SURVEY AND SUMMARY
50-UTR RNA G-quadruplexes: translation regulation
Anthony Bugaut1and Shankar Balasubramanian1,2,3,*
1Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW,2Cancer Research
UK, Cambridge Research Institute, Li Ka Shing Center, Cambridge CB2 0RE and3School of Clinical Medicine,
University of Cambridge, Cambridge CB2 0SP, UK
Received November 7, 2011; Revised January 17, 2012; Accepted January 19, 2012
RNA structures in the untranslated regions (UTRs)
of mRNAs influence post-transcriptional regulation
of gene expression. Much of the knowledge in this
area depends on canonical double-stranded RNA
elements. There has been considerable recent ad-
vancement of our understanding of guanine(G)-rich
nucleic acids sequences that form four-stranded
structures, called G-quadruplexes. While much
ofthe research hasbeen
G-quadruplexes, there has recently been a rapid
emergence of interest in RNA G-quadruplexes, par-
ticularly in the 50-UTRs of mRNAs. Collectively,
these studies suggest that RNA G-quadruplexes
exist in the 50-UTRs of many genes, including
genes of clinical interest, and that such structural
elements can influence translation. This review
features the progresses in the study of 50-UTR
RNA G-quadruplex-mediated translational control.
It covers computational analysis, cell-free, cell-
based and chemical biology studies that have
quadruplexes in both cap-dependent and -inde-
pendent regulation of mRNA translation. We also
discuss protein trans-acting factors that have
been implicated and the
RNA motifs have potential as small molecule
target. Finally, we close the review with a perspec-
tive on the future challenges in the field of 50-UTR
RNA G-quadruplex-mediated translation regulation.
While the discovery of canonical DNA double helix struc-
ture comprising Watson–Crick base pairing has provided
the basis for our understanding of the genetic code (1), it is
becoming increasingly evident that non-Watson–Crick
interactions between bases and non-canonical nucleic
acids structures have importance in biology. More than
40 years before the elucidation of the DNA double
helix, Bang (2) reported the observation that guanylic
acid (GMP) forms gels at high concentration in aqueous
solution. Fifty years later, Gellert and co-workers (3) col-
lected X-ray fiber diffraction data revealing that the struc-
tural basis for this phenomenon was the formation of
regular hydrogen-bonded helices based on the assembly
of tetrameric units, now know as guanine (G)-quartets.
A G-quartet is formed by four G bases arranged in a
square planar cyclic hydrogen-bonding pattern, where
each guanine is both the donor and acceptor of two
hydrogen bonds, providing a central site where the
oxygen lone pair of the carbonyl groups can coordinate
with metal cations (Figure 1A). Beyond GMP monomers,
G-quartets can also arise intermolecularly between G-rich
strands or intramolecularly within some G-rich nucleic
acid sequences. When several G-quartets can form prox-
imally within a single strand of nucleic acids they can
stack upon each other, by mean of p–p interactions, to
form a 3D structure, called a G-quadruplex (Figure 1B)
(4). Such structures are further stabilized by physiologic-
ally abundant monovalent cations, particularly K+or
Na+, which fit in or coordinate between the G-quartets.
From DNA to RNA G-quadruplexes
G-quadruplex formation by biologically relevant nucleic
acid sequences remained largely unexplored until it was
discovered that the ends of human chromosomes, the
telomeres, are composed of tandem repeats of a G-rich
DNA sequence d(TTAGGG) (5). It was subsequently
demonstrated by NMR spectroscopy that the single-
stranded G-rich 30overhang of human telomeres is
prone to form intramolecular G-quadruplex in vitro (6).
In addition, a key paper by Zahler et al. (7) demonstrated
an interplay between Oxytricha nova telomeric sequence
*To whom correspondence should be addressed. Tel: +44 1223 336347; Fax: +44 1223 336913; Email: email@example.com
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Published online 20 February 2012 Nucleic Acids Research, 2012, Vol. 40, No. 114727–4741
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
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G-quadruplex formation and telomerase (i.e. the enzyme
responsible for the elongation of chromosome ends during
DNA replication) function in vitro, which stimulated
further consideration of the potential function of DNA
G-quadruplex in biology. Owing to the strong associations
between telomerase over-expression and human cancers
(8), extensive research effort has been devoted to investi-
gate the relevance of G-quadruplex formation in telomere
maintenance (9,10). This has led to the proposal of a new
anticancer strategy based on small molecule agents that
could target and stabilize G-quadruplex structures at telo-
meres with a view to inhibit telomerase function and thus
impart cell death (11,12).
