Download A global picture of tRNA genes in plant genomes

The Plant Journal (2011) 66, 80–93
doi: 10.1111/j.1365-313X.2011.04490.x
THE PLANT GENOME: AN EVOLUTIONARY VIEW ON STRUCTURE AND FUNCTION
A global picture of tRNA genes in plant genomes
Morgane Michaud, Valérie Cognat, Anne-Marie Duchêne* and Laurence Maréchal-Drouard*
Institut de Biologie Moléculaire des Plantes, UPR 2357-CNRS, Université de Strasbourg, 12 rue du Général Zimmer,
F-67084 Strasbourg Cedex, France
Received 30 September 2010; accepted 4 January 2011.
*
For correspondence (fax +33 3 88 61 44 42; e-mail anne-marie.duchene@ibmp cnrs.unistra.fr or [email protected]).
SUMMARY
Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization
and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and
distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous
in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like
tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes
or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we
identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa
nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for
tRNATyr and tRNAMete. By contrast, in green algae, more than half of the tRNA genes contain an intron. This
suggests divergent means of intron acquisition and the splicing process between green algae and land plants.
Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in
flowering plants. The only exceptions are tRNAGly–snoRNA and tRNAMete–snoRNA cotranscripts in dicots and
monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by
RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and
plastidial tRNA gene populations is also provided.
Keywords: alga, angiosperm, non-coding RNA, genome evolution, organelles, SINE.
INTRODUCTION
Transfer RNA (tRNA) is among the most ancestral RNA in the
world. Its role in cell life is essential as it represents the
interface between the genetic code and protein. In addition
to its essential function in translation, several recent studies
have shown that tRNA is a multi-functional molecule that
is involved in many processes of cellular metabolism
(Francklyn and Minajigi, 2010). Furthermore, tRNA-derived
RNAs appear to be used in the RNA silencing pathway, and
are a major source of short interspersed short interspersed
nuclear elements (SINEs; Bermudez-Santana et al., 2010;
Phizicky and Hopper, 2010). It is postulated that all tRNA
genes (tDNAs) derive from an ancestral ‘proto-tRNA’ (Eigen
et al., 1989). During evolution, a full set of tRNA genes was
generated as the result of numerous mutation, duplication
and re-organization events. However, little is known about
the organization, distribution and expression of tRNA genes
or tRNA-related sequences on a genome-wide scale in
eukaryotes, and comparative studies are rare (Goodenbour
80
and Pan, 2006; McFarlane and Whitehall, 2009; BermudezSantana et al., 2010). With the completion of several plant
genomes, we now have the opportunity to study their
complete tRNA gene sets.
Using the results of whole-genome sequencing projects,
the software tRNAscan-SE, and the few experimental datasets available, we have analysed the genomic content and
distribution of tRNA genes and tRNA-related genes in five
flowering plants (Arabidopsis thaliana, Medicago truncatula, Populus trichocarpa, Oryza sativa and Brachypodium
distachyon) and one green alga (Chlamydomonas reinhardtii). We first manually curated the lists of tDNAs
extracted from each genome, correctly annotating pseudogenes as well as organellar-like tDNAs. As a result of this
analysis, a new family of short interspersed tRNA-related
SINEs was identified in P. trichocarpa. This enabled us
to present here the most up-to-date set of ‘true’ tDNAs
present within plant nuclear genomes. We then provide a
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd
tRNA genes in plants 81
comprehensive comparative view of tRNA gene composition and organization. Emphasis is given to the 5¢ and 3¢ ciselements involved in nuclear tRNA gene expression. The
existence of co-transcribed tRNAs and intron-containing
tRNAs is also discussed. The content, composition and
expression of tRNAs in mitochondrial and chloroplastic
genomes are also assessed to provide a full picture of tRNA
gene sets in these organisms.
TRNA SEQUENCE DETECTION AND ANALYSIS
All available tRNA gene sequences from five angiosperms
(A. thaliana, At; M. truncatula, Mt; P. trichocarpa, Pt; O. sativa, Os; B. distachyon, Bd) and one green alga (C. reinhardtii, Cr) were used. These species were selected as they
possess the most annotated genomes in terms of their
tDNAs. When necessary, some data were also retrieved from
two other species: the green alga Ostreococcus tauri and the
moss Physcomitrella patens. The nuclear sequence data
were obtained from the Arabidopsis Information Resource
(TAIR, http://www.arabidopsis.org/), the Medicago truncatula sequencing resources http://www.medicagohapmap.
org/?genome and the US Department of Energy Joint
Genome Institute (http://www.jgi.doe.gov/) for the other
plants. Although we recently annotated the Chlamydomonas tDNA sequences using version 3.0 of the genome
release (Merchant et al., 2007; Cognat et al., 2008),
version 4.0 is now available with an updated list of tDNAs.
Organellar genome sequences were obtained from the
Genbank database (http://www.ncbi.nlm.nih.gov/) or from
the Organelle Genome database (http://gobase.bcm.umontreal.ca/). A full list of the accession numbers for the genes is
given in Table S1. The existence of tRNAs was first predicted
by using the highly efficient automated analysis program
tRNAscan-SE version 1.21 (Lowe and Eddy, 1997; http://
lowelab.ucsc.edu/tRNAscan-SE/) using default parameters.
However, to minimize the number of false positives, we also
manually curated the lists of tDNAs extracted from each
genome. By means of the manual curation, we correctly
annotated a few genes or pseudogenes that were misannotated, correctly annotated numerous non-expressed
mitochondrial or plastidial tDNA sequences inserted into
the nuclear genome and recognized by tRNAscan-SE,
re-annotated some tRNAs in terms of amino acid and codon
recognition due to the presence of post-transcriptional
anticodon modifications, and identified new tRNA-derived
SINEs that could not be distinguished from true tRNAs by
tRNAscan-SE. For manual curation, the following parameters were taken into account: constraints on the sequence
and secondary structure of tDNAs, conserved upstream and
downstream transcriptional sequence elements, and BLAST
alignments (http://blast.ncbi.nlm.nih.gov/Blast.cgi). As an
result of manual curation, a new family of 35 SINEs
(Figure S1) was discovered in the poplar nuclear genome,
although they are assigned as tRNALeu genes by tRNAscanSE and described as such in the genomic tRNA database
(http://gtrnadb.ucsc.edu/; Chan and Lowe, 2009). This new
family is described below. Another example of the benefits
of manual curation is reduction of previous over-estimates
of the number of elongator tRNAMet (tRNAMete) genes in rice
(http://gtrnadb.ucsc.edu/). Some of these are either mitochondrial or plastidial tDNAs inserted into the nuclear genome, but they lack the regulatory transcription elements
required for nuclear tDNA expression. Currently, there is no
evidence regarding whether an organellar-like tDNA integrated into a plant nuclear genome can become functional.
