The AlkB domain of mammalian ABH8 catalyzes hydroxylation of 5-methoxycarbonylmethyluridine at the wobble position of tRNA

Article abstract of DOI:10.1002/anie.201001242

Family ties: The AlkB family of nonheme iron, α-ketoglutarate (αKG)-dependent dioxygenases is involved in biological processes such as DNA/RNA repair and obesity. The AlkB domain of ABH8 is shown to catalyze the hydroxylation of a modified uridine (mcm

Full text of DOI:10.1002/anie.201001242

Angewandte
Chemie
DOI: 10.1002/anie.201001242
RNA Modification
The AlkB Domain of Mammalian ABH8 Catalyzes Hydroxylation of
5-Methoxycarbonylmethyluridine at the Wobble Position of tRNA**
Ye Fu, Qing Dai, Wen Zhang, Jin Ren, Tao Pan,* and Chuan He*
The ABH8 protein (also called ALKBH8) is a member of the
AlkB (alkylated DNA repair protein) family of nonheme
iron/ a-ketoglutarate (aKG)-dependent dioxygenases.[1]
Other members of this protein family are known to catalyze
oxidative demethylation to repair damaged DNA/RNA.[2]
AlkB-like proteins exist in viruses, bacteria, and eukaryotic
species.[3] Nine mammalian homologues of AlkB (ABH1-
ABH8, and FTO, a protein associated with fat mass and
obesity) have been identified through bioinformatics stud-
ies.[1b,4] Four of the homologues (ABH1, ABH2, ABH3, and
FTO) show demethylation activity for N-methylated bases, in
either DNA or RNA, through the hydroxylation of the N-
methyl group and subsequent release of an aldehyde from the
oxidized intermediate.[5] ABH2, which repairs m1A, m3C, and
eA (1,N6-ethenoadenine) in dsDNA, guards mammalian
genomic DNA against methylation damage.[6] ABH3 appears
to be involved in RNA repair.[7] FTO was first identified in a
fused-toes malformation phenotype resulting from a mouse
genome deletion,[8] and was later shown to significantly affect
energy homeostasis and lead to obesity.[9] FTO has the highest
activity in demethylating m3T in ssDNA and m3U in
RNA;[4,10] however, the link between its biochemical activity
and physiological phenotype is yet to be discovered. Aside
from ABH1, ABH2, ABH3, and FTO, the biochemical
activities of the other AlkB homologues are still unknown.
Very recently Tet1, another iron(II)/aKG-dependent dioxy-
genase, has been shown to hydroxylate the 5-methyl group of
m5C in dsDNA to form 5-hydroxymethylcytosine (hm5C) in
certain neural and stem cells. This discovery has fueled
speculation that this modification is important in epigenet-
ics.[11]
ABH8 has been identified in high eukaryotes. This protein
contains an N-terminal RNA recognition motif (RRM) and a
predicted C-terminal methyltransferase domain fused to the
central AlkB homologous domain (Figure 1a). This config-
uration is conserved from insects (e.g., B. mori), to worms
(e.g., C. elegans), to humans. Together with the observation
that ABH8 is exclusively located in the cytoplasm, the
potential role of ABH8 in the regulation of RNA function
through controlled methylation/demethylation has been sug-
gested.[1a] However, the demethylation of methylated bases
by ABH8 has never been reported. We have cloned,
expressed, and purified several constructs of truncated
ABH8, having both RRM and AlkB domains, from both
humans (hABH8) and mice (mABH8), and tested potential
demethylation activity against m1A, m3C, m1G, m3T, m6A, and
eA. No activity was observed under standard conditions with
iron and aKG cofactors (see the Experimental Section; data
not shown).
