Unique tRNA Introns of an Enslaved Algal Cell
Oliver Kawach, Christine Voß, Jonci Wolff, Katalin Hadfi, Uwe-G. Maier, and Stefan Zauner
Cell Biology, Philipps-University Marburg, Karl-von-Frisch Staße 8, 35032 Marburg, Germany
Nucleomorphs are remnant nuclei of eukaryotic, secondary endosymbionts exclusively found in cryptophytes and chlorarachniophytes. The nucleomorph of the cryptophyte Guillardia theta codes for 36 transfer RNA (tRNA) genes, 15 of them
predicted to contain introns and 1 pseudo-tRNA. Some of the predicted intervening sequences are manifested at positions
not known in Eukarya, even tRNAs with more than one intron were suggested. By isolating reverse transcriptase–
polymerase chain reaction products of the spliced tRNAs we verify the processing of all predicted intron-harboring tRNAs
and demonstrate the splicing of the smallest introns (3 nt) investigated so far. However, the spliced intervening sequences
are in some cases shifted in respect to the predicted ones. Moreover, we show that introns, if inserted into the B-box
of tRNA genes in the nucleomorph of cryptophytes, mimic promoter regions and do not abolish transcription by RNA
polymerase III. Consequently, internal nucleomorph-encoded tRNA promoter regions are in some cases dissected from
the sequence of the mature tRNAs. By reanalyzing tRNA introns of a recently sequenced red algae we furthermore show
that splicing of introns at unusual positions may be introduced in cryptophytes by its secondary endosymbiont. However,
in contrast to the rest of the symbiont genome, introns are not minimized in quantity but are instead scattered along the
tRNA genes.
Introduction
It is generally accepted that the genetic system of
eukaryotes is related to that of Archaea and seems to be different from the transcription-translation machinery of Bacteria (Martin and Müller 1998; Martin et al. 2001). Such
a difference can be observed in the splicing mechanisms
of transfer RNA (tRNA) introns as well. These types of
introns are common in all three domains, Bacteria, Archaea,
and Eukarya (Marck and Grosjean 2002), but Bacteria
evolved self-splicing tRNA introns, whereas tRNA introns
from Archaea and Eukarya were processed in an enzymatic
catalyzed reaction (Abelson, Trotta, and Li 1998). tRNA
introns, if present, could be located at different positions
in Archaea (Belfort and Weiner 1997) and are restricted
to a conserved position between the first and second base
3# of the anticodon in Eukarya (Marck and Grosjean
2002) (fig. 1). Moreover, as recently reported, red algae
may encode tRNAs with introns, which are predicted at
positions outside the conserved position near the anticodon
(Matsuzaki et al. 2004) (fig. 1). In Bacteria self-splicing
group I introns are described, which are located 3# of the
anticodon or within the anticodon (Kuhsel, Strickland,
and Palmer 1990; Xu et al. 1990; Reinhold-Hurek and Shub
1992; Biniszkiewicz, Cesnaviciene, and Shub 1994) (fig. 1).
Several algae evolved by the enslavement of a phototrophic eukaryotic cell by another, except heterotrophic eukaryote (secondary endosymbiosis) (Delwiche and Palmer
1998; McFadden 1999; Maier, Douglas, and Cavalier-Smith
2000). During the evolution of a ‘‘cell within a cell,’’ the symbiont organelles were reduced or eliminated. As in some secondary evolved algae the reduction led to a plastid surrounded
by additional membranes, and an intermediate stage in the
reduction of the symbiont organelles is found in cryptophytes
and chlorarachniophytes (Cavalier-Smith 2002; Stoebe and
Maier 2002). Here a remnant cytoplasm of the eukaryotic
symbiont including its pigmy nucleus, the nucleomorph, is
maintained. The nucleomorph genome of one cryptomonad,
Key words: Guillardia theta, Cyanidioschyzon merolae, tRNA,
nucleomorph, secondary endosymbiosis.
E-mail: [email protected].
