Group Title: BMC Genomics
Title: RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes
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Title: RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes
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Language: English
Creator: Grosjean, Henri
Gaspin, Christine
Marck, Christian
Decatur, Wayne
de Crécy-Lagard,Valérie
Publisher: BMC Genomics
Publication Date: 2008
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Abstract: BACKGROUND:Naturally occurring RNAs contain numerous enzymatically altered nucleosides. Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics) depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms of life. If the genomic sequences of the RNA molecules can be derived from whole genome sequence information, the modification profile cannot and requires or direct sequencing of the RNAs or predictive methods base on the presence or absence of the modifications genes.RESULTS:By employing a comparative genomics approach, we predicted almost all of the genes coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original predictions.CONCLUSION:The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms. This may result from the specific lifestyle of halophiles that require high intracellular salt concentration for survival. This salt content could allow RNA to maintain its functional structural integrity with fewer modifications. We predict that the few modifications present must be particularly important for decoding, accuracy of translation or are modifications that cannot be functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This analysis also guides future experimental validation work aiming to complete the understanding of the function of RNA modifications in Archaeal translation.
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Research article

RNomics and Modomics in the halophilic archaea Haloferax volcanic:
identification of RNA modification genes
Henri Grosjean1,2,3, Christine Gaspin4, Christian Marck5, Wayne A Decatur6
and Valerie de Crecy-Lagard* I


Address: 'Department of Microbiology and Department of Microbiology and Cell Science, University of Florida, Gainsville, FL-32611, Florida,
USA, 2IGM, Universite de Paris-sud, UMR 8621, Orsay, F 91405, France, 3CNRS, IGM, Orsay, F-91405, France, 4National Institute of Agronomical
Research, Biometrics and Artificial Intelligence Department, Chemin de Borde-Rouge, Auzeville BP 27, 31326 Castanet-Tolosan, France, 5Institut
de Biologie et de Technologies de Saclay (iBiTecS), Commissariat a l'Energie Atomique (CEA), Gif sur Yvette, F-91191, France and 6Department
of Biochemistry and Molecular Biology, University of Massachussets, Amerherst, MA 01003, Massachusetts, USA
Email: Henri Grosjean henri.grosjean@igmors.u-psud.fr; Christine Gaspin Christine.Gaspin@toulouse.inra.fr;
Christian Marck christian.marck@cea.fr; Wayne A Decatur wdecatur@biochem.umass.edu; Valerie de Crecy-Lagard* vcrecy@ufl.edu
* Corresponding author


Published: 9 October 2008
BMC Genomics 2008, 9:470 doi:10.1186/1471-2164-9-470


Received: I July 2008
Accepted: 9 October 2008


This article is available from: http://www.biomedcentral.com/1471-2164/9/470
2008 Grosjean et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Naturally occurring RNAs contain numerous enzymatically altered nucleosides.
Differences in RNA populations (RNomics) and pattern of RNA modifications (Modomics)
depends on the organism analyzed and are two of the criteria that distinguish the three kingdoms
of life. If the genomic sequences of the RNA molecules can be derived from whole genome
sequence information, the modification profile cannot and requires or direct sequencing of the
RNAs or predictive methods base on the presence or absence of the modifications genes.
Results: By employing a comparative genomics approach, we predicted almost all of the genes
coding for the t+rRNA modification enzymes in the mesophilic moderate halophile Haloferax
volcanii. These encode both guide RNAs and enzymes. Some are orthologous to previously
identified genes in Archaea, Bacteria or in Saccharomyces cerevisiae, but several are original
predictions.
Conclusion: The number of modifications in t+rRNAs in the halophilic archaeon is surprisingly
low when compared with other Archaea or Bacteria, particularly the hyperthermophilic organisms.
This may result from the specific lifestyle of halophiles that require high intracellular salt
concentration for survival. This salt content could allow RNA to maintain its functional structural
integrity with fewer modifications. We predict that the few modifications present must be
particularly important for decoding, accuracy of translation or are modifications that cannot be
functionally replaced by the electrostatic interactions provided by the surrounding salt-ions. This
analysis also guides future experimental validation work aiming to complete the understanding of
the function of RNA modifications in Archaeal translation.


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Background
Post-transcriptional modification of transfer and ribos-
omal RNAs is essential for their cellular activities as core
molecules of the translation apparatus. To date, the chem-
ical structure of more than one hundred RNA modifica-
tions have been identified in all domains of life [1-3]. In
transfer RNAs, modified nucleotides are found predomi-
nantly within the 3D-core of molecules and in the antico-
don arm, especially at the wobble position 34 and at
position 37, 3' adjacent to the anticodon (conventional
numbering of tRNA positions is as defined in [4], http://
www.tRNA.uni-bayreuth.de). These particular modifica-
tions allow the molecules to adopt the canonical L-shaped
conformation and modulate interactions with various
interacting macromolecules such as aminoacyl:tRNA-syn-
thetases, initiation, elongation and termination factors,
mRNA and/or elements of the decoding and peptidyl-
centers of the ribosome (reviewed in [5-10]). In ribos-
omal RNAs, modified nucleotides are located mostly in
regions corresponding to the functional centers of the
ribosome [11-14]. Their location suggests a role in accu-
racy and efficiency of translation, however the specific
function of each modified nucleoside is still largely
unknown. This lack of knowledge stems from peculiarities
of the rRNA molecule itself: it is a large molecule (molec-
ular mass between 1.5 to 3.9 MDa); some nucleotides are
only partially modified and their functions) are most cer-
tainly dependent on a network of synergistic interactions
with different elements of the ribosome, including other
modified nucleosides that may act cooperatively. Never-
theless, function has been attributed to modified nucleo-
sides in rRNA in a few cases [13,15-22].

Difference in profile and type of RNA modifications
(Modomics) is one of the criteria that distinguish the
three kingdoms of life. While universal modifications
such as T, msU, t6A or mlG are found in a large numbers
of archaeal, bacterial and eukaryal tRNAs, each kingdom
has a set of signature modifications. For examples mimG,
G+, m22Gm, ac6A or m1' are typical of archaeal tRNAs,
while yW, mcmSU and manQ, or k2C, mo5U and m6t6A
are typical of tRNAs from Eukarya or Bacteria respectively
(for review see Figure 8.1 in [23]). The same conclusion
applies for modified nucleotides in rRNAs (see [24]; httpg
/people.biochem.umass.edu/foumierlab/snomadb/
main.php).

In Archaea, our knowledge of the diversity of RNA modi-
fications is largely founded on the lifework of Jim McClos-
key and Pamela Crain, who analyzed bulk tRNA and
rRNA preparations from a phylogenetically diverse set of
Archaea. The technique used combined separation of
nucleosides of bulk RNA RNase hydrolysate by liquid
chromatography, followed by comparison of the derived
modified nucleosides to synthetic ones by mass spectrom-


etry techniques [25] (for more recent development of the
technique, see [26] and references therein). However, to
date Haloferax volcanii, a Halobacteriaceae that lives opti-
mally at 42C in the presence of 1.5-2.5 M NaCl [27], is
the only Archaea for which both the chemical identities
and positions of almost all modified ribonucleosides have
been mapped for nearly the whole set of the 52 sequenced
tRNAs with distinct anticodons [28,29]. In addition, 13
tRNA sequences of two closely related mesophilic halo-
philes are available, Halobacterium cutirubrum (12
sequences) and Halococcus morrhuae (one sequence) [4].
For H. volcanii ribosomal RNAs, the type and position of
modifications are available in the case of the 16S RNA
[30,31 ], but not for the 23S nor the 5S RNAs. These can be
inferred from studies on another closely phylogenetically
related halophilic Archaea Haloarcula marismortui [32,33].
However, while the RNA modifications have been
mapped in RNAs of halophiles, including H. volcanii, the
identity of the genes that code for the corresponding RNA
modification enzymes remains largely ignored.

Using a comparative genomic analysis method, that we
have recently applied to the only other organism with an
almost complete set of sequenced tRNAs, the pathogenic
bacteria Mycoplasma capricolum [34], we set out to predict
all the RNA modification genes in the halophilic archae-
aon H. volcanii. Some were easily predicted by homology
with experimentally validated RNA modification genes
from other organisms, while a few are original predictions
based on comparative genomic analysis [35] (not based
on homology). This computation work provides predic-
tions that can now guide the experimental validation
work with the goals of elucidating the role of RNA modi-
fications in Archaeal translation, and ultimately obtaining
a better understanding of the emergence of this extraordi-
nary complex enzymatic machinery during evolution.

Results and Discussion
Post-transcriptional modification of RNA
Modification pattern of tRNAs
Thanks to the "tour de force" of Gupta [28,29], a list of
almost all of the modified nucleosides present in the dif-
ferent tRNAs of Haloferax volcanii, a typical mesophilic
halophile, is available. Figure 1A shows their distribution
(identity and location) in the general 2D-cloverleaf struc-
ture of tRNA, while Figure 1B shows their positions in a
schematic 3D-architecture model. The modifications that
are unique to archaeal tRNAs are shown in gray. For exam-
ple G+-15 (for Archaeosine), C*-34 (for a lysidine-type of
nucleoside) and m1'-54 are unique to all archaeal tRNAs
analyzed so far, both by their chemical structure and their
positions in the nucleic acid, while others such as m22G-
10, T-22, T-52, Cm-56 and mil-57 (I for inosine) are
unique only because of their position, rather than their
chemical structure (see in [1,4], reviewed in [23]). Since


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3'

5' O

0-0
0-0
0-0
0-0
0-0


3D-core


T-loop L T-stem


Figure I
Type and location of modified nucleosides in tRNAs of H. volcanii.(A) 2D-Cloverleaf representation of tRNA. Group
of nucleosides in boxes are present at the same location but in different isoacceptors species. Modified nucleosides in white
and in a gray background are uniquely present at that position in archaeal tRNAs so far. Those indicated in black are also found
in tRNAs from Bacteria and/or Eukarya. See text for references on Abbreviations. The large gray box including the m'P con-
taining branch and the G+ containing branch encompass the interacting parts of the tRNA molecule that forms the 3D-core.
(B) Schematic representation of tertiary interactions in tRNA structure. Each nucleoside involved in stacking or base pairing
with another nucleotide within the 3D-core (gray background box) is represented by a rectangle. Other parts of the tRNA
(anticodon branch and amino acid stem are represented by lines. Inside the large gray rectangle are the elements that contrib-
ute to the 3D interaction, allowing an L-shaped spatial conformation to be formed from the 2D cloverleaf structure.


tRNA modification enzymes are usually site-specific, we
expect the corresponding genes to be different from those
in the other kingdoms. In terms of chemical structure
(and not their position), the modified nucleosides m22G,
m5C and mlI found in tRNAs of H. volcanii are character-
istic of eukaryal rather than eubacterial tRNAs, while D
(for dihydrouridine), I (for inosine), m5U, i6A (i for iso-
pentenyl), Q (for queuosine) and m7G, which are com-
mon in tRNAs of Bacteria and Eukarya, are absent in H.
volcanii (but not necessarily in other Archaea see Fig 8.1
in [23]). Also, as mentioned in the Methods section, mlG


at position 9 and m5C at positions 50-52 are found in
tRNAs from another halophile, H. cutirubrum, but absent
in these positions in all H. volcanii tRNAs.

As illustrated in Figure 1B, the modified nucleosides in
tRNA can be classified in two categories: those that are
present in the 3D-core (gray background) and presumably
implicated mostly in the formation and/or the control of
flexibility of the L-shaped molecule (reviewed in [9,36]);
and those present in the decoding region (anticodon hair-
pin), implicated in the efficacy and accuracy of interaction


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with selected amino-acyl tRNA synthetases (reviewed in
[7]) and the various codons within the mRNA:ribosome
complex (reviewed in [5,8]).

