Selective Enzymatic Demethylation of N2,N2-Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing

Article abstract of DOI:10.1002/anie.201700537

The abundant Watson–Crick face methylations in biological RNAs such as N¹-methyladenosine (m¹A), N¹-methylguanosine (m¹G), N³-methylcytosine (m³C), and N²,N²-dimethylguanosine (m²₂G) cause significant obstacles for high-throughput RNA sequencing by impairing cDNA synthesis. One strategy to overcome this obstacle is to remove the methyl group on these modified bases prior to cDNA synthesis using enzymes. The wild-type E. coli AlkB and its D135S mutant can remove most of m¹A, m¹G, m³C modifications in transfer RNA (tRNA), but they work poorly on m²₂G. Here we report the design and evaluation of a series of AlkB mutants against m²₂G-containing model RNA substrates that we synthesize using an improved synthetic method. We show that the AlkB D135S/L118V mutant efficiently and selectively converts m²₂G modification to N²-methylguanosine (m²G). We also show that this new enzyme improves the efficiency of tRNA sequencing.

Full text of DOI:10.1002/anie.201700537

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201700537
German Edition: DOI: 10.1002/ange.201700537
Sequencing Methods
Selective Enzymatic Demethylation of N2,N2-Dimethylguanosine in
RNA and Its Application in High-Throughput tRNA Sequencing
Qing Dai+, Guanqun Zheng+, Michael H. Schwartz+, Wesley C. Clark, and Tao Pan*
Abstract: The abundant Watson–Crick face methylations in
biological RNAs such as N1-methyladenosine (m1A), N1-
methylguanosine (m1G), N3-methylcytosine (m3C), and
cDNA synthesis. To alleviate this problem, it would be highly
desirable to identify a third demethylase with enhanced
activity toward m2 G in RNA and apply it for tRNA
2
N2,N2-dimethylguanosine (m2 G) cause significant obstacles
sequencing.
2
for high-throughput RNA sequencing by impairing cDNA
synthesis. One strategy to overcome this obstacle is to remove
the methyl group on these modified bases prior to cDNA
synthesis using enzymes. The wild-type E. coli AlkB and its
D135S mutant can remove most of m1A, m1G, m3C modifica-
Previously, Zhu et al. showed that mutating active-site
residues of an RNA/DNA demethylase switched its deme-
thylation activity from m1A to m6A modification.[4] We have
also shown that an AlkB D135S mutant demethylates m1G
much more efficiently than the wild-type enzyme.[3] There-
fore, we reasoned that further mutagenesis of residues around
the active site of AlkB might produce a demethylase with
tions in transfer RNA (tRNA), but they work poorly on m2 G.
2
Here we report the design and evaluation of a series of AlkB
mutants against m2 G-containing model RNA substrates that
higher activity for our target m2 G modification.
2
2
we synthesize using an improved synthetic method. We show
that the AlkB D135S/L118V mutant efficiently and selectively
Unlike m1A, m1G and m3C which only have one methyl
group in their structures, m2 G has two methyl groups at the
2
converts m2 G modification to N2-methylguanosine (m2G). We
exocyclic amine (Figure 1A). For sequencing purposes,
2
also show that this new enzyme improves the efficiency of
tRNA sequencing.
t
RNAs decode mRNA codons and are essential for cells. In
humans, tRNA abundance is tissue dependent[1] and tRNA
expression and mutations are known to be associated with
neurological pathologies and cancer development and pro-
liferation.[2] Efficient and quantitative tRNA sequencing
methods are crucial for biological studies of tRNA. However,
standard sequencing methods are very ineffective for tRNA-
seq, primarily due to the high levels of four Watson–Crick
face methylations, m1A, m1G, m3C and m2 G that impair
2
cDNA synthesis. Recently, we reported an efficient and
quantitative high-throughput tRNA sequencing method
(DM-tRNA-seq) by using a highly processive thermostable
group II intron reverse transcriptase, and importantly, two
demethylases that remove most base methylations and thus
significantly improved the tRNA sequencing efficiency: for
example, the amount of longer and full-length cDNA was
substantially increased after the treatment of tRNA with
demethylases.[3] Despite this success, our sequencing data
Figure 1. AlkB mutant design for m2 G demethylation. A) Chemical
2
structure of m2 G. B) Location of m2 G modification in tRNA tertiary
2
2
structure (PDB 4tna). The m2 G residue is in orange, and the two
2
methyl groups are in blue. C) View of AlkB active site stereochemistry
model with m2 G coordination based on protein data bank (PDB) ID
2
3BIE. m2 G is labeled in cyan.