Mol. Biol. Evol. 22(8):1694–1701. 2005
doi:10.1093/molbev/msi161
Advance Access publication May 4, 2005
Ó The Author 2005. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
Guillardia theta, was sequenced, thereby lightning up a reduced eukaryotic genome organized in three small chromosomes (Zauner et al. 2000; Douglas et al. 2001). This
miniaturized genome is maintained to encode genes whose
products have important functions in the plastid. To synthesize these functions, a transcription and translation apparatus
has to be expressed in the secondary symbiont cytoplasm.
By scanning the nucleomorph genomic sequence for
tRNA-encoding genes, we showed that 35 transfer DNAs
(tDNAs) should be encoded by the nucleomorph (Douglas
et al. 2001). However, the nucleomorph should not be
autonomous in respect to tRNAs due to a missing tRNA
for glutamate (Douglas et al. 2001). In addition, we described one tRNA intron, located at a position otherwise
not known from Eukarya (Zauner et al. 2000).
In the paper at hand, we show that splicing of tRNA
introns, which are unique for eukaryotic organisms in respect to its localization, lead to bona fide tRNAs. In this
respect, splicing of tRNA introns, located 3# or 5# of the
anticodon, in the variable loop, TwC-loop, or D-loop, is
demonstrated. Moreover, we demonstrate splicing of the
smallest eukaryotic introns (3 nt) and show that insertion
in the internal promoter regions of tRNA genes do not abolish the transcription and splicing of tRNAs. Our data indicate that the ability to splice tRNA introns located at unique
positions within the nucleomorph genome was most likely
the legacy of the red algae progenitor. However, in the
nucleomorph genome tRNA introns were shortened, but
in contrast to other noncoding chromosomal elements,
spread over most possible tRNA gene localizations, and
contradict therefore the general tendency of streamlining
the nucleomorph genome.
Materials and Methods
Reverse Transcriptase–Polymerase Chain Reaction and
Sequencing
RNA from G. theta was isolated and reverse
transcriptase–polymerase chain (RT-PCR) reactions were
done according to manufacturer’s instructions (MBI
Fermentas, St. Leon-Rot, Germany). Primer sequences are
given in supplementary table 1 (Supplementary Material online). RT-PCR products were isolated from 17% denaturing
tRNAs of cryptomonads
1695
FIG. 1.—Comparison of intron positions in the nucleomorph, other eukaryotes, Bacteria, and Archaea. Arrows show intron positions. The thick arrow
in the scheme of nucleomorph tRNA indicates the region from which a ‘‘variable loop’’ is removed by splicing. Data for the intron positions of Archaeaspecific introns are from Marck and Grosjean (2002); for eubacterial group I introns see text.
polyacrylamid gels and cloned into pGEM-TÒ (Promega,
Mannheim, Germany). Individual clones were sequenced
on a LI-COR 4200 sequencer using labeled M13-20 standard primers. In some cases, we detected additional splicing
variants harboring insertions-deletions as well as exchanges
of bases (‘‘editing’’). In the case in which the variants were
isolated only one time or show no secondary structure specific for tRNAs, they were not included in our analyses.
Instead, they were considered as PCR artifacts or as improper transcripts, which were detected in nucleomorphspecific messenger RNAs (mRNAs) in another study as well
(Fraunholz, Moerschel, and Maier 1998).
tDNA Detection and Intron Prediction
tDNA detection and intron prediction were done
with tRNAscan-SE provided at http://www.genetics.wustl.
edu/eddy/tRNAscan-SE/ using relaxed parameters (cove
only). tDNA promotor prediction was performed with
POLIIISCAN provided at http://wwwmgs.bionet.nsc.ru/
mgs/programs/poliiiscan/.
Characterization of Cotranscription
Cotranscription of tRNA genes with upstream-located
genes was identified in the case of hlip-tRNA LeuUAG
and uce-E2-tRNA PheGAA in expressed sequence tag
clones. Two further cotranscribed tRNAs were isolated by
RT-PCR experiments. For these, specific primers for the
upstream-located gene (snrpD and tcpZ1) and for the
tRNA genes (tRNA ValUAC and tRNA IleUAU) were used
for RT-PCR experiments, respectively.
