Identification of a selective polymerase enables detection of N 6-methyladenosine in RNA

Article abstract of DOI:10.1021/ja4105792

N⁶-methyladenosine (m⁶A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extr

Full text of DOI:10.1021/ja4105792

Communication
pubs.acs.org/JACS
Identification of a Selective Polymerase Enables Detection of
N6‑Methyladenosine in RNA
Emily M. Harcourt,† Thomas Ehrenschwender,† Pedro J. Batista,‡ Howard Y. Chang,‡ and Eric T. Kool*,†
†Department of Chemistry, Stanford University, Stanford, California 94305, United States
‡Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California
94305, United States
S
* Supporting Information
methylation extent of 20−90% at one site. Modern RNA
ABSTRACT: N6-methyladenosine (m6A) is the most
sequencing techniques have advanced our ability to identify
transcripts modified by m6A. Using massively parallel
abundant mRNA modification and has important links to
sequencing and m6A-selective antibodies, two groups recently
human health. While recent studies have successfully
identified thousands of mammalian RNA transcripts
containing the modification, it is extremely difficult to
identify the exact location of any specific m6A. Here we
have identified a polymerase with reverse transcriptase
activity (from Thermus thermophilus) that is selective by up
to 18-fold for incorporation of thymidine opposite
unmodified A over m6A. We show that the enzyme can
be used to locate and quantify m6A in synthetic RNAs by
analysis of pausing bands, and have used the enzyme in
tandem with a nonselective polymerase to locate the
presence and position of m6A in high-abundance cellular
RNAs. By this approach we demonstrate that the long-
undetermined position of m6A in mammalian 28S rRNA is
nucleotide 4190.
identified thousands of modified mRNAs and ncRNAs from
mice and humans and confirmed the consensus sequence of
RRACU for the general location of methylated adenines.24,25
After completion of the present work, Liu et al. reported a new
method for detecting and quantifying m6A at a specific site
using multiple enzymatic steps.26 However, the ability to
interrogate the methylation status of any specific adenine at
nucleotide resolution, without painstaking digestion analysis,
has remained elusive.
The ability to locate m6A modifications in RNAs at
nucleotide resolution will no doubt aid in understanding their
function. Polymerase enzymes offer a possible mechanism for
locating modifications due to their sterically sensitive active
sites. Notably, polymerase selectivity has previously been
harnessed to detect m6A in DNA via single-molecule
sequencing.27 An early attempt at a related single-molecule
sequencing technique for RNA has also been described,28 but
employed an enzyme with low selectivity (HIV-RT; see below),
and will need further development before it is practical.
Another class of DNA-processing enzymes, ligases, can also be
sensitive to structure, and Dai et al. describe a technique in
which ligation of complementary DNAs occurs more favorably
in the presence of A than m6A.29 However, the conditions were
tuned carefully for the specific reaction, and no attempt was
made to detect m6A in an actual sample of cellular RNA.
We postulated that there might exist a polymerase enzyme
with substantial selectivity against m6A, and that such an
enzyme might be harnessed for site-specific detection of the
modification. We carried out a screen of enzymes with reverse
transcriptase activity, monitoring their ability to extend a
radiolabeled DNA primer by incorporating thymidine triphos-
phate (dTTP) opposite either A or m6A in an RNA template
(Figure 1). The data showed that only recombinant Thermus
thermophilus DNA polymerase I (Tth DNA pol) showed strong
selectivity (61% vs 15% primer extension) among the enzymes
tested. This DNA polymerase is known to act as a reverse
transcriptase in the presence of Mn2+.30
n the quest to understand cellular function at the molecular
level, the study of post-transcriptional modification of RNA
I
is of vital interest. In particular, N6-methyladenosine (m6A, 1) is
a relatively abundant modification in the mRNA of higher
eukaryotes and some viruses.1 Although its discovery in mRNA
occurred decades ago,2 there has been renewed interest in m6A
due to the finding that it acts as a substrate for fat mass and
obesity-associated protein (FTO),3 an oxidative demethylase
which has been linked to obesity and the regulation of
homeostasis;4−6 and for AlkBH5, an oxidative demethylase in
the same family.7
Identifying the function of m6A modifications has proved
challenging.8−12 Early work using enzymatic digestion and
radiolabeling led to the discovery of the consensus sequence
RAC (R = A or G) for m6A13−18 and the identification of
specific modified sites in Rous sarcoma virus RNA19−21 and in
bovine prolactin mRNA.22,23 This research also showed that
methylation at any particular site can be incomplete, with a
Since Tth DNA pol showed selectivity in the context of one
specific template sequence under one set of conditions, we
Received: October 16, 2013
Published: December 11, 2013
© 2013 American Chemical Society
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Communication
modified. Additionally, 5′ G and 3′ C both appear to decrease
enzyme efficiency to a small degree. Most notably, the
selectivity of the Tth polymerase in the consensus methylation
site context (GAC) is 4−6.4-fold, thus supporting the notion
that the enzyme’s selectivity may be useful for identifying the
most common occurrences of m6A in naturally occurring
RNAs.
