Research paper
RNA Biology 8:5, 802-805; September/October 2011; © 2011 Landes Bioscience
An RNA G-quadruplex in the 3' UTR
of the proto-oncogene PIM1 represses translation
Amit Arora and Beatrix Suess*
Institut für Molekulare Biowissenschaften; Johann Wolfgang Goethe-Universität Frankfurt; Frankfurt/Main, Germany
Key words: PIM1, proto-oncogene, quadruplex, RNA, translation
Abbreviations: UTR, untranslated region; G, guanine; CD, circular dichroism; qRT-PCR, quantitative RT PCR
We have identified a conserved G-quadruplex forming sequence in the 3' untranslated region (UTR) of the protooncogene PIM1. Circular dichroism and thermal denaturation studies revealed that the PIM1 mRNA guanine-rich
sequence forms a highly stable intramolecular G-quadruplex structure. Reporter gene assay demonstrated that the PIM1
RNA G-quadruplex represses translation. It is the first experimental evidence of an RNA G-quadruplex structure located
in the 3' UTR that may act as posttranscriptional regulator.
Introduction
biological significance, we decided to characterize PIM1 mRNA
G-rich sequence in more detail.
We performed biophysical experiments using a synthetic RNA
oligonucleotide to determine the structure formation in PIM1
RNA G-rich sequence. Circular dichroism (CD) spectroscopy of
PIM1 RNA showed a characteristic spectrum with a positive peak
at 265 nm and a negative peak at 240 nm indicating the existence
of a parallel G-quadruplex structure1,9,10 (Fig. 1A). C3'-endo type
of sugar pucker in RNA generally disfavors syn-conformation in
guanosines making it difficult for RNA quadruplexes to adopt
anti-parallel topology21 unlike DNA quadruplexes which can
adopt different topologies.22-28 A mutant in which several guanines have been replaced by adenines did not show any characteristic signature for quadruplex formation (Fig. 1A). An increase
in the CD signal with the increase in salt concentration also
indicated the formation of G-quadruplex structure (Fig. S1A).
Thermal difference spectra 29 of G-rich sequence with a characteristic negative peak at 295 nm and two positive peaks at 243
and 275 nm further supported the formation of a PIM1 RNA
G-quadruplex structure unlike the mutated sequence which
lacked such specific signatures (Fig. S1B).
Next, we recorded UV annealing and melting curves in the
presence of 1 mM KCl which resulted in a reversible sigmoidal
transition with a hypochromic shift at 295 nm characteristic of
G-quadruplex structure formation,30 with a Tm value of 72.0 ±
1.0°C (Fig. 1B). In contrast, for a mutated sequence no such
transition and shift was observed (Fig. S2A). At higher salt concentrations, the RNA could not be unfolded indicating a very
stable quadruplex structure (Fig. S2A). Moreover, the RNA
showed reversible sigmoidal transition at 295 nm with a Tm value
of 52.0 ± 1.0°C even in the absence of K+ which is further indicative of quadruplex stability (Fig. S2A). Concentration dependent
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Recently, RNA G-quadruplex structures located in the 5' UTR
of various mRNAs have been shown to act as translational
repressor.1-11 In addition, RNA G-quadruplex structures can
also stimulate mitochondrial transcription termination12 and
regulate alternative splicing in TP53 intron 3.13 In addition, a
G-quadruplex close to a cleavage sites in the 3' UTR of insulinlike growth factor II mRNA has been proposed and its structure
formation has been demonstrated in vitro.14 But, bioinformatics
analysis have not only predicted putative G-quadruplex forming sequences in the 5' UTRs and close to mRNA processing
sites but also within the 3' UTR of many mRNAs.15,16 In most
of the vertebrates, 3' UTR already constitute an attractive target
for extensive posttranscriptional gene regulation via regulatory
proteins/factors17 and microRNAs.18 Till date, nothing much
is known about a possible role of G-quadruplex structure in 3'
UTR mediated regulation. In this study, we for the first time
have demonstrated that G-quadruplex in the 3' UTR of PIM1
mRNA represses protein synthesis.
