2
'
-
O
-methyl-5-formylcytidine (f
5
Cm), a new modified nucleotide at the `wobble' position of two cytoplasmic tRNAs
Leu
(NAA) from bovine liver
2 ' - O -methyl-5-formylcytidine (f 5 Cm), a new modified nucleotide at the `wobble' position of two cytoplasmic tRNAs Leu (NAA) from bovine liver
Jean-Paul
Païs de Barros
,
Gérard
Keith
1
,
Chakib El
Adlouni
1,+
,
Anne-Lise
Glasser
w
,
Gérard
Mack
,
Guy
Dirheimer
1
and
Jean
Desgrès*
Laboratoire de Biochimie Médicale, Equipe de Recherche Biochimie-Biologie Cellulaire, Faculté de Médecine et Centre Hospitalier Universitaire, Université de Bourgogne, 21034
Dijon
,
France
and
1
Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Unité Propre de Recherche no. 9002, Institut de Biologie Moléculaire et Cellulaire du CNRS et Université Louis Pasteur, 67084
Strasbourg
,
France
Received December 28, 1995
;
Revised and Accepted March 4, 1996
ABSTRACT
The nucleotide analysis of a cytoplasmic tRNA
Leu
isolated from bovine liver revealed the presence of an unknown modified
nucleotide N. The corresponding N nucleoside was isolated by different
enzymatic and chromatographic protocols from a partially purified preparation
of this tRNA
Leu
. Its chemical characterization was determined from its chromatographic properties, UV-absorption spectroscopy and mass spectrometric measurements, as well as
from those of the borohydride reduced N nucleoside and its etheno-trimethylsilyl derivative. The structure of N was established as 2
'
-
O
-methyl-5-formylcytidine (f
5
Cm), and its reduced derivative as 2
'
-
O
-methyl-5-hydroxymethylcytidine (om
5
Cm). By sequencing the bovine liver tRNA
Leu
, the structure of the anticodon was determined as f
5
CmAA. In addition, the nucleotide sequence showed two primary structures
differing only by the nucleotide 47c which is either uridine or adenosine. The two slightly differing bovine liver tRNAs
Leu
(f
5
CmAA) are the only tRNAs so far sequenced which contain f
5
Cm. The role of such a modified cytidine at the first position of the anticodon
is discussed in terms of decoding properties for the UUG and UUA leucine
codons. Recently, precise evidence was obtained for the presence of f
5
Cm at the same position in tRNAs
Leu
(NAA) isolated from rabbit and lamb liver. Therefore, the 2
'
-
O
-methyl-5-formyl modification of cytidine at position 34 could be a general
feature of cytoplasmic tRNAs
Leu
(NAA) in mammals.
INTRODUCTION
The primary structures of several cytoplasmic mammalian isoaccepting tRNAs
Leu
are already known as carrying the following anticodons: IAG from bovine liver (
1
) and cow mammary gland (
2
), CAG from cow mammary gland (
3
), and MmAA from rat Morris hepatoma (
4
) and human HeLa cells (
5
) where the unknown nucleotide called Mm was described as a 2'-
O
-methylated modified pyrimidine.
In order to determine whether or not this modified nucleotide Mm was identical
to the already published 2'-
O
-methyl-5-carbamoylmethyluridine (ncm
5
Um) found in yeast tRNA
Leu
(ncm
5
UmAA) (
6
,
7
), we purified a bovine liver tRNA
Leu
corresponding to the above tRNAs
Leu
(MmAA), and analyzed its modified nucleotides. Some preliminary studies showed
the presence of an unknown nucleotide that could be located at the `wobble'
position of the anticodon. This unknown nucleotide, that we called N, behaved
on thin-layer or high performance liquid chromatography like a 2'-
O
-methylated modified pyrimidine, as previously described for Mm in tRNAs
Leu
(MmAA) from rat Morris hepatoma (
4
) and human HeLa cells (
5
).
We report here on the isolation of the unknown N nucleoside, and its structure determination by the combined means of thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) with real-time UV-absorption spectroscopy, and gas chromatography-mass spectrometry (GC-MS) analysis (
13
,
14
). By improving some of the previously used sequencing methods (
8
-
12
), the complete nucleotide sequence of the tRNA
Leu
isolated from bovine liver was further determined. The latter is also reported
in this paper.
Preliminary studies recently conducted in our laboratories have shown that N nucleotide was also present in pure tRNAs
Leu
(NAA) isolated from rabbit and lamb liver. The coding properties of this modified nucleotide located at the `wobble' position of these mammalian tRNAs
Leu
are discussed.
MATERIALS AND METHODS
Purification of bovine tRNA
Leu
(NAA)
Total bovine tRNA was prepared according to Fournier
et al.
