LC-MS based quantification of 2′-ribosylated nucleosides Ar(p) and Gr(p) in tRNA

Article abstract of DOI:10.1039/c1cc11011j

RNA nucleosides are often naturally modified into complex non-canonical structures with key biological functions. Here we report LC-MS quantification of the Ar(p) and Gr(p) 2′-ribosylated nucleosides in tRNA using deuterium labelled standards, and the fir

Full text of DOI:10.1039/c1cc11011j

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LC-MS based quantification of 20-ribosylated nucleosides Ar(p) and
Gr(p) in tRNAw
David Pearson, Antje Hienzsch, Mirko Wagner, Daniel Globisch, Veronika Reiter,
¨
Dilek Ozden and Thomas Carell*
Received 21st February 2011, Accepted 13th March 2011
DOI: 10.1039/c1cc11011j
RNA nucleosides are often naturally modified into complex
non-canonical structures with key biological functions. Here
we report LC-MS quantification of the Ar(p) and Gr(p)
20-ribosylated nucleosides in tRNA using deuterium labelled
standards, and the first detection of Gr(p) in complex fungi.
RNA possesses a number of chemically modified bases that are
involved in key biological processes, such as codon-anticodon
binding and stabilization of the RNA structure.1–5 To date,
over 100 modified RNA nucleosides have been identified from
natural sources.6 The functions and biosyntheses of these
interesting structures have been elucidated to some extent;
however, few studies have attempted to quantify natural levels
of modification or to investigate the biological relevance or
regulation of these levels. In order to address these issues, we
Fig. 1 20-phosphoribosylated nucleosides Ar(p) 1 and Gr(p) 2 and
the corresponding dephosphorylated forms Ar 3 and Gr 4 resulting
from enzymatic RNA digestion.
have begun to quantify the modified bases using an LC-MS
based quantification method using isotope labelled standards.7
The Ar(p)
1 and Gr(p) 2 nucleosides are unusual
20-phosphoribosylated modifications of the canonical bases
adenosine (A) and guanosine (G) (Fig. 1).8–10 Both nucleosides
have been detected at position 64 (in the T arm) of initiator
tRNAMet in yeasts and plants, and appear to distinguish this
tRNA from elongator tRNAMet, which is unmodified in this
position.11,12 The nucleosides are also postulated to generally
occur in fungi based on tRNA sequences,11 but have not yet
been detected in complex fungi. Both nucleosides possess a
characteristic 20-phosphoribose modification; however, under
typical digest conditions needed to detect these compounds,
the phosphate group is hydrolysed to give the simpler Ar 3 or
Gr 4 nucleoside. Here we report syntheses of deuterium
labelled analogues in order to characterize the distribution
of these specially modified bases in various tissues and in
particular in higher fungi. Our study shows that in the higher
fungi investigated by us it is Gr(p) and not Ar(p) that is
present in the tRNAs in this position, suggesting that Gr(p) is
more commonly utilised.
Syntheses of both nucleosides have been reported involving
Vorbruggen glycosylation reactions between protected A or G
nucleosides and an appropriately reactive ribose.13–15 Based
on these available synthetic routes, we decided that deuterated
ribose would be the most practical precursor for a practical
late stage incorporation16 of the label. Scheme 1 shows the
most practical retrosyntheses of 3, revealing that step (a) is the
best option for incorporation of the (cost) limiting labelled
reagent. Late stage incorporation of an isotope labelled base
(adenine or guanine) was expected to be more synthetically
challenging, and was therefore not attempted. As an isotope
labelled ribose, we chose 5,5-d2-ribose 5 (Fig. 2), which can be
Center for Integrated Protein Science (CiPSM) at the Department of
Chemistry, LMU Munich, Butenandtstrasse 5–13, 81377 Munich,
Germany. E-mail: thomas.carell@cup.uni-muenchen.de;
Fax: +49 892 1807 7756; Tel: +49 892 1807 7750
w Electronic supplementary information (ESI) available: Fig. S1,
experimental data and NMR spectra. See DOI: 10.1039/c1cc11011j
Scheme
1 Retrosynthesis of Ar showing possible stages for
incorporation of an isotope label. Potentially labelled starting materials
are coloured in red.
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Fig. 2 Isotope-labelled ribose 5 and target deuterated nucleosides 6
and 7. Labelled atoms are coloured in red.
Scheme 3 Synthesis of d2-Gr 7.
on the strategy reported for the non-deuterated nucleoside.13
However, in our hands this reaction gave consistently poor
yields. Instead, the simpler 1-Ac-2,3,5-Bz-protected d2-ribose
16 was used (Scheme 3).14 Several strategies were investigated
for the effective preparation of this precursor. The most
effective was determined to be methylation of the 1-hydroxyl
group of d2-ribose using Dowex 50 cation exchange resin
(H+ form) in MeOH,19 followed by benzoylation then acetylation
20
in AcOH/Ac2O/H2SO4 to give protected ribose 16. This
precursor was then reacted with protected guanosine 1721
and SnCl4 to give 18 in 52% yield. Protecting groups were