2
showed that the m2 G at position 26 of tRNAs was not
2
effectively demethylated and caused a significant stop in
removal of one methyl group to N2-methylguanosine (m2G)
should be sufficient for highly efficient cDNA synthesis by the
thermophilic reverse transcriptase.[3] Indeed, we found that
[*] Dr. Q. Dai[+]
Department of Chemistry, The University of Chicago
Chicago, IL 60637 (USA)
Dr. G. Zheng,[+] Dr. M. H. Schwartz,[+] Dr. W. C. Clark, Prof. T. Pan
Department of Biochemistry & Molecular Biology
The University of Chicago
m2 G blocks the reverse transcriptase (RT) reaction of
2
thermostable group II intron reverse transcriptase while it
fully reads through m2G modification (Figure S1 in the
Supporting Information). m2 G is present in about half of
2
human tRNAs at position 26 at the junction between the D-
Chicago, IL 60637 (USA)
E-mail: taopan@uchicago.edu
arm and the anticodon arm,[5] as indicated in the tertiary
structures of tRNA (Figure 1B). m2 G26 can form non-
[+] These authors contributed equally to this work.
2
canonical base pairing with A44, which has a destabilizing
effect on RNA duplexes, as revealed by the crystal structure
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201700537.
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of an RNA duplex with m2 G:A pairs.[6] The m2 G26 residue is
buried in the structure, making it particularly difficult to be
Several methods have been reported for preparing m2G
2
2
and m2 G derivatives[10] but the most straightforward one used
2
demethylated. The co-crystal structure of E. coli AlkB with
a “one-step” reductive amination reaction to methylate the
exocyclic amine directly.[10b] We attempted to combine the
reductive amination with Beigelmanꢀs protecting strategy to
synthesize both phosphoramidites 7a and 7b at the same
time. We started our synthesis from guanosine 1 by selective
protecting 3’,5’-hydroxyls with di-tert-butylsilyl group and 2’-
hydroxy with TBDMS in a “one-pot” reaction to give
intermediate 2 in 75% yield.[9] Protection of O6 using p-
nitrophenylethyl group by the treatment of 2 with p-nitro-
phenylethanol in the presence of triphenylphosphine (Ph3P)
and diisopropyl azodicarboxylate (DIAD) gave 3 in 92%
yield.[11] The reductive amination reaction between the
exocyclic amine in 3 with paraformaldehyde and sodium
cyanoborohydride (NaBH3CN) in acetic acid at 408C resulted
in sequential methylation of exocyclic amine to give a mixture
of 4a and 4b. Depending on the amount of paraformaldehyde
and NaBH3CN used, either 4a or 4b could be obtained as
major products. All the protecting groups in intermediate 3
remained stable under the acidic conditions. Treatment of 4a/
4b with hydrogen fluoride pyridine removed the 3’,5’-di-tert-
butylsilyl group quantitatively while keeping 2’-TBDMS
intact to give intermediates 5a/5b. 5’-Tritylation gave the
mixture of 6a/6b. Although two types of methylated deriv-
atives could be readily separated by column chromatography
at 4a/4b or 5a/5b stage, it is more efficient to separate 6a and
6b by column chromatography. Subsequent 3’-phosphityla-
a nucleic acid substrate[7] was used to model the m2 G base
2
into the active site (Figure 1C). AlkB D135S mutant we used
previously for DM-tRNA-seq works efficiently toward m1G,
in which the shorter side chain of S135 not only creates more
room to accommodate guanosine but also forms the crucial
hydrogen bond to stabilize guanosine in the active site. We
reasoned that shortening the side chain of amino acids within
the van der Waals ratios of the methyl groups on m2 G would
2
allow better accommodation for m2 G, while leaving the rest
2
of the active site intact would potentially enhance the activity
towards m2 G. Therefore, three mutants, Arg210-to-Lys,
2
Leu118-to-Val, and Met61-to-Ser were prepared.
To test the activity of these potential m2 G demethylases,
2
we synthesized three 9mer RNA oligos as demethylase
substrates. The oligo sequence mimics the human tRNAPhe
sequence around the position of 26 (5’-GAGCXUUAG, X =
m2 G, m2G or G) (Scheme 1). It was generally believed that
2
tion 6a and 6b under standard conditions provided m2 G
2
phosphoramidite 7a and m2G phosphoramidite 7b in 88%
and 85% yield, respectively.