Next we asked whether the Tth enzyme could be used in a
quantitative sense to evaluate the degree of methylation at a
specific site. To test this, we mixed known ratios of m6A-
containing RNAs with their A-containing counterparts and
measured the yield of dTTP incorporation at a fixed time point.
The percent extension of the primer in this RNA context was
linearly proportional to the amount of m6A present (Figure 2),
suggesting that the polymerase can be used in quantitative
evaluations of the extent of methylation at one position.
Figure 1. Screen of polymerase selectivity for incorporation of dTTP
opposite A or m6A in an RNA template. (A) Sequences of RNA
template/DNA primer used in screen. (B) Autoradiogram showing
primer extension (p+1 band) in the presence of A or m6A. Products
were resolved on a 20% polyacrylamide denaturing gel using a32P-5′-
labeled primer.
tested whether variations in temperature, time, and buffer
composition might enhance selectivity (see Supporting
Information (SI)). In particular, Mn2+ is known to decrease
enzyme selectivity;31 however, we found that Mn2+ was
required for reverse transcriptase activity in Tth DNA pol.
We next investigated whether this selectivity would extend to
other sequence contexts. A 24mer template sequence was
chosen from the 3′-untranslated region (3′-UTR) of the eef 2
gene, which was found to be highly expressed and highly
modified in mouse tissue and mouse embryonic stem cells.25,32
The template was synthesized containing either A or m6A, and
the bases on either side of the A/m6A were varied
systematically to allow a comparison of sequence context
effects (see SI for details). In addition, several of the native
sequence contexts in which m6A has been reported to occur
were also synthesized. Single nucleotide incorporation kinetics
were determined for each sequence containing A and m6A
using steady-state methods33 (Table 1).
Figure 2. T insertion is correlated to the relative amount of m6A at
target position. 5'-AGXCUGCCACAUGCUGCACAGUGC-3' was
used as the template RNA at 1 μM concentration with varied ratios
of m6A:A at the target position. Error bars show standard deviations
from five trials.
Table 1. Steady-State Incorporation Efficiency for Insertion
of dTTP Opposite A or m6A in Synthetic RNAs by Tth DNA
pol in Varied Sequence Contexts
We proceeded to carry out experiments to test whether this
enzyme could be employed in probing methylation in RNAs
extracted from mammalian cells. Since the amount of RNA and
its secondary structure are likely to affect primer extension
efficiencies, we introduced two control strategies. The first of
these involves the use of two simultaneous primers (one
adjacent to the probed methylation site and one nearby but
adjacent to a nucleotide with known methylation status).
Comparison of these two should account for the amount of a
given RNA present in a sample. The second control makes use
of the same primers with a nonselective enzyme, avian
myeloblastosis virus reverse transcriptase (AMV RT). AMV
RT was selected because it showed consistent ratios relative to
Tth DNA pol across a range of RNA concentrations. Insertion
of T at a site of interest could then be compared both to the
control site and to incorporation by a control nonselective
enzyme.