To isolate complementary DNAs (cDNAs) covering
hlip (high light-induced protein), our cDNA library was
screened with a gene-specific probe. Positive clones were
sequenced as described above.
Results
The Nucleomorph Is Expected to Encode tRNAs with
Unique Introns
As predicted by tRNAscan-SE (Lowe and Eddy
1997), 36 tRNAs and 1 related pseudogene should be
encoded in the nucleomorph genome of G. theta. This lead
to a set of tRNAs offering all amino acids with the exception of a tRNA glutamate (Glu, E), which seems to be missing in the nucleomorph genome of G. theta (Douglas et al.
2001). The predicted tRNAs show proper anticodons
flanked 5# by a T and 3# by a purine, as it is common
in eukaryotes except for initiator tDNA iMetCAU in higher
eukaryotes (Marck and Grosjean 2002).
As in other eukaryotes, the tRNA genes (tDNAs) are
dispersed in the nucleomorph genome and not clustered as
1696 Kawach et al.
FIG. 2.—Sequence of tRNA genes and processed tRNAs. The sequence of tRNA genes (tDNAs) and the processed tRNAs are shown. The first line in
the columns indicates the tDNA. Green positions show the B-box, blue the A-box, and red the anticodon. The predicted introns are shown in bold letters.
Value of the A- and B-box refers to the F value predicted by POLIIISCAN. (i) 5 intermediate form; (i2) 5 second intermediate form. tRNAs are indicted
in the three-letter code. The red colored A refers to the appearance of a tRNA GluUUC transcript with an additional A at position 53. (See also supplementary table 2, Supplementary Material online.)
it is found predominately in bacteria (Marck and Grosjean
2002). Furthermore, 3#-CAA is not part of the tDNA
(Tomita and Weiner 2001), and A- and B-boxes, representing the internal promoter, can be identified in the predicted
tDNAs (Spargue 1995) (fig. 2).
Further on tRNAscan-SE (Lowe and Eddy 1997) predict introns at the highly conserved position between the
first two bases 3# of the anticodon in eukaryotes. In addition
the prediction program indicates introns at unusual positions (fig. 2). Out of the 36 nucleomorph-encoded tRNAs,
15 were predicted to encode at least one intervening sequence. These potential nonsplicesomal introns vary in
length from only 3 up to 24 nt (fig. 2). Some of them would
be unique for the domain Eukarya in respect to its localization and should emphasize the unique nature of some
nucleomorph-encoded tRNA genes: seven of the tDNA
introns are predicted to be located in the conserved position
between the first two bases 3# of the anticodon, seven in the
D-loop, and eight in the TwC-loop (fig. 1). Interestingly, six
tDNAs should encode pre-tRNAs with more than one intron. Five of them are predicted to harbor one intron in
the D- and another one in TwC-loop, which would represent
a unique arrangement of intron insertions into a eukaryotic
tDNA (fig. 1). Surprisingly, some of the introns are inserted
into the B-box of tRNA genes, which would interrupt the
promotor structure and therefore suppress transcription of
these genes by RNA polymerase III (fig. 2).
Furthermore, nucleomorph chromosomes are built up
of terminal repeats bordering a single-copy region (Zauner
et al. 2000). Housekeeping genes located within the singlecopy regions show an average A/T content of 77%. The A/T
content of the tDNAs is remarkably lower (55%), reflecting
the structural constrains of the two- and three-dimensional
structures of the tRNAs. On the other hand A/T content
is increasing in the predicted tRNA introns significantly
(81% A/T), which shows the preference of A/T in noncoding regions.
tRNAs of the Symbiont of Cryptophytes are
Encoded in the Nucleomorph
As the extremely condensed nucleomorph genome of
the cryptophyte G. theta has only a limited coding capacity
(Douglas et al. 2001), it is possible that tRNAs are imported
from the host compartments to supplement missing functions as it is known from other organisms (Tan et al.