Phosphoramidite 7a or 7b was then incorporated into
RNA oligos with similar efficiency as the commercial
unmodified guanosine phosphoramidite to prepare the
oligos ON-m2 G and ON-m2G. As a control, we also
2
synthesized an unmodified oligo ON-G with the same
sequence. After deprotection and HPLC purification, the
structures of three RNA oligos were confirmed by Maldi-
TOF MS, which showed 14 Dalton difference in mass between
Scheme 1. Improved syntheses of m2G and m2 G phosphoramidites
ON-m2 G and ON-m2G and between ON-m2G and ON-G
2
2
and their incorporation into RNA to prepare demethylase substrates.
i) a) (t-Bu)2Si(OTf)2, DMF; b) imidazole, TBDMS-Cl, 908C, 75%.
ii) p-nitrophenylethanol, Ph3P, DIAD, 1,4-dioxane 1008C, 92%.
iii) (HCHO)x, NaBH3CN, AcOH, 408C. iv) HF·Py/THF. v) DMTr-Cl/Py.
vi) (i-Pr)2NP(Cl)OCH2CH2CN, CH2Cl2, (i-Pr)2NEt, 1-methyl-imidazole.
vii) RNA synthesis and deprotection.
(Figure 2B).
We tested the demethylation activities of the AlkB mutant
proteins using these RNA oligos as substrates. Oligo ON-
m2 G (1 nmol) was incubated with each enzyme in the
2
reaction buffer containing 300 mm KCl, 2 mm MgCl2, 50 mm
of (NH4)2Fe(SO4)2·6H2O, 300 mm a-ketoglutarate (a-KG),
2 mm l-ascorbic acid, 50 mgmLÀ1 BSA and 50 mm MES buffer
(pH 5.0) for 2 h at room temperature, and then quenched by
the addition of 5 mm EDTA. RNA oligos in the reaction were
recovered by ethanol precipitation and dissolved in water.
Part of samples were digested to free nucleosides by treat-
ment of nuclease P1 and alkaline phosphatase and then
analyzed by HPLC (Figure 2A), while the rest of samples
were directly analyzed by Maldi-TOF MS (Figure 2B). As
shown in Figure 2A, the D135S/L118V mutant gave the best
the protection of O6 of guanosine with p-nitrophenylethyl
(NPE) group would not only lower the polarity and increase
the solubility of guanosine derivatives to facilitate their
purification, but also enhance the nucleophilicity of the
exocyclic amine.[8] Subsequent conversion of 2-amine to 2-
methylamine and 2-dimethylamine were accomplished by
converting 2-NH2 to 2-F, followed by substitution of 2-F with
methylamine and dimethylamine. The use of fluoride reagent
to introduce 2-F precluded the application of Beigelmanꢀs
optimal silyl protecting strategy for phosphoramidite syn-
thesis[9] and therefore several extra steps were needed for
protecting group exchanges.
results among the five candidates. The m2 G peak disap-
2
peared completely with the appearance of m2G accordingly;
suggesting that one methyl group from m2 G was fully
2
removed. Other enzymes such as the wild-type AlkB,
2
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We then tested whether AlkB D135S/L118V mutant
improves m2 G demethylation in tRNA. Like our previous
2
DM-tRNA-seq method,[3] we used the combination of three
demethylases, wild-type, D135S and D135S/L118V to treat
tRNA samples from HEK293T cells and constructed
sequencing libraries to evaluate the effect of AlkB D135S/
L118V mutant on high-throughput tRNA sequencing. As
controls, we also sequenced the same samples without
demethylase treatment or treated with just two demethylases,
wild-type and D135S. To evaluate the effect of the D135S/
L118V demethylase on m2 G, we applied a quantitative
2
measure, modification index (MI) which is the sum of the
fraction of sequencing reads derived from RT mutations
(from G to A/C/T in this case) and stop at the m2 G26 in each
2
tRNA[5b] (Figure 3A). Removal of the m2 G methyl group
2
Figure 2. Evaluations of a series of AlkB mutants for efficient m2 G 5’-
2
GAGCXUUAG, X=m2 G (ON-m2 G), m2G (ON-m2G) or G (ON-G).