sequence
context
efficiency, X = A
(Vmax/Km)
efficiency, X = m6A
(Vmax/Km)
A/m6A
ratio
UXC
0.86 0.17
0.58 0.15
1.22 0.17
0.95 0.61
1.13 0.77
1.39 0.26
1.51 0.61
1.87 1.25
1.24 0.32
3.52 0.94
0.10 0.04
0.14 0.03
0.19 0.04
0.10 0.10
0.14 0.14
0.13 0.03
0.10 0.02
0.11 0.06
0.10 0.05
0.33 0.22
8.6
4.0
GXC
(G)GXC
GXU
6.4
9.1
CXU
8.2
CXG
10.4
15.6
17.5
13.1
10.6
AXG
AXA
(G)AXC
UXA
The RNA templates containing A show 4- to 18-fold better
enzyme efficiency with Tth pol than templates with the
corresponding sequence containing m6A. Overall, then, differ-
ences in context produce moderate to negligible differences in
selectivity. The UAA sequence context is processed with higher
efficiency than other sequences, whether or not adenine is
Human rRNA is known to contain two m6A modifications,
one at position 1832 in the 18S subunit, and one at position
4189 or 4190 in the 28S subunit.34 On the basis of ratios to
nearby unmodified bases after enzymatic digestion, these sites
are reported to consist only of the modified base (100%
m6A).35,36 Primers were designed to interrogate these three
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Communication
sites, as well as two other sites known to contain A (1781 in
18S; 4984 in 28S). Total RNA was extracted from 293T cells,
and the RNAs were probed with primer sets (see SI). As seen
in Figure 3, results showed less than 20% incorporation of T by
plasmid and overexpressed in 293T cells. Two primers were
designed, one to detect the known modification site and one to
detect a nearby adenosine (see SI). The putative m6A site
showed significantly lower incorporation than the nearby
control A site. As an additional control, we performed in vitro
transcription of the bPRL transcript in the absence of m6A,
allowing us to compare the primer incorporation at the site of
interest when only A was present. At the high enzyme
concentrations initially used, no difference was seen between
the total cellular RNA and the in vitro transcribed RNA. Use of
a lower enzyme concentration, however, did reveal a significant
difference in the m6A:A ratio (0.75 0.09 for IVT RNA vs 0.43
0.04 for total cellular RNA; all extension products were
included in quantification), confirming the presence of m6A in
the mRNA expressed in 293T cells.
In summary, we have characterized a commercially available
polymerase that discriminates m6A from A in all tested
sequence contexts. We have used it to detect m6A in abundant
cellular RNAs. While further development is needed before this
method is robust enough for detection and quantification of
m6A in lower abundance mRNAs, it seems likely that the
inherent selectivity of this enzyme will prove useful in the
development of future m6A analysis techniques.
Figure 3. Identification of methylated adenine sites in human rRNA.
Primers were designed to probe for m6A at three sites in rRNA.
Incorporation at these sites by Tth pol was compared to incorporation
by Tth pol at known nonmethylated control sites and to incorporation
by nonselective AMV RT.
ASSOCIATED CONTENT
* Supporting Information
Experimental methods and materials, additional data, and
characterization of synthetic intermediates. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
Tth DNA pol at the 1832 m6A site and the 4190 m6A site
relative to controls, consistent with complete methylation in
our quantitative experiments with synthetic RNAs. In contrast,
we observed a high degree of incorporation (∼120%) by Tth
pol relative to AMV RT at the 4189 site. As a result, we can
assign the previously undetermined site of methylation in
human 28S RNA as 4190 and not at the neighboring adenine at
4189.
AUTHOR INFORMATION
Corresponding Author
kool@stanford.edu
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Finally, we attempted to detect m6A in a cellularly expressed
mRNA (Figure 4). We chose a known site in the 3′-UTR of the
bovine prolactin (bPRL) transcript.23 This is the only precisely
mapped m6A site in a mammalian mRNA; native levels of
modification are estimated to be ∼20%, and this presents a
challenging case for detection.23 The 3′-UTR was cloned into a
■
We thank the National Institutes of Health (GM068122) for
support. E.M.H. is supported by a Graduate Research
Fellowship (DGE-1147470) from the NSF. T.E. is supported
by a Postdoctoral Research Fellowship from the DAAD. P.J.B.
is the Kenneth G. and Elaine A. Langone Fellow of the Damon
Runyon Cancer Research Foundation; H.Y.C. is an Early
Career Scientist of Howard Hughes Medical Institute.
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