2002; Esseiva et al. 2004). In order to prove if tRNAs of
the secondary endosymbiont originate exclusively from
nucleomorph genes or from a second gene located in the
cell nucleus, we amplified exemplarily four tRNA genes
(tDNAs for AlaUGC, CysACA, LeuUAG, and PheGAA) from
genomic DNA of G. theta. As the tDNAs for AlaUGC are
predicted to encode one intron and the genes for tRNA
CysACA and PheGAA even two (see later), primers were
designed such that neither a tDNA with nor a possible
nuclear-encoded tDNA without the predicted intron(s) is
favored in PCR reactions. These experiments generated
solely nucleomorph-specific amplificates thereby demonstrating that only nucleomorph-specific tDNA serves as
matrix for spliced tRNAs. As, therefore, no indications
for a nucleus-encoded symbiont-specific tRNA exist, our
experiments exclude an import of nucleus-encoded tRNAs
into the symbiont in the analyzed cases.
Introns of the Anticodon Loop are Spliced by the
Secondary Endosymbiont
In order to prove whether the eukaryotic symbiont of
cryptophytes is able to excise the predicted introns or the
tRNAs of cryptomonads
1697
FIG. 3.—Scheme of conserved tRNA bases found in the processed nucleomorph-specific tRNAs. Clamps indicate the predicted internal base pairing
in the tertiary structure. Lines 1–3: conventionally used numbering system for tRNA positions. Line 4: secondary structure of the tRNA cloverleaf model.
Solid boxes indicate the anticodon (AC). V: variable loop positions 44–48 (X: UGAGGUAAUCA; X1: GGUUACAACC; X2: GGGUACUACCU). The
putative intron in MetCAU not found to be spliced is indicated as A*(UUGUACGCG).
intron-harboring tDNAs are pseudogenes, we performed
RT-PCRs. As control, tRNA ArgUCG and tRNA GlnCUG,
whose tDNAs show no evidence for introns, were amplified
from G. theta total RNA. As expected, both tRNAs are collinear with the genomic sequence (fig. 2). Next, we amplified the nucleomorph tRNAs ArgCCU, ArgUCU, TrpCCA, and
TyrGUA (fig. 2). The tDNAs possess a predicted intron at the
conserved position between the first two bases 3# of the
anticodon. The RT-products confirmed that introns are encoded in the tDNAs and that they are removed in the mature
tRNAs. The predicted localization of the introns concurs
with the obtained RT-products in the tRNAs ArgUCU and
TrpCCA. Surprisingly, the introns of ArgCCU and TyrGUA
are shifted downstream in respect to its predictions. In
the case of ArgCCU the intron is also extended for four
bases (fig. 2). Therefore, introns in the tDNAs for ArgCCU
and TyrGUA are inserted at positions otherwise not known
from Eukarya. However, the mature tRNAs show bona fide
anticodons as well as the conserved sides for the threedimensional structure (fig. 3). Interestingly, the anticodon
stem in tRNAs ArgCCU and TyrGUA shows one mismatch.
At the moment, we cannot decide whether this secondary
structure leads to a nucleomorph-specific functional tRNA
or is a characteristic of nonfunctional tRNA.
Pseudogenes, the Missing tRNA for Glutamate and
Introns 5# of the Anticodon
In nucleomorph chromosome III one stretch of bases is
predicted to encode a pseudogene with some similarities to
a potential tDNA (fig. 2). No anticodon was predicted precisely in this genomic region, but we noticed that conserved
bases necessary for three-dimensional folding as well as
possible A- and B-boxes for transcription initiation are
present. Characterization of the maturated transcript of this
region showed a bona fide tRNA LeuUAA (fig. 2), which is
created by removing a 10-nt-long intron 5# of the anticodon
and a 3-nt one in the TwC-loop. The latter is one of the
smallest introns identified so far and, interestingly, integrated within the B-box (see later).