2
2
A) HPLC traces of the digestion products after ON-m2 G was treated
2
with demethylases showed that AlkB D135S/L118V mutant quantita-
tively converts m2 G to m2G. B) Comparison of MALDI-TOF MS of
2
unmodified RNA oligo ON-G, m2G-containing RNA oligo ON-m2G,
m2 G-containing RNA oligo ON-m2 G, and ON-m2 G which was treated
2
2
2
with AlkB D135S/L118V mutant confirmed that the demethylation
product of ON-m2 G is ON-m2G instead of ON-G.
2
Figure 3. Application of AlkB D135S/L118V to high-throughput tRNA-
seq. A) tRNA-seq heatmap result showing the modification index (MI)
change at position 26 in tRNAs with known m2 G sites. 2DM=wild-
2
D135S or D135S/R210K mutant showed limited activities
while D135S/M61S mutant showed no activity at all toward
type AlkB + D135S; 3DM=wild-type AlkB + D135S + D135S/L118V.
The higher the MI value, the higher the fraction of modifications.
Therefore, decreased modification fraction corresponds to lower MI
value or more intense green color for the corresponding tRNA
isoacceptor. B) Full MI plot for the full-length mitochondrial tRNAIle.
m2 G. The Maldi-TOF MS of the sample treated with D135S/
2
L118V mutant also showed complete disappearance of the
peak at m/z 2922 and appearance of a new peak at 2908 which
is identical to that of oligo ON-m2G, indicating that one of two
methyl groups in the m2 G oligo ON-m2 G was removed
MI at the m2 G nucleotide is 0.972, 0.343, 0.178 for untreated (black),
2
+2DM (red), and +3DM (blue), respectively.
2
2
completely. No peak identical to that of ON-G was observed
(m/z = 2894), again indicating that only one methyl was
removed. Indeed, when we treated ON-m2G with AlkB
D135S/L118V mutant, both HPLC and Maldi-TOF showed
no reaction, further confirming that AlkB mutants can only
would result in increased cDNA synthesis through this
nucleotide and incorporation of the correct nucleotide.
Therefore, reduced level of m2 G26 would decrease the MI
2
values at this position. Indeed, treatment using the three-
remove one methyl group from m2 G. The shorter side chain
demethylase mixture (+ 3DM) reduced MI values for the
2
of Val118 compared with original Leu118, which sits directly
majority of all known tRNAs with known m2 G26 modifica-
2
opposite to methyl groups on m2 G, results in more space for
tions at greater extent compared to using the two-demethy-
2
m2 G to fit in the active site to be demethylated.
lase mixture (+ 2DM). Because m2 G26 is located in a very
2
2
We also used a 9mer oligo containing an m1G modifica-
tion[3] to test whether the AlkB D135S/L118V mutant can also
demethylate m1G efficiently. Unlike AlkB D135S mutant
which demethylated m1G quantitatively, we observed that
AlkB D135S/L118V mutant had only limited activity toward
m1G, suggesting that AlkB D135S and D135S/L118V mutants
can have distinct substrate selectivity (Figure S2). To obtain
the optimal result in tRNA sequencing, it is therefore best to
use a three-demethylase mixture: wild-type AlkB (to remove
m1A and m3C), D135S (to remove m1G) and D135S/L118V
structured region of tRNA (Figure 1B), the absolute deme-
thylation fraction is still substantially lower than the m1A,
m1G and m3C modifications shown in the previous study.[3]
Among the cytosolic tRNAs, tRNAThrs, tRNATrp and tRNATyr
show a greater extent of MI reduction than other tRNAs
(Figure 3A); this result may be attributed to the variable
stabilities of cytosolic tRNAs that carry many modifications
of distinct types at distinct sites. Consistent with the idea that
the low absolute m2 G demethylation fraction is related to
2
tRNA structure, the mitochondrial tRNAIle has the highest
(to remove m2 G).
level of MI value reduction (Figure 3B), because mitochon-
2
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drial tRNAs are markedly less structured than the cytosolic
tRNAs.[5b]
[1] K. A. Dittmar, J. M. Goodenbour, T. Pan, PLoS Genet. 2006, 2,
e221.
[2] a) S. Kirchner, Z. Ignatova, Nat. Rev. Genet. 2015, 16, 98 – 112;
b) J. A. Abbott, C. S. Francklyn, S. M. Robey-Bond, Front.
Genet. 2014, 5, 158.