(
9
). It was further chromatographed on BD-cellulose column (
10
). The fraction containing the tRNA
Leu
(NAA) was detected by dot-blot hybridization (
12
) with a probe complementary to the 3'-end (nucleotides 59-73) of already sequenced tRNAs
Leu
(MmAA) from rat Morris hepatoma (
4
) and human HeLa cells (
5
). This partially purified preparation of tRNA was used for the structure
determination of the unknown N nucleoside, as described below. Further
purification steps of tRNA
Leu
(NAA) were carried out by chromatography on Sepharose 4B column (
10
), followed by two dimensional polyacrylamide gel electrophoresis in non-denaturating conditions (
8
,
11
). To obtain a tRNA suitable for sequencing, the tRNA
Leu
(NAA) was further purified on a denaturing polyacrylamide gel at 55-60oC, in order to remove partially cut molecules (
10
,
11
).
Isolation of the unknown N nucleoside
The N nucleoside was isolated from the partially purified preparation of bovine liver tRNA
Leu
(NAA), using the analytical procedure previously described for the isolation of
other unknown nucleosides (
13
,
14
). This procedure can be summarized as follows:
(i) nuclease P1 digestion followed by bacterial alkaline phosphatase (BAP) treatment of the tRNA
Leu
(NAA) sample (~50
A
260
units in 200 [mu]l water); (ii) isolation and desalting by HPLC of a dinucleotide resistant
to nuclease P1 action; (iii) combined hydrolysis by snake venom
phosphodiesterase (SV-PDE) and BAP of the dinucleotide sample, leading to the liberation of two
nucleosides; (iv) isolation and desalting of N nucleoside by HPLC; and (v)
drying of N nucleoside under vacuum at room temperature.
Analysis, isolation and desalting of the ribonucleosides by HPLC were performed
using the chromatographic experimental conditions previously described (
13
,
15
-
17
).
Mass spectrometry
The conversion of N nucleoside (~3 [mu]g) to trimethylsilyl (TMS) derivative, and the electron impact-mass spectrometry (EI-MS) of silylated N by gas chromatography-mass spectrometry (GC-MS) analysis, using a Hewlett Packard GC-mass spectrometer, model 5971A, were
carried out as previously reported (
7
,
13
,
14
). Identical GC-MS conditions were also applied on the TMS and etheno-trimethylsilyl (etheno-TMS) derivatives of the reduced product resulting from
borohydride treatment of N nucleoside.
Borohydride reduction of N nucleoside
Borohydride reduction of N nucleoside was performed according to Matsuda
et al.
(
18
). To ~1 [mu]g of the dry N nucleoside were added 100 [mu]l 5 mg/ml aqueous natrium borohydride solution (NaBH
4
) and 100 [mu]l 20 mM orthophosphoric acid. The mixture was incubated for 1 h at 65oC and further analyzed by HPLC. The resulting product was isolated,
desalted and dried as described above for the isolation of N nucleoside. The
dry compound was finally converted either to TMS derivative, or to etheno-TMS derivative for structure characterization by GC-MS analysis.
Etheno-trimethylsilyl (etheno-TMS) procedure
The etheno-TMS derivatives of nucleosides were prepared using an adaptation of the procedures previously described by others (
19
-
22
). A 50% aqueous chloracetaldehyde solution was adjusted to pH 4.0 using 2 M sodium hydroxide solution. To 50 [mu]l of aqueous solution containing ~1 [mu]g of N nucleoside were added 10 [mu]l of the above chloracetaldehyde solution, and the mixture was
stirred overnight at room temperature. In order to isolate the resulting
product (i.e. the etheno derivative), and to remove the remaining
chloracetaldehyde, the above mixture was chromatographed on HPLC, using the
same chromatographic conditions as for the HPLC analysis of tRNA nucleosides (
13
,
14
). After drying under vacuum at room temperature, the pure etheno derivative was
submitted to TMS derivatization, and it was further characterized by GC-MS analysis.
Sequencing procedures
To sequence the tRNA
Leu
(NAA), and to characterized the modified nucleotides therein, we used the random
single hit sequencing
32
P post-labelling procedure that has already been described (
11
). Prior to sequencing, the highly purified tRNA (~5 [mu]g in 150 [mu]l) was renatured by heating for 5 min at 65oC in presence of 20 mM MgCl
2
, and cooled down slowly step by step: 10 min at 37oC followed by 10 min at room temperature. To remove the excess of MgCl
2
, the sample was desalted by centrifugation on Sephadex G25 columns poured into
1 ml syringes (
11
).
After desalting, the sample was dried and hydrolyzed in 10 [mu]l deionised formamide at 95oC for 4 min (
10
,
11
). The digestion products were labelled by T4 polynucleotide kinase and [[gamma]-
32
P]ATP, deproteinized by phenol, and desalted on Sephadex G25 as described above.