As mentioned in Douglas et al. (2001), we noticed that
the nucleomorph of G. theta should encode at least one
tRNA for the whole set of amino acids with the exception
of glutamate (Glu, E), for which no nucleomorph-located
tDNA could be identified. On the other hand, we detected
two tDNAs encoding a tRNA for cysteine. One of them,
CysACA, is anticipated to harbor an 18-nt insertion at the
conserved intron position 3# of the anticodon (fig. 2). However, the RT-PCR product of the predicted tRNA CysACA
differs remarkably in respect to the intron positioning and
anticodon prediction. As indicated in figure 2, splicing of
a 10-nt-long intron containing the predicted anticodon
occurred. This creates a potential tRNA with a ‘‘new’’ anticodon prediction for GluUUC harboring an additional
intron located in the conserved position 3# of the UUC anticodon. This second intron is spliced as well (fig. 2). Thus,
the missing tRNA providing glutamate is hidden in a tDNA
predicted to encode tRNA CysACA.
The tDNA GluUUC is not only exceptional by harboring two introns. By studying the genomic version as well as
the different intermediate forms, we detected that after
splicing the 5# intron three different forms exist. In one,
the intron 3# of the anticodon is spliced, in the second
an additional a G to A transversion was detected, and in
the third form an A is inserted (supplementary table 2, Supplementary Material online). However, in the tRNA from
which both introns are spliced, only the additional A is
present (fig. 1). As the different forms were isolated in independent experiments several times, a complicated maturation of this tDNA GluUUC may be indicated by the
different intermediates.
Single Introns Located Within the TwC- or
the D-Loop
Previously, we have shown that the symbiont-specific
tRNA SerAGA, whose gene is encoded in the nucleomorph
of G. theta, contains two introns (Zauner et al. 2000). One is
located 3# of the anticodon and the other one in the D-loop;
the latter one was the first description of an intron in this
1698 Kawach et al.
loop in a eukaryotic encoded tDNA. An extended in silico
search for further unusual tRNA introns suggested five nucleomorph-specific intron positions otherwise not known
from eukaryotes: three in the TwC-loop (tDNA AlaUGC,
IleUAU, and LeuCAA) and two in the D-loop (tDNA AsnGUU
and ValAAC) (fig. 2). We amplified the spliced molecules of
these tDNAs. The tRNAs AlaUGC, IleUAU, and LeuCAA
were processed as predicted (fig. 2). Interestingly, as in
tRNA LeuUAA, a 3-nt intron is correctly spliced from the
primary transcript of tRNA AlaUGC. In all three genes,
although the predicted introns are located within the Bbox, these tDNAs are transcribed. In the case of the Dloop–located introns of tRNAs AsnGUU and ValAAC the
intron prediction differs from in vivo situation. Whereas
in the tRNA AsnGUU the intron is shifted for one base,
the intron of tRNA ValAAC is shifted and extended by 1
nt. Moreover, we detected in the tDNA ValAAC an additional
intron, which is again 3-nt-long and inserted into the B-box
region (fig. 2).
Splicing of tRNAs with Multiple Predicted Introns
As shown above, the nucleomorph harbors tDNAs including introns that are unique for the domain Eukarya in
respect to its localization. tRNAScan-SE predicts further
nucleomorph-specific tDNAs, which may harbor more
than one intron. This is true for two tDNAs (PheGAA and
CysGCA) which are predicted to encode two introns, one
in the D- and one in the TwC-loop, as well as for elongator
tDNA eMetCAU with a prediction of three introns, one in the
conserved position 3# of the anticodon, one in the D-, and
one in the TwC-loop.
In the case of tDNA PheGAA our experiments show
that both introns are processed. Interestingly, both introns
were discovered to be shifted two bases to the 5#; the TwCloop intron is furthermore extended for one base. Again,
the TwC-loop intron is located within the B-box (fig. 2).