Results and Discussion
We identified a guanine (G)-rich sequence 277-nucleotides
downstream of the stop codon in the 3' UTR of human PIM1
mRNA using Quadfinder.19 The sequence is highly conserved
both among different species and with respect to its position as
shown in Table 1. PIM1 is a highly conserved serine/threonine
protein kinase, which is involved in the control of cell growth,
differentiation and apoptosis.20 Overexpression of PIM1 promotes tumor progression and therefore constitutes an interesting anti-drug target.20 Inspired by the high conservation and
*Correspondence to: Beatrix Suess; Email: [email protected]
Submitted: 03/08/11; Revised: 03/29/11; Accepted: 03/30/11
DOI: 10.4161/rna.8.5.16038
802
RNA Biology
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research paper
Research paper
Table 1. Conservation of G-rich sequence in 3' UTR of PIM1 mRNA
Organism
Sequence (5'-3')a
NCBI ID
Positionb
Homo sapiens
GGGGGU---GGGGGG---UGGGGG--UGGG
NM_002648.3
277
Macaca mulatta
-GGGGU---GGG GGGCGGUGGGGG--UGGG
XM_001113117.1
278
--GGGU---GGGGGGCGGUGGGGG--UGGG
NM_174144.2
274
Felis catusc
GGGGGU---GGGGGGGGGAGGGGGGAGGGG
NM_001009322.1
281
Mus musculus
--GGGCC--GGGGGGGGGGGGGGGGGGGGG
NM_008842.3
311
Rattus norvegicus
--GGGUGUUGGGGGGGGAGGUGGGAGUGGG
NM_017034.1
222
Consensus
--GGG----GGGGGG----G-GGG---GGG
Bos taurus
c
The nucleotides displayed in bold are conserved across different organisms. Dashes represent gaps in the alignment. The nucleotides in normal text
differ from the putative G-quadruplex forming sequence in humans. bThe number of nucleotides downstream of stop codon in the mRNA and before
the first G of the putative G-quadruplex forming sequence. cThese organisms possess additional putative G-quadruplex forming sequences in the 3'
UTR of PIM1 mRNA.
a
under physiological concentration of KCl whereas the mutated
RNA does not.
A biological characterization of the PIM1 RNA G-quadruplex
was performed to specify its function in the control of gene
expression. Both a 22-nucleotide G-quadruplex forming and the
mutated sequence were cloned into the 3' UTR of Renilla luciferase (Rluc) reporter gene downstream of the stop codon and named
as PIM1GQ and PIM1GQm, respectively. In addition, to determine the influence of flanking nucleotides, a 100-nucleotide long
fragment of the original PIM1 3' UTR carrying the G-quadruplex
centered and its respective mutant were constructed and named
as GQ100UTR and GQm100UTR, respectively. We have chosen a 100-nucleotide long region of PIM1 3' UTR to maintain
the local structural environment of the G-quadruplex structure
and to determine the influence of flanking nucleotides. We did
not use the complete 3' UTR to minimize a possible influence of
additional regulatory elements on the reporter gene asssay. HeLa
cells were transiently transfected with the plasmids carrying the
constructs and an additional firefly luciferase (Fluc) reporter gene
for normalization of transfection efficiency. The luciferase activity was measured. In parallel quantitative RT PCR (qRT-PCR)
was performed to determine the level of mRNA.
Figure 2A showed that the expression of Rluc increased twofold when the G-quadruplex structure was mutated (shaded
bars) whereas the mRNA level remained unaffected (open
bars). The same effect occurred when we not only analyzed the
isolated G-quadruplex structure but also a 3' UTR fragment
bearing the G-quadruplex structure within its natural context
(Fig. 2B). Taken together, all these results demonstrated that
the G-quadruplex structure did not affect the mRNA level but
influenced the translation efficiency. Our data clearly indicates
that the G-quadruplex structure located in the 3' UTR of the
proto-oncogene PIM1 has an inhibitory effect on translation.
Therefore, we postulate that the G-quadruplex is a regulatory
element, which might be responsible for the onset of tumorigenesis. Factors, which can destabilize the G-quadruplex structure,
like helicases or small molecules, may lead to tumor progression.