Moreover, some tRNAMete correspond to isoleucine tRNAs
that have been wrongly annotated as methionine tRNA
genes due to the presence of a CAT anticodon. The C at the
first position of the anticodon is post-transcriptionally
modified to a lysidine derivative, enabling the tRNA to be
isoleucine-specific (Weber et al., 1990). Thus, using both
tRNAscan-SE prediction software and manual curation, we
have performed the most thorough survey on plant tRNA
genes to date (summary in Table 1). Although a few ‘true’ or
‘false’ tRNA genes may inevitably have escaped our analysis, this probably represents a very tiny percentage of all
Table 1 Overview of the number of tDNAs present on the six plant nuclear genomes studied here
A. thaliana
M. truncatula
P. trichocarpa
O. sativa
B. distachyon
C. reinhardtii
All
True
Pseudo
Organellar
637
691
652
723
615
329
599
452
562
516
479
256
14
148
38
14
30
2
24 (23m, 1p)
91 (33m, 58p)
17 (2m, 15p)
193 (24m, 166p, 3m/p)
106 (5m, 101p)
0
Others
35
71
Introns
81
20
30
27
30
161
‘All’ indicates the number of tDNAs detected using tRNAscan-SE. ‘True’ indicates the number of tDNAs considered as likely to be expressed after
manual curation (i.e. subtraction of pseudogenes, SINEs and organellar-like tDNAs). ‘Pseudo’ indicates the number of tDNAs considered as
pseudogenes. ‘Organellar’ indicates the number of mitochondrial- or plastidial-like tDNAs inserted into plant nuclear genomes. The number of
mitochondrial-like (m) or plastidial-like (p) tDNAs is given in parentheses. In O. sativa, ‘m/p’ indicates the number of tRNA gene sequences for
which the origin (m or p) has not been unambiguously determined. ‘Others’ indicates the number of SINEs. ‘Introns’ indicates the number of
expressed tDNAs containing an intron.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
82 Morgane Michaud et al.
For each of the six genomes included in our survey, we
determined the number of tDNAs and tRNA pseudogenes
as well as the number of organellar tRNA genes inserted
into the nuclear genomes (Table 1). We cannot exclude the
possibility that a few of the transferred organellar tRNA
genes became functional once integrated into the nuclear
genome, but so far none of them has been shown to be
functional (Tian et al., 2007). In the absence of any experimental evidence, we therefore considered them as unexpressed genes and they were not considered as ‘true’
tDNAs. The number of true tDNAs does not greatly differ
from one plant species to another for those that we analysed. In flowering plants, this number ranges between 500
and 600, whereas the green alga C. reinhardtii has only
approximately 250. This limited variation in the green
lineage has been reported previously, and contrasts with
the situation in the animal kingdom where the number of
nuclear tDNAs varies drastically (e.g. a few hundred in
monkeys and apes, but more than 100 000 in cows and
rats; Bermudez-Santana et al., 2010). With the exception of
the nuclear genome of A. thaliana, we noted an expected
correlation between the number of tDNAs and genome
size (Figure 1a). This result is also apparent when looking
at the number of tDNAs per chromosome (Figure 1b); a
mean of 1.5 tDNAs are present per Mb per chromosome.
In general, single tDNAs are mostly evenly distributed
along chromosomes from all plants studied in this analysis. Furthermore, tRNA isoacceptor genes are also randomly dispersed on the various chromosomes (Figure S2).
In general, tDNA clusters are rare in plant nuclear
genomes, with only a few exceptions, notably in A. thaliana (Figure 1). In A. thaliana, chromosome 1 contains two
large clusters of tDNAs due to extensive gene duplications
(Theologis et al., 2000). One cluster contains 27 tandem
tRNAPro genes, and the second contains 27 tandem repeats
of tRNATyr–tRNATyr–tRNASer genes. These clusters are
mainly responsible for the large number of tDNAs per Mb
found for chromosome 1. The other four chromosomes
also have a higher number of tDNAs per Mb (mean 4)
compared to the other plant species. Overall, this explains
the larger number of tRNAs in A. thaliana, and may reflect
a stronger general tendency to gene duplication in this
species compared to other plants. However, clustering
does also occur in other plants, but not at the same level.
For example, in Brachypodium, the four copies of the
tRNAIle(TAT) gene are all present on chromosome 1. This
suggests that, in some cases, the number of tDNAs
implemented by gene duplication is facilitated within the
Nb of tRNA genes
NUCLEAR TRNA GENE ORGANIZATION
(a)
700
600
500
400
300
200
100
0
At
Bd
Os
Pt
Mt
Cr
0
200
400
Genome size (Mb)
600
(b)
Nb of tRNA genes/Mb
tRNA genes, and should not affect the interpretation of the
data. The complete curated sets of extracted tRNA genes
from the species used for the analyses are available at http://
www-ibmp.u-strasbg.fr/Drouard/tRNA.html.
10
8
6
4
2
0
0
1
2
3
4 5 6 7 8
Chromosome
9 10 11 12
Figure 1. Number of tDNAs on nuclear genomes of flowering plants.
(a) Correlation between tDNA number and genome size in A. thaliana (At),
M. truncatula (Mt), Populus trichocarpa (Pt), O. sativa (Os), B. distachyon (Bd)
and C. reinhardtii (Cr).
(b) Number of tDNAs per Mb in each chromosome. At, black diamond; Mt,
grey triangle; Os, black square; Bd, cross.
same chromosome. However, other constraints probably
exist, as very large clusters were only observed in
A. thaliana genome.
In contrast to the overall correlation between tDNA gene
content and genome size, there is no such correlation for the
number of tRNA pseudogenes (Table 1). Over the course of
evolution, mitochondrial (mt) and plastidial (pt) DNA fragments have become integrated into the nuclear genomes
of flowering plants (Lin et al., 1999; Tian et al., 2007; and
references therein). Following these DNA transfer events,
variable amounts of tDNAs originating from the endosymbiotic organelles are found in nuclear genomes of higher
plants (Table 1), and show between 95 and 100% sequence
identity with their organellar counterparts. The number of
organellar tDNA insertions varies greatly from one plant
species to another (none in green algae, 17 in P. trichocarpa
and 193 in O. sativa). With the exception of Arabidopsis,
the number of plastidial tDNAs is higher than the number of
mitochondrial ones. Mitochondrial tDNAs are mainly localized on a single chromosome, for example A. thaliana
chromosome 2, which contains 75% of the mitochondrial
genome, M. truncatula chromosome 1 and O. sativa chromosome 12, due to large but rare mitochondrial DNA
fragment insertion. In contrast, plastidial tDNAs are mostly
scattered across all chromosomes (Table S2) and are found
as short (100–200 bp) or large DNA insertions (up to
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
tRNA genes in plants 83
10 000 bp). This suggests that most tDNA transfers occurred
through a DNA-mediated pathway rather than an RNAmediated pathway. Moreover, two means of insertion of
plastidial tDNAs into rice nuclear DNA have been proposed.
Either the transfer is direct (from the plastidial DNA to the
nuclear DNA), or indirect via the mitochondrial genome (i.e.
first a transfer from the chloroplast to the mitochondria and
then from the mitochondria to the nucleus; Tian et al., 2007).