In addition to studies on G-quadruplex formation at
telomeres, computational searches have been performed
to identify G-rich sequences with potential to form
G-quadruplex structures (putative quadruplex sequence,
PQS) in the human genome and other genomes (13–26).
Such bioinformatics studies revealed a prevalence of PQSs
in genomes with a notable enrichment in gene promoter
regions, indicating possible roles for DNA G-quadruplex-
forming sequences as regulatory elements of transcription
(17–20,22,24). This association of PQSs with gene pro-
moters was consistent with an original hypothesis of
Simonsson et al. (27) linking G-quadruplex formation
Subsequently, Hurley and co-workers (28) showed that
mutational destabilization of the C-MYC promoter
quadruplex led to a 3-fold increase in the transcription
activity of a plasmid-based reporter assay. Data have
continued to accumulate in the literature in support of
transcription–associated roles for DNA PQSs, particularly
for oncogenes, and the potential for such motifs to act as
drug targets (29).
While much attention in the G-quadruplex field has
been focused on DNA, in the 1990s, there were early
reports of G-quadruplex formation from biologically
relevant RNA molecules. For example, a 19-nt oligo-
nucleotide derived from Escherichia coli 5S RNA was
found to form an extremely stable K+-stabilized tetra-
meric aggregate in vitro that was dependent on a UG4U
sequence motif at its 30end (30). The authors noted ‘This
complex is so stable that it would be surprising if similar
structures do not occur in nature’. Based on 2D
NMR spectra and molecular dynamics data, the UG4U
of the C-MYCgene.
stacked G-quartets and at least one U-quartet (31,32).
Otherearly studies linked
G-quadruplex structures to biological events in viruses,
suchHIVor herpes simplex
four-stranded RNA structures formed from G-rich se-
quences were proposed to be involved in the processing
and translational control in human transcripts (35–43). A
noteworthy recent example of the potential importance of
RNA G-quadruplex emerged from the discovery of ?100-
to 9000-nt G-rich telomeric repeat-containing RNAs
(TERRA), which arise from RNA polymerase II tran-
scription of the C-rich strand human DNA telomeres
(44,45). TERRA transcripts are thought to play a key
role in chromatin remodeling and in the regulation of tel-
omerase activity (46). Unsurprisingly, G-rich oligonucleo-
tides derived from TERRA sequence have been shown to
adopt G-quadruplex structures in vitro (47–54). While syn-
thetic oligonucleotide probes having human TERRA
sequence and light-switching pyrene moieties at their
ends have been used to show G-quadruplex formation in
living cells (55), proof of G-quadruplex structures in
native TERRA transcripts in vivo and details of their func-
tional significance are still in need of robust experimental
support. In addition to TERRA, G-quadruplex formation
has also been proposed at the 50extremity of human tel-
omerase RNA (hTR) (56). Interestingly, two recent
studies demonstrated that hTR is bound in vivo by the
RHAU/DHX36 protein, which is an RNA helicase that
exhibits G-quadruplex substrate preference (57,58).
Numerous biophysical studies have shown that RNA
sequences form G-quadruplex structures that are thermo-
dynamically much more stable than their DNA counter-
parts (59–64). Some structural explanations of the higher
stability of RNA versus DNA G-quadruplexes have
recently been provided by X-ray crystallography studies
structures from human telomeric repeats (53,54). In
RNA G-quadruplexes, the ribose C20hydroxyl groups
participate in an extended network of hydrogen bonds,
backbone atoms, O40sugar oxygen and H-bond acceptors
N2 groups of quartet-forming guanines (Figure 1C). As
compared to DNA,these
phosphate and oxygen
Figure 1. Schematic representations of (A) a G-quartet arrangement, (B) a G-quadruplex nucleic acids structure and (C) the intermolecular hydrogen
bonding network (dash lines) between the ribose C20hydroxyl groups and the O40sugar oxygens of RNA G-quartet-forming residues [adapted from
(53), PDB: 3IBK].