Recently, it has been shown that even single nucleotide mutations (SNPs) often co-localize with G-quadruplex structure
completely abolishing its function and may be the reason for
tumor onset.2
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Figure 1. (A) CD spectra of 1 μM PIM1 RNA G-quadruplex (○) and the
mutated (◆) sequence in 10 mM Tris-HCl, pH 7.5 in the presence of
1 mM KCl at 20°C. (B) UV annealing (○) and melting (△) profile of 1 μM
PIM1 RNA G-quadruplex forming sequence in 10 mM Tris-HCl, pH 7.5
in the presence of 1 mM KCl at 295 nm with a cooling and heating rate
of 0.2°C min-1. Standard deviation (±) in melting temperature (Tm) was
calculated from three independent experiments.
melting experiments (1 to 10 μM) indicated the formation of
intramolecular G-quadruplex structure as the Tm was found to
be independent of concentration (data not shown). CD melting
of PIM1 RNA G-quadruplex structure (Fig. S2B) in the presence of 1 mM KCl was also found to be in accordance to the
UV melting data. Thus, PIM1 RNA G-rich sequence folds into
stable, parallel intramolecular G-quadruplex structure in vitro
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RNA Biology
803
In summary, in the present study we for the first time demonstrated that a G-quadruplex structure located in the 3' UTR of an
mRNA leads to repression of protein synthesis. It consequently
belongs to the class of cis-acting element of structure-anchored
repression.31 It will be interesting to see if further factors are
involved in regulation and which steps of translation may be
targeted. Further on, the identification of factors responsible for
G-quadruplex dysfunction is of profound interest as potential
targets in cancer therapy.
Materials and Methods
PIM1 RNA G-quadruplex forming sequence (PIM1G RNA)
and mutated sequence (PIM1GM RNA) were purchased from
Dharmacon (USA) and their sequence is presented as follows:
PIM1G RNA: 5'-GGG GGU GGG GGG UGG GGG UGG
G-3'
PIM1GM RNA: 5'-GAG AGU GAG GAG UGA GAG UGA
G-3'
RNA concentration was determined using an extinction coefficient of 22.24 x 104 M-1cm-1 and 24.41 x 104 M-1cm-1 for PIM1
RNA G and PIM1 RNA GM, respectively. DNA oligonucleotides for PCR and cloning were purchased from PURIMEX and
Eurofinns MWG. Chemicals were purchased from Roth GmbH.
All solutions were prepared in autoclaved RNase free MilliQ
water from Millipore.
UV and CD spectroscopy. Prior to CD experiments, RNA
sample at 1 μM concentration for both the guanine rich and
mutated sequence was heated to 95°C and cooled down to 20°C at
a 0.2°C min-1 in 10 mM Tris-HCl, pH 7.5 with KCl (0–50 mM).
CD experiments were performed at 20°C using a JASCO J-715
spectropolarimeter equipped with a Peltier temperature controller. CD scans were taken from 220 to 320 nm in triplicates each
with two accumulations and their average was calculated. A CD
spectrum of the buffer was recorded and subtracted from the
spectrum obtained for the RNA-containing solution. Data were
zero-corrected at 320 nm; all the CD spectra are presented till
300 nm.
We prepared RNA samples at 1 μM concentration in
10 mM Tris-HCl, pH 7.5 with KCl (0 mM–50 mM). UV
annealing and melting studies were carried out on a JASCO
UV-visible spectrophotometer equipped with a Peltier temperature controller. Samples were heated to 95°C and cooled down
to 20°C at a 0.2°C min-1 temperature gradient and absorption
data recorded at 295 nm were collected every 0.5 min on both
annealing and melting steps. Both the annealing and melting
cycles were performed in duplicate in three independent experiments. Folded fraction (α) was calculated and Tm was calculated
using dα/d(1/T) method for the intramolecular equilibrium as
described in the literature.32
Construction of plasmids. Complementary DNA oligonucleotides (PIM1 Gs + PIM1 Gas and PIM1 GMs + PIM1 GMas,
respectively) encoding the 22-nucleotide long G-rich sequence
of the PIM1 3' UTR and the respected mutated sequence were
annealed and inserted into the XhoI restriction site of the psiCHECK-2 plasmid (Promega) downstream of the Renilla
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Figure 2. Dual luciferase reporter assay (shaded bars) and qRT-PCR
analysis (empty bars) (A) PIM1GQ and PIM1GQm (B) GQ100UTR and
GQm100UTR reporter constructs. The reporter assay data is presented
as a ratio of Renilla luciferase/firefly luciferase (Rluc/Fluc) luminescence
values. qRT-PCR data is presented as ratio of C T values for Renilla luciferase/firefly luciferase mRNA. Results were normalized to PIM1GQ (A) and
GQ100UTR (B) constructs respectively. Error bars represent the standard
deviation and were calculated from three independent experiments for
both reporter gene assay and qRT-PCR experiments.