NUCLEAR TRNA ISOACCEPTOR AND ISODECODER GENES
The anticodon triplet recognizes an mRNA codon and represents one of the main functional regions of a tRNA molecule. Plants use the universal genetic code in the three
compartments in which translation occurs, and the 61 sense
codons are linked to the 20 standard amino acids. For each
amino acid, up to five tRNAs with distinct anticodons (tRNA
isoacceptors) are required. The various tRNA isoacceptors
charging the same amino acid constitute one family. As
shown in Figure S3, the number of tRNA isoacceptors ranges from 45 to 47 in the six species studied. Among these
species, 45 tRNA isoacceptors are evolutionary related. The
presence of one or two supplementary tRNA isoacceptors
in some plant species is either due to orphan tDNAs of
unknown origin (in P. trichocarpa, O. sativa and B. distachyon), or the existence of a tRNASec gene in C. reinhardtii.
Selenocysteine (Sec) is a rare amino acid that is found in
several selenoproteins. It is recognized as the 21st amino
acid in the genetic code, and its codeword, UGA, is normally
(b)
45
40
35
30
25
20
15
10
5
0
y = 0.3646x + 6.0016
R 2 = 0.70195
Gly
Leu
Ala
Arg
Lys
Met
Phe
Cys
Asp
Thr
Ile
Glu
Gln Asn
Trp
His
0
Val
20
A. thaliana
40
60
80
50
Isoaccepting tRNA
gene copy number
(a)
Isoaccepting tRNA
gene copy number
a stop codon (Rao et al., 2003). In eukaryotes, selenoproteins
are found in animals but are absent in fungi or higher plants.
However, Sec-containing proteins were found in the green
algae C. reinhardtii (Novoselov et al., 2002). As incorporation of selenocysteine into proteins requires a specific tRNA
designated tRNASec, such a tRNA is present in C. reinhardtii
but is absent in higher plants.
In various organisms (e.g. bacteria, yeast, Drosophila
melanogaster or Caenorhabditis elegans), the number of
tRNA genes gives a good approximation of the abundance
of each tRNA isoacceptor, which, in turn, is correlated with
the amino acid frequency (Duret, 2000). To study whether
there is also such a gene-dosage effect in plants, we
analysed the relationship between the number of tRNA
genes charging one amino acid, and the frequency of the
corresponding amino acid for four of the higher plants (a
codon usage table is not available for B. distachyon) and for
the green algae C. reinhardtii. A good correlation between
the number of tRNA genes and the frequency of the amino
acids was observed for all plants analysed (Figure 2 and
Figures S4 and S5). This is in agreement with a similar
analysis performed using the rice nuclear genome (Itoh
et al., 2007). The best correlation obtained was for the green
alga C. reinhardtii, which may reflect the lower complexity
of its nuclear genome. In the case of A. thaliana, a weak
correlation was obtained when all amino acids were considered (R2 = 0.16, Figure S5A). This is due to the existence
of three tDNA families (Tyr, Ser and Pro) that are highly
y = 0.4245x + 6.2807
R 2 = 0.70376
40
30
Val
Met
20
Trp
10
0
100
0
Amino acid frequency/1000
Ala
Gly
Tyr
Cys His
20
Pro
Gln
Ser
Lys
Ile
Asn
Phe
Asp
Thr
O. sativa
40
60
80
100
Amino acid frequency/1000
(d)
30
20
10
A. thaliana
0
0
20
40
Codon frequency/1000
60
Isoaccepting tRNA
gene copy number
Isoaccepting tRNA
gene copy number
(c)
Arg
Leu
Glu
30
20
10
O. sativa
0
0
20
40
60
Codon frequency/1000
Figure 2. tRNA isoacceptors and tRNA isodecoders in plant nuclear genomes.
(a, b) Correlation between the number of tRNA gene copies specific for each amino acid and the frequency of occurrence of the same amino acid in A. thaliana (a)
and O. sativa (b). In A. thaliana, three amino acids, Ser, Tyr and Pro, were excluded from this analysis. For additional information, see Figures S4 and S5. The
frequency of amino acids was estimated by counting the number of corresponding codons available on the codon usage database (http://www.kazusa.or.jp/codon/).
(c, d) Correlation between the number of tRNA gene copies specific for their cognate codons and the frequency of occurrence of the same codon(s) in A. thaliana (c)
and O. sativa (d). The frequency of codons was estimated by counting the number of corresponding codons available on the codon usage database (http://
www.kazusa.or.jp/codon/).
Graphs were produced using the data presented in Table S3.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
84 Morgane Michaud et al.
amplified on chromosome 1. When the three families are
excluded from the analysis (Figure 2), or when the numbers
of tDNA copies present on chromosome 1 are subtracted
from the total number of tDNA copies for the corresponding
amino acid (Figure S5B), the correlation significantly
increases (R2 = 0.70). According to in vitro expression
analysis, most of the tRNAs belonging to these three gene
families are very probably expressed (Stange et al., 1991;
Yukawa et al., 2007). However, two questions remain to be
answered: do the numbers of tRNASer, tRNATyr or tRNAPro
genes correlate with the abundance of the corresponding
tRNAs, and/or are these tRNAs involved in other processes
than translation in A. thaliana?
Another question is whether the correlation observed
between the number of tRNA genes and the frequency of
the corresponding amino acid also holds for the number of
tRNA isoacceptor genes and the codon usage. The decoding
properties of individual tRNA species have not been studied
in detail in higher plants. Furthermore, only a few sequences
are available at the RNA level, restricting our knowledge on
modified nucleotides present at the first position of the
anticodon and codon/anticodon recognition. The influence
of codon/anticodon interactions and modified bases on
codon usage has been well studied in bacteria (Ran and
Higgs, 2010) but not in plants, and only educated guesses
can be made regarding which isoacceptor tRNAs read which
codons. It is usually accepted that, in vivo, most tRNAs can
translate more than one codon, and some codons can be
translated by more than one isoacceptor tRNA. Nevertheless, assuming that a codon is preferentially read by only
one tRNA isoacceptor (see Table S3), we analysed the
relationship between the number of isoacceptor tRNA genes
and the codon usage in A. thaliana and O. sativa. As shown
in Figure 2, a very good and unexpected correlation exists
for both plants. This suggests the existence of a tRNA
isoacceptor gene-dosage effect with very simple rules to
optimize translation.
Full genome sequencing has allowed us to identify
another level of complexity: that some differences are found
elsewhere in the sequence of tRNA isoacceptors. Recently,
these tRNAs were defined as tRNA isodecoders by Goodenbour and Pan (2006). For A. thaliana, the various sequences
for tRNAs belonging to the same group of tRNA isoacceptor
can be visualized at http://www.inra.fr/internet/Produits/TAARSAT/. The number of tRNA isodecoder genes (i.e. the
number of distinct tRNA sequences) has been determined
(Figure S3). In higher plants, this number varies between
147 and 194, and is of the same order of magnitude as in
animals (Goodenbour and Pan, 2006). In the green alga
C. reinhardtii, only 83 tRNA isodecoder genes were found.
However, as shown in Figure S3, the number of tRNA
isodecoders is correlated with the number of tRNA genes
within a nuclear genome, and suggests that tRNA isodecoders derive from neutral evolutionary drift. In general, among
each tRNA isoacceptor family, tRNA sequences are highly
conserved within the same species but also between
species. Interestingly, in higher plants, some tRNA isoacceptors show much more sequence fluctuation than others
(i.e. their number of tRNA isodecoders is high). For example,
of 50 plant tRNAPhe sequences, 48 are identical and only
two show nucleotidic divergence (with 2 and 8 nucleotide
differences, respectively, compared to the 48 other
sequences). For tRNAGln(CTG), for which the number of
tRNA isodecoders is higher, tRNA sequences are less
conserved among the tRNA isoacceptor family (Figure S6).