The labelled digest was denatured (5 min at 95oC in loading buffer), and directly loaded onto pre-heated (by pre-electrophoresis at 1500 V for 2 h) denaturing 15%
polyacrylamide gels containing 10% deionised formamide and 7 M urea. To keep
the fragments in denatured form during loading, the buffer in the wells was
heated by injecting, prior to loading, hot (95oC) running buffer. Separation of the fragments was performed at 1500 V.
RESULTS
Isolation of the unknown N nucleoside
HPLC nucleoside analysis of the partially purified bovine liver tRNA
Leu
(NAA) after exhaustive nuclease P1 digestion followed by BAP hydrolysis (Fig.
1
a) gave the following modified nucleosides (in moles per mole of tRNA): D, 1.6; [psi], 4.3; m
1
A (+m
6
A), 1.0; m
5
C, 1.2; T, 1.0; Um, 1.0; m
1
G, 0.9; ac
4
C, 0.7; m
2
G, 1.8; m
2
2
G, 1.1; Cm, 0.1; i
6
A, 0.1; m
7
G, 0.3; Gm, 0.3; and a peak eluted at 53 min retention time, and which should
correspond to the peak N mentioned at 31 min retention time on the HPLC
chromatogram of nucleosides from bovine tRNA
Leu
previously published elsewhere (
23
). These results suggested that the tRNA
Leu
(NAA) preparation was contaminated with several cytoplasmic tRNAs (tRNA
Ser
, tRNA
Phe
, ...).
Characterization of N
42
as a modified 2
'
-
O
-methylcytidine by mass spectrometry
The EI-mass spectrum of N
42
as TMS derivative is presented in Figure
3
a, and several ion series are summarized in Table
1
. The ion peaks at m/z 501, 486, 469 and 398 were assigned to the molecular ion
M
+
and to the fragments M
+
-CH
3
, M
+
-CH
3
OH and M
+
-CH
2
OSi(CH
3
)
3
, respectively. Resulting from N-C glycosylic bond cleavage and fragmentations in the sugar moiety (
26
), the ion peaks at m/z 341, 312, 284, 268, 251, 240, 223 and 212 were assigned
to contain the base moiety (B) of 210 Da plus portions of the sugar moiety.
Finally, numerous ions such as m/z 290, 258, 187 and 159 were characteristic of
ribonucleosides having a 2'-
O
-methylated ribose.
DISCUSSION
In terms of primary structures, there are two cytoplasmic tRNAs
Leu
(f
5
CmAA) in bovine liver that differ only by the nucleotide 47c which is either
uridine or adenosine, whereas they carry the same f
5
CmAA anticodon. Among all the tRNAs so far sequenced, these bovine tRNAs
Leu
are the first tRNAs which contain 2'-
O
-methyl-5-formylcytidine (f
5
Cm). However, a closely-related nucleoside, the 5-formylcytidine (f
5
C), has been already described in the tRNA
Met
isolated from mitochondria of the same mammalian tissue (bovine liver) (
24
,
25
), and of the nematode
Ascaris suum
(
29
).
When comparing the primary structures of the two bovine tRNAs
Leu
(f
5
CmAA) with those of the already published mammalian tRNAs
Leu
(NAA) from rat Morris hepatoma (
4
) and human HeLa cells (
5
), several differences are displayed for some nucleotides of the extra loop: (i)
in rat Morris hepatoma, N44 is mentioned to be an unknown modified U, but not
Um, (ii) N47c is either A or U in both bovine liver and rat Morris hepatoma,
whereas this heterogeneity in position 47c is replaced by a unique G47c in human HeLa cells, and (iii) G47j is absent from both Morris hepatoma and
HeLa cells, while C45 is absent from only Morris hepatoma. These extra loop
differences between the previously published primary structures of cytoplasmic
tRNAs
Leu
(MmAA) from rat Morris hepatoma (
4
) and human HeLa cells (
5
), and those of tRNAs
Leu
(f
5
CmAA) from bovine liver determined in the present paper, are underlined in the
following schematic in-line models:
43
44
46
47
47c
47j
48
Morris hepatoma
G
N
([Psi]/U)
C C G
(
U/A
)
A U G G A G
m
5
C
HeLa cells
G
Um C
[Psi]
C C G
G
A U G G A G
m
5
C
Bovine liver
G
Um C
([Psi]/U)
C C G
(
U/A
)
A U G G A G
G
m
5
C
However, the most noteworthy feature of the mammalian tRNAs
Leu
(NAA) is the presence of a new modified nucleotide found at their `wobble'
positions. This new nucleotide was partially characterized in rat Morris
hepatoma as a probably C-5 substituted Cm, called Mm (
4
), and as the same Mm modified nucleotide in human HeLa cells (
5
), while it has been fully identified as f
5
Cm in the present paper. These results suggest that the N34 nucleotides carried
by the cytoplasmic tRNAs
Leu
(NAA) of the three above mammalian species could have a unique chemical
structure: f
5
Cm. To argue for this hypothesis, studies were recently conducted in our
laboratories on other purified tRNAs
Leu
(NAA) isolated from rabbit and lamb liver. Using our analytical procedure, we
obtained precise evidence for the presence of f
5
Cm in the `wobble' position of these tRNAs. Therefore, the 2'-
O
-methyl-5-formyl hypermodification of cytidine at position 34 could be a
specific feature of the cytoplasmic mammalian tRNAs
Leu
(NAA).