The identification of a modified nucleotide does not
imply it is present in a one to one ratio with the RNA mol-
ecule. Indeed, the presence and final chemical structure of
certain modified nucleotides, particularly the hypermodi-
fled ones, may vary according to the physiological con-
straints of the cell (aerobic/anaerobic conditions,
temperature, availability of intermediate metabolites or
cofactors of the modification enzymes, various metabolic
stress conditions; discussed in: [37-41]). The A-15, C-34,
U-52 and U-54 residues in some H. volcanii tRNAs were
reported to be only partially modified into G+-15, ac4C-34
(ac for acetyl), T-52 or T-54/mil-54 respectively, giving
rise to distinct iso-tRNA species that sometimes can be
separated by liquid chromatography or 2D-gel electro-
phoresis [28]. When a modification requires multiple
modification enzymes like min-54, G+-15 and few U-34
derivatives (see below), only intermediate products may
exist under certain physiological conditions. However, the
genes corresponding to all of the expected modified
nucleotides (present or not) in the cellular tRNA popula-
tion should be present in the genome.

Modification pattern of rRNA
In their early work, Gupta and Woese identified four posi-
tions with modified bases in H. volcanii 16S RNA[30].
These were later confirmed [31] and identified as acp3U-
910 (position 966 by E. coli numbering; acp for 3-amino-
3-carboxypropyl, thus an amino acid) in hairpin 31
located in the 3' major domain, m6A-1432 (position
1500) in helix 44, the tandem m62A-1450 and m62A-1451
(positions 1518 and 1519) in hairpin 45 and a modified
cytidine (C*) of still unknown structure (MW:330.117 as
determined by mass spectrometry) at position 1352
(1404) in helix 44 in the 3' minor domain ofSSU RNA.
Their locations are shown in the schematic 2D-structure
(Figure 2A) and 3D-structure (Figure 2B) of 16S rRNA.

The characteristic pair of tandem dimethylated adenosine
(m62A m62A) is universally present at analogous positions
in rRNA of all organisms examined so far. These are
located at the interface of the two ribosomal subunits
[11,12,14] and their formation may serve as a checkpoint
in quality control of ribosome biogenesis [42-44]. Like-
wise, acp3U-910 (966) in hairpin 31 appears to be nearly
universally modified, although the type of base and corre-
sponding modification vary from one organism to
another: m2G 3'-adjacent to a msC in 16S RNA of both E.
coli and Thermotoga maritima [45], m22G 3'-adjacent to
m5C in Thermus thermophilus [46], ml-acp3U in Dro-
sophila melanogaster SSU RNA and designated as unknown
modified nucleoside in SSU RNA of other organisms,


mostly archaeons [31]. This modified nucleotide is above
the P-site-bound tRNA and directly contacts the antico-
don stem-loop of tRNA at position 34 [47-51], and is also
often modified (see Figure 2A and Additional file 1). Sev-
eral studies indicate this nucleotide is important in decod-
ing genetic information, particularly at the step of
initiation [21,22,52].

Helix 44 is the dominant structural component of the 30S
subunit interface. It's upper end lies just below where the
mRNA transverses the subunit in the P site [53,54]. This
portion forms a significant intersubunit bridge while at
the same time is directly functionally important for effi-
cient and accurate decoding since two bases, at least in the
E. coli ribosome (bases 1492 and 1493) flip out of an
internal loop in this region [53,54]. This allows the mon-
itoring by direct contact of the mRNA-tRNA base pairing
in the A site, a conformational transition facilitated by the
binding of aminoglycoside antibiotic, e.g. paromomycin,
to a pocket in the major grove of the top of helix 44 [55].
Modified nucleoside m6A-1432 (1500 E. coli numbering)
at the bottom of helix 44 is present in SSU RNA of most
(if not all) Archaea, and only a few Eukarya, but never in
Bacteria (for references see [31]). It is also termed a
'decoding site nt' [49] because it is present in the function-
ally significant region of helix 44, adjacent to a critical
intersubunit bridge (B2a). Contrary to the others above,
the unknown N-330 (C* 1352; 1404 E. coli numbering) is
found in Archaea and in many Bacteria, but not in
Eukarya. While it directly contacts paromomycin bound
to helix 44, its function remains an enigma and its chem-
ical structure remains to be elucidated.

Modification pattern of large subunit rRNAs
No data are available for H. volcanii 23S RNA modifica-
tions. We therefore used the analysis performed in the
closely related organism Haloarcula marismortui
[32,33,56] that led to the identification of modified
nucleotides at eight positions. Their locations in the gen-
eralized schematic 2D and 3D-stucture of 23S rRNA are
shown in Figures 2C and 2D.

Three T residues are present: two of which, T-1956 and
T-1958, are located at universally conserved positions
(1915 and 1917; E. coli numbering) in helix 69 loop of
domain IV. The helix 69 stem-loop contacts A- and P-site
tRNAs, contributes to bridge regions B2a and B2b of 23S
rRNA, is involved in translation termination, contacts
ribosome recycling factor, plays an active role in dissocia-
tion of subunits at the end of translation, and is important
for subunit association [17,33,49,57-62]. Specifically, T-
1956 (1915) contacts the D stem of tRNA in the A site
(positions 11 and 12) and T-1958 (1917) is immediately
adjacent to bridge B2a contacts, as well as direct contacts
to A-site tRNA; they are important for the conformational


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A


C


B 3, D
P site
E site
A site M


Figure 2 (see legend on next page)


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Figure 2 (see previous page)
Distribution in the ribosome of modified nucleosides in Halobacteriaceae rRNA.(A) A schematic of secondary
structure of H. volcanii 16S rRNA with the locations of the various modified nucleosides indicated with darkened circles. The
helices in which these appear are numbered according to the designations used for E. coli (B) The locations of modified nucle-
osides of H. volcanii 16S rRNA are highlighted in a crystal structure of the small ribosomal subunit derived for Thermus ther-
mophilus (PDB entry 2j00; portion of 70S) by showing full atomic volume (van der Waals radii) darkened on a backbone of the
rRNA. The decoding center region is indicated by shading where the anti-codon stem loops of the A, P, and E-site tRNAs sit.
(C) A schematic of secondary structure of H. marismortui 23S rRNA with the locations of the various modified nucleosides
indicated with darkened circles. (D) The locations of modified nucleosides of H. marismortui 23S rRNA are highlighted in a
crystal structure of the large ribosomal subunit derived for Thermus thermophilus as described above. The peptidyl transferase
active site is indicated by shading where the acceptor stems of the A- and P-site tRNAs sit. The location of the T residue of H.
salinarum is highlighted with an open circle in (C) and lighter gray atomic volume in (D). In (B) and (D), the subunit interface is
towards the front.


flexibility of helix 69 and their loss affects subunit associ-
ation, dissociation, and translation termination [17,58-
61]. The peptidyl transferase center of the large ribosomal
subunit is the rRNA that directly surrounds the active site
of peptide bond formation and is made of the rRNA of the
central loop and proximal nucleotides of Domain V, as
well as the A-loop (loop of hairpin 92) and P-loop (loop
of hairpin 80, Figure 2) [63]. The third P is conserved only
in eukaryotes and is located at position 2621 (2586 E. coli
numbering) between hairpin 90 and 93 in the central
loop of domain V, immediately adjacent to rRNA contacts
with the P-site tRNA (terminal A of CCA at position 76)
[49,63].

Three 2'-O-ribose methylations were also found: one Gm
is located at position 1950 (1909) in the structurally con-
served helix 69 of domain IV contacting nucleotide 12 of
the tRNA in the P site [49], while the conserved tandem
UmGm, is located at positions 2587/2588 (2552/2553 E.
coli numbering) in hairpin 92, also called A-loop or pep-
tidyltransferase loop of domain V, that contacts acceptor
end of tRNA in the A-site of the ribosome. In the case of
H. marismortui, mutagenesis studies [64] and X-ray crystal-
lography [63] demonstrated the existence of base pairing
between Gm-2588 (2553) and C-75 of CCA end of tRNA
in the A-site of the ribosome, thus implicating a role in the
peptidyl transfer reaction.

Lastly, two base methylations (mlA, a modified nucleo-
side bearing a positive charge and m3U) are present
respectively at position 628 (571) in hairpin 25.1 of
domain II and at position 2619 (2584) of domain V,
between hairpins 90 and 93. The importance of each of
these two modified nucleotides (not modified in E. coli)
for maintenance of the archaeal ribosome architecture or
translation is not known. However, since m3U- 2619
(2584) is in the central loop of Domain V and immedi-
ately adjacent to rRNA contacts with P-site tRNA[63], a
fact revealed early on by affinity labeling results showing
E. coli 2584 adjacent the CCA end of P-site RNA [65,66],


this suggest a role in peptidyltransferase activity. Moreo-
ver, the absence of the U-methylation in the homologous
position in H. salinarium 23S rRNA confers resistance to
sparsomycin, and antibiotic that normally binds to the
peptidyl transferase center [67].

In domain V of 23S RNA of H. salinarium, an additional
T-2606 (2580) at the bottom of helix 90, not present in
rRNA of H. marismortui, has been reported [32]. However,
as no homolog of the bacterial RluC-type, or the eukaryo-
tic Pus5p-type of enzymes responsible for the formation
of this T in the LSU rRNA of respectively E. coli and S. cer-
evisiae can be found in the genome H. volcanii (see below),
its presence in the 23S rRNA of H. volcanii is highly
unlikely.

The case of 5S rRNA
As no modifications were detected in the 5S rRNA of the
two halophilic archaea H. halobium and H. marismortui
[68,69], we predicted that H. volcanii would also lack
modifications in this rRNA. A 2'O-methylcytosine (Cm) at
a conserved C-position (position 32) has been reported
only in the 5S rRNA of the thermophiles Sulfolobus acido-
caldarius [69] and S. solfataricus [68], while in the hyper-
thermophilic Pyrodictium occultum the base at the same
location (position 35 in P. occultum) is further acetylated
into ac4Cm. Both derivatives ac4C and ac4Cm coexist,
indicating incomplete modification of C-35 under the
conditions the cells were grown before extraction of the
RNA [68,69]. The same is true for other modified nucle-
otides in the 16+23 S rRNAs.

A complete inventory of tRNA genes (tRNomics)
The genome ofHaloferax volcanii, strain D2 (4,012,900 nt)
comprises one chromosome (2,847,757 bp) in several
identical copies (up to 20 [70]) and four smaller plasmids
(pHV1:85,092 bp, pHV2:6,359 bp, pHV3:437,906 bp and
pHV4:635,786 bp). All tRNA genes are located on the
chromosome. This mesophilic halophile exhibits the typ-
ical archaeal tRNA set [71] which is characterized by 46


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distinct anticodons able to read all 61 sense codons (see
details below). The extra G nucleotide at position 0 of
tDNA-His (GTG) is encoded in the genome but none of
the CCA 3' terminal sequence of tDNAs are present. The
list of tDNA sequences in linear and cloverleaf forms is
given in Additional file 2. Remarkably the sequences of
each mature tRNA (as sequenced by Gupta [28,29]) and
corresponding tDNAs as identified above perfectly match.
The only sequences of mature tRNAs that are missing
from Gupta's analysis are those specific for tRNA-Val
(anticodon UAC), tRNA-Ser (UGA), tRNA-Thr (UGU),
tRNA-Gln (UUG), tRNA-Arg (UCU) and tRNA-Arg
(CCU), all but one harboring a T34 wobble base in the
corresponding tRNA gene. As stated in the original work
[28], the missing tRNAs probably correspond to minor
isoacceptor species that co-migrated with one of the major
species and therefore were impossible to isolate and
sequence.