A simple explanation for these differences may be that the
set of sequence determinants to ensure correct L-shaped
structure and charging of a tRNA by a specific amino acid
varies from one tRNA isoacceptor to the other.
Each tRNA contains a few sets of nucleotides that are
required for tRNA recognition by the cognate aminoacyltRNA synthetase. These identity elements are mostly located
at the two distal extremities of tRNAs: the anticodon loop
and the acceptor stem (Giege et al., 1998; Giege, 2008). It is
generally assumed that similar aminoacylation rules are
used in the various eukaryotes. Only a limited amount of
data are available concerning such identity elements in
plants. In most cases of aminoacylation, studies revealed
that plant identity elements are similar to those found in
other organisms (Carneiro et al., 1994; Dietrich et al., 1996;
Giege et al., 1998; Ulmasov et al., 1998; Delage et al., 2003;
Wu et al., 2007), but some differences were also observed
(Salinas et al., 2005). For example, the G3.U70 base pair was
identified as an identity element in Escherichia coli, Saccharomyces cerevisiae and Thermus thermophilus tRNAAla
(Giege et al., 1998). In accordance with these results, mutation of G3.U70 to G3-C70 in A. thaliana tRNAAla inhibits its
alanylation (Carneiro et al., 1994). In tRNAGly, the nucleotide
at position 73 discriminates between prokaryotes (U73)
and eukaryotes (A73). However, in plants, the mitochondrialencoded tRNAGly contains U73 and the three nuclearencoded tRNAGly have A73. Surprisingly, the active
mitochondrial glycyl-tRNA synthetase (GlyRS), which is
similar to prokaryotic GlyRSs, is able to glycylate both
the mitochondrial-encoded and nuclear-encoded tRNAsGly
(Salinas et al., 2005).
An important question for the future is whether the
differential sequence flexibility observed in the tRNA isoacceptor gene families only reflects selection pressure and/or
whether some tRNA isodecoders perform distinct functions.
Analysis of various biological samples coupled to microarrays as well as aminoacylation studies on tRNA isodecoders will enable such studies to be initiated.
NUCLEAR TRNA GENE EXPRESSION
Nuclear tDNAs are transcribed by RNA polymerase III (PolIII). Their promoters are typically PolIII type 2, and consist of
conserved sequence elements within the transcribed region.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
tRNA genes in plants 85
5¢-TGGCNNAGTGG-3¢ and 5¢-GGTTCGANNC-3¢ for A and B
boxes, respectively. These sequences are highly conserved
in A. thaliana tDNA sequences, with consensus sequences
of 5¢-Tg/aGC/tNNAGTg/tG-3¢ and 5¢-G/aGTTCGANNC-3¢,
respectively (Figure S7). The A box is found at positions
8–18, and the B box is 30–60 nucleotides downstream of the
A box, depending on the presence of introns and the length
of the variable loop in tRNAs. In most cases, the B box is
located 13–22 nucleotides upstream of the end of the mature
tRNA.
These elements, called A and B boxes, are highly conserved
in eukaryotes, partly because they encode the tRNA D and
T loops (Galli et al., 1981; Hofstetter et al., 1981). Upstream
elements were also found to be important for transcription
of many tRNA genes. For example TATA box-like sequences
were shown to greatly contribute to transcription efficiency
in many organisms (Hamada et al., 2001; Schramm and
Hernandez, 2002; Giuliodori et al., 2003; Dieci et al., 2006,
2007). In plants, analysis of the 50 nucleotides immediately
upstream of 68 tDNAs has shown a high proportion of A and
T nucleotides in this region, the presence of TATA-like motifs
and the prevalence of CAA triplets (Choisne et al., 1998).
AT-rich regions and TATA-like sequences appear to be
important for a high level of expression, and CAA triplets
correspond to transcription initiation sites (Ulmasov and
Folk, 1995; Yukawa et al., 2000). We have therefore searched
in more detail for such motifs in photosynthetic organisms.
Frequency of AT and TATA-like elements upstream
of tDNAs
The 50 nucleotides upstream of tDNAs appear to be enriched
in A and T residues compared to tDNA sequences. Moreover, there is a clear difference between flowering plants
(approximately 70% AT in dicots and monocots) compared
to the green alga C. reinhardtii (<50%; Figure 3a). However,
these values should be viewed with caution because the AT
content varies between genomes. For example, Chlamydomonas is only 36% AT-rich, compared with 64% in Arabidopsis. Consequently, the increase in the percentage of AT in
A and B boxes
The transcriptional internal control regions, called A and B
boxes, are highly conserved among eukaryotes, particularly
in the plant kingdom. The consensus sequences are
(b)
%
100
At
(64%)
Mt
(67%)
Pt
(67%)
Os
(59%)
Bd
(54%)
Cr
(36%)
75
50
25
0
–50
–40
–30
–20
–10
Nucleotides position
0
0
5’ –50
–40
–30
–1 3’
–10
–20
(d)
60
30
50
25
% of sequences
% of sequences
(c)
1
Bits
(a)
40
30
20
15
10
5
10
0
20
0
0
1
2
3
Number of CAA motifs
4
–15
–12
–9
C position
–6
–3
Figure 3. Upstream region (from )50 to )1) of tDNAs.
(a) AT frequency. The sequences were analysed in stretches of five nucleotides. On the x axis, 0 represents the sequence of mature tRNA from positions 1–5, )5
represents the upstream region from positions )1 to )5, and )50 represents the sequence from )46 to )50. The percentage in parentheses represents the AT
percentage in the whole genome.
(b) WEBLOGO analysis (http://weblogo.berkeley.edu/) of the upstream region of tDNAs in flowering plants.
(c) Number of CAA triplets in flowering plants.
(d) Position of the C residue in the most proximal CAA triplet.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
86 Morgane Michaud et al.
tDNA upstream regions compared to whole genomes is
roughly the same in all species. In flowering plants, the
percentage of AT is particularly high between positions )25
and )35, with a mean of 83% (Figure 3b). TATA-like
sequences are found in this position upstream of many PolIII
class III genes and some class II genes (Dieci et al., 2007).
TATA-like sequences in direct orientation (as defined
by PLACE; http://www.dna.affrc.go.jp/PLACE/signalup.html)
are found in the region between )20 and )40 of 18% of
upstream sequences in flowering plants, but are absent in
the green alga. In this context, Chlamydomonas resembles
animals, in which TATA boxes upstream of tDNAs do not
occur frequently (Ulmasov and Folk, 1995).