In summary, we have identified an AlkB mutant (D135S/
L118V) that can efficiently remove one methyl group from
the m2 G to convert it to m2G. Unlike m2 G, m2G does not
2
2
block RT reaction under our sequencing conditions, resulting
in improved tRNA sequencing efficiency. Different AlkB
mutants showed demethylation selectivity on different base
methylations, suggesting a role of the active-site residues in
substrate recognition and the possibility to find other AlkB
mutants to selectively demethylate other base modifications
such as dimethyl adenosine. Besides the application of these
demethylases in tRNA sequencing, these selective demethy-
lases may also serve as useful tools for developing single-base
resolution sequencing methods for base methylations in other
RNA species such as mRNA.[12]
[3] G. Zheng, Y. Qin, W. C. Clark, Q. Dai, C. Yi, C. He, A. M.
Lambowitz, T. Pan, Nat. Methods 2015, 12, 835 – 837.
[4] C. Zhu, C. Yi, Angew. Chem. Int. Ed. 2014, 53, 3659 – 3662;
Angew. Chem. 2014, 126, 3733 – 3736.
[5] a) J. Edqvist, K. B. Straby, H. Grosjean, Biochimie 1995, 77, 54 –
61; b) W. C. Clark, M. E. Evans, D. Dominissini, G. Zheng, T.
Pan, RNA 2016, 22, 1771 – 1784.
[6] P. S. Pallan, C. Kreutz, S. Bosio, R. Micura, M. Egli, RNA 2008,
14, 2125 – 2135.
[7] C. G. Yang, C. Yi, E. M. Duguid, C. T. Sullivan, X. Jian, P. A.
Rice, C. He, Nature 2008, 452, 961 – 965.
[8] P. Rife, C. S. Cheng, P. B. Moore, S. A. Strobel, Nucleosides
Nucleotides 1998, 17, 2281 – 2288.
[9] V. Serebryany, L. Beigelman, Tetrahedron Lett. 2002, 43, 1983 –
1985.
Acknowledgements
[10] a) K. Piecyk, R. E. Davis, M. Jankowska-Anyszka, Bioorg. Med.
Chem. Lett. 2012, 22, 4391 – 4395; b) M. Sekine, M. Aoyagi, M.
Ushioda, A. Ohkubo, K. Seio, J. Org. Chem. 2005, 70, 8400 –
8408; c) N. D. Abeydeera, C. S. Chow, Bioorg. Med. Chem. 2009,
17, 5887 – 5893; d) M. Sekine, T. Satoh, J. Org. Chem. 1991, 56,
1224 – 1227.
[11] K. Kamaike, K. Kinoshita, K. Niwa, K. Hirose, K. Suzuki, Y.
Ishido, Nucleosides Nucleotides Nucleic Acids 2001, 20, 59 – 75.
[12] a) D. Dominissini, S. Nachtergaele, S. Moshitch-Moshkovitz, E.
Peer, N. Kol, M. S. Ben-Haim, Q. Dai, A. Di Segni, M. Salmon-
Divon, W. C. Clark, G. Zheng, T. Pan, O. Solomon, E. Eyal, V.
Hershkovitz, D. Han, L. C. Dore, N. Amariglio, G. Rechavi, C.
He, Nature 2016, 530, 441 – 446; b) X. Li, X. Xiong, K. Wang, L.
Wang, X. Shu, S. Ma, C. Yi, Nat. Chem. Biol. 2016, 12, 311 – 316.
This work was supported by National Institute of Health
grants in USA (K01 HG006699 to Q.D., and RM1HG008935
to T.P.). We thank Dr. Matthew Eckwahl-Sanna for helping
edit the figures.
Conflict of interest
The authors declare no conflict of interest.
Keywords: AlkB mutant · demethylase · methylguanosine ·
reverse transcriptase reaction · tRNA sequencing
Manuscript received: January 16, 2017
Revised: February 10, 2017
Final Article published: && &&, &&&&
4
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Communications
Sequencing Methods
An AlkB mutant has been identified that
efficiently removes one methyl group
Q. Dai, G. Zheng, M. H. Schwartz,
from N2,N2-dimethylguanosine (m2 G) to
2
W. C. Clark, T. Pan*
&&&& — &&&&
convert into N2-methylguanosine (m2G).
Unlike m2 G, m2G does not block reverse
2
Selective Enzymatic Demethylation of
N2,N2-Dimethylguanosine in RNA and Its
Application in High-Throughput tRNA
Sequencing
transcription reaction under sequencing
conditions, resulting in improved tRNA
sequencing efficiency.
Angew. Chem. Int. Ed. 2017, 56, 1 – 5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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