRNAs (11).

Northern blot analysis using C. merolae total
RNA verified that permuted tRNA genes areindeed transcribed in the cell (fig. S3). Next, toexamine whether these tRNAs are aminoacylated, we performed Northern blot analysis ofacid-urea gels with use of total RNA prepared

Fig. 2. Aminoacylation
analysis of tRNAiMet(CAU).
Total C. merolae RNA prepared under acidic conditions
was separated on an acidurea gel directly (lane 1) orafter deacylation (lane 2). In
vitro methionylation reaction
mixtures were loaded onto
the gel (lanes 3 to 9).
Deacylated total RNA (lanes
3 to 5) or in vitro transcribed tRNAiMet with [wild type (WT), lanes 6 and 7] or without (DCCA, lanes 8 and 9)
the CCA sequence was used.

Fig. 3. RT-PCR amplification of tRNAGln(CUG) and a model for the maturation of the permuted pretRNAs. (A) 5′ and 3′ primers used for RT-PCR are indicated as solid and broken arrows, respectively.
(B) PCR products amplified from cDNA of pre-tRNAGln and from cDNA generated from one (×1) or
two (×2) rounds of reverse transcription around a circular RNA intermediate or a circularized
mature tRNAGln are indicated. Lane M, DNA molecular weight markers (F×174 DNA-Hae III digest).
(C) Maturation of the pre-tRNA starts with processing of a BHB motif (boxed) by the tRNA-splicing
machinery, resulting in formation of a circular RNA intermediate. The intervening sequence isremoved by RNase P and tRNase Z, then followed by the CCA addition.

452

19 OCTOBER 2007

VOL 318SCIENCEfrom C. merolae cells under acidic conditions.
Putative aminoacylated forms, which migrate
more slowly than the deacylated forms, weredetected, showing that these tRNAs could beaminoacylated in vivo (Fig. 2, lanes 1 and 2, andfig. S4). An in vitro aminoacylation analysis
showed that recombinant C. merolae methionyltRNA synthetase (MetRS) methionylates tRNAiMet
in total RNA preparations (Fig. 2, lanes 3 to 5).
An in vitro transcribed tRNAiMet with the 3′ terminal CCA sequence was methionylated, although with less efficiency (Fig. 2, lanes 6 to 9).
Thus, tRNA molecules expressed from permutedtRNA genes are aminoacylated and are likely toparticipate in protein synthesis.
What is the processing mechanism of the pretRNA for these unusual tRNA genes? To clarify
the processing pathway, we detected processing
intermediates by reverse transcription polymerase chain reaction (RT-PCR) with two different
sets of primers (Fig. 3A), followed by sequencing
analysis of those RT-PCR products (Fig. 3B andfig. S5). Analysis of the tRNAGln(CUG) verified
the sequences of the pre-tRNAGln with a circularly permuted structure in which the leader
sequence, the 3′ half, the intervening sequence,the 5′ half, and the trailer sequence were alignedin this order (Fig. 3B, lane 1, and fig. S5A).
Interestingly, we also detected a circular RNA
intermediate in which the leader and trailer
sequences were removed and the resulting ends
were ligated, while the intervening sequence wasretained (Fig. 3B, lane 2, and fig. S5B). The
existence of the circular RNA intermediate wasconfirmed by the generation of a PCR productrepresenting two rounds of reverse transcription
around the circular RNA (Fig. 3B, lane 2). The
3′-terminal CCA sequence is added posttranscriptionally in eukaryotes (12). To determine theterminal sequence of the mature tRNAGln, we
performed RT-PCR with total RNA circularizedby T4 RNA ligase. The sequence of the maturetRNAGln, in which the extra sequences are
removed and the CCA sequence is added to the
3′ terminus of the acceptor stem, was verified(Fig. 3B, lane 3, and fig. S5C). As summarized in
the model presented in Fig. 3C, maturation of the
pre-tRNA probably starts with processing of theleader and trailer sequences, resulting in formation of the circular RNA intermediate. This
processing step is most likely carried out by thetRNA-splicing machinery because the sequencesadjoining the processing sites potentially form a
BHB motif, which is the dominant recognition
element for nuclear and archaeal tRNA-splicing
endonucleases (11). The intervening sequence isthen removed, possibly by ribonuclease (RNase)
P and tRNase Z (13, 14), followed by the CCA
addition, to generate the correct termini. Thismodel would be common to the permuted tRNA
genes, because the circular RNA intermediate
was detected for all 11 genes.
How could permuted tRNA genes havearisen? Permuted noncoding RNA (ncRNA)
genes have been reported for Trypanosoma