CAA motifs
The )1 to )10 region is particularly rich in A residues but also
in C residues (Figure 3b), and CAA triplets have been shown
to be transcription initiation sites in tobacco (Nicotiana
tabacum; Yukawa et al., 2000). In A. thaliana, 78% of tDNAs
have one or more CAA motifs in the )1 to )50 region
(Figure 3c), with the most proximal CAA motif starting at )6,
)5 or )4 (Figure 3d). In agreement with this result, 65% of
sequences in flowering plants have at least one CAA triplet
in the )1 to )10 region, but this motif is only found in 13% of
C. reinhardtii sequences.
Downstream element
PolIII terminates transcription at short runs of T residues (at
least four; Dieci et al., 2007). The presence of short stretches
of T residues downstream of plant tDNAs has been reported
previously in Arabidopsis (http://www.inra.fr/internet/Produits/TAARSAT/). Its importance in PolIII transcription termination has been shown experimentally (Ulmasov and
(a)
Folk, 1995; Yukawa et al., 2000). In addition, the poly(U) tail
is specifically recognized by the La protein (Wolin and
Cedervall, 2002), and plays an important role in stabilizing
the primary tRNA transcript. In C. reinhardtii, a short poly(T)
tail is present 1–10 nucleotides downstream of the predicted
3¢ end on two-thirds of the tDNAs but is absent on the
remaining third. In the latter case, lack of such a tail is
probably due to the presence of polycistronic tRNAs, a situation that is usually not found in eukaryotes, in particular
higher plants (Cognat et al., 2008). Indeed, in the five flowering plants studied here, a short run of T residues (4–15) has
been found in the first 25 bp downstream of 90–97% of the
tDNAs. In most other cases, such as several tRNATrp genes,
the poly(T) stretch is found further downstream than the first
25 nucleotides. Finally, a few tRNA genes do not have any
poly(T) domain in the near vicinity. They correspond to
tRNAs that are co-transcribed with small nucleolar RNAs
(snoRNAs). These snoRNAs contain a so-called C/D box, and
play an essential role in guiding the methylation of
ribosomal RNAs and a few other RNAs in eukaryotes. Families of di-cistronic tRNAGly–snoRNA genes and di-cistronic
tRNAMete–snoRNA genes have been found on the Arabidopsis and rice nuclear genomes, respectively (Kruszka et al.,
2003). We searched for di-cistronic tRNA–snoRNA genes
in the five higher plants, and confirmed the widespread
occurrence of these di-cistronic genes (Figures S8 and S9).
Interestingly, the tRNAGly–snoRNA gene family was only
identified in dicots (i.e. A. thaliana, M. truncatula and
P. trichocarpa, with 9, 14 and 13 members, respectively) and
the tRNAMete–snoRNA gene family was only identified in
monocots (i.e. O. sativa and B. distachyon, with seven
members in each plant; Figure 4). No other tDNAs associated to a snoRNA gene were retrieved using our criteria, i.e.
tRNAGly
Mt
Pt
At
C
(b)
tRNAMete
Os
Bd
D
Figure 4. Sequence alignment of di-cistronic tRNA–snoRNA genes present in flowering plant species.
(a) Sequence alignment of representative tRNAGly–snoRNA co-transcripts in A. thaliana, M. truncatula and P. trichocarpa.
(b) Sequence alignment of representative tRNAMete–snoRNA co-transcripts in O. sativa and B. distachyon.
The C and D conserved motifs of snoRNA sequences are indicated. Arrowheads indicate the putative 5¢ and 3¢ ends of snoRNA genes. Note the stretches of T
residues present downstream of snoRNA gene sequences.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
tRNA genes in plants 87
the presence of C/D boxes and poly(T)s downstream of the
snoRNA gene. Whether the variants are all expressed and
whether they are used to methylate the same rRNA sites
requires experimental validation.
Analysis of tRNA gene expression
Although sequence elements required for tRNA gene
expression in plants have been characterized (see above),
only a handful of datasets are available regarding in vivo
expression of plant tRNA genes. In C. reinhardtii, it has been
shown that the most abundant tRNAs recognize the most
frequent codons, whereas tRNAs of low abundance recognize rare codons (Vinogradova et al., 2009), but a complete
analysis has not been performed. Similarly, a global
expression profile on higher plant tRNAs is not yet available,
although a considerable number of small non-coding RNA
libraries have been obtained using deep sequencing technology. However, such libraries are usually restricted to
15–35 nucleotide long RNAs: classically full-length tRNAs
are excluded from the analyses and only fragments of
tRNAs are retrieved as by-products. In the near future, it
would be of interest to use such deep sequencing
approaches to perform a global analysis of tRNA expression
from various plant tissues or under various environmental
conditions.
INTRON-CONTAINING TRNA GENES
Only two families of higher plant nuclear-encoded tRNAs,
tRNAMete and tRNATyr, have so far been reported to contain
introns. In vitro maturation assays on mutated tRNATyr or
tRNAMete have revealed the importance of the nucleotide
sequence and also the pre-tRNA structure on the maturation
(a)
tRNAMete
tRNATyr
A
A
C
C
C
C
A
A
C G
G C
C G
G C
G C
G C
A U
A C
G
C G
C
U A
C G
G C
U A
A
U U
CG
UG
U G A C UCGA U
UGA
UGGCC U A
UGGCC
CUCUC U A
G
C GCGA
U
U
G
G G
ACUGG
GAGAG
ACUGG U
GAGC
G
GCGC
U
C
CUA
UC
U
G
C
UC
U
C
G
GC A
U
U
C
U
G
U
G
U
U GA
A G
G
A U G
A U G
G C
G C
A
C
G
A C
G
U
C
A UA U
C
U U AA
G
C GAU
C GU
U
A
U A
G C
C G
G
C
C G
U
A G
C
A
UA
C
A
G
A
A
U AG
UC
G
AU
....
...
.
...
...
.
...
..
.....
....
...
.
...
...
.
...
..
.....
efficiency of introns (Stange et al., 1992; Akama et al., 1997,
2000). In the predicted pre-tRNA structures, the 5¢ and 3¢
cleavage sites are present in single-stranded RNA regions
separated by 3–5 bp (Figure 5a; Stange et al., 1991; Akama
et al., 2000). These structural features have been shown to
be important for in vitro intron splicing of tRNATyr and
tRNAMete (Stange et al., 1992; Akama et al., 2000). Other
experiments have demonstrated the deleterious effect of a
cytidine residue at the 5¢ or 3¢ intron borders on intron
maturation (Akama et al., 1997, 2000), and involvement of
the D stem and the variable loop in pre-tRNATyr intron
splicing (Stange et al., 1992).