4728 Nucleic Acids Research, 2012,Vol.40, No. 11
contribute to the increased thermodynamic stability of
RNA G-quadruplexes by both decreasing the entropic
cost associated with the number of ordered water mol-
ecules in the RNA G-quadruplex grooves and enhancing
the favorable enthalpic contribution to the free energy
of RNA G-quadruplex formation. Another noteworthy
differenceis that whereas
G-quadruplex structures are generally highly polymorphic
(65,66), currently available biophysical data indicate that
intramolecular RNA G-quadruplexes have a preference to
adopt a parallel-stranded conformation regardless of the
sequences and the experimental conditions (60–64).
However, it should be noted that such data have been
predominantly obtained from circular dichroism (CD)
spectroscopic measurements on rather short synthetic
RNA sequences with closely related tandem repeat
G-rich patterns. Recently, a high-resolution NMR struc-
ture of 1:1 complex between a 36-nt G-rich in vitro selected
RNA aptamer and a peptide from the RGG domain of the
human fragile X mental retardation protein (FMRP) has
revealed the formation of a three-G-quartet RNA
G-quadruplex structure adopting an unprecedented fold,
which contains a mixed-junction quartet that connects
the quadruplextoa flanking
Due to the relative paucity of RNA G-quadruplex
studies, it is conceivable that further structural variations
in RNA G-quadruplex structures will be discovered in
RNA G-quadruplexes in the 50-UTRs of mRNAs
transcribed RNA molecules are relatively unconstrained
and can readily fold intramolecularly to form a wide
variety of nucleic acid structures, which ultimately
dictate the fate and function of the RNA. A particular
example is the formation of secondary and tertiary struc-
tures within the 50untranslated regions (UTRs) of
mRNAs, which has been shown to play important roles
in the post-transcriptional regulation of gene expression
(68–72). Whereas much of the scientific history of this
field has been based on RNA structures that depend on
canonical Pu/Py base pairs, numerous other non-covalent
interactions can exist between the nucleotides that
constitute a molecule of RNA, including the formation
of G-quadruplexes. In 2007, we reported an RNA
G-quadruplex-forming sequence within the 50-UTR of
the NRAS human proto-oncogene and demonstrated
that formation of the RNA G-quadruplex inhibits
protein expression in vitro (73). At the same time, a com-
putational search of all annotated 50-UTRs of the human
transcriptome, in the latest available version of the
Ensembl database at that time (version 40, NCBI build
36), identified ?3000 50-UTRs that contained one or more
RNA PQSs, including several other proto-oncogenes (73).
This allowed usto propose
G-quadruplex formation could be a more general mech-
anism to regulate mRNA translation (73). In support of
this proposition, a more detailed follow-up bioinformatics
study revealeda significant
G-quadruplex-forming motifs in the 50-UTRs of human
comparedto genomic double-helical DNA,
genes as compared to the whole transcriptome (74).
Moreover, a recent bioinformatics analysis revealed a
similar prevalence and distribution for RNA PQSs in the
genome of the plant Arabidopsis thaliana (75).
In the past few years, there has been a rapid emergence
of interest on RNA G-quadruplexes in the 50-UTRs of
mRNAs. Collectively, these studies suggest that RNA
G-quadruplexes exist in many mRNAs, including genes
of clinical interest for human diseases, and that such struc-
tures play a role in regulating the level of translation
of their host gene into the corresponding protein
product. In addition, recent publications have provided
proof-of-concept for the inhibition of translation by
small molecules that target G-quadruplexes in the
50-UTR of RNA transcripts. While recent reviews have
progresses in RNA G-quadruplex (66,76,77), herein we
discuss in detail the collective advancements in the
emerging area of 50-UTR RNA G-quadruplex-mediated
translational control. Specifically, we cover the experimen-
G-quadruplexes in cap-dependent and -independent regu-
lation of mRNA translation, plus the putative protein
trans-acting factors that might be involved in 50-UTR
G-quadruplex-mediated translation regulation, and also
the evidence that such RNA motifs may serve as molecu-
lar targets for synthetic small molecules to affect gene ex-
pression. Finally, we close the review with a perspective on
the future challenges for this field.