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REPORTSREPORTStRNA genes might have developed via acommon process with the split-tRNA genes of
Nanoarchaeum equitans (18). Further investigation should provide a hint about how to evaluate
the evolution of tRNA genes in the early
eukaryote.
References and Notes
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Trojan Horse Strategy inAgrobacterium Transformation:
Abusing MAPK Defense Signaling
Armin Djamei,1*† Andrea Pitzschke,1† Hirofumi Nakagami,1†‡ Iva Rajh,1 Heribert Hirt1,2
Nuclear import of transfer DNA (T-DNA) is a central event in Agrobacterium transformation ofplant cells and is thought to occur by the hijacking of certain host cell proteins. The T-DNA–
associated virulence protein VirE2 mediates this process by binding to the nuclear importmachinery via the host cell factor VIP1, whose role in plants has been so far unknown. Here weshow that VIP1 is a transcription factor that is a direct target of the Agrobacterium-induced
mitogen-activated protein kinase (MAPK) MPK3. Upon phosphorylation by MPK3, VIP1 relocalizes
from the cytoplasm to the nucleus and regulates the expression of the PR1 pathogenesis-related
gene. MAPK-dependent phosphorylation of VIP1 is necessary for VIP1-mediated Agrobacterium
T-DNA transfer, indicating that Agrobacterium abuses the MAPK-targeted VIP1 defense signaling
pathway for nuclear delivery of the T-DNA complex as a Trojan horse.
igher eukaryotes recognize microbes
through pathogen-associated molecular patterns (PAMPs) and activate theinnate immune response in animals and plants(1). The perception of PAMPs leads to rapid activation of host defense mechanisms, including the activation of mitogen-activated protein

H1

Department of Plant Molecular Biology, Max F. Perutz
Laboratories, University of Vienna, Dr.-Bohr-Gasse 9, 1030
Vienna, Austria. 2URGV Plant Genomics Laboratory, 2 Rue
Gaston Crémieux, 91057 Evry, France.
*Present address: Department of Organismic Interactions,
Max-Planck-Institut für terrestrische Mikrobiologie, Karlvon-Frisch-Strasse, 35043 Marburg, Germany.
†These authors contributed equally to this work.
‡Present address: Plant Immunity Research Team, RIKEN Plant
Science Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 2300045, Japan.

kinases (MAPKs), production of reactive oxygen species, and subsequent induction of defenserelated genes. However, virulence factors ofsuccessful pathogens can inhibit PAMP-elicitedbasal defenses (2). In specific cases, plants have
evolved resistance proteins specialized to detect these pathogen-derived virulence factors
or their effects on host targets. As a consequence, a hypersensitive response (HR) occursthat includes localized cell death and the arrestof pathogen spread (3, 4).
Agrobacterium transforms plants by transporting a single-stranded copy of the transfer
DNA (T-DNA) from its tumor-inducing Ti plasmid into the host cell and integrating it into thehost cell genome. The agrobacterial transformation process is mediated by Vir (virulence) proteins. The T-DNA strand that is exported into

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SCIENCEVOL 318

14. H. Schurer, S. Schiffer, A. Marchfelder, M. Morl, Biol.
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(2005).
20. Supported by Japan Society of the Promotion of Science(JSPS) Fellowships (7473 to A.S. and 1924 to N.Y.), byGrants-in-Aid for Creative Scientific Research (17GS0314
to Y.S. and 16GS0304 to F.K.) from JSPS and Ministry of
Education, Culture, Sports, Science and Technology,
respectively, by grants from the Yamagata Prefectural
Government and Tsuruoka City in Japan (toM.T., A.K., J.S., and N.Y.), and by the Frontier project
Adaptation and Evolution of Extremophile (to Y.S.).