Our survey shows that all higher plant tRNAMete and
tRNATyr genes contain an intronic sequence between positions 37 and 38 of their tRNA sequences. Analysis of the 185
tRNAMete and tRNATyr and their intronic sequences reveals:
(i) striking similarities in the sequence and structure of the
anticodon stem and the variable loop (Figure 5a), (ii) a
variation in intron size (6–22 nucleotides), and (iii) limited
sequence conservation in introns (Figure 5b). The intron
sequences in the pre-tRNAMete family are more conserved
than in the tRNATyr family. Sixty-two of the 65 tRNAMete
introns have a GCT motif at the 5¢ end of the intron and a
GAGT motif one nucleotide upstream of the 3¢ end of the
intron. The GAG nucleotides are involved in the base pairing
necessary to maintain the structure of the pre-tRNAMete,
which is a prerequisite for intron splicing (Figure 5a; Akama
et al., 2000). In the tRNATyr family, only a T at the first
position of the intron and the G residue 2–4 nucleotides
before the 3¢ end of the intron are conserved (Figure 5b). The
T residue appears to be important for formation of pseudouridine at the second position of the anticodon (Akama et al.,
(b)
tRNATyr
At
Mt
Pt
O
s
B
d
T AGACG . . . . . . . . . CAGA T T .
T TCT T T AA . . . . . . . CAGAC . .
T GGG T GA A A T CC T GA T GGC C . .
T T G T T G . . . . . . . . . A GG T A . .
T T TGAAG . . . . . . . . CAGA T T A
T
G
tRNAMete
At
Mt
Pt
O
s
B
d
GC T A C . . . T GA GT G
GC T T C . . . T GA GT A
GC T T C . . . T GA GT T
GC T A T . . . T GA GT T
GC T T A A A GCGA GT G
GC T
GA GT
Figure 5. Intron-containing tDNAs in flowering plant nuclear genomes.
(a) Cloverleaf secondary structures of Arabidopsis intron-containing pre-tRNAMete and pre-tRNATyr. Intron cleavage sites are indicated by arrows. Anticodons are
indicated by black dots. Nucleotides in the anticodon stem and variable loop region that are conserved between the tRNAMete and tRNATyr gene families are shown
against a blue background. Nucleotides within intron sequences that are conserved within the tRNAMete or tRNATyr family are shown against green and grey
backgrounds, respectively.
(b) Alignments of the intron sequences present in pre-tRNATyr and pre-tRNAMete of flowering plants. For simplicity, only one representative sequence per nuclear
genome is presented. Full sequence alignments are available upon request. The conserved nucleotides are underlined.
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
88 Morgane Michaud et al.
1997). Experimental analyses are required to determine
the role of these conserved domains on intron splicing
efficiency.
In addition to the above intron-containing tRNAs, a
putative tRNAAla in O. sativa and a putative tRNAThr in
B. distachyon contain an intron between positions 37 and
38, but of a much larger size (129 and 132 nucleotides,
respectively). These tRNA genes (Figure S10) are orphan
genes and are not present in the other higher plant nuclear
genomes. The B. distachyon tRNAThr gene is lacking one
nucleotide in the anticodon stem. Nevertheless, the A and B
internal boxes as well as the upstream CAA triplet and a
downstream poly(T) stretch are present in both tDNAs.
Whether these two tRNAs are expressed and functional is
questionable and awaits experimental evidence.
In green algae, the situation appears different. In C. reinhardtii, of the 256 tRNA genes detected in the genome, 161
have an intron (Merchant et al., 2007). The intron size is
more variable compared to higher plants, and ranges from
9 to 56 nucleotides. Alignment of these intronic sequences
does not reveal any obvious conservation at the nucleotide
level, and nothing is known about the pre-tRNA structure
in C. reinhardtii at present. The tRNAMete and tRNATyr genes
also contain an intron in C. reinhardtii (Figure S11). Mature
sequences of these two tRNA families present in the
anticodon stem and the variable loop, the characteristics
found in higher plants. Although the tRNATyr intron shows
similarities in size and sequence to those of higher plants
(T at position 1 and a G residue 2–4 nucleotides before the
intron’s 3¢ end), tRNAMete introns do not share the GCT and
GAGT motifs and are much longer in size compared to
tRNAMete introns in higher plants. In the green alga O. tauri,
of 48 tRNA genes identified, 16 contain an intron, whereas
of the 432 tDNAs identified in the moss P. patens, only
tRNAMete and tRNATyr genes contain an intron. As a whole,
flowering plants and bryophytes appear to have evolved
differently from the green alga kingdom with respect to the
presence of introns in tRNA genes.
TRNA-RELATED SINES
Short interspersed nuclear elements (SINEs) are mobile
non-autonomous elements that are found in a wide variety
of eukaryotic genomes and are transcribed by PolIII. The
best known family of SINEs is the Alu family (Kramerov and
Vassetzky, 2005). SINE retroelements use the enzymatic
machinery of autonomous long interspersed elements
(LINEs) for propagation by retrotransposition. The SINE/
LINE partnership is based on common 3¢ regions or poly(A)
tails, and a general model of SINE and LINE retroposition
has been proposed by Sun et al. (2007). Although most
SINEs are ancestrally derived from tRNA genes (or in rare
cases from 7SL or 5S RNAs), the typical tRNA cloverleaf
structure is not apparent. SINEs derived from tRNAs have a
composite structure comprising a tRNA-related 5¢ portion
followed by a tRNA-unrelated region. In all cases, the A and
B boxes corresponding to the internal promoter are present
in the tRNA-related portion. A eukaryotic phylogenetic tree
of SINE structures has been tentatively constructed (Sun
et al., 2007). The authors proposed that SINE RNAs started
as simple structures with a single tRNA-related segment.
Then either this segment is amplified, or short or long tRNAunrelated segments are added to the original molecule. The
first SINE described in plants, p-SINE1, was found in the
O. sativa Waxy gene (Umeda et al., 1991). In A. thaliana and
Brassica species, various families of tRNA-related SINEs,
called RathE1, RathE2, RathE3, S1 and S2 have been identified (Pelissier et al., 2004). SINEs are very short (<500 bp)
and have no conserved sequence, making research into
them very difficult. Despite these difficulties, SINEs have
now been identified in many plant species, and are usually
dispersed randomly in nuclear genomes (for review, see
Deragon and Zhang, 2006). SINEs are usually not present in
micro-organisms, and Chlamydomonas is the first unicellular organism in which such elements have been identified
(Merchant et al., 2007). Remarkably, the three SINE tRNArelated families identified in this green alga have retained an
authentic tRNA structure, resembling tRNAArg(CCG),
tRNAAsp(AUC) and tRNAAsp(GUC), as well as an intron
between positions 37 and 38 of the tRNA sequences. However, in vivo, no mature tRNA from the three types of SINE
RNAs was detected, demonstrating that the correct cloverleaf folding required by tRNA-processing enzymes is not
present, and that Chlamydomonas tRNA-related SINE RNAs
do not fold like their ancestral molecule, as already demonstrated for other SINEs (Pelissier et al., 2004; Cognat et al.,
2008). Completion of the poplar whole-genome sequencing
project enabled us to identify a new tRNA-related SINE
family of 35 members in P. trichocarpa (Figure 6 and Figure
S1). Surprisingly, as for Chlamydomonas, the tRNA-related
portion can be folded into a mostly perfect cloverleaf tRNA
structure with an anticodon AAG (theoretically specifying
leucine). The tRNA-unrelated portion is very well conserved
among the family, and is terminated by a poly(A)-rich region
followed by a poly(AT)-rich domain. The 35 members of the
family are randomly dispersed on the various scaffolds
(Figure S1). Identification of this new family of SINEs represents an additional example of the complexity of these
elements, and their probable importance in both the evolution and organization of plant nuclear genomes. In most
cases, their function remains unknown and needs to be
elucidated.