50-UTR RNA G-QUADRUPLEXES IN TRANSLATION
The translation of an mRNA into its protein product
involves three major steps: initiation (assembly of a
ribosome on the mRNA platform), elongation (protein
synthesis) and termination (disassembly of the ribosome)
(78). Initiation is believed to be the rate limiting and
most regulated step of the whole process. The canonical
model for eukaryotic translation initiation (also called a
‘scanning’ mechanism) can be briefly described by: (i) the
assembly of a pre-initiation ribosomal complex (43S) at a
modified nucleotide cap analog that forms the mRNA 50
end, (ii) scanning of the complex along the 50-UTR of the
mRNA until it reaches the initiator AUG codon and (iii)
release of the initiation factors (eIFs) and recruitment of
the 60S ribosomal subunit to form a competent ribosome
(80S) that proceeds to protein synthesis. Alternatively,
certain mRNAs have the capacity to use another form
of translation initiation that does not involve the cap
nucleotide analog, but rather relies on internal ribosome
entry sites (IRESs) (79). Translation initiation by IRES
involves a mechanism in which translation of specific
mRNAs is carried out using non-coding RNA se-
quences/structures that substitute the 50cap and some
eIFs. This alternative mechanism for translation initiation
is particularly important under circumstances where
cap-dependent translation is compromised, such as cell
stress, growth, differentiation, mitosis, apoptosis or viral
infection. Both translation initiation mechanisms are
Nucleic Acids Research, 2012,Vol.40, No. 114729
influenced by 50-UTR RNA structural elements, including
G-quadruplex structures (Figure 2).
50-UTR RNA G-quadruplexes in cap-dependent
Several studies have demonstrated that thermodynamic-
ally stable RNA hairpin structures in the 50-UTRs of
mRNAs can impair eukaryotic cap-dependent translation
by compromising the assembly of the translation initiation
machinery at the 50cap of the mRNA and/or by perturb-
ing its scanning process toward an AUG translation start
codon (80–83). However, the variety of RNA structures
may be substantially broader than stem–loop formation,
and could involve a wide range of non-Watson–Crick
hydrogen bonding interactions, including Hoogsteen
pairing between guanine bases that lead to the formation
of G-quadruplex structures. Given that folded RNA
under near physiological
suggests that they are likely to form in vivo.
In 2001, Moine and co-workers provided a first glimpse
of the potential of an intramolecular 50-UTR RNA
G-quadruplexes to influence translation efficiency. They
identified a 35-nt RNA G-quadruplex structure, as
determined by footprinting experiments, within the
RGG-coding region of the FMRP as a specific binding
site for the FMRP protein to its own mRNA (39).
When this sequence was inserted in the 50-UTR of a
luciferase reporter mRNA, this resulted in a 1.5-fold
decrease in in vitro translation efficiency as compared to
control mRNAs, either lacking or containing an incom-
plete G-quadruplex sequence.
Subsequently, in 2007, we reported the presence of an
RNA G-quadruplex-forming sequence naturally occurring
in the 254-nt 50-UTR of the p21 GTPase encoding human
NRAS mRNA (73). This motif is conserved, in both its
sequence and its position relative to the translation start
site, across the 50-UTRs of human, chimpanzee, macaque,
mouse, rat and dog NRAS genes. Using CD spectroscopy
and UV-melting experiments, we demonstrated that the
18-nt G-rich sequence folded into a RNA G-quadruplex
in vitro. We then inserted the NRAS 50-UTR in front of
the Firefly luciferase reporter gene and directly down-
stream of a minimal T7 promoter in a plasmid vector,
and generated control constructs that either lacked the
PQS or contained mutations that disrupt G-quadruplex
mRNAs in rabbit reticulocyte lysates revealed that the
RNA G-quadruplex structure in its natural context
within the NRAS 50-UTR inhibits translation by ?80%
(73). This result suggested that native RNA G-quadruplex
structures in 50-UTRs could act as regulatory elements of
In a following publication, the same year, Wieland and
Hartig (84) provided evidence consistent with RNA
G-quadruplex formation in bacteria. Using artificial ra-
tionally designed G-rich elements introduced adjacent to
the Shine–Dalgarno sequence of a GFP reporter gene,
they demonstrated that sequences with potential to fold
into four-stranded structures inhibited gene expression in
Escherichia coli, presumably by abrogating access to the
ribosome binding site. Importantly, the levels of GFP ex-
pression correlated well with the thermal stabilities of the
inserted G-quadruplex structures, and G-quadruplex
structures of moderate stabilities were shown to behave
as thermo-regulators of GFP expression in bacteria.
Although further work is needed to address the existence
and importanceof naturally
quadruplex elements in prokaryotic translation initiation,
these findings suggested that RNA G-quadruplex-forming
sequences could be useful as tools in synthetic biology.