An important consequence of the splicing reaction in the
D-loop is shown in figures 2 and 3. The conserved GG
at positions 18 and 19 in respect to the mature tRNA,
necessary for correct three-dimensional interaction of the
tRNA (Marck and Grosjean 2002), is missing in the gene
structure as well as in the potential tRNA after the removal
of the predicted intron. However, splicing the authentic
intron creates this conserved twin G, thereby indicating
that the tDNA PheGAA encodes a tRNA with a correct
three-dimensional structure.
tRNAScan-SE detects two tDNAs specific for cysteine
in the nucleomorph of G. theta. As mentioned above, one of
these tDNAs is transcribed and spliced to a tRNA with
specificity for glutamate. Therefore, the other tDNA,
predicted as specific for cysteine (tRNA CysGCA) should
provide this amino acid. The in silico data predict that the
gene for this tRNA contains two introns, one in the
TwC- and the other in the D-loop (fig. 2). In our experiments, three introns were found to be spliced: one is
located within the D-loop, one was predicted as the variable loop, and the third is again a 3-nt intron located within the B-box. Thus, our findings indicate a tDNA with
three introns at positions otherwise not known from
eukaryotes.
Whereas the initiatior tRNA iMetCAU is encoded by
the nucleomorph without any in silico-detectable intron,
elongator tRNA eMetCAU is predicted to harbor three introns. RT-PCR analyses confirmed that the introns located
in the D- (shifted and extended for one base) and TwC-loop,
which is located within the B-box, are removed from the
pre-tRNA (fig. 2). Moreover, we detected intermediates,
from which only the D-loop intron is spliced, whereas
the intron inserted in the TwC-loop is still present. To
our surprise no products were detected from which the third
intron is removed. As this intron seems to be the most common one in this tDNA, we repeated the experiment with
different primers. By analyzing more than 100 different
transcripts none was detected in which the intron at the conserved position is removed. Therefore, no mature tRNA
eMetCAU was verified. This result could reflect either very
small amounts of the mature tRNA or technical problems,
which did not occur in our analyses of all other nucleomorph-specific tDNA introns. Another possibility is that
the host provide the elongator tRNA, thereby getting the
control of the translation in the symbiont compartment.
The Enigmatic Introns in the B-box
Our analyses of the mature nucleomorph-specific
tRNAs and their encoding genes clearly demonstrate that
in seven cases introns are inserted within the B-box. It
is known that cotranscription of genes is common for
nucleomorphs (Gilson and McFadden 1996; Fraunholz,
Moerschel, and Maier 1998). Having this in mind, a hypothesis to explain the presence of introns in the B-box
is cotranscription of tRNA genes with upstream-located
protein-encoding ones. This would change a RNA polymerase III transcript into one which is expressed by the RNA
polymerase II. In order to prove this, we determined exemplarily the cotranscription of four tRNAs with upstream
sequences. These were the genes for hlip (high lightinducible polypeptide) and the downstream-located, intron-less tRNA LeuUAG, the gene for the small nucleolar
ribonucleoprotein D and tRNA ValUAC (one intron in the
D-loop), T-complex protein 1, zeta subunit, and tRNA
IleUAU (one intron in the B-box), and finally the gene for
a ubiquitin-conjugating enzyme E2 and the downstreamlocated tRNA PheGAA (two introns). Cotranscription was
detected in all examples. Nevertheless, this could speak either for the correctness of the hypothesis or for an extended
transcription at the 3# end of protein-encoding mRNAs. As
in some cases where the tRNAs overlap with their upstream-located protein-encoding gene (fig. 4), we analyzed
the maturation of the primary transcripts. In the case of the
cotranscript of hlip with tRNA LeuUAG, the upstream gene
overlaps by two bases with tRNA LeuUAG. One would expect that the mature transcript of hlip and a complete tRNA
could be generated by an endonucleolytic step and subsequent polyadenylation (fig. 4). An endonucleolytic cut 5# of
the GA from the stop codon TGA of hlip would generate the
correct 5# end of the tRNA, and subsequent polyadenylation of the 3# end of the hlip-transcript would then generate
a TAA stop codon (fig. 4). Therefore, a hlip-transcript with
the secondarily created stop codon (TAA) should be detectable. However, by analyzing 12 cDNAs specific for
tRNAs of cryptomonads
1699
inserted into the B-box mimic the promoter region with
high predicted values (fig. 2). Therefore, the encoded sequence of promoter regions is not part of the mature tRNAs,
which is unique for eukaryotes.