ORGANELLAR TRNA GENES
In plants, translation occurs in the cytosol but also in
mitochondria and plastids. These two organelles originate
from endosymbiotic a-proteobacteria or cyanobacteria,
respectively. Both organelles have a genome, but many
genes have been lost or transferred to the nucleus during
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
tRNA genes in plants 89
(a)
tRNA-related
A
Internal region
Poly AT
B
Poly A
75
110
15
40
(b)
Figure 6. tRNA-related SINEs in P. trichocarpa.
(a) Schematic representation of the structure of the putative tRNALeu-related
SINE members. The A and B boxes (dark grey) within the tRNA (black)
required for transcription, and the poly(A)-rich (pale grey) and poly(AT)-rich
(medium grey) regions are indicated. The size (in nucleotides) of each domain
is given.
(b) Cloverleaf structure of a representative tRNA-like region of the putative
tRNALeu-related SINE transcripts. The location of the intron-like sequence
is indicated. The three black dots correspond to the anticodon. Blue and red
dots correspond to U–A or U.G and C–G pairings respectively. Thirty-five
tRNA-related SINE sequences were identified (Figure S1).
evolution. The few remaining genes encode proteins and
structural RNAs (all rRNAs and some tRNAs).
In A. thaliana, O. sativa and P. trichocarpa chloroplast
genomes, 37, 38 and 39 tDNAs have been annotated,
respectively. These genes correspond to 30 tRNA isoacceptor species (Hiratsuka et al., 1989; Sato et al., 1999; http://
genome.ornl.gov/poplar_chloroplast/). In Chlamydomonas,
only 30 tDNAs are present on the plastidial genome,
corresponding to 25 tRNA isoacceptor species (Maul et al.,
2002). In all cases, the various tRNAs are sufficient to allow
protein synthesis, and it is generally assumed that the
chloroplast genomes encode all tRNAs required for translation. In agreement with this hypothesis, knockout of
tRNAAsn(GUU) or tRNACys(GCA) tobacco plastidial genes
showed that these two genes are essential for plant development, and that the absence of plastidial tRNAAsn or
tRNACys cannot be compensated for by import of their
cytosolic counterparts into chloroplasts (Legen et al., 2007).
However, it appears that some tRNA genes have been lost
on the plastidial genome of a few organisms, such as the
parasitic non-photosynthetic plants Epifagus virginiana
and Oronbranche minor (Wolfe et al., 1992; Lohan and
Wolfe, 1998) and some dinoflagellates (Howe et al., 2008).
Although it has not been shown, it is tempting to speculate
that these missing tRNAs are imported from the cytosol into
plastids.
Overall, 17–29 tDNAs have been identified in plant mitochondrial genomes, with the notable exception of Chlamydomonas mitochondria, which contain only three tDNAs
(Vinogradova et al., 2009). Transfer RNA genes corresponding to Ala, Arg, Leu, Thr and Val are missing in all sequenced
angiosperm mitochondrial genomes. Nuclear-encoded
tRNA import has been shown to compensate for
the deficiency of mitochondrial tDNAs (Schneider and
Maréchal-Drouard, 2000; Duchêne et al., 2009) (see below).
Another characteristic of mitochondrial genomes is acquisition of tDNAs of another origin, particularly plastidial origin.
As an example, in Arabidopsis, 11 tRNA species called
‘native’ tRNAs are encoded by authentic mitochondrial
genes, and four others are chloroplast-like tRNAs. The
remaining tRNAs, corresponding to seven amino acids, are
imported from the cytosol (Duchêne and Maréchal-Drouard,
2001). Similar data have been obtained in numerous plant
species (Rainaldi et al., 2003).
Expression of organellar tDNAs
Plastids have two types of RNA polymerases: plastidencoded eubacterial-type RNA polymerase (PEP), and
nuclear-encoded phage-type RNA polymerases (NEPs).
Standard PEP promoters resemble E. coli promoters, with
consensus sequences at positions )35 (TTGACA) and )10
(TATAAT). NEP-dependent promoters are of two types.
Some (type I) have a YRTA motif, and others (type II) have
no identified consensus. Some tRNAs are co-transcribed
with other RNAs. For example, the four rRNA molecules are
co-transcribed with tRNAIle, tRNAAla and tRNAArg from
an NEP and/or a PEP promoter in higher plants (LealKlevezas et al., 2000; Shiina et al., 2005). Transcription of
tRNAGlu(UUC) and tRNAVal(UAC) genes is controlled by a
PEP promoter in A. thaliana (Hanaoka et al., 2003).
Mitochondrial genes are transcribed by two NEPs, and
many promoter regions have a CRTA consensus sequence
(Fey et al., 1999; Kühn et al., 2005). Numerous promoters
are scattered along the genome, and multiple promoters for
a single gene are also common. Indeed transcription of
protein, rRNA or tRNA genes in mitochondria appears to be
a relaxed process with little control or modulation (Holec
et al., 2006).
Import of nuclear-encoded tRNAs into mitochondria
To provide an active mitochondrial translation system and
to compensate for the lack of mitochondrial-encoded tRNAs,
some nuclear-encoded tRNAs used in the cytosolic transla-
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
90 Morgane Michaud et al.
tion machinery are imported into mitochondria. Gene loss
and tRNA import acquisition occurred very early during
plant evolution (Akashi et al., 1996; Vinogradova et al.,
2009). The number and identity of imported tRNAs vary from
one plant to another. Import of tRNAs corresponding to Ala,
Arg, Leu, Thr and Val occurs in all known angiosperms, but
gene loss and import of the corresponding tRNA can also
occur in individual species such as tRNATrp in Arabidopsis
(Salinas et al., 2008, Duchêne et al., 2009) or tRNASer in
Helianthus annuus (sunflower; Ceci et al., 1996). The most
detailed surveys in higher plants have been performed for
potato (Solanum tuberosum; Maréchal-Drouard et al., 1990;
Brubacher-Kauffmann et al., 1999), wheat (Triticum aestivum; Glover et al., 2001) and Chlamydomonas mitochondria (Vinogradova et al., 2009). Various tRNA isoacceptors
are usually found in mitochondria, although the extent of
their steady-state levels in the organelle may vary (Glover
et al., 2001; Vinogradova et al., 2009). Comparing tRNA
isoacceptors in C. reinhardtii, the extent of mitochondrial
localization was shown to be highly variable. For instance,
tRNAGly(CCC), tRNAVal(CAC), tRNALeu(CAG) are mainly
cytosol-specific, tRNAGly(GCC), tRNAVal(AAC) are equally
present in both compartments, whereas tRNAVal(UAC) and
tRNALeu(CAA) are mainly localized in mitochondria. As discussed below, the observed differences might reflect the
need for mitochondria to optimize translation.