The first demonstration of translation inhibition by
a 50-UTR RNA G-quadruplex in living eukaryotic
cells was published in 2008 (85). The authors investigated
within the 719-nt 50-UTR of the mRNA of the human
Zic-1. UV melting and CD spectroscopy experiments
indicated that this sequence is likely to adopt a
highly stable RNA G-quadruplex structure under near
physiological ionic conditions. Using an established
thein vitro transcribed
Figure 2. Schematic illustration of the possible roles of 50-UTR RNA G-quadruplex formation in cap-dependent and cap-independent regulation of
translation initiation. Red light indicates translation inhibition. Green light indicates translation enhancement.
4730 Nucleic Acids Research, 2012,Vol.40, No. 11
dual-luciferase plasmid-based assay, the authors showed
that the 27-nt G-rich fragment of the UTR repressed
protein synthesis in HeLa cells by ?80% while a
mutated version of the sequence, which did not form a
stable quadruplex structure, did not influence translation.
As identical levels of mRNA were detected using quanti-
tative RT-PCR, it was concluded that the decrease in
protein synthesis was due to repression of translation
rather than a consequence of reduced transcription. In
addition, western-blot analysis revealed that expression
of Zic-1 from a plasmid vector was strongly reduced by
a 73-nt fragment of the UTR containing the G-quadruplex
Subsequent to these reports, additional studies have
shown the presence of G-quadruplex-forming sequences
within the mRNA 50-UTRs of several human genes
(Table 1). A comparable strategy, based on biophysical
analysis ofthe G-rich sequence,
reporter gene-based expression
employed to confirm the capability of the sequences to
fold into RNA G-quadruplex structures and to modulate
translation (Table 1). The genes studied included the
matrix metalloproteinase MT3-MMP (86), the estrogen
receptor ESR1 (87), the apoptotic regulator BCL2 (88),
the telomere shelterin protein TRF2 (89) and the
a-secretase ADAM10 (90). Down-regulation of translation
sequences containing four to six G3tracts and oligo(U)
loops of various length has also been reported (91). This
study indicated that both loop-length and the number of
G3repeats could influence in cellulo protein expression.
Both shorter loop-lengths and increased numbers of G3
tracts correlated with greater reductions in expression of
a luciferase reporter gene in mammalian cells, which also
correlated with the thermal stabilities of the RNA
G-quadruplexes. In our own studies, we have explored
the effect of RNA G-quadruplex position and stability
in the NRAS 50-UTR on protein synthesis (92). We
observed a marked difference in translation inhibition by
the G-quadruplex-forming sequence depending on its
position relative to 50cap of the NRAS 50-UTR. We
found that the NRAS G-quadruplex motif is inhibitory
of in vitro translation only when it is located sufficiently
close to the 50cap (within ?50–100nt), illustrating the
importance of studying 50-UTR RNA G-quadruplex-
forming sequencesin their
drawing conclusions on their biological function.
Recently, Beaudoin and Perreault (93) have provided
an in-depth analysis on naturally occurring 50-UTR
RNA G-quadruplex structures, which included in silico,
in vitro and in cellulo experiments. Extending on a bio-
informatics study previously reported (74), a database
of all 50-UTR PQS in the protein-coding genes of 18 dif-
ferent organisms was constructed. Next, PQSs within the
50-UTRs of nine human genes encoding proteins involved
in various biological processes were further investigated
(Table 1). First, their abilities to fold into RNA
G-quadruplex structures in vitro were evaluated using
CD and also by the application of in-line probing, a tech-
nique originally developed in the to study riboswitches
(94). Based on these experiments, six sequences were
observed to form G-quadruplex. Each of them was
shown to significantly inhibit translation in their natural
context within the full-length 50-UTR, using plasmid-
Comparative analysis of the nine PQSs revealed that
the three sequences that failed to form a G-quadruplex
structure contained significantly more cytosines than
those that folded into G-quadruplex. The authors
proposed that the large number of cytosines most likely
increases the propensity of those sequences to adopt stem-
loop structures based on canonical GC base pairing, thus
competing out G-quadruplex formation. This hypothesis
was then substantiated through an elegant mutagenesis
studyin which several
introduced to lower the G/C ratios and to destabilize the
stem–loop structures. In all three cases, mutations were
identified that rescued G-quadruplex formation in vitro,
and in one case the C/A change was shown to induce
translation repression. These results raise questions on
whether any single nucleotide polymorphisms (SNPs)
within 50-UTRs map to RNA PQSs and therefore have
potential to influence function. Through a bioinformatic
search a total of 143 SNPs within 116 50-UTR RNA PQSs
were found in the human transcriptome (93). One SNP,
consisting of a G-to-C substitution, was identified within
one of the initially studied candidates, namely the
AASDHPPTT 50-UTR PQS (Table 1). This SNP was
then shown to be detrimental to RNA G-quadruplex for-
mation by in-line probing, and was demonstrated to
increase the in cellulo translation efficiency of a reporter
gene as compared to the wild-type (G-quadruplex-
forming) sequence (93). This result suggests that SNPs
within 50-UTR RNA G-quadruplex-forming sequences
can in principle cause differential
in HEK 293 cells.