finally removed, again using TBAF then NH3/MeOH to give
d2-Gr 7 in 30% yield after RP-HPLC purification.
Following successful preparation of deuterium labelled Ar
and Gr, LC-MS quantifications of Ar(p) and Gr(p) were
carried out using these standards and our reported method.7
Briefly, the isotope labelled nucleosides were added to
unknown nucleoside samples from enzymatic digestion of
tRNA, LC-MS was measured, then the integrals of the
LC-MS peaks for the labelled and natural nucleosides were
compared to quantify the natural nucleosides. Initially,
calibration curves comparing the LC-MS integrals for the
labelled and natural nucleosides13 were determined (Fig. S1,
ESIw). These give excellent linear fits, confirming the
applicability of the deuterated standards to quantification of
nucleoside samples. Subsequently, LC-MS quantifications of
Ar(p) and Gr(p) in tRNA isolated from various tissues were
carried out (Table 1).
Scheme 2 Synthesis of d2-Ar 6.
synthesized in 6 steps17 and is also commercially available, for
the preparation of our target deuterated nucleosides 6 and 7
(Fig. 2).
In order to allow optional incorporation of the phosphate
group present in natural Ar(p), we initially undertook the
synthesis of d2-Ar starting with a 5-Pac (phenoxyacetyl)
protected ribose15 (Scheme 2). Here, d2-ribose 8 was protected
with a MMTr (monomethoxytrityl) group at the 5-hydroxyl
group, then Bz (benzoyl) groups at the 1, 2 and 3 hydroxyl
groups to give protected ribose 9. Subsequently, the MMTr
group was removed under acid conditions, at which point
the a and b anomers of the product 10 were separated.
The 5-hydroxy group of 10b was then protected with a
phenoxyacetyl group to give fully protected d2-ribose 11.
The key step, Vorbruggen glycosylation of 11 with the
30,50-protected A derivative 1218 was carried out in moderate
yield (60%), and finally all protecting groups were removed
with TBAF followed by NH3/MeOH to give d2-Ar 6 in 43%
yield after RP-HPLC purification.
To our surprise, we observed that the distribution of the
nucleotides Ar(p) and Gr(p) strongly varies between the
investigated species. Ar(p) was only detected in Saccharomyces
cerevisiae, while neither of the two nucleosides were found in
Table 1 Levels of Ar(p) and Gr(p) in various cell and tissues. n.d.,
not detected
Ar(p) (per 100
tRNA molecules)
Gr(p) (per 100
tRNA molecules)
Species/cell type
E. coli
n.d.
1.25 ꢀ 0.03
n.d.
n.d.
1.55 ꢀ 0.07
2.4 ꢀ 0.1
1.25 ꢀ 0.06
n.d.
S. cerevisiae
C. albicans
C. nebularis
F. fomentarius
S. scrofa
n.d.
n.d.
n.d.
n.d.
Initially, the same strategy was attempted for d2-Gr 7 using
protected ribose 11 as the Vorbruggen coupling partner, based
HeLa
n.d.
n.d.
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bacteria (Escherichia coli) or in mammalian cells (Sus scrofa
and HeLa). The nucleoside Gr(p) in contrast is widely
distributed. Most importantly we for the first time investigated
the modified nucleosides in complex fungi such as Clitocybe
nebularis and Fomes fomentarius. Here we detected only Gr(p),
indicating that this modification is more commonly used to
distinguish between initiator and elongator tRNA. In both
higher fungi the nucleoside Ar(p) was clearly not present since
not even traces could be detected. This result is in agreement
with a study in plants and other yeast species showing that
Gr(p) is of widespread use.8
Notes and references
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The detected levels of Ar(p) and Gr(p) are relatively low
(1–3 modifications per 100 tRNA molecules) compared to the
levels of other modified nucleosides.7 Analysis of a previous
quantification of yeast tRNA22 reveals that initiator tRNA
makes up approximately 2.5% of all tRNA present in cells.
Because we detected the modification at a level between 1 and
3%, our results show that even in complex fungi, the
modification is likely found only in the initiator tRNA and
that it is absent in all other tRNA molecules present in the cell.
The quantitative data therefore support the idea that
the modifications Ar(p) and particularly Gr(p) are key
components of initiator tRNA needed to correctly start the
translational process.
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In summary, we report the synthesis of isotope labelled Ar
and Gr and performed the first quantitative analysis directly in
higher fungi. The results support the idea that Gr(p) is more
widely distributed and that both modifications are only
present in initiator tRNA.
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The authors acknowledge Prof. Wolfgang Steglich
(LMU Munich) for providing samples of C. nebularis
and F. fomentarius, and the Alexander von Humboldt
Foundation for a postdoctoral fellowship to D.P. We thank
the Deutsche Forschungsgemeinschaft (grant numbers
CA275/8-4, SFB 749) and the Excellence Cluster CIPSM for
financial support.
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5198 Chem. Commun., 2011, 47, 5196–5198
This journal is The Royal Society of Chemistry 2011