Subsequently, we noticed through sequence alignment
that the methyltransferase domain of ABH8 is homologous to
the yeast Trm9 protein (Figure 1a and Figure 1 in the
Supporting Information), which is a methyltransferase for
several yeast tRNAs. Trm9 catalyzes the methylation of 5-
carboxymethyluridine (cm5U) to form 5-methoxycarbonyl-
methyluridine (mcm5U), and methylation of of 5-carboxyl-
methyl-2-thiouridine (cm5s2U) to give 5-methoxycarbonyl-
methyl-2-thiouridine (mcm5s2U) at the anticodon wobble
position.[12] This tRNA modification modulates the decoding
selectivity of the tRNA for a subset of codons, thereby
affecting translation in a codon-specific manner;[13] this data
prompted us to propose that the Trm9-like domain in ABH8
may carry out a similar methylation in tRNA, and that the
AlkB domain in ABH8 may add another modification at the
same anticodon wobble nucleotide. Therefore, the AlkB
domain in ABH8 might modify either cm5U or mcm5U in
tRNAs. After searching the known tRNA modifications, we
noticed that 5-(S)-carboxyhydroxymethyluridine ((S)-
chm5U) and 5-(S)-methoxycarbonylhydroxymethyluridine
((S)-mchm5U), which are the a-hydroxylation products of
cm5U and mcm5U, respectively, had already been identified at
the anticodon wobble position of tRNAGly(U*CC) (U* is the
modified uridine) in the silkworm, Bombyx mori (Fig-
ure 1b,c).[14] The ABH8 gene is conserved among silkworms
and mammals (see Figure 2 in the Supporting Information),
and the sequence of tRNAGly(U*CC) is also conserved (see
Figure 3 in Supporting Information). Therefore, we set out to
test the idea that the AlkB domain of ABH8 in high
[*] Y. Fu, W. Zhang, Dr. J. Ren, Prof. C. He
Department of Chemistry, The University of Chicago
929 East 57th Street, Chicago, IL 60637 (USA)
Fax: (+1)773-702-0805
E-mail: chuanhe@uchicago.edu
Homepage: http://he-group.uchicago.edu
Dr. Q. Dai, Prof. T. Pan
Department of Biochemistry and Molecular Biology
The University of Chicago
929 East 57th Street, Chicago, IL 60637 (USA)
E-mail: taopan@uchicago.edu
Prof. T. Pan, Prof. C. He
Institute for Biophysical Dynamics, The University of Chicago (USA)
[**] This work was supported by the U.S. National Institute of Health
(GM071440 to C.H.) and by an NIH EUREKA award (GM088599 to
C.H. and T.P.). Q.D. was supported by the Chicago Biomedical
Consortium (CBC). W.Z. was partially supported by the American
Recovery and Reinvestment Act NIGMS Administrative Supple-
mentary grant (3R01M071440-05S1). We thank Dr. Leslie M. Hicks
for performing mass spectrometry analysis and Dr. Stephen B. H.
Kent for the use of the LC mass spectrometer.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001242.
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The two 17-mer RNAs
were treated with purified
mABH81À340 in the presence
of iron and aKG. The RNAs
before and after the reaction
with mABH8 were digested
with nuclease P1 and alkaline
phosphatase, and the resulting
single nucleosides were ana-
lyzed by HPLC methods.
Whereas mABH81À340 did not
show activity against cm5U in
the 17-mer RNA, it catalyzed
the hydroxylation of mcm5U in
the 17-mer RNA (Figure 2a).
The peak representing the
product having the added hy-
droxy group was observed in
the high-resolution mass spec-
trum
(HRMS
calcd.,
Figure 1. Distinct domains and proposed function of ABH8. a) ABH8 contains three domains: the N-
333.09286; found, 333.09311;
Figure 2b). MS/MS analysis of
the enzymatic product shows
the exact fragmentation pat-
tern as seen for the synthesized
mchm5U standard, thereby
confirming the location of the
hydroxy group (see Figures 7
and 8 in the Supporting Infor-
terminal RNA recognition motif (RRM; blue), the middle iron/aKG-dependent AlkB domain (red), and the
C-terminal methyltransferase domain (green). The C-terminal domain is homologous to a tRNA
modification methyltransferase Trm9 in yeast. b) The anticodon wobble-base U*34 in higher eukaryotes
tRNAGly(U*CC) can be hypermodified. c) Examples of hypermodification on U*34: 5-carboxymethyluridine
(cm5U), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonylhydroxymethyluridine
(mchm5U). d) The proposed sequential modification of the wobble U*34 in a 17-mer RNA that contains
the anticodon stem–loop of tRNAGly(U*CC) by the two catalytic domains of ABH8.