50-UTR RNA G-quadruplexes in cap-independent
Most examples in literature have described 50-UTR RNA
PQSs that inhibit translation, leading to the proposal that
50-UTR RNA G-quadruplex are ‘predictable’ inhibitory
elements of gene expression (91). However, there are
cases where RNA G-quadruplex formation has been
shown to actually promote translation. In 2003, Bonnal
et al. (40) reported an analysis of the cis-acting elements
defining a IRES present within the 50-UTR of the human
fibroblast growth factor 2 (FGF2) mRNA that revealed a
G-quadruplex motif as a structural determinant of IRES
activity. Here, chemical and enzymatic footprinting
experiments were employed to probe the structure of the
484-nt FGF2 mRNA 50-UTR and an RNA G-quadruplex
structure composed of five G-quartets was identified.
Deletion analysis using bicistronic plasmid constructs in
human liver adenocarcinoma SK-Hep1 cells demonstrated
that the G-quadruplex-forming sequence (Table 1) is part
of a 176-nt RNA module that is necessary and sufficient to
confer IRES activity. While the RNA sequences and struc-
tural features of cellular IRESs remain largely unknown
(79), this study provided the first example of an RNA
G-quadruplex structure involved in an IRES element.
Nucleic Acids Research, 2012,Vol.40, No. 11 4731
Table 1. RNA G-quadruplex-forming sequences identified within human mRNA 50-UTR that were experimentally shown to modulate translation efficiency
RNA G-quadruplex-forming sequence (50to 30)
Evidence for RNA
In vitro/in cellulo
In vitro (RRL)
Zinc finger protein Zic-1
In cellulo (HeLa)
Matrix metallopeptidase 16
In cellulo (HeLa)
Estrogen receptor a
In vitro (RRL)
Estrogen receptor binding site
associated antigen 9
In cellulo (HEK293)
Frizzled family receptor
In cellulo (HEK293)
BarH-like 1 homeobox protein
In cellulo (HEK293)
Neural cell adhesion molecule 2
In cellulo (HEK293)
Thyroid hormone receptor a
In cellulo (HEK293)
In cellulo (HEK293)
Apoptosis regulator Bcl-2
In cellulo (MCF10A,
Telomeric repeat binding factor 2
In cellulo (293T)
In vitro (RRL)
fibroblast growth factor 2
Enzymatic and chemical
In cellulo (SK-Hep1)
vascular endothelial growth factor
In cellulo (HeLa)
a% change in protein expression a reporter gene (luciferase or GFP) induced by the G-quadruplex-forming-sequence when compared to constructs where the sequence has been deleted or
mutated. # indicates a reduction and " indicates an increase in protein expression.
bFootprinting experiments performed in the native context of the full-length 50-UTR.
4732 Nucleic Acids Research, 2012,Vol.40, No. 11
In a more recent study, Morris et al. (95) have provided
structural and functional evidence that a G-rich sequence
within the 50-UTR of human vascular endothelial growth
factor (hVEGF) mRNA adopts an RNA G-quadruplex
structure that is essential for IRES-mediated transla-
tion initiation. The hVEGF mRNA possesses a long
(?1000-nt), GC-rich 50-UTR that harbors two separate
IRESs. Using RNase T1 and dimethylsulfate to probe to
the 293-nt IRES-A structure, a 17-nt G-rich sequence
(Table 1) was identified that adopts a G-quadruplex struc-
ture. Mutational analysis of the hVEGF IRES-A in the
context of a bicistronic dual-luciferase reporter vector
demonstrated that RNA G-quadruplex formation is
essential for cap-independent translation initiation in
HeLa cells. However, it was notable that not every
mutation tested led to a complete loss of IRES activity.