Six tRNA genes are present in two copies, raising the total
number of tRNA genes from 46 to 52. Among these six
pairs, five are perfect duplicates (from positions 1 to 73),
while the two tDNA-Gly (GCC) differ by the two base
pairs 4-69 and 5-68 (CG and TA versus TA and CG,
respectively) as previously noted [28]. Three of these
tRNA pairs are organized in direct tandems with a short
distance between the two genes: 2 x tDNA-Gly (GCC), 12
nt; 2 x tDNA-Asp (GTC), 29 nt; 2 x tDNA-Val (GAC), 45
nt probably revealing a recent gene duplication. The two
tDNA-Ala (TGC) are each embedded in the two copies of
the ribosomal operon (between 16S and 23S rRNA
genes). Other tDNAs are randomly distributed through-
out the genome; the next closest distance between two
tDNAs being 96 nt.

As only one gene exists for the majority of tRNAs harbor-
ing each a distinct anticodon, large differences must exist
either in the expression levels of individual tDNAs, or in
the half-life of individual mature tRNAs (or both). Indeed
the steady state concentrations within the cell of the major
tRNAs (reading most used codons) must be higher than
those of minor tRNAs (reading rare codons). The regula-
tion of the expression of the different tDNAs is yet to be
elucidated in H. volcanii and in all other Archaea (dis-
cussed in [71]). Its is possible that tRNA stability depends
on factors similar to those identified in yeast (reviewed in
[721).

Only three genes carry introns and in contrast with many
other archaea (see [73]), all are found at the canonical
position 37/38. The three genes, tDNA-Met (ATG) (intron
of 75 nt), tDNA-Gln (CAA) (intron of 31 nt) and tDNA-
Trp (intron of 103 nt), display a nearly perfect hBHBh'
motif [73] with the so-called h helix being the anticodon
stem and the so-called h' helix being 3-, 8- and 2-bp long,


respectively (see [73] and Additional file 3). Pre-tRNA-Trp
is unique as it contains the C/D and C'/D' boxes that allow
methylation of 2' hydroxyl of the ribose at positions 34
and 39 in the intron sequence [74-77] see also below).

As always in Archaea and Bacteria but not in Eukarya [71],
three different tDNAs bearing the (CAT) anticodon are
present: the initiator tDNA-Met (CAT), the elongator
tDNA-Met (CAT) and the tDNA-Ile (CAT). In this last
case, the final identity of the mature functional tRNA-Ile
depends on post-transcriptional modification of C-34
into an as yet unknown modified C-derivative (see
below).

Codon decoding strategy
The sequences of the 46 tRNAs harboring a distinct anti-
codon (or tDNA when the sequence of mature tRNA is not
available) are listed in Figure 3 from the wobble base at
position 34 to nucleotide-39 (the proximal first base pair
of the anticodon stem). This figure allows us to define the
codon decoding strategy in a halophilic archaeon. It
appears that: i) a systematic 'A-34 sparing' strategy is
found, allowing the decoding of all pyrimidine ending
codons (NN.U/C) by one tRNA harboring a G34. N'N'
anticodon (N stating any of the 4 canonical nucleotides,
N' its complementary Watson-Crick counterpart and G-34
is never post-transcriptionally modified). The presence of
G-34 in tRNA-Arg (GCG) of the four codons decoding box
is remarkable as the corresponding nucleoside is always
an A-34 (in fact post-transcriptionally deaminated into
inosine-34) in all Eukarya and most Bacteria [71]; ii) no
'C-34 sparing' strategy is used, that would require a U34-
containing tRNA to decode a codon ending with G-3,
while in the majority of Bacteria such a situation is fre-
quent (see [711). Thus in H. volcanii, the only wobbling-
type case of decoding during translation of the mRNA is
between a G34-containing tRNA and a codon ending with
a U-3. An acetyl group is present on N4 of C-34 in many
C34-containing tRNAs, and many of these tRNAs seem to
be only partially modified [281. The presence of ac4C at
the wobble position of tRNAs is unique to Archaea, with
the exception of the elongator tRNA-Met (ac4C.AU) in E.
coli [781. However, the same modification has been found
at position 12 in the D-arm of some tRNA-Leu and tRNA-
Ser molecules of S. cerevisiae [791 and in the 5S rRNA of
some thermophilic archaea (see above). This modified
nucleotide exhibits an exceptional conformational rigid-
ity when embedded in an RNA molecule [ 80,811. Its pres-
ence in the wobble position probably allows better
binding of the tRNA to the cognate codon, possibly helps
the tRNA to discriminate against codons ending with A
[781 and to aid in phase maintenance during translation;
iii) the rare isoleucine AUA codon is translated by a minor
tRNA-Ile, like in all bacteria. It harbors a unique type of
modified cytidine able to discriminate against the Met-


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Am. Codon anticodon loop Am. codon anticodon loop Am. Codon Anticodon loop Am. codon anticodon loop
Ac Ac coo Ac Ac
Phe UUU / Ser UCU / Tyr UAU / Cys UGU /
2 UUC GAA. A0>2 4 UCC GGA.AAG 2 UAC I GUA.flA^ >2 2 UGC GCA.M AG
Leu UUA CAA. UCA (UGA.AAG) Stop UAA /// Stop UGA ///
2 UUG UAA.A" UCG C GA.AA3 Stop UAG /// Trp UGG I MCA. AUm
Leu CUU / Pro CCU / His CAU / Arg CGU /
4 CUC GAG.MG 4 CCC M GGG. UC 2 CAC GUG.AG 4 CGC GCG. AG
CUA 0DAG. AC CCA UGG. UG Gin CAA (UUG.GAG) CGA [MCG. AC
CUG CAG.JGC CCG Ir.gG 2 CAG MUG.MGG CCG CCG.WAC
Ile AUU / Thr ACU / Asn AAU / Ser AGU /
3 AUC GUA. AC 4 ACC GGU. AG 2 AAC GUU.WAC 2 AGC GCU.WAC
AUA MAU. AC ACA (UGU.AAU) Lys AAA 8 UU. AC Arg AGA (UCU.AAU)
Met AUG BAU.WA ACG CGU. AG 2 AAG U. 2 AGG (CCU.AAC)
Val GUU / Ala GCU / Asp GAU / Gly GGU /
4 GUC GAC.AUG >2 4 GCC GGC.AUG 2 GAC GUC.ACG >2 4 GGC GCC.AUG > 2
GUA (UAC.AAG) GCA UGC.AAG >2 Glu GAA UC. AG GGA [CC.AAG
GUG CAC.ACG GCG CGC.AAG 2 GAG FaUC.ACG GGG CCC.AAG
Meti AUG CAU.AAC

Figure 3
Decoding strategy in H. volcanii. The various sense codons of mRNA (from 5' to 3') are boxed according to their corre-
spondence with one of the 20 amino acids. In each decoding box containing 1, 2, 3 or 4 synonymous codons are indicated the
corresponding sequences of anticodon loop in tRNA (from nucleotide at the wobble position 34 to nucleotide at position 39,
on the 3' side of the anticodon, the three first bases being the anticodon). A dash line means no tRNA with strictly comple-
mentary codon exists. The modified nucleotides are indicated in white under gray background. Abbreviations are the conven-
tional ones as defined in [4] except for symbol C* in the case of one tRNA-lle (C*AU) which correspond to a yet unknown
modified cytosine at position 34. Likewise, symbol !U in the wobble position of several tRNAs correspond to a yet experimen-
tally unidentified uridine derivative. In the case of tRNA-GIn, tRNA-Lys and tRNA-Glu, !U probably correspond to a mcm5s2U
or a similar type of U-derivative (for details see text). Symbol in front of a sequence means a Cm is present at position 32,
while symbol # note the presence of an unexpected A instead of the usual pyrimidine C or U at position 32. No inosine has
been found at the wobble position of any tRNA. The sequences indicated between brackets and in italics correspond to the
tDNA sequence only. A number >2 on the right of the anticodon sequence means there exist 2 genes harboring the same anti-
codon on the genome. In all other cases, only one single gene exists (no redundancy). There is no tRNA-Sel/Sec coding for
selenocysteine in H. volcanii. For more details see Additional files I and 2.


AUG codon. In E. coli, (and all Bacteria and eukaryotic
mitochondria), this C-34 residue is always modified into
lysidine (k2C, [82], reviewed in [83]); while in Archaea,
the chemical structure of the modified cytosine-34
remains to be identified ([84] see also below); iv) due to
lack of sequence information about many of the mature
U34-containing tRNAs the identification of the chemical
nature of the modified U (indicated as U* in the original
works of Gupta and '?U' in Figure 3) will require the dis-
covery of potential U-34 modifying enzymes in the
genome of H. volcanii (see below) or additional analytical
experiments; v) without exception, three isoacceptor
tRNAs are always used to decode four synonymous
codons in the four codons decoding boxes and two isoac-
ceptor tRNAs for decoding the two purine-ending codons
(NN.G/A) in the split codon boxes. Thus altogether 45
elongator tRNA and one additional initiator tRNA-Met are
required to decode the 61 sense codons in H. volcanii.
From the early work of Bayley and Griffiths [85], it is
known that accuracy of translation of synthetic homopol-
ymers by extracts of the extreme halophilic bacterium H.


cutirubrum, and probably all halophiles, requires the pres-
ence of very high salt concentration (up to 4 M).

Genes coding for transfer RNA modification enzymes
(Modomics)
Biochemical analysis using as substrate T7-transcripts of
tRNA genes lacking all the modified nucleosides, allows
enzymatic activities for producing pseudouridine and sev-
eral base-methylated derivatives in tRNA, such as m'A-57,
mil-57, Cm-56, m'P-54, m5C-49 and m22G-26 to be dem-
onstrated in cell-free extracts of H. volcanii [86], but none
of the corresponding genes were identified. Only recently
were the genes coding for the multiprotein complex that
use guide RNA to methylate the 2'-hydroxyl of cytosine-34
and uridine-39 in H. volcanii tRNA-Trp characterized
[74,75]. In other Archaeal species (mainly in M. jannaschii
and P. furious or P. abyssi), genes coding for several tRNA
modification enzymes have been not only identified, but
also experimentally validated. These were used to easily
predict the H. volcanii orthologs with good confidence
(Table 1). These include the enzymes that introduce the


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Table I: Predictions of H. volcanii tRNA modifications genes


Mod. tRNA (see Additional
file I)


10 m2G/m22G GI-4/RI, Dl, QI, E -2,
HI, PI-3
13 T RI, DI, QI, El-2, GI-2,
HI, Me, Mi, PI-3,
SI,TI,VI-2
15 G+ R2-3, NI, CI, 12, LI-5,
KI-2, Me, Mi, FI, PI, P3,
SI-3,TI-2,WI,YI, V2
22 T Me

26 m2G m22G A3, R3, L4/A2,R2,11-2, LI-
3,L5,KI 2,S I-3,TI-2, WI
28 T II

32 Cm KI-2,WI, YI


34 Cm Me,WI

?mcm5s2U? R3, E2, G2, K2, L5


cmosU
C*


ac4C QI, El, KI,PI, SI
37 t6A NI,11-2, KI-2, Me not Mi,
S3, TI-2
m'G RI-3, CI, QI, E2, HI, LI-
5, FI, PI-3, W I,YI

38-40 P38,P39 38 = Pl/39 = L2, L4, Kl,
Me, FI, SI,YI
39 Um WI
39-40 m5C 39 = LI/40 = 11

48,49 mSC Almost all except L4, D I,
QI, HI, Mi
52 T KI
54 and 55 T All


mIP
56 Cm


all except QI,HI


57 mlA

m'l All harboring A57 except
HI


Prediction method


Homology with P. abyssi
PAB1283*[177]
Homology with E. coli
TruD*[178]

Homology with M.
jannaschii MJ0436*[87]

Cbf5 without Guide
RNA?
Homology with P. furious
protein PF1871*[179]
Cbf5 without Guide
RNA?
Homology with E. coli
protein TrmH*[91].