eukaryotes catalyzes the hydroxylation of cm5U or mcm5U
into chm5U or mchm5U, respectively, in tRNAGly(U*CC)
(Figure 1d).
mation). The chemically synthesized diastereomers of the
nucleoside mchm5U can be separated, and the absolute
A truncated form of mABH8 (1-340) containing the RRM
and AlkB domain with an N-terminal His6 tag was expressed
and purified from E. coli (see Figure 4 in the Supporting
Information). This construct (mABH81À340) gives the most
stable ABH8 protein, therefore it was then employed in our
activity studies. The conserved iron-binding ligands in ABH8
are His237, Asp239, and His292 as determined from sequence
alignment with the AlkB family proteins (see Figure 2 in the
Supporting Information). Two mutant forms of mABH81À340
,
in which either His237 or Asp239 was mutated to Ala, were
also constructed. The mutant proteins were expressed and
purified in the same way as the wild-type mABH81À340. To
evaluate the potential modification of cm5U or mcm5U at the
wobble base of tRNAGly(U*CC), we chemically synthesized
the phosphoramidite of mcm5U (see Figure 5 in the Support-
ing Information). By using solid-phase synthesis, the phos-
phoramidite was incorporated into a 17-mer stem–loop RNA,
corresponding to the sequence of the anticodon stem–loop of
tRNAGly(U*CC) conserved in high eukaryotes, with mcm5U
at the U* position. After using different deprotection
strategies, the 17-mer RNA oligonucleotides containing
either the mcm5U or cm5U modification were obtained
(Scheme 1 and Figure 5 in the Supporting Information).
These 17-mer RNA oligonucleotides were purified by
HPLC methods and characterized as having the correct
mass by using MALDI-MS methods (see Figure 6 in the
Supporting Information).
Scheme 1. Synthesis of mcm5U phosphoramidite (5), and incorpora-
tion of mcm5U and cm5U into a tRNAGly(U*CC) anticodon stem–loop
oligomer sequence (RNA-I and RNA-II). Reaction conditions: a) Ac2O,
Py, RT, overnight; b) BzCl, DMAP, NEt3, 0.5 h; c) CH2(COMe)2, DBU,
THF, RT, overnight; d) NaOMe, MeOH, 508C, 1 h; e) DMTr, Py, RT,
overnight; f) TBDMSCl, Im, Py, RT, 10 h; g) (iPr2N)P(Cl)OCH2CH2CN,
iPr2NEt, THF, RT, 4 h; h) standard solid-phase RNA synthesis and
subsequent deprotection under ultramild conditions (see the Support-
ing Information). Bz=benzoyl, DBU=1,8-diazabicyclo[5.4.0]undec-7-
ene, DMAP=4-dimethylaminopyridine, Im=imidazole, Py=pyridine,
TBDMS=tert-butyldimethylsilyl, THF=tetrahydrofuran.
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activity and modifies mammalian tRNA. Thus, members of
this family of proteins could also perform modification
functions on nucleic acids. ABH8 has been shown to affect
cancer progression.[16] This protein is highly expressed in
bladder cancer, and over-expression of ABH8 was proposed
to effect an increased reactive oxygen species (ROS) pro-
duction inside these cancer cells. In yeast, the tRNA-
modification function of Trm9 changes the codon preference
and alters the translational efficiency of mRNAs with
increased abundance of cognate codons read by the modified
tRNAs.[13b] In B. mori, it has been shown that the additional
hydroxylation modification of mcm5U into mchm5U has a
significant impact upon the affinity of tRNAGly(U*CC) to
GGA and GGG codons.[17] This oxidation modification by
ABH8 may therefore affect the translation of certain
mRNAs.