Only a quadruple mutant, lacking sufficient Gs to adopt
an intramolecular G-quadruplex structure, was close to
being completely inactive. Analysis of the primary
sequence (GGAGGAGGGGGAGGAGGA) suggested
G-quadruplexes are theoretically possible by using differ-
ent combinations of the five G-stretches (underlined). In
principle, such redundancy can still allow G-quadruplex
formation despite the introduced mutations. The authors
proposed that the hVEGF 50-UTR PQS could act as a
promote G-quadruplex structure formation and to tune
IRES-mediated translation initiation.
50-UTR RNA G-QUADRUPLEXES AND PROTEINS
The number of reported RNA G-quadruplex-binding
proteins is still relatively scarce compared to DNA
G-quadruplex-binding proteins (96,97), and most of the
biological data that link protein binding to 50-UTR
RNA G-quadruplexes with biological functions are
mostly correlative rather than definitive.
The FMRP is possibly the best-studied RNA G-
quadruplex-binding protein. FMRP is an RNA-binding
protein whoseaberrant expression
common form of intellectual disability, the fragile X
syndrome (FXS) (98). An in vitro selection for RNA mol-
ecules that preferentially bind to the FMRP identified
a large number of sequences containing PQSs (37),
and indeed when FMRP-containing ribonucleoprotein
complexes were immunoprecipitated from mouse brain
?70% of the associated mRNAs contained a PQS (38).
Of particular importance in the regulation of FMRP ex-
pression is the demonstration that FMRP binds to its own
mRNA via a G-quadruplex structure in the coding region
(39). In addition, a large number of putative FMRP
mRNA targets have been identified, which contain com-
putationally predicted G-quadruplex motifs. Experimental
evidence for the involvement of FMRP and related
proteins in G-quadruplex-mediated mRNA metabolism
has recently been reviewed (99). At least two mRNA
targets of FMRP harbor one or more PQS in their
50-UTR, namely the MAP1B and the PP2A genes
(37,100,101). In both cases, absence of FMRP was
is linkedto a
associated with elevated levels of protein expression, sug-
gesting that FMRP binds to the 50-UTR G-quadruplex
structures to repress translation, possibly by stabilizing
the G-quadruplex structure and thus perturbing transla-
tion initiation (100,101).
In a recent study, we proposed that another class of
translational regulatory protein, Pat1, selectively binds
to RNA G-quadruplex structures (102). The Pat proteins
are conserved RNA-binding proteins that have been
shown to participate in processing (P)-bodies formation,
which are ribonucleoprotein cytoplasmic foci involved in
translation repression and mRNA decay (103). As in the
case of FMRP, the Xenopus Pat proteins, xPat1a and
xPat1b, as well the human Pat1b, were shown to prefer-
entially bind poly r(G) in vitro (102). In competition assays
with xPat1a and xPat1b, we found that an oligonucleotide
of the NRAS RNA G-quadruplex sequence (Table 1),
used as model RNA G-quadruplex-forming sequence,
but not a mutated sequence that is unable to form a
G-quadruplex, could efficiently compete for poly r(G)
G-quadruplex, but not the mutated sequence, was able
to isolate xPat1a protein from a Xenopus oocytes lysate
using streptavidin-coated beads. Altogether, these results
suggest that Pat1 proteins preferentially bind G-quartet
containing RNA structures. Interestingly, other identified
protein components of P-bodies, as for example, the yeast
protein Stm1 and the exoribonuclease XRN1p, have been
shown to preferentially interact with RNA G-quadruplex
structures in vitro (104,105). However, more work is still
needed to elucidate whether such interactions between
proteins and 50-UTR RNA G-quadruplexes is actually
associated with P-body formation.
Because of the high thermodynanic stability of RNA
G-quadruplexes in vitro, it is likely that resolving such
structures in vivo would require specialized helicases.
RHAU (also known as DHX36 or G4R1) is a member
of the human DEAH-box family of RNA helicases that
has been shown to bind with high affinity to RNA
G-quadruplex in vitro and to unwind G-quadruplex struc-
tures more efficiently than double-stranded nucleic acids
(106,107). RHAU has been shown to associate with
mRNA in cellulo (108), and was identified as the main
source of tetramolecular RNA G-quadruplex-resolving
activity in HeLa cell lysates (107). In a recent study,
Lattmann et al. (58) over-expressed RHAU in HeLa
cells and employed RNA immunoprecipitation coupled
with genome-wide microarray analysis (RIP-chip) to
identify and quantify RNAs associated with the RHAU
helicase. RHAU was found to be associated with 106
RNAs, of which >50% contained at least one PQS.