Hv orfa


HVO_0156

HVO_0658


HVO_2001




HVO_0236



HVO 2906


COG


Comments b


1041


I Z2Z


I IQ8


See text

2DUL

See Text


aFib +sRNA* [75]


Homology with yeast
Trm9* [180], Elp3* [125].
Tucl* [123]

Prediction this work

Prediction this work
Homology M. maripaludis
protein MMP0186*[181]
Homology with M.
jannaschii MJ0883*[ 182]

Homology with Yeast
Pus3* [101]
aFib + sRNA [75]
Homology with P. abyssi
PAB1947* [1 10]
Homology with P. abyssi
PAB1947* [110]
Cbf5 without sRNA?
Cbf5 without sRNA? and/
or homology to P. furious
PF 1 39 (PsuX) [98]
Prediction [93]
Homology with P. abyssi
PAB 1040* [94]
Homology with P. abyssi
PAB0283* [111]
Prediction this work


HVO_0574 +
HVO_2888 +
HVO_0580
?
HVO 0339 or
HVO 0697
HVO_2736
HVO_0253

HVO_0929


HVO_1852


HVO_1594

HVO_1594


HVO 2493
HVO_ 1979

HVO_1989
HVO_ 1173

HVO_1383

HVO 2747


2226 1243 0037


?
1571 or 2047

1444
0009

0336


See Additional files
4 and 5
See text


see text
See text

See text
2EQA

2FRN


See Additional file 4
0144

0144

See text
0103 1258 See text 2V9K


See text 2QMM
203A

I YB2

See text; 215H


aRefSeq annotation in archaea.ucsc.edu/; bif the structure of an Archaeal member of the family is available the PDB code is given ; *Experimentally
verified.


m2G/m22G-10, m'G-37, and m1A-57 modifications (ref-
erences are given in Table 1). A protein homologous to the
key enzyme transglycosyltransferase (TGT) responsible
for the insertion of the G+ precursor preQ0 in M. jannaschii
tRNA [87] is also found in H. volcanii. The genes involved
the synthesis of preQ0 and in the conversion of preQ0 to


G+ after its insertion in tRNA, are not known in any
Archaeal organisms to date. They are currently being iden-
tified in our laboratory at the University of Florida in
Gainesville and will be described elsewhere. Another set
of tRNA modification enzymes that introduce the T-13,
m2G/m22G-26 and t6A-37 modifications respectively can


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be predicted by homology with yeast and/or E. coli exper-
imentally validated orthologs (Table 1). For the 12
remaining modifications, the prediction process is less
straightforward because the homology scores with the
experimentally validated yeast or bacterial homologs are
too low, paralog families complicate the analysis or the
corresponding gene has not been identified in any species.
These are discussed separately below.

CmIUm residues
In H. volcanii tRNA, 2'-0-methylation of ribose occurs in
four positions, 32, 34, 39 and 56 (Figure 1A). As stated
above, Cm-34 and Um-39 in the anticodon branch of
tRNA-Trp are formed by the guide RNA machinery that
includes the Fibrillarin enzyme (aFib) and accessory pro-
teins Nop56/58 and L7Ae [88-90], all encoded in the
genome of H. volcanii (Table 2). The RNA antisense bear-
ing the C/D and C'/D' boxes is part of the pre-tRNA
sequence and includes the long intron of 103 nt, a situa-
tion that exists also in pre-tRNA-Trp from at least 29
archaea (see Additional file 4) [76,77]. The mechanism by


Table 2: Predictions of H. volcanii rRNA modifications genes


which the 'intronic' antisense sequence acts in vivo in cis to
'self induce the 2'-0-methylations of C-34 and U-39 in
pre-tRNA-Trp, or in trans by acting on an other molecule
of pre-tRNA-Trp, is still an open question. However in
vitro experiments favor a trans-acting box C/D snRNA
guided mechanism [75]. In the case of intron-containing
tRNA-Met, Cm-34 is also guided by a sRNA (see Additional
file 5) but here the C/D antisense RNA is not intronic but
exonic as described for the C/D box sRNA sR49, which
was predicted to guide the modification of Cm-34 in the
tRNA-Met of Pyrococcus [74]. We identified 18 sRNA can-
didates to guide the modification of Cm-34. We found a
candidate in the genome of H. walsbyi for which no tRNA-
Met containing an intron could be identified in the
genomic sequence available at NCBI. Thanks to the target
region, we also identified the tRNA-Met containing the
intron. Our analysis reveals that sRNA guiding formation
of 2'-0-methyl ribose at position 34 and 39 in pre-tRNA-
Trp is always intronic, while the formation of the same
Cm-34 in pre- tRNA-Met is always exonic. In both cases,
part of the intron sequence is involved in base pairing


Modification Prediction method


Hv orfa COG Commentsb


5S
None
I6SC
910 (966)
1352 (1404)
1432 (1500)
1450+1451 (1518+1519)


23S Data From H. mansmortuiD
628 (571)

1950 (1909)
1956+1958 (1915+1917)
2587 (2552)
2588 (2553)
2619 (2584)
2621 (2586)


None


acp3U
C*= N330
m6A
m62A



m'A

Gm
T,T
Um
Gm
m3U


Guide RNA protein
machinery
Cbf5
Gar lp
NoplO


L7Ae
Fibrillarin

Nop56/58


Prediction this work

Prediction this work
Homology with M.jannaschii
RsmA* [137]


Prediction, weak homology with
E. coli RImA [146]
aFib+ sRNA
Cbf5+ sRNA
Homology with E. coli RImE [138]
Prediction RImE?
Prediction, this work and [93]
Cbf5+ sRNA



Homology P. funosus Cbf5* [183]
Homology P. furious Gar I p* [ 183]
Homology P. funosus Nop 10*
[183]
Homology S. solfataricus L7* [184]
Homology with S. solfatancus aFib*
[184]
Homology S. solfataricus Nop56/
58* [184]


HVO_
?
HVO_
HVO_


0390 2016
?
1475 2263
2746 0030


HVO_0309 2226



HVO_0180 1189
HVO_0180 1189
HVO 2565 2016


HVO_2493 0103
HVO_ 1108 3277
HVO_0698 2260

HVO_2737 1358
HVO_ 1669 1889

HVO_I670 1498


See text, I Q7H
See text
See text, I QAN


See Additional file 8
See Additional file 8

See text
See text, I K3R
See Additional file 8


2AUS, 2RFK, 2APO
2HVY, 2EY4 2RFK
2AUS

IPXW, 2FC3, I RLG, ISDS, 2QA4
I NT2, I PRY I G8S, I FBN, I G8A

I NT2


aRefSeq annotation in archaea.ucsc.edu/;bif the structure of an Archaeal member of the family is available the PDB code is given ; *Experimentally
verified; c H. volcanii numbering corresponding E. coli numbering given in brackets; d H. marismortui positions corresponding E. coli position in
brackets


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Position


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with sRNA, thus 2'-0-methylation at ribose in position 34
and 39 in pre-tRNA-Trp and in position 34 in pre-tRNA-
Met have to occur before intron splicing [77].

Remarkably, Halobacteria show more degenerated C, C',
D and D' boxes and a longer region between D' and C'
boxes (19 to 21 pb) than other orders (4 to 10 pb). In con-
trast, the insertion of Cm-32 found in the anticodon loop
of four tRNAs (two specific for Lys, one for Tyr and one for
Trp) and of Cm-56 found in all H. volcanii tRNAs (with no
exceptions; see Additional file 1), is almost certainly cata-
lyzed by non RNA guided enzymes. Indeed, a solid
homolog of the TrmH (YhfQ) protein that has been found
to catalyze the formation of Xm-32 in E. coli [91], is
present in the genome of H. volcanii (Table 1). It is the
only member of the SpoU family [92,93] found in this
organism. For Cm-56 in the T-loop, a strong homolog of
the P. abyssi protein found to catalyze this reaction in vitro
([94] and reviewed in [95]), can be identified in the
genome ofH. volcanii (Table 1).

residues
Apart from T-13 which is most certainly modified by the
TruD ortholog (HVO_0658, belonging to COG0585, see
table 1), seven other P residues? are present in H. volcanii
tRNA at positions 22, 28, 38, 39, 52, 54 and 55 (Figure 1).
T-55 is a universal modification inserted in yeast by
Pus4p [96] and in E. coli by TruB [97], both belonging to
the same COGO103. The only homolog of these two pro-
teins that can be identified in the H. volcanii genome is
Cbf5p, which is the catalytic subunit of the guide machin-
ery (see below). However, recent work from different lab-
oratories have shown that in vitro, Cbf5p can modify U-54
in tRNA, as well as in rRNA, in a guide-independent fash-
ion, the enzymatic reaction being stimulated by the pres-
ence of NoplOp [98-100]. PsulOp from P. furious, that is
not part of the TruB/Cbf5p family of proteins (COG0103)
but is instead a member of the COG1258 family (Table
1), can also introduce the P-55 modification in archaeal
tRNAs in vitro. This observation has been validated by
complementation experiment using an E. coli truB mutant
[98 ]. It is however still not clear which of the two enzymes
(Cbf5p and/or PuslOp) is responsible for the formation
of T-55 (as well as of Psi-54) in Archaeal tRNAs in vivo. As
discussed in [99], the possibility exists that each of the two
P-55 forming enzymatic systems act on distinct sets of
tRNAs. It is worthy of mention that no PsulOp homolog
is found in N. equitans, whereas a genes coding for Cbf5p
and Nopl0O homologs are detected (see "Archaeal rRNA
modification" subsystem in the SEED database for
sequences). Unfortunately, no evidence for the presence
or absence of P-55 in any of the tRNAs, or of T in rRNA
is available for this organism.


Other quasi universal P. modifications are T-38/39 of the
anticodon branch inserted in yeast by Pus3p [101] and in
E. coli by TruA [102], both members of the COG0101
family. Only one protein of this family could be identified
in H. volcanii (Table 1). Its homology with both the E. coli
and the yeast GOGO101 members is quite low but multi-
ple sequence alignments using clustalw [103] confirmed
that the critical TruA specific active site consensus
sequence (XXXRTD) [104] is conserved in the Haloferax
protein. No homologs of yeast Pusp 1- 9 [105] or E. coli
TruABCD (reviewed in [13]) could be identified in the H.
volcanii genome, leaving P 's at positions 22, 28 and 52
with no corresponding candidate pseudouridine syn-
thase. The corresponding uridines are located in helical
regions (at least in the fully mature tRNA), thus perhaps
relatively inaccessible to the enzyme, and these are
isomerized to P only in one tRNA species, whereas the
other P modifications, at least those in positions 38, 54
and 55 are located in loops and found in several tRNAs
(see Additional file 1). This led us to suppose they may be
introduced at a very early stage of the tRNA biosynthesis,
possibly still during transcription and when the cloverleaf
structure of the nascent pre-tRNA is not yet formed,
potentially by the RNA-guided machinery including
Cbf5p, NoplOp, Garlp and L7Ae homologs (Table 2).
However no H/ACA snRNA could be identified in H. vol-
canii. This could be due to a high divergence of H/ACA
RNA structures in H. volcanii or, as discussed above in the
case of P-55 formation, to a guide RNA independent
pseudouridylation by the Cbf5p/NoplOp dependent
machinery acting during transcription on an as yet unfin-
ished tRNA molecule composed of stems and loops. One
cannot however rule out that an as yet unidentified pseu-
douridine synthase family is present in this organism.

m5C residues
Four positions 39, 40, 48, 49 are modified to msC in H.
volcanii. In yeast, Trm4p is not site-specific and introduces
this modification at several positions in tRNA molecules
[106]. Members of this huge family of proteins
(COGO 144) are however difficult to annotate by sequence
alone as some also modify rRNA [107-109]. Recently one
of the five COG0144 members from P. abyssi (PAB1947)
was found to catalyze in vitro the formation of msC at sev-
eral positions in tRNA, including positions 48 and 49
[110]. H. volcanii has just one member of this family
(HVO_1594) that is highly similar to PAB1947, and
ribosomal RNA of this organism does not contain any
m5C (see Figures 2A-D). Hence, it is highly probable that
HVO_1594 is the only RNA:msC methyltransferase that
modifies the four cytosines found in the sequenced tRNAs
of H. volcanii (Figure 1). The presence of additional m5C
residues at positions 50-52 in some tRNAs of H. cutiru-
brum probably also result from the action in this organism



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of a unique multi-site specific tRNA:msC methyltrans-
ferase.