luciferase stop codon. The plasmids were named PIM1GQ and
PIM1GQm, respectively.
The sequences of the DNA oligonucleotides are as follows:
PIM1 Gs: TCG AGG GGG TGG GGG GTG GGG GTG GG
PIM1 Gas: TCG ACC CAC CCC CAC CCC CCA CCC CC
PIM1 GMs: TCG AGA GAG TGA GGA GTG AGA GTG AG
PIM1 GMas: TCG ACT CAC TCT CAC TCC TCA CTC TC
The sequences underlined are the part of XhoI restriction site
used for cloning. For the cloning of the 100-nucleotide long fragment of PIM1 3' UTR, human genomic DNA was isolated from
HeLa cells using DNAeasy kit (Qiagen). The 100-nucleotide
long fragment of PIM1 3' UTR was PCR amplified using specific
forward primer (5'-3'): AAC ACA CTC GAG TTC TTC TCA
TAG GTG TCC AGC A and reverse primer (5'-3'): AAC ACA
GCG GCC GCA TGA TGA AGG AAA CAG TTC CA and
human genomic DNA as a template. Restriction sites are underlined. The PCR amplified 100-nucleotide fragment was inserted
into the XhoI-NheI restriction sites of the psiCHECK-2 plasmid
(Promega) downstream of the Renilla luciferase stop codon. The
plasmid was named as GQ100UTR. Next, site directed mutagenesis was performed using forward primer (5'-3'): GAG AGT
GAG GAG TGA GAG TGA GTC AGA ACC CTG CCA
TGG and reverse primer (5'-3'): GGG ATA TTT CAG AGT
CCA G to mutate the G’s into A’s to construct GQm100UTR.
Mutagenesis reaction was performed using standard protocol
RNA Biology
Volume 8 Issue 5
from site directed mutagenesis kit (Finnzymes). All plasmids
were verified by sequencing (SRD GmbH).
Cell culture. HeLa cells were grown at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco’s modified
Eagles medium (DMEM) supplemented with 10% (v/v) fetal calf
serum (FCS) (Biochrom AG), 2 mM L-glutamine, non-essential
amino acids and the antibiotics 100 μg/ml streptomycin (PAA)
and 100 U/ml penicillin (PAA). HeLa cells were seeded with an
approximate cell density of 40,000 cells per well in 24-well plate
for reporter assay and an approximate cell density of 500,000
cells per well in 6-well plate for RNA isolation.
Transient transfection and dual luciferase reporter
assay. Transfection was carried out in 24-well plates using
LipofectamineTM 2000 (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after transfection, Dual Glow
luciferase reporter assay were performed according to the manufacturer’s instructions using Dual-Glow ® Luciferase Assay System
(Promega). The firefly and Renilla luciferase activities were measured in 96-well plate (Greiner bio-one) with a TECAN Infinite
M200 luminometer using one-second integration time. Standard
deviation was calculated from three independent experiments.
Quantitative RT-PCR (qRT-PCR) assay. Total cellular RNA was isolated from transfected HeLa cells using the
RNeasy mini kit along with RNase-free DNase to remove
DNA contamination (QIAGEN). RNA was further subjected
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Acknowledgments
A.A. is a Humboldt Post Doctoral Fellow. B.S. acknowledges
the funding by the Aventis foundation and the DFG (Cluster of
excellence: Macromolecular complexes). We thank Jens Kurreck
for providing psiCHECK-2 vector.
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Fluc reverse: GTC GAA AGA CTC TGG CAC GAA GTC G
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Fluc forward: GAC TAC CAG GGC TTC CAG AGC ATG T
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