With regard to codon-anticodon interactions, the role and the significance of f
5
Cm in the two cytoplasmic bovine tRNAs
Leu
(f
5
CmAA) remain to be determined. However, they can be discussed in terms of
decoding properties for the leucine codons, as compared with those previously described in different eucaryotic species,
like yeasts and plants.
According to the genetic code, the leucine codons are located in two codon
boxes: the CUN box which contains only leucine codons, and the UUN box which is
shared between two UUPy phenylalanine codons and two UUPu leucine codons. In
yeast, the latter two corresponding anticodons are carried by two tRNAs
Leu
(XAA) which differ by 31 nucleotides in their primary structures, and by the
length of their variable loop: 15 versus 13 nucleotides (
6
,
30
). In addition, the X modified nucleotide located at the `wobble' position of
yeast tRNAs
Leu
(XAA) is either m
5
C (
30
), or ncm
5
Um (
7
), which restrict the recognition of the leucine codons to UUG or UUA,
respectively (
7
). An identical situation, i.e. two different tRNAs reading each one of the UUPu
leucine codons, could exist in the two cytoplasmic tRNAs
Leu
(ZAA) isolated from plants (
31
,
32
). However, the Z modified nucleotides in position 34 of these tRNAs have not
yet been identified.
Regarding the bovine liver cytoplasm, we show in the present paper that two
slightly differing isoacceptor tRNAs
Leu
carry the same anticodon, f
5
CmAA. The question then arises as to whether the modified nucleotide f
5
Cm could give to these two tRNAs
Leu
the ability to translate both the UUG and UUA leucine codons. As compared with
the conformational properties described for the structurally-related f
5
C nucleotide of bovine mitochondria tRNA
Met
(
25
), f
5
Cm should take a rigid C3'-endo form similar to that of f
5
C, and that rigidity could even be re-enforced by the 2'-
O
-methylation on the ribose moiety (
33
,
34
). In addition, it should also present an intramolecular hydrogen bond between
the carbonyl of the 5-formyl group and the 4-amino function (
25
). These conformational properties would avoid f
5
Cm in the first position of the anticodon to form base pairs with U and C (
33
,
35
,
36
). Therefore, one function of f
5
Cm in bovine tRNAs
Leu
(f
5
CmAA) would be to prevent an incorrect recognition of the UUU and UUC
phenylalanine codons. Another function of f
5
Cm could be to restrict the decoding property of the two cytoplasmic bovine
tRNAs
Leu
(f
5
CmAA) to the only UUG leucine codon, similarly to what has been shown for f
5
C by Takemoto
et al.
(
37
) in
in vitro
translation studies on tRNA
Met
(f
5
CAU) from bovine mitochondria.
Therefore, and similarly to the situation mentioned above for the yeast and
plant species, a second cytoplasmic bovine liver tRNA
Leu
(NAA), with N different from f
5
Cm, would be involved in the decoding of the UUA leucine codon. Investigations
to detect such a tRNA
Leu
in bovine and other mammalian species are underway in our laboratories. Other
studies are devoted to confirm or not the f
5
Cm structure for the unknown Mm nucleotide previously mentioned at the `wobble'
position in the tRNAs
Leu
(MmAA) from rat Morris hepatoma and human HeLa cells (
4
,
5
). The aim of these studies would be to accurately define the coding function(s)
of this new modified cytidine, and to clarify the translation process of both
the UUG and UUA leucine codons in mammals.
ACKNOWLEDGEMENTS
The expert technical assistance of C. Fix in preparing and sequencing tRNA
Leu
samples is warmly acknowledged. We are grateful to A. Athias who conducted the
MS studies in the Laboratoire de Biochimie des Lipoprotéines (Université de Bourgogne, Dijon, France) managed by Prof. P. Gambert. We
are also indebted to Dr B. Rayner, Université de Montpellier II, for technical advice in the borohydride reduction
procedure on nucleosides, and Prof. J. A. McCloskey, University of Utah, for
fruitful discussions in mass spectrometry. This research had financial support
from Association pour la Recherche sur le Cancer, Ministère de la Recherche et de la Technologie, Ligue Bourguignonne contre le
Cancer, and Centre National de la Recherche Scientifique.
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