157 and m'157
In H. volcanii, the only inosine (deaminated adenosine, in
the form of m1l) residue is found at position 57 of the
majority of tRNAs (Figure 1, see also Additional file 1).
Enzymatic formation of the doubly modified mlI occurs
in two strictly sequential steps. The first step is the meth-
ylation of A-57 catalyzed by the tRNA:m'A methyltrans-
ferase of the P. abyssi TrmI family (COG2519, [111])
(Table 1). Then deamination of m1A-57 occurs by a
tRNA:m'A-specific deaminase [86,112], that is different
from other tRNA deaminases such as Tadip and Tad2p/
Tad3p catalyzing the formation of inosine from adenos-
ine in position 37 and 34 respectively in S. cerevisiae
tRNAs [113-115] or TadA catalyzing the site-specific for-
mation of inosine-34 exclusively in tRNA-Arg (anticodon
AGC)[116], as we could not identify any homologs of
these families in the H. volcanii genome. We searched for
protein families specifically conserved in all Archaea but
absent in Eukarya, with RNA binding domains. One can-
didate is the COG 1491 family. It is annotated as an RNA-
binding protein as the structure of the A. fulgidus family
member (PDB: 2I5H) showed that the N-terminal
domain is similar to many nucleic acid binding protein
with the presence of a characteristic S1 domain [117].
Analysis of a clustalw sequence alignment reveals a highly
conserved histidine residue in a motif [R,K] [L,M]H
[A,S,T,Q,M]L [E,Q,N] (Figure 4A) that is similar to the
adenosine deaminase "motif I" found in all adenosine
deaminases [113].

mFri'
The methylation at position 54 is a hallmark of Archaea,
except in Thermococcales where m5U54 is found [118].
COG1901 proteins that are part of SPOUT superfamily
have been predicted as candidates for this missing methy-
lase [93] and genes of this family do indeed cluster with
PsulOp in several genomes (Figure 4A). However, it is
present in organisms that are expected to have msU and
not m1' at this position such as the Pyrococci [1181. Exper-
imental validation is required to ascertain the function of
this putative Y-dependent methyltransferase (work in
progress).

Modified uridine-34 derivatives
As a rule U34-harboring tRNAs belonging to the split
codon boxes corresponding to Leu (UAA), Gln (UAG),
Lys (UUU), Glu (UUC) and Arg (UCU) need to discrimi-
nate for the NN-Purine codons and not miscode for the
NN-pyrimidine codons. Only certain types of modified U-
34 can perform this task [119](reviewed in: [120]). In
contrast, tRNAs bearing unmodified U-34 are able to
decode codons ending with purines and pyrimidines,


such as those found in the four codon decoding boxes as
in Mycoplasma for example [34]. As expected a modified
U! (of which the chemical identity remains to be deter-
mined) was identified in naturally occurring tRNAs of H.
volcanii specific for Lys (U!UU) and Glu (U!UC) [28,29].
However for tRNA-Leu (?UAA) and tRNA-Arg (?UCU), the
identity of U-34 is yet to be determined as these tRNAs
remain to be sequenced (Figure 3 and Additional file 1).
Curiously, U-34 of H. volcanii tRNA-Leu (UAA) was
reported not to be modified, while the U-34 residue in
tRNA-Leu (?UAG), tRNA-Arg (?UCG) and tRNA-Gly
(?UCC) belonging to the four codons boxes appears to be
(Figure 3). Unexpectedly, U-34 in tRNA-Pro (UGG) and
tRNA-Ala (UGC) is not modified [28,29], while the corre-
sponding four codons decoding boxes contains two other
tRNAs (one with G-34 and the other one with C-34) able
to decode the other codons, except the one ending with A
(see Figure 3). Thus the pattern of modified/unmodified
U-34 in H. volcanii tRNAs is non-canonical and the exact
chemical nature of the U! in the different tRNAs of H. vol-
canii remains an enigma [as well as in the few other
archaeal tRNAs sequenced so far [4].

In the case of tRNAs specific for Gln, Lys and Glu in H. vol-
canii, a thiolated U-34 derivative should exist, as for their
bacterial and eukaryal counterparts (for examples see
[121,122]). Indeed, in the genome of H. volcanii, a gene
homolog to eukaryal Tucl belonging to COG0037 is
found. In S. cerevisiae, this Tucl protein has recently been
shown to be involved in the formation of s2U-34 in yeast
cytoplasmic tRNAs [123]. Also, clustering of Tucl with
IscS and IscU, the two proteins required for donating the
thio compound (Figure 4B) strengthens the prediction
that this family of proteins do participate in the formation
of thiolated compounds. However, analysis of the H. vol-
canii genome suggests that ?U34 in tRNAs, as in many
other archaeal tRNAs, is more complex than just a s2U.
Indeed, homologs of the yeast Trm9p methylase and of
the radical SAM enzyme Elp3 are also found in H. volcanii
genome (Table 1). Trm9p is the yeast mcmsU/mcm5s2U
tRNA carboxyl methyltransferases [124] and Elp3 is in
yeast part of the elongation complex comprised of 6 pro-
teins Elpl-6 that have all been shown to have a role in the
formation of mcmS(s2)U [1251. In vivo the pleiotropic
effect of mutations in the yeast Elp genes appear to be due
to the absence of the modified base in tRNA [126]. How-
ever, out of the six eukaryal Elp proteins only homologs of
Elp3 can be found in Archaea. This protein is part of the
radical SAM family [127]. In S. solfataricus, the Elp3 and
Trm9p encoding genes are also clustered (Figure 4B). Tucl
is present in all sequenced genomes of Archaea, Elp3 is
lacking only in N. equitans, and Trm9p homologs are
found only in a limited subset of Archaea (see "Archaeal
tRNA modification" subsystem referenced in the methods
section). Taken together, the data suggest that the type of


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A


Methanococcoides burtonii


Sulfolobus solfataricus P2



Thermoplasma acidophilum


Methanospirillum hungatei JF- 1


- 3 COG1491 1 2 5 6



4


COG1901



7 COG1444


Tucl IscS IscU




Trm9 Elp3


Natronomonas pharaonis



Sulfolobus solfataricus P2


Ferroplasma acidarmanus


COG 1571 Trm5
Picrophilus torridus DSM 9790


Methanococcus maripaludis S2 Tgt LiT Tru


Figure 4
(A) Clustering of COG 149 I1, COG 1901 and COG 1444 with translation gene. I = KsgA [Dimethyladenosine trans-
ferase (EC 2.1.1.-)]; 2 = HemK (Methylase of polypeptide chain release factors); 3 = L21 p (LSU ribosomal protein L21 p); 4 =
PsuX (Pus 10 family see [98] and text); 5 = YhfQ (methylase potential involve in Cm32 methylation of tRNA, see text); 6 =
S3Ae (SSU ribosomal protein S3Ae);7 = Efl b (Translation elongation factor I beta subunit) (B) Clustering of Tucl, Elp3 and
Trm9 family genes with sulfur transfer enzymes encoding genes (see text for abbreviations).(C) Clustering of COG 1571 with
RNA processing genes Tgt = tRNA-guanine transglycosylase; LigT = 2'-5' RNA ligase (EC 6.5.1.-); Trm5 = tRNA (Guanine37-
N I)-methyltransferase (EC 2.1.1.31); TruD = tRNA pseudouridine 13 synthase (EC 4.2.1.-). The full analysis is available in the
"Archaeal tRNA modification" and "Archaeal rRNA modification" subsystems in the SEED database.




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I


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U-34 modification in H. volcanii tRNAs belonging to two
codon split decoding boxes is similar but not identical to
the mcm5s2U derivative found in eukaryal tRNAs.

Gupta proposed that mosU-34 may exist in H. volcanii
tRNAs[28]. In bacteria it has been shown that chorismic
acid is a precursor to mosU-34 formation through the
hosU intermediate, with the product of cmoB catalyzing
the conversion of hosU-34 to mosU-34 [128]. CmoB pro-
tein is part of the methyltransferases type 11 family http:/.
/www.ebi.ac.uk/interpro/IEntry?ac=IPR013216 that is
difficult to annotate because it is so widespread. A distant
homolog of CmoB was indeed identified (Table 2) and
could be the potential moSU synthase but this prediction
is not very robust and absolutely requires experimental
validation. The genes that are responsible for the forma-
tion ofho5U have not been identified in any organism.

ac4C-34
Is found only at position 34 in many H. volcanii tRNAs. In
certain Archaea and Eukaryotes it has also been detected
in ribosomal RNA (see above). The only known enzyme
involved in ac4C formation is yeast Tanl (YGL232W [79])
that does not have any homolog in H. volcanii. However it
was predicted that Tanl binds tRNA and carries the recog-
nition determinants but must function in complex with
yet unidentified acetylation enzymes [79], reviewed in
[105]. One enzyme family COG1444, that contains an
ATPase domain fused to an acetyltransferase domain was
identified as a potential candidate. Genes of this family
cluster with translation genes (Figure 4A). In yeast, the
homolog (YNL132W) is essential [129], whereas the E.
coli ypfl homolog is not [130], and has recently been
shown to be responsible for ac4C formation in tRNA initi-
ator in E. coli [131].

Lysidinelk2 C34 homolog
Finally, like Bacteria, all Archaea but N. equitans have a
minor tRNA-Ile (CUA) [71,84]. This requires the modifi-
cation of C-34 to a C*. Otherwise this tRNA-Ile would be
charged erroneously with methionine [132], reviewed in
[83]. In all bacteria except in Mycoplasma mobile [34], a lys-
inyl group is inserted by the ATP-dependent TilS family of
enzymes [133], but in Archaea the structure of C-34 mod-
ification is still not known, and no tilS gene has been
found in H. volcanii. Potential candidates for a gene cod-
ing for an enzyme catalyzing the selective modification of
C-34 in tRNA-Ile (CAU) should be found in all Archaea
but N. equitans and should also be absent in the genomes
of E. coli and S. cerevisiae. Using the OrthoMCL phyloge-
netic distribution search tool [134], we identified 10 pro-
tein families that conform to the above criteria. We favor
two candidates in this list for missing C*-synthase genes:
i) the nucleic acid binding protein-OB fold family
(COG1571) that contains a potential RNA binding


domain, and ii) the COG2047 family, annotated as an
ATP-grasp superfamily. Both these families cluster with
tRNA modification genes in several Archaea (Figure 4C)
and follow the expected phylogenetic distribution (see
"Archaeal tRNA modification" subsystem in SEED).