The Red Alga Dowry
FIG. 4.—Alternative transcription model of tRNAs in the nucleomorph of Guillardia theta. The hlip gene overlaps with tDNA for LeuUAG
at the 3# end by two bases. After endonucleolytic processing the transcript,
the disrupted stop codon of the hlip-transcript could be restored by polyadenylation. The two arrows labeled with Met indicate putative start codons of orf101 downstream of the tDNA for LeuUAG.
the hlip-sequence (from which 10 represent independent
transcripts as demonstrated by different 3# nontranslated regions) we exclusively detected the unmodified cotranscriptional form. Thus, the ‘‘cotranscription hypothesis’’ is
attractive, but most likely wrong. Hereupon we analyzed
the intron sequences again and identified that the introns
The genetic system of eukaryotes is more related to
archaea than to that of bacteria (Martin and Müller 1998;
Martin et al. 2001). This is also seen in the presence of
tDNA introns in archaea and eukaryotes, which are rare
and of another type in bacteria. Due to projects on the nucleomorph of G. theta and the genome of the red alga Cyanidioschyzon merolae we noticed that contrary to earlier
views, introns in tDNA can be similarly distributed in eukaryotes than in archaea. As red algae and the symbiont of
the cryptophytes have a common ancestor (Van de Peer
et al. 1996), similarities in respect to intron structure and
localization can be expected. Therefore, we have reanalyzed the red algal data and confirm that some of the
indicated C. merolae tDNA introns, which are generally
longer than the nucleomorph G. theta ones, should be positioned at unique sites within tRNA genes, too (table 1).
The predicted tDNA introns from C. merolae are inserted
into the variable loop and the D- and TwC-stems. Otherwise, no introns were predicted in the anticodon loop except
Table 1
Comparison of Guillardia theta and Cyanidioschyzon merolae
G. theta Nucleomorph
AC
Intron
(Number)
Length (bp)/
Position
Arg
Arg
Arg
Pro
Pro
Ala
Asn
Met (elongator)
CCU
ACG
UCG
CCG
CGG
UGC
GUU
CAU
1
0
0
0
0
1
1
3
Met (initiator)
Ser
Ser
Tyr
Trp
Phe
Thr
Pro
Ala
Ile
Leu
Leu
Cys
CAU
CGA
AGA
GUA
CCA
GAA
UGU
AGG
AGC
GAU
CAA
UAA
GCA
0
0
2
1
1
2
—
—
—
—
1
1
2
Glu
Val
Arg
Val
Ile
UUC
GUU
UCU
UAC
UAU
2
2
1
1
1
12/cons. 1 1
n.d.
n.d.
n.d.
n.d.
3/T/C-loop
9/D-loop
10, 9, 8/D-loop, cons.,
T/C-loop
n.d.
n.d.
10, 7/D-loop, cons.
7/cons. 1 4
8/cons.
9, 8/D-loop, T-loop
—
—
—
—
24/T/C-loop
3/5# Anticodon
10, 10, 3/D-loop, T/C-loop,
variable loop
10, 8/5# Anticodon, cons.
10, 3/D-loop, T/C-loop
7/cons.
9/D-loop
6/T/C-loop
tRNA
C. merolae
Intron
Length (bp)/
Position
1
2
1
1
1
0
1
2
4/T-stem
32, 3/cons., variable loop
16/cons.
12/cons.
15/cons.
n.d.
11/cons.
30, 19/D-loop, cons.
0
1
1
1
1
2
1
1
1
1
—
—
—
n.d.
50/cons.
11/cons.
15/cons.
12/cons.
28, 34/D-loop, cons.