Supporting Online Materialwww.sciencemag.org/cgi/content/full/318/5849/450/DC1
Materials and Methods
Figs. S1 to S5
References and Notes

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mitochondrial small subunit (SSU) ribosomal
RNA (rRNA) (15) and a bacterial transfer
messenger RNA (tmRNA) (16) that function ina two-piece form, in contrast to the C. merolaetRNAs reported here that function in a one-pieceform. The permuted rRNA and tmRNA genes
are hypothesized to have arisen by a gene
duplication that formed a tandem repeat,followed by the loss of the outer segment ofeach copy (15, 17). Even if a circular permutation
occurred in tRNA genes, most of the resulting
permuted genes would not be retained in the
genome because of the failure of expression or
the loss of functional structure of the RNA. In C.
merolae, however, permuted tRNA genes might
have persisted in the genome because of the useof the upstream promoter by the transcription
system and processing of the circularly permuted
pre-tRNA into a canonical tRNA molecule by thetRNA-splicing machinery. Considering that C.
merolae is an early rooted eukaryote and that the
BHB motifs would play a pivotal role in thetRNA processing, it is possible that the permuted

29 May 2007; accepted 31 August 2007
10.1126/science.1145718

plant cells has the VirD2 protein covalently attached to its 5′ end. The VirE2 protein, which istranslocated into plant cells independently of the
T-DNA strand, associates with the T strand in
the plant cell (5) before nuclear import mediatedby cellular karyopherin a, which binds to VirD2.
Nuclear import is further facilitated by the hostprotein VIP1, which functions as an adaptor (6),but whose cellular function is as yet unknown.
PAMPs activate a variety of plant MAPK
cascades (7), which make a major contribution to the host defense responses. In Arabidopsis thaliana, PAMPs such as flagellin activate
at least three MAPKs—MPK3, MPK4, and
MPK6—resulting in altered expression of various stress-responsive genes (8–10). So far, thedirect downstream targets of these plant MAPKs
are largely unknown. In this study, we showthat Agrobacterium triggers the activation of several MAPKs, including MPK3, and we identify
VIP1 as a target of MPK3.
A yeast two-hybrid screen was performed
in order to find interactors with MPK3. Clones
carrying full-length cDNAs of VIP1 (VirE2interacting protein At1g43700) (11) were repeatedly isolated. The VIP1-MPK3 interaction in yeast
was confirmed by its ability to induce anotherreporter gene, lacZ, encoding for b-galactosidase(Fig. 1A). In order to test whether VIP1 alsointeracts with other MAPKs, targeted yeast twohybrid interaction experiments were performed
with VIP1 and representatives of all the A. thaliana
MAPK subfamilies (MPK2, -3, -4, -5, -6, -7, -16,and -17). VIP1-MAPK interaction was specificfor MPK3, because neither MPK6, the closest
homolog of MPK3, nor any of the other MAPKs
tested interacted with VIP1 (Fig. 1A).
To substantiate these results, we transiently expressed MPK3 and VIP1 in Arabidopsis

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Reports: “Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon
merolae” by A. Soma et al. (19 October, p. 450). The last sentence on p. 452 referred to anincorrect subunit. The sentence should begin “Permuted noncoding RNA (ncRNA) genes havebeen reported for Tetrahymena mitochondrial large subunit (LSU) ribosomal RNA (rRNA)(15).”

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30 NOVEMBER 2007

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CORRECTIONS & CLARIFICATIONSERRATUM

Post date 21 December 2007

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Reports: “Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon
merolae” by A. Soma et al. (19 October 2007, p. 450). The last sentence on p. 452 referred to
an incorrect subunit. The sentence should begin “Permuted noncoding RNA (ncRNA) genes
have been reported for Tetrahymena mitochondrial large subunit (LSU) ribosomal RNA (rRNA)(15)…”

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21 DECEMBER 2007

1