Genes coding for ribosomal RNA modification enzymes
Very few Archaeal enzymes involved in the modification
of ribosomal RNA have been experimentally characterized
to date with the exception of the guide rRNA methylation
and peudouridylation machinery enzymes (see for exam-
ples: [100,135,136]). However thus far little investigation
has been performed in the case of halophilic organisms,
including H. volcanii. The analysis below (Table 2, and ref-
erences therein) is hence mainly based on comparative
genomics predictions that await experimental validations.

m62A
Among modification enzymes acting on rRNA that can be
easily identified in H. volcanii by sequence homology is
the archaeal member of the RsmA/Dimlp family. This
enzyme introduces four methyl groups in the conserved
tandem adenosine in hairpin 45 of the 16S RNA to form
m62A m62A (positions 1450 and 1451; 1518 and 1519, E.
coli numbering). The function of the M. jannaschii RsmA
ortholog was experimentally confirmed [137]. A strong
homolog of this enzyme is found in H. volcanii (Table 2).

2'-0-methylated derivatives
Likewise, a homolog of RrmJ (or RImE belonging to COG
1189) that catalyzes the formation of the quasi universally
conserved Um at position 2552 of the hairpin 92 of bacte-
rial 23S RNA [138] in E. coli (Um-2587 in H. marismortui)
is also found in H. volcanii (Figure 2C and Table 2). In S.
cerevisiae a site-specific Mrm2 enzyme introduces the
same modification in the mitochondrial 21S (Um-2791)
[139] but a guide RNA (SnR52) machinery is responsible
for the equivalent cytoplasmic yeast 28S rRNA methyla-
tion (Um-2921) [140]. In this later case, Spblp (of COG
1189 as Mrm2 and RImE) can also catalyze the formation
of Um-2921 (2552 E. coli numbering) even if its normal
function is to catalyze the 2'-O-methylation of adjacent G-
2922 [141]. Since the snRNP-dependent formation of Um-
2921 occurs within the nucleus at an early step of the
rRNA maturation process, and the action ofSpbl enzyme
proceed later within the cytoplasm, only if U-2921 has
not previously been fully modified in the nucleus, can
Spblp then complete the reaction [140]. In the case of U-
2587 (2552) and/or G-2588 (2553) methylation in 23S
RNA of H. volcanii, searches for potential guide RNA have
been unsuccessful by using both pattern matching
approaches and the dedicated SnoScan software, while in
P. abyssi, a sR25 C/D box sRNA was predicted to guide the
methylation of U-2669 (U-2552 in E. coli) [136]. Failure
to detect the guide in the halophiles might be due to a


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divergent structure of the snRNAs in these organisms or
could reflect the real absence of such guide RNAs for these
particular methylation targets. The possibility exists that
the halophilic RImE homolog, identified above, is a
multi-site specific enzyme and catalyzes both the forma-
tion of Um-2587 and Gm-2588. A precedent for such a sit-
uation is found in the enzymatic formation of mlA at
both positions 57 and 58 of tRNA by the Pyrococcale TrmI
enzyme [ 111], while the bacterial and eukaryal homologs
(TrmI and Trm6p respectively) are strictly site-specific and
methylate only A-58 in tRNAs [142,143]. Another possi-
bility is that Halophiles have multiple paralog copies
(from 3-6 copies, see 'Archaeal RNA subsystem") of
COG3269 family that contain the RNA binding TRAM
(TRM2 And MiaB, domain) [144] and one of these
enzymes could be responsible for the formation of Um-
2921.

We found at least one C/D box sRNA candidate to guide
the 2'O-methylation of the ribose at position G-1950
(1909 in E. coli). Homologous sequences were found in
25 archaeal genomes (Additional file 7). Moreover, our
results suggest that in Pyrococcus, the sR41 orphan C/D
box sRNA http / /lowelab .ucsc.edu/snoRNAdb/Archaea
Pyro-annote.html could modify the equivalent of G-1950
position.

Pseudouridines
No homologs of the multiple known E. coli pseudourid-
ine synthases that modify rRNA (for review see [13])
could be identified in H. volcanii. As demonstrated for
rRNA of Eukarya and some Archaea, such as S. solfataricus,
A. fulgidus or P. furious and P. abyssi, T residues could also
be introduced by the guide RNA machinery and indeed,
all the enzymes needed are presents in the H. volcanii
genome (Table 2). In Eukaryotes, the equivalent of P-
1956 and 1958 are modified by the same H/ACA sRNA,
respectively U19 in Human and snR191 in Yeast. In
Archaea the equivalent of T 1956 was proposed to be
modified in Pyrococcales and A. Fulgidus respectively by
Pf7 in P. furious and Afu4 in A. Fulgidus [145]. Recently a
combination of in silico and experimental work identified
seven H/ACA involved in pseudouridylation of rRNA in P.
abyssi while a total of 17 P residues were detected [100].
Some of these sRNA modify several positions in rRNA but
clearly not all the 17 P residues are accounted for, and for
certain positions (such as T-2603 of P. abyssi rRNA) the
modification can be introduced in vitro by the Cbf5p/
NoplOp dependent complex in the absence of any guide
RNA [100]. Indeed T-2016 (P-1956) was introduce by
the Pf7 homolog (Pab40) and the Afu-4 H/ACA sRNA in
vitro but the modification equivalent to T-1958 was not
[100]. Pf7 contains three hairpin motifs, namely Pf7-
stem-I, Pf7-stem-II and Pf7-stem-III, each one able to
guide a modification. The in silico approach used in the


present analysis allowed to identify two H/ACA sRNA
hairpins, respectively HP1 and HP2, candidate to the
modifications of T-1956, T-1958 and T-2621. We did
not find the homolog of Pf7-steml hairpin, which is con-
sistent with the absence of a fourth modification in H.
marismortuii. HP2 is clearly the homolog of Pf7-stemIII
and HP1 appear to be the homolog of Pf7-stemlI.
Remarkably in H. volcanii and other halobacteria, both
hairpins are conserved but are separated by a long spacer
whereas they are adjacent in thermococcales. HP2 would
be able to guide P 1956 and P 1958 by forming alterna-
tive structures around the position to modify (see Addi-
tional file 8B) while the HP1 could target T-2621 (see
additional file 8A). Remarkably in P. abyssi, this last mod-
ification was not found experimentally [100] whereas
Pab40 could adopt an alternative structure able to target
this position (see Additional file 8A). Finally we did not
find the homolog of Pf7-I and Pf7-II in the Crenarchaeote
Ignococcus hospitals. Certainly, the modification targeted is
not present.

m'A
This methylated adenosine is located in hairpin 25.1
(position 628) of Domain II (Figure 2C). A weak
homologs of RImA that introduces a mlG in the E. coli
large subunit in position 745 [146] can be identified
(Table 2) and is a possible candidate for the formation of
mlA, even if it is a different purine base. Indeed, during
evolution, an enzyme able to methylated N1 in guanosine
might have adapted to methylation of N1 in adenosine,
exactly as an ancient C5-methylated enzyme has derived
to become a C5-methyltransferase of uridine by simply
changing few aminoacids in the active site in order to
accommodate U instead of C [147,148].

m3U
This N3 methylated uridine (position 2619 in 23S RNA,
2584 in E. coli) is located between hairpins 92 and 93 (Fig-
ure 2C). A good candidate for the missing m3U inserting
enzyme in H. volcanii is the protein belonging to
COG2106 (Table 2). Indeed, analyzing the SPOUT family
enzymes, Bujnicki and coll. [93] found that COG1385,
exemplified by E. coli RsmE that introduces the m3U mod-
ification in 16S RNA [149], has a complementary phylo-
genetic distribution to the COG 2106 family found in
Archaea and eukaryotes. Moreover, genes encoding
COG2106 proteins are inserted in operons encoding for
ribosomal proteins in phylogenetically diverse Archaea
such as the Pyrococci, Archeoglobus fulgidus and H. sali-
narium (data not shown).

m6A
This methylated exocyclic NH2 of adenosine is located at
position 1432 (1500 E. coli numbering) of 16S RNA in
helix 44 in the 3' minor domain (Figure 2A). Compilation


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of modification data in the SSU RNA modification data-
base [3] shows that the m6A modification found in the H.
volcanii 16S RNA can also be found at the same position
in S. solfataricus and in three eukaryotes Homo sapiens,
Xenopus laevis and Rattus norvegicus. By searching for genes
that are present in these four organisms (and that are gen-
erally annotated as methylansferase) we identified the
COG2263 family. Annotated as RNA methyltransferase or
N6-DNA-methylase, members of this family are present in
most archaea and many eukaryotes. The structure of the
COG2263 member PH1948 was determined in complex
with S-AdoMet [150] and revealed that this protein was a
structural homolog of ErmC' (pdb :1QAN) that confers
resistance to macrolides by introducing an N6-methyla-
tion at adenine 2058 (as E. coli numbering) of 23S rRNA
[151]. We propose that the H. volcanii COG2263 member
(Table 2) is also involved in m6A formation but in the 16S
RNA, not the 23S RNA.

acp3U
This uridine bearing a 3-amino-3-carboxypropyl group on
N3 of uridine is located at position 910 (966 E. coli num-
bering) of hairpin 31 in 3'major domain of 16S RNA (Fig-
ure 2A). It is modified to mlacp3P. in all eukaryotes
analyzed so far but is never present in small RNA subunits
of bacteria that always have a non modified G (or m2G)
in this position [3]. Using the phylogenetic pattern tool of
the OrthoMCL database [134] we searched for genes that
are conserved in mammals, S. cerevisiae and all Archaea
but absent in all bacteria. A large collection of genes fol-
low this pattern (89 altogether), most of them are ribos-
omal proteins and other translation related genes. One
candidate stood out as a potential acp3U inserting
enzyme, the COG2016 family. Proteins of this family are
found in all sequenced Archaea and eukaryotes and con-
tain a C-terminal PUA domain (Pseudo Uridine synthase
and Archaeosine transglycosylase) that is often involved
in RNA binding [152]. The yeast member of the
COG2016 family, YER007C-A, has been shown to associ-
ate with ribosomes and a null mutant has clear translation
defects [153].

Beside the putative genes identified above, a few other
genes corresponding to as yet unidentified modified
nucleotides need to be discovered, such as for the cur-
rently unidentified C*(N330) derivative located at posi-
tion 1352 in hairpin 44 in the 3' minor domain SSU RNA
(Figure 2A). N-330 is also found at the same position in
the bacteria Thermotoga maritima [45]. Lastly, while the
possibility is meager, one or two additional modified
nucleotides might still exist in rRNAs of H. volcanii.
Indeed, the full lengths of the 16S and 23S (1472 nt and
2922 nt respectively) of H. volcanii or of H. marismortui
have not been explored, only the most critical regions


where the probability was high to discover conserved or
semi-conserved nucleotides have been investigated.

Conclusion
The archaeaon Haloferax volcanii has the particularity of
being a 'salt-loving' prokaryote that lives in the mildly hot
and hypersaline environment of the Dead Sea (40- 50 C,
1.5-3M NaCl) where it was first isolated [27,154]. Life at
such high salt concentrations is energetically costly.
Indeed, to insure the osmotic balance between the cytosol
and the high salt environment in which they thrive, halo-
philes have to accumulate and maintain high concentra-
tions of solutes. These are mainly inorganic ions, such as
KC1 that can reach molar concentrations or Mg2+, but var-
ious organic osmotic solutes such as glycerol, trehalose
and/or glycine betaine are also used [154,155]. As there
are no visible compartments in the Haloferax cell [156],
this lifestyle requires the adaptation of the entire intracel-
lular enzymatic machinery, including RNA maturation
and mRNA translation processes. Indeed, at high salt con-
centrations, the high molecular weight rRNA and the
majority of proteins from non halophilic prokaryotes sim-
ply precipitate (reviewed in [157]).