In summary, we have presented herein the identification
of a unique biochemical activity of ABH8 distinct from those
of other proteins from the AlkB family. The discovery of the
tRNA modification chemistry catalyzed by the AlkB domain
of ABH8 may help elucidate the exact cellular function of
ABH8 and its potential roles in cancer. Furthermore, studies
on the structure and function of ABH8 are ongoing in our
laboratory.[18]
Experimental Section
Figure 2. Hydroxylation of mcm5U in a 17-mer tRNAGly(U*CC) antico-
don stem–loop. a) The chemically synthesized 17-mer RNA oligonu-
cleotide resembling the tRNAGly(U*CC) anticodon stem–loop was
treated with mABH81À340 in the presence of iron(II) and a-KG. The
HPLC traces show the appearance of a peak for the product (S)-
mchm5U when mcm5U is converted in the presence of the enzyme.
b) HRMS data of the product (S)-mchm5U confirms the addition of a
hydroxy group to mcm5U. MES=2-(N-morpholino)ethanesulfonic acid.
Assays for the hydroxylation reaction of RNA oligonucleosides: In a
typical reaction, the RNA substrate (1 nmol) was added to 50 mL of
50 mm 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.5).
The solution containing RNA oligonucleotides was heated at 558C
for 3 min, and then slowly cooled to room temperature, after it had
been was cooled in an ice bath. The following components were then
added to reach the target concentration: KCl (100 mm), MgCl2
(2 mm), RNasin (0.2 UmLÀ1, Promega), l-ascorbic acid (2 mm), a-
KG (300 mm), (NH4)2Fe(SO4)2·6H2O (150 mm), and mABH8
(1 nmol). The reaction mixture was incubated for 12 h at 168C. The
reaction was quenched by the addition of ethylene diamine tetra-
acetic acid (EDTA) to a final concentration of 1 mm.
configuration at the asymmetric C51 atom was determined by
comparing the CD spectra of the diastereomers (see Figure 9
in the Supporting Information) with the previously reported
CD spectra of the same compounds.[15] The S diastereomer,
which was discovered in B. mori., shows the same HPLC
retention time when compared to the product obtained from
the ABH8-catalyzed reaction, whereas the R diastereomer
does not (see Figure 10 in the Supporting Information),
suggesting that the enzymatic hydroxylation of mcm5U by
ABH8 is also stereoselective. Three different control experi-
ments—1) in the absence of either iron or aKG cofactors,
2) with the active site mutation or either mABH81À340 H237A
or mABH81À340 D239A, and 3) with other AlkB family
proteins such as ABH2, ABH3 or FTO, all of which were
run under the same conditions—failed to produce (S)-
mchm5U (data not shown). Thus, this reaction is specific to
ABH8 and requires iron(II) and aKG cofactors.
RNA digestion and HPLC assay: After the hydroxylation
reaction, 10% of 3m NaOAc pH 5.3 was added to the reaction
mixture, after which isopropanol (2.5 times the volume of the crude
mixture) was added to precipitate the 17-mer RNA. The pallet was
washed with 75% ethanol once and then dried by air. The resulting
pallet was dissolved in 100 mL Milli-Q H2O and then digested into
nucleosides using nuclease P1 and alkaline phosphatase. The
digestion solution was analyzed by using a high-performance liquid
chromatography (HPLC) system having
a C18 column (150 ꢀ
4.6 mm). The eluent comprised buffer A1 (water containing 5 mm
ammonium acetate) and buffer B1 (5 mm ammonium acetate, 60% of
acetonitrile, 40% water and 0.01% trifluoroacetic acid (TFA)); A1/
B1 ratios ranged from 100:0 to 90:10 (v/v) with a flow rate of
1 mLminÀ1 at room temperature. The detection wavelengths were set
at 260 nm and 280 nm. The fraction containing the digested product
was collected and lyophilized for HRMS and MS–MS analysis. For
LC–MS analysis, the digestion solution was run on the same C18
column using buffer A2 (water containing 0.1% TFA) and buffer B2
(acetonitrile containing 0.08% TFA).
The AlkB family of proteins are thought to be engaged in
oxidative de-modification (e.g., demethylation) in biological
systems. We show herein that ABH8 exhibits hydroxylase
Received: March 1, 2010
Revised: May 24, 2010
Published online: June 25, 2010
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www.angewandte.org
8887

Communications
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Keywords: enzyme models · hydroxylation · oxidoreductases ·
RNA structures · tRNA
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