33/cons.
12/cons.
16/D-loop
14/cons.
—
—
—
—
—
—
—
—
—
—
—
—
—
NOTE.—Comparison of intron-harboring tRNA-genes from the nucleomorph of G. theta and the red alga C. merolae. Indicated
are the tRNAs, their anticodons (AC), the number of introns per tDNA in G. theta, and the predicted ones in C. merolae. The fourth
and sixth columns show the lengths and positions of the introns; n.d. indicates no introns detected; cons., (conserved) means that the
intron location is between positions 37 and 38. Dashes indicate that no tRNA is detectable neither in the nucleomorph of G. theta
nor in the nucleus of C. merolae.
1700 Kawach et al.
the conserved intron localized 3# of the anticodon. There is
also no intron prediction concerning the B-box region. A
comparative analysis showed that none of the tDNAs with
a unique intron localization encoded by the nucleomorph
genome of G. theta has a counterpart in C. merolae (fig.
1 and table 1). Taken together, the nucleomorph-specific
tDNA introns are shorter than the red algal ones, but occupied additional localizations within the tRNA genes.
Similar to the nucleomorphs of cryptophytes, red algae
genomes are poor in splicesomal introns and in comparison
to other eukaryotes enriched in tRNA introns. Therefore,
the presence and unique distribution of nucleomorphspecific tRNA introns indicate a red alga dowry. Nevertheless, it was unexpected that the nucleomorph genome on the
one hand streamlines and minimizes its genome, but on the
other hand tolerates introns within nearly all positions of its
tRNA genes. This may be explained either by the selfish
nature of these introns or by structural-functional reasons.
Structural-functional reasons of tRNA introns are discussed
for Archaea. Here intervening sequences could play an essential role in tRNA maturation, especially in the modification of pre-tRNAs by intron-dependent tRNA modification
enzymes (Motorin and Grosjean 1999). Moreover, introns
may also be essential in the stepwise folding of the tRNAs
by avoiding false pairing in tRNA maturation (Dennis,
Omer, and Lowe 2001).
Conclusions
The complete nucleomorph sequence of the cryptomonad G. theta exhibited a streamlined eukaryotic genome
with a gene density similar to bacterial genomes (Douglas
et al. 2001). Such a reduction of noncoding sequences is
manifested e.g., in splicesomal introns as shown by the detection of only 17 splicesomal introns in approximately
450 nucleomorph-specific genes or open reading frames
(Douglas et al. 2001). One exception of genome streamlining is shown in this paper. As mentioned, 15 of 36 tDNAs
plus one region which is annotated as a pseudogene encode
tRNAs with one or more introns. Moreover, most of these
introns are unique for Eukarya in respect to its localization
and size (fig. 2). Therefore, the cryptophytic symbiont had
successfully evolved additional mechanisms to detect and
process these unusual tRNA introns. This capacity may be
bequeathed from the red alga progenitor of the secondary
symbiont of cryptophytes, as seen for tRNA introns detected in a recently sequenced red algae. Both the existence
of introns and the evolution of uncommon targeting mechanisms for a splicing apparatus seem to be contradictory for
a streamlined genome. Thus, the existence of introns in nucleomorph-coded tRNAs in the conserved position 3# of the
anticodon as well as those at unique positions should have
structural and/or functional reasons or may indicate selfish
DNA. Anyway, biochemical and structural analyses on the
unusual introns of the secondary endosymbiont will be of
major importance and will show new mechanisms of RNA
metabolism. Moreover, new genome projects on other protists will highlight if unusual intron insertions are common
to other organisms. Such a situation could imply that earliest eukaryotes, like the red algae, once harbored introns
outside the 3# conserved region of the anticodon, which
were differentially lost in the course of evolution in most
eukaryotes studied so far.
Supplementary Material
Supplementary tables 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
Our research is supported by the Deutsche Forschungsgemeinschaft (SFB-TR1).
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Geoffrey McFadden, Associate Editor
Accepted April 22, 2005