Here we combine the identification of the whole set of
functional tRNAs, including the presence of modified
nucleosides (tRNomics), with the identification of most
of the corresponding RNA modification enzymes (Modo-
mics) in H. volcanii. This analysis allows to address: i) the
peculiarities of the decoding strategies used by H. volcanii
to read the 62 (61+1 initiator) sense codons of mRNAs;
and ii) to emphasize the relative low number of modifica-
tions in halophilic t+rRNAs. This work is a logical contin-
uation of a similar tRNomics analysis of fully sequenced
genomes from the three kingdoms of life [71,73,158],
later extended to the Modomics analysis of Mollicutes, a
family of parasites that underwent a drastic reduction of
their genomes during evolution [34]. On an evolution
point of view, Halobacteriales like the euryarchaeon H.
volcanii, and other distantly related organisms able to
grow at salt concentrations above 100 g/L (1.7 M NaCl),
such as certain Methanosarcinales (Archaea), Flavobacte-
ria, Cyanobacteria and Proteobacteria (Bacteria) or a few
Flagellated, Ciliates and Fungi (Eukarya), are all located
on a relatively 'recent' branches of the small subunit rRNA
based phylogenetic tree (see Figure 1 in [154]). Thus,
emergence of halophilic organisms likely results from an
adaptive-type of cellular evolution from a non-halophilic
ancestor arising independently several times during the
evolution of the three domains of life.

The detailed mechanism by which mRNAs are accurately
decoded without slippage by the ribosome in an
extremely halophiles is largely ignored. The only pub-
lished study using cell-free system from Halobacterium


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cutirubrum shows that incorporation of radiolabeled
amino-acids into polypeptides under the direction of syn-
thetic polyribonucleotides, follows the same decoding
rules found in non halophilic organisms, but that the
accuracy of amino acids incorporation was dependent on
the presence of various salts at high concentrations (KC1,
NaCi, NH4C1 [85]). This lead to the conclusion that the
codon recognition processes are only secondarily depend-
ent on ionic interactions and that the effect of salts was
probably to enable all the macromolecular components
to assume their correct secondary and tertiary configura-
tion, a conclusion that is evident nowadays.

What is clear from the present work, is that the 52 (45
elongators + 1 initiator + 6 duplicants) tRNAs found in H.
volcanii that read the 62 universally used sense codons
(61+1 initiator) are typical of the Archaea that have been


analyzed to date with a few minor differences discussed
above ([71] and unpublished data). What is more inter-
esting is that H. volcanii uses only 16 different types of
modified nucleotides at 18 positions in the 46 mature
tRNA isoacceptors, while both E. coli and S. cerevisiae use
at least 28 different types of modified nucleotides at 20
and 35 different positions respectively [4,71]. As far as the
type and position of modified nucleoside in tRNAs, the
archaeon H. volcanii resembles Eukarya in some ways and
Eubacteria in others (Table 3). The cases where an identi-
cal modification is found at the same position in the three
kingdoms are rare (indicated in bold Table 3). The modi-
fications that are archaeal specific by their chemical struc-
ture and/or their positions in tRNAs are also not
numerous (underlined in Table 3). Examples include G-
15, ac4C-34, mlT-54, Cm-56 and mil-57 (Fig 1). Phylo-
genetic and structural analysis of the transglycosylase TGT


Table 3: Type and location of tRNA modifications of representative organisms belonging to the three domains of life Archaea (A),
Eubacteria (B) and Eukarya (E)


H. volcanii M. capricolum.


E. coli


S. cerevisiae


E (B)

S. cer (mito)


T
Cm, Um
Um, s2U
mnm5U
k2C, cmoSU
ac4C, I
m6A, ms2i6A
m I G, t6A, m6t6A


T
T

T
acp3U



m5U
T


m2G/m22G, T
T
T
T
Cm, ', m3C
Cm,Um,Gm,s2U
mcm5U
cm5U, m5C

i6A
m'G, t6A
mll, yW

TV


For H. volcanii and yeast mitochondria [S. cer(mito)] all modifications are listed whereas for E. coli, M. capricolum and yeast cytoplasm those that are
relevant for the comparison are indicated (for more details see [4]); modifications present in A,B,E are in bold; modification that appear specific to
H. volcanii (and most certainly to Archaea) are in underlined bold and.


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Kingdom Position


10
12
13
15
17/20
22
26
28


m2G/m22G

T
G+


m2G/m22G
T


Cm
Cm, ?s2U
?mcm5U
C*, U*
ac4C

m'G, t6A




m5C

m5C
m5C



Cm
mil


Cm, Um, s2U
cmnm5U
k2C

m6A
m'G, t6A


m2G/m22G
T
T
T


cmnm5U
U*?
-?
i6A
m'G, t6A


P?


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catalyzing the incorporation of precursor of Archaeosine
(G+) into tRNA, points to a common evolutionary origin
with the present-day enzyme catalyzing the formation of
queuosine at position 34 in many bacteria and higher
eukaryotes [159], a typical case of divergent evolution. In
contrast, the enzymatic formation of ml at position 57 in
archaeal tRNA involves a totally different set of enzymes
than those needed to catalyze the formation of the same
modification at position 37 in eukaryal tRNA-Ala [86],
this time a case of convergent evolution. Our analysis has
raised several questions that await experimental follow-
up. Several predictions such as those for the genes
involved in mul or ac4C formation need to be validated.
The nature of s2U-34 derivative that was predicted from
the comparative genomic analysis but was not found in
the initial tRNA sequencing work [28,29] has to be iden-
tified. We failed to find any gene coding for putative
(multi) site-specific RNA pseudouridine synthase(s), nor
for 'classical' box H/ACA guide RNAs with that catalyze
the formation of pseudouridines at positions 22, 28 and
52. As point out above, it might well be that these 'P 's are
formed at very early step of the tRNA maturation (possibly
during transcription) by the non RNA guide Cbf5p/
NoplOP/Garl complex.

The type and location of modified nucleotides found in
16S rRNA of H. volcanii and in the 23S rRNA of the closely

Table 4: Type and location of rRNA modifications of representative
Eubacteria (B) and Eukarya (E)


phylogenetically related H. marismortui were compared to
those found in E. coli and S. cerevisiae (Table 4). There
again the surprising feature in halophiles is the paucity of
rRNA modifications with only 4 different modified nucle-
otides in 5 positions in the 16S rRNA (out of 1472 nt) and
6 in 8 positions in the 23S rRNA (out of 2922 nt). In E.
coli there are 16 different types of modified nucleotides
within 35 positions of the 16+23S rRNAs and in S. cerevi-
siae 18+23+5S rRNAs contain at least 8 different modified
nucleotides located at more than 100 positions [12,13].
Only a few of these modifications are found in all the
three biological domains in rRNA analyzed to date from
(in bold in Table 4). Without exception, they are located
in critical functional domains of the RNA molecules, e.g.
in the decoding center of the SSU rRNA (Figure 2B) and
near the peptidyl transferase center of LSU rRNA (Fig 2D)
manifesting their functional importance in various
aspects of the dynamic process or mRNA translation (as
discussed above in the data section). Their importance is
further supported by the fact that the genes coding for the
corresponding enzymes, as well as the sRNA guided mod-
ification machinery allowing the formation of these con-
served t+rRNA modifications, are also remarkably
conserved among the different domains of life, except for
Gm-2588, mlA-628 and acp3U-910 that are present in
eukaryotic rRNA and absent in bacterial rRNA (see Table
4).

organisms belonging to the three domains of life Archaea (A),


pos A


E (B)


H. volcanii


acp3U-9 10


1352 C*= N330-1352


1432
1450
1451
LSU
628
1950
1956
1958


m6A
m62A
m62A
H. marismortui
m'A
Gm
T
T


2587 Um

2588 Gm
2619 m!U
2621 T


Here, the numbering of the specific organisms is given; modifications present in A,B,E are in bold; modification that appear specific to H. volcanii/H.
mansmortui (and most certainly to Archaea) are in underlined bold; for M. capricolum the presence of the modification has been predicted from the
presence of the corresponding gene [34] ; for H. volcanii and yeast mitochondria [S. cer(mito)] all modifications are listed whereas for E. coli, M.
capricolum and yeast cytoplasm those that are relevant for the comparison are indicated, however, in few cases, is listed a given modification in E.
coli or yeast rRNA that has no equivalent in H. volcanii but is present in the vicinity of a modified nucleotides found H. volcanii.


Page 18 of 26
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M. capricolum.


Enz for m2G


Enz for m62A
Enz for m62A



Enz for T
Enz for T
Enz for Gmr
Enz for Umr


E. coli


m2G-966
m4Cm- 1402
C- 1404
m7G-1407
A-1500
m62A- I5 18
m6 2A-1519

U-571
C- 1909
m3P- 1915
- -1917
Gm-2251
Um-2552
P-2580
G-2553
U-2584
U-2586


S. cerevisiae


m'acp3P-l 189
Cm-1638



m62A- 1780
m62A- 1781


mIA-645
A-2252
P-2258
p-2260
Gm-2619
Um-2921
U-2949
Gm-2922
U-2953
U-2955


S. cer (mito)


Gm-2270
Um-2791
P-2918


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A clear positive correlation has been observed between the
total number of ribose methylation sites, the eventual cor-
responding number of methylation guide sRNAs and the
optimal temperature at which an organism is growing,
suggesting an important role of this type of modification
in RNA stabilization (reviewed in [160]). Clearly as the
number of 2'- 0-methyl ribose is exceptionally low in
rRNA of halophiles, the rules guiding the faithful matura-
tion of rRNA molecule, as well as the stabilization of their
quaternary structure within the ribosome, might differ
from other Archaea (psychrophiles, mesophiles and
hyperthermophiles). Of note also is the absence of
polyamines in extreme halophiles, as the slight amount of
polyamines that can be detected actually originate from
the culture medium (Oshima Tairo, personal communi-
cation). Polyamines, like Mg2+ and other ions stabilize
nucleic acids (reviewed in [161]) and also facilitate pro-
tein synthesis [162,163]. The 3D structure of the large 50S
subunit of H. marismortui has been solved to 2.4 Angstrom
resolution [164]. Analysis of the structure reveals a great
number of monovalent and divalent ions as well as water
molecules that are critical for the formation and stabiliza-
tion of that rRNA structure. Hence, we propose that the
presence of high concentration of salts (mono- and diva-
lent) in the cytosol ofH. volcanii has allowed the elimina-
tion of numerous rRNA and tRNA modifications as well as
of polyamines biosynthesis, whose 'global' functions are
to allow faithful maturation of pre-t+rRNAs and/or to sta-
bilize the mature t+rRNAs and their association with other
proteins (e.g. quaternary structure in the case of ribos-
ome). If the functional replacement of many RNA modi-
fication by salts had indeed occurred, then modified
nucleotides remaining in t+rRNA of halophilic organism
must serve purposes other than stabilization of RNA
architecture, such as decoding, accuracy of translation or
other functions that cannot be functionally replaced by
the electrostatic interactions provided by the surrounding
salts. This hypothesis is corroborated with the fact that
most, if not all of the modified nucleoside found in H. vol-
canii/H. marismortui rRNA are among the most evolution-
ary conserved modified nucleosides along organisms of
the three biological domains (Table 4 and discussed
above in data section). They are also among those we have
pointed out as being the most refractory to reductive evo-
lution in Mycoplasma [34]. This would suggest that the
modifications remaining in H. volcanii tRNA are also crit-
ical for functions that cannot be replaced by salt and we
are currently mutating all the corresponding genes to
address the functional of these modifications in vivo.

This tRNomics and Modomics analysis of H. volcanii rein-
forces the necessity to integrate the knowledge of both
t+rRNA sequences and modifications in order to under-
stand the decoding properties of a given organisms. For
most organisms this information can be derived only


from comparative genomic analysis as sequence informa-
tion of mature RNAs are lacking. However, to predict the
presence or absence of modified nucleotides just from the
analysis of the encoded genes is still quite dangerous and
requires the type of systematic analysis performed here as
a foundation in order to analyze other Archaeal genomes
and understand of the function of RNA modification in
Archaeal translation and its evolutionary importance.