Many studies have been and are still being performed to
understand the tRNA import process, in particular to determine the specificity and targeting determinants, and to
identify mitochondrial membrane channels. The anticodon,
D and T loops have been identified as import determinants
in plant mitochondria. At the level of the mitochondrial outer
membrane, the voltage-dependent anion channel and some
components of the protein import machinery (TOM complex) have been shown to be essential for tRNA import
(Salinas et al., 2008; Duchêne et al., 2009). However, elucidating the tRNA mitochondrial import process is still a huge
challenge.
Organellar tRNAs and codon usage
In many organisms, there is a good correlation between
codon usage and the level of corresponding tRNAs.
Adjustment of the tRNA population to codon usage has been
demonstrated in chloroplasts (Pfitzinger et al., 1987), and
the concentration of various tRNAs is not controlled by gene
dosage (Maréchal-Drouard et al., 1993). Plastidial genomes
of photosynthetic plants usually encode 30 tRNA species
and do not import tRNAs from the cytosol. According to
standard rules, 32 tRNAs are required to read all codons
of the universal genetic code (Crick, 1966). However, the
existence of relaxed wobble rules has been proposed
(Percudani, 2001). In particular, the presence of a modified
uridine at the first position of the anticodon enables a
single tRNA to decode the four codons of a codon box. For
example, Pfitzinger et al. (1990) showed that, due to a
modified uridine at position 34, plastidial tRNAAla or tRNAPro
are able to decode the four-codon family. In addition, by
producing knockout mutants for the plastidial tRNAGly(UCC)
or tRNAGly(GCC) genes, Rogalski et al. (2008) demonstrated
that tRNAGly(UCC) containing an unmodified U in the anticodon can read all four glycine codons due to ‘superwobbling’, whereby the U can pair with the four nucleotides.
Similarly, tobacco plants from which the tRNAVal(GAC) gene
had been deleted from the plastidial genome showed
normal development (Corneille et al., 2001). As no import of
cytosolic tRNAVal occurs in these transplastomic plants, the
GUN codon family is probably recognized by the only
remaining plastidial tRNAVal(UAC) (P. Maliga, S. Corneille,
Waksman Institute of Microbiology Rutgers, The State
University of New Jersey, USA, and L. Maréchal-Drouard,
unpublished data).
In the plant mitochondrial translation system, wobble
pairing rules for codon–anticodon recognition have not been
established, and it is difficult to draw precise conclusions
on the minimal set of mitochondrial tRNAs required for an
active translation machinery. However, using classical
wobble rules, it is worth noting that, in Chlamydomonas
mitochondria, a good correlation was found between the
steady-state levels of imported tRNAs and the frequency of
the cognate codon, not only in mitochondria but also in the
cytosol (Vinogradova et al., 2009). Furthermore, it is possible to estimate the steady-state levels of potato mitochondrial tRNAs fractionated using 2D-PAGE (Maréchal-Drouard
et al., 1990) and to compare their level to the plant mitochondrial codon usage (L. Maréchal-Drouard, unpublished
data). Leucine codons are the most frequently used mitochondrial codons (approximately 12.5%) and imported
tRNAsLeu are the most abundant mitochondrial tRNAs
(approximately 15%). Codons corresponding to cysteine
and tryptophan are the least frequently used (approximately
1.7% for each amino acid), and are recognized by mitochondrial-encoded tRNAs that are present in low amounts in the
organelle (approximately 1.5% for each tRNA). There are
also tRNAs corresponding either to mitochondrial-encoded
(e.g. tRNAPhe) or nuclear-encoded (e.g. tRNAVal, tRNAArg)
species that are present in equal amounts and recognize
codons similarly used in the mitochondria (5–7%). In addition, it should be noted that, although the plant mitochondrial codon usage does not fluctuate greatly, the origin of the
mitochondrial tRNA population varies significantly from one
plant species to another. Thus, it appears likely that no
correlation exists between the origin (mitochondrial or
nuclear) of a mitochondrial tRNA and the frequency of the
cognate codons.
CONCLUSION
Analysis of tDNAs among various plant genomes highlights
many common characteristics, but also some differences
ª 2011 The Authors
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 80–93
tRNA genes in plants 91
within angiosperms or between angiosperms and green
alga. A good correlation was observed between the number
of tDNAs and genome size. The number of isoacceptors is
also nearly identical in all species and correlates with codon
usage. However, Arabidopsis breaks these rules due to
the very high numbers of three tRNA isoacceptor genes. The
various genomes are not homogenous with regard to
the number of pseudogenes, organellar-like tDNAs or SINEs.
Whether these elements are a source of genetic instability in
plant nuclear genomes will need to be assessed in the future.
Flowering plants share a common transcriptional and
post-transcriptional organization (single transcription units,
upstream and downstream signals, types of introns) and are
clearly different from the green alga Chlamydomonas. The
great flexibility in tDNA organization and transcription generates the ubiquitous, highly structured tRNA molecules.
However, the presence of a large number of tRNA isodecoder
genes raises a question regarding their potential function: are
they simply the result of neutral mutations during evolution,
or do they play an important role within the cell? This comprehensive analysis provides a basis for further studies, and
the next challenges will be to explore the influence of this
flexibility on genome maintenance and expression.
ACKNOWLEDGEMENTS
We thank Lee Tuddenham (Architecture et Réactivité de l’ARN, UPR
9002 CNRS, Strasbourg, France) for thorough assessment of our
manuscript. M.M. holds a fellowship from the French Ministère
Délégué à l’Enseignement Supérieur et à la Recherche. This work
was supported by the Centre National de la Recherche Scientifique
and the University of Strasbourg.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Sequence alignments of the tRNA-related SINEs found
in P. trichocarpa.
Figure S2. Distribution of tDNAs on B. distachyon and A. thaliana
chromosomes.
Figure S3. Number of tRNA isoacceptors and tRNA isodecoders per
nuclear genome, and ratio between the number of tRNA isodecoders and the number of tRNA genes.
Figure S4. Correlation between the number of tDNA copies specific
for each amino acid and the frequency of occurrence of the same
amino acid in M. truncatula, P. trichocarpa and C. reinhardtii.
Figure S5. Correlation between the number of tRNA gene copies
specific for each amino acid and the frequency of occurrence of the
same amino acid in A. thaliana.
Figure S6. Number of tRNAPhe(GAA) or tRNAGln(CTG) isodecoders
per number of tDNAs.
Figure S7. A and B box sequences in A. thaliana tDNAs.
Figure S8. tRNAGly–snoRNA co-transcripts in A. thaliana, M. truncatula and P. trichocarpa.
Figure S9. tRNAMete–snoRNA co-transcripts in O. sativa and B. distachyon.
Figure S10. Cloverleaf structure of the two orphan tDNAs found in
O. sativa and B. distachyon.
Figure S11. Alignment of pre-tRNATyr and pre-tRNAMete from
C. reinhardtii.
Table S1. Accession numbers or websites from which tDNAs were
retrieved for plant genomes.
Table S2. Number of mitochondrial or plastidial-like tDNAs per
chromosome.
Table S3. Nuclear tRNA gene content and codon frequency in
A. thaliana and O. sativa.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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