Methods
tRNA genes searches in the H. volcanii genome
The complete genome of H. volcanii DS2 (April 2007
(haloVolcl) assembly) was obtained using the UCSC
Archaeal Genome Browser http://archaea.ucsc.edu/cgi-
bin/hgTracks?hgsid=84889&chromInfoPage=[165]. The
full set of tRNA genes (tDNAs) was first identified by
searching the nucleotide sequence corresponding to all
the archaeal-type conserved tRNA cloverleaf structures
(for details see [71]). Verification with tRNAscan-SE[166]
disclosed two more genes displaying anomalously low
Cove score values. Close examination of the sequences
revealed the presence of an anomalous G at position 58
(instead of the universal A58) in elongator tDNA-Met
(CAT) (Cove score: 54.0); this remarkable sequence
exception is confirmed by the tRNA sequencing [29]. The
other exception is a G at position 8 (instead of the univer-
sal pyrimidine T8/C8) in tDNA-Thr (TGT) (Cove score:
44.6). This tRNA however was not sequenced, but one can
observed in this tRNA that base 14, which is usually
paired with base 8 (Watson-Crick A-T pair), is also excep-
tionally G instead of A suggesting a Hoogsteen G8-G14
base pair. The complete list of the 52 tDNAs ofH. volcanii
tabulated in a linear, as well as in a cloverleaf representa-
tions is given in Additional file 2. These 52 genes corre-
spond to 46 different tRNAs (different anticodons) since
6 genes are present in two copies (the two copies oftDNA-
Gly (GCC) slightly differ in the amino acid stem only).
Only three genes bear introns: tRNA-Trp (CCA), tRNA-
Gln (TTG) and tRNA-Met (CAT) for details see text
below.

Compilation of mature tRNA sequences harboring
modified nucleotides
The linear sequences of the 41 naturally occurring mature
tRNAs of H. volcanii, as sequenced by Gupta [28,29] are
listed in Additional file 1 (including the two variants of
tRNA-Gly (GCC)). From comparison with the other fully
sequenced tRNAs, the presence of many modified nucle-
otides in these tDNAs can however easily be inferred.
Beside C* in the minor tRNA-Ile (C*AU) and U* in sev-
eral U34-containing tRNAs, the chemical structures of all
modified nucleotides are known. The probability of
unknown modified nucleotides remaining in one of the
six unsequenced tRNAs of H. volcanii is small. Analysis of
the 12 additional sequences from other mesophilic halo-


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philes, reveals the presence of msC at positions 50 of
tRNA-Thr (GGU), position 51 of tRNA-Val (CAC+GAC)
and position 52 of tRNA-Arg (GCG), as well as of mlG at
position 9 in tRNA-Val (CAC) and probably also in tRNA-
His (GUG) and tRNA-Gln (CUG) in H. cutirubrum. These
last modifications are not found in any of the sequenced
tRNAs of H. volcanii.

Mining genes coding for RNA modification enzymes
Most of the comparative genomic analysis to identify
putative RNA modification genes was performed in the
integrative SEED database[ 167] at http://anno-
3.nmpdr.org/anno/FIG/subsys.cgi. Results are made
available in the "Archaeal tRNA modification" and the
"Archaeal rRNA modification subsystem" on the publicly
available server http://theseed.uchicago.edu/FIG/
index.cgi. Microbes online [168] was also used for cluster-
ing analysis and mining co-expression data. The phyloge-
netic pattern searches were performed using the signature
search tool on the NMPDR server [169], the COG phylo-
genetic pattern search at NCBI ([170], hittp-/
www.ncbi.nlm.nih.gov/COG/old/phylox.html), the
ortholog table tool at the MBGD database [171], the phy-
logenetic search query forms of OrthoMCL [134] or of the
Integrated Microbial Genome (IMG) database [172].
Genome specific BLAST searches [173] were also per-
formed at NCBI http://www.ncbi.nlm.nih.gov/sutils/
genom table.cgi. Phylogenetic distribution of any given
gene family was obtained through the IMG database
[172]. Information on the presence of a given modifica-
tion in RNA was essentially extracted from the RNA mod-
ification database [174], the tRNA database [4], the small
rRNA modification database [3] and the 3D ribosomal
modification map database [14] (for corresponding http,
see above in Introduction section). Databases for rRNA
and snoRNA that are involved in RNA-guided modifica-
tions are located at. http://people.biochem.umass.edu/
foumierlab/snornadb/main.php[241 and http://lowe
lab.ucsc.edu/snoRNAdb/. Additional information was
extracted from specific articles cited throughout the text.

Mining genes coding for C/D and HIACA boxes RNA guide
of RNA modifications
In archaea, C/D box sRNA contains four short conserved
sequence motifs called the C box (RUGAUGA), D' box
(CUGA), C' box (UGAUGA) and D box (CUGA), and one
or two antisense elements. Each antisense element is 8-
12nt long, is located immediately upstream of box D or
D', and shows conserved complementarities spanning the
site of modification. Each antisense element is the deter-
minant of the site-specificity of the methylation site which
is always the nucleotide of the target sequence paired to
the fifth sRNA nucleotide upstream from the D(D') box
(See Additional file 6, and [160]). Archaeal H/ACA sRNA
are composed of one, two, or three stem-loop structure


[145,175,176]. Each of these stem-loop structures can be
described by two stems separated by an internal loop, a K-
turn motif, and a conserved ANA (generally ACA) motif at
the 3' end. The internal loop is composed of two single
stranded regions which are complementary to a target
region around the modified nucleotide. The target region
itself encompasses two regions able to form the duplex by
forming RNA-RNA interactions with the internal loop.
These two regions are separated by UN, U being the urid-
ine which will be converted into a pseudouridine (see
Additional file 6

The C/D box and H/ACA box sRNAs responsible for a
given set of modifications were searched by using PatScan
and Darn! http://carlit.toulouse.inra.fr/Darn/. In princi-
ple, the knowledge of presence of 2'-O-methyl derivatives
as well as of P in RNA is of great help to identify potential
sRNAs. However, as Halobacteria may use non canonical
type of sRNAs, the task is not simple. Despite this, for C/
D box sRNA, we used a signature describing half of a C/D
box sRNA containing a C (C') box motif, a short spacer,
the antisense region and a D (D') box motif. The antisense
region was modeled as a motif complementary to the
sequence spanning four nucleotides before and after the
target position. Each candidate was then extended either
at its 5' or 3' end to obtain a complete sRNA sequence. In
some cases, it was necessary to degenerate the signature
(including one or two errors in C, C', D, D' and antisense
regions) to obtain a good sRNA candidate. The same strat-
egy was used for H/ACA sRNA. For H/ACA sRNA, the ini-
tial signature contained the characteristics of a stem-loop
structure with the stem down to the pocket, the two 3-5
nt antisense elements surrounding the residue to modify,
a K-turn (K-loop) motif and an ANA motif situated 13-16
nucleotides from the T residue. For each candidate found,
we searched for homologous sequences by combining
pattern matching approaches and similarly searches
(using NCBI-Blast against complete genomes of archaea at
http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi. Only
candidates found in inter-coding sequences and showing
strong homology evidence were kept as good candidates.

Comparison with known sRNA was performed by using
data from the literature and available databases http/
people.biochem.umass.edu/fournierlab/snoma db/
main.php, http://www-snoma.biotoul.fr/,. http://bio
inf. scri. sari. ac.uk/cgi-bin/plant snorna/home and http/
lowelab.ucsc.edu/snoRNAdb/).

Nomenclature
All tRNA genes and mature tRNA (with their anticodon in
brackets) are designated as this example: tDNA-Ile (GAT)
and tRNA-Ile (GAU) respectively. The conventional num-
bering system for tRNA positions and the symbols used
for the modified nucleosides are those adopted in the


Page 20 of 26
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BMC Genomics 2008, 9:470







http://www. biomedcentral.com/1471-2164/9/470


tRNA database [4]. The number after a ribonucleotide
(symbolized by A, U, G, C) or its modified counterpart Additional File 3
corresponds to its position in the tRNA molecules. In the The hBHBh' structure of the introns in pre-tRNA-Met (CAU), pre-
case of rRNA, unless otherwise specified, numbers corre- tRNA-Gln (UUG) and pre-tRNA-Trp (CCA) in Haloferaxvolcanii.
spond to the equivalent position in the E. coli rRNA. Only Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
nucleoside C* is unconventional. As discussed below, C* 2164-9-470-S3.ppt]
found at the wobble position 34 of H. volcanii tRNA-Ile
(anticodon C*AU) corresponds to a yet incompletely Additional File 4
characterized, probably 'lysidine-type' cytosine, while at sRNAs of Haloferaxvolcanii predicted to modify Cm34 and Um39 in
position 1342 (1404 E. coli numbering) of H. volcanii 16S tRNA-Trp.
rRNA, C* corresponds to another uncharacterized C- Click here for file
derivative of a molecular mass of 330.117 Da (N-330). [http://www.biomedcentral.com/content/supplementary/1471-
Detailed chemical structures, scientific and common 2164-9-470-S4.doc
names corresponding to each indicated modified nucleo- Additional File 5
side and as well as of the corresponding RNA modifica- sRNA of Haloferaxvolcanii predicted to ,1dify Cm34 in tRNA-Met.
tion enzymes can be found at h1tt1P// Click here for file
library.med.utah.edu/RNAmods/ and at http://modo [http://www.biomedcentral.com/content/supplementary/1471-
mics.genesilico.pl 2164-9-470-S5.doc]

Abbreviations Additional File 6
COG: Cluster of Orthologous Group; ORF: open reading Representation of C/D box and H/ACA sRNA in archaea.
Click here for file
frame; SAM or S- AdoMet: S-Adenosyl-L-Methionine; SSU: [http://www.biomedcentralcom/content/supplementary/1471
small subunit; LSU: large subunit; PTC: peptidyl trans- 2164-9-470-S6.ppt]
ferase center; NCBI: National Center for Biotechnology
Information; RNP: ribonucleprotein; Nt: nucleotide. Additional File 7
sRNA of Haloferaxvolcanii predicted to modify Gm1934 (Gm1950)
Authors' contributions in 23S rRNA.
HG and VdC-L designed the study and coordinated the Click here for file
analysis. HG carried out the analysis of the tRNA [http://www.biomedcentral.com/content/supplementary/1471-
H d t t a o t 2164-9-470-S7.doc]
sequences, CM searched and analyzed the tDNA
sequences, CG searched and analyzed the sRNA Additional File 8
sequences, WD did the analysis of the rRNA modification H/ACA sRNA sequences of Haloferaxvolcanii and some homologous
in the context of the Ribosome structure, VdC-L predicted sequences.
all the modifications genes and drafted the manuscript. Click here for file
All authors read and approved the final manuscript. [http://www.biomedcentral.com/content/supplementary/1471-
2164-9-470-S8.doc]
Additional material Additional File 9
Legends for Additional files
Additional File 1 Click here for file
Sequences of the 41 mature tRNAs + 6 tDNA covering the whole [http://www.biomedcentral.com/content/supplementary/1471-
decoding set of Haloferax volcanii. 2164-9-470-S9.doc
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2164-9-470-Si.doc]
Acknowledgements
Additional File 2 We wish to thank Jonathan Eisen for making the sequence of Haloferax vol-
Sequences of the 52 tDNAs of Haloferax volcanii and Genetic Code canii publicly available before publication, lan K. Blaby for improving the
coverage, manuscript and Jean-Pierre Rousset for stimulating discussions. This work
Click here for file was supported by US Public Health Service grant number GM 19351 to
[http://www.biomedcentral.com/content/supplementary/1471- Maurille J. Fournier (W.D) and the National Science Foundation grant
2164-9-470-S2.doc] number MCB-05169448 to V dC.L.

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