Furthermore, the PQS density per transcript showed a
small but significant correlation with the level of RNA
enrichment by RHAU. These findings provide clear indi-
cations that RNA G-quadruplexes are physiologically
relevant targets of RHAU in cells. Of particular note
was the observation that nearly three-quarters of the
identified PQSs were found within the 50- and/or
30-UTRs of mRNAs. Even though no role for RHAU in
translation regulation has yet been demonstrated so far,
this raises the interesting possibility that RHAU could be
Nucleic Acids Research, 2012,Vol.40, No. 11 4733
involved in G-quadruplex-mediated translational control,
or other aspects of RNA metabolism such as splicing or
turnover. Recently, the human DHX9 helicase, which
displays a high similarity with RHAU, has also been
shown to bind and unwind RNA G-quadruplexes
in vitro (109).
FXS is genetically characterized by expansion of a CGG
repeat in the 50-UTR of the FMR1 gene, which is thought
to cause loss of FMRP. Fry and co-workers (110) have
provided some evidence that the CGG repeats can
modulatory function. They showed that a 99-nt r(CGG)
sequence positioned upstream of a luciferase reporter
gene in a FMR1 promoter-driven plasmid repressed trans-
lation in HEK293 cells. However, normal translation effi-
ciency could be restored upon co-expression of CBF-A
and hnRNP A2 proteins, which were previously shown
to destabilize bimolecular
(111,112). These results suggest that a balance between
G-quadruplex formation through the r(CGG)n repeats
in the 50-UTR FMR1 mRNA and endogenous G-
50-UTR RNA G-QUADRUPLEXES AS SMALL
Achieving control of gene expression by using agents that
target nucleic acids represents a major goal in the life
sciences. One such approach is to target and inhibit
mRNA function(s). Oligonucleotide-based agents such
as antisenses,aptamers and,
interfering RNAs represent promising molecules for the
silencing of mRNAs; however their poor pharmacological
properties still represent an issue. Thus, interventions
based on ‘drug-like’ synthetic small molecules could
provide an alternative strategy to overcome the disadvan-
tages associated with nucleic acids-based approaches, par-
ticularly with a view to ultimately translating chemical
biology studies towards therapeutics (113).
Small molecules that bind to structural elements within
the 50-UTRs of mRNAs have been explored with a view to
interfering with gene expression at the translation level
(114). In eukaryotic translation studies, examples have
mainlybeen limited to
molecule-binding RNA aptamers. Artificial systems were
produced by inserting the aptamer sequences upstream of
reporter genes. These systems were shown to be responsive
to the small molecules, and provided proof-of-principle
for small molecule-mediated translation inhibition by tar-
geting structural elements in the 50-UTRs of mRNA
(115,116). These studies have provided proof-of-principle
that small molecules known to bind to defined structural
elements within the 50-UTRs of eukaryotic mRNAs can be
used to control the translation rate of transcripts.
cellular mRNAs with small molecules relates to iden-
tifying RNA structural elements that are amenable to
selective molecular recognition. The special structural
features of G-quadruplexes and the discovery of RNA
numerous genes, including several proto-oncogenes, led
us to propose that such RNA motifs could be suitable
targets for small molecules (73).
In principle small molecules targeting of 50-UTR RNA
G-quadruplex structures could modulate the mRNA
translation according three mechanisms: (i) by stabilizing
the RNA G-quadruplex structure and thus impairing
the assembly and/or the scanning process of the 43S
ribosomalcomplex; (ii) by destabilizing
G-quadruplex structure and thus stimulating translation
or (iii) by interfering with a biologically essential RNA
G-quadruplex/protein binding event.
luciferase system, we evaluated the translational effect of
several small molecules from our ligand collection that
had previously been established to exhibit selective
double-stranded DNA. In this study, we compared the
in vitro translation efficiencies of both the wild-type
NRAS 50-UTR and a control 50-UTR in the presence of
increasing concentration of the G-quadruplex ligands
(117). The control mRNA
50-UTR. In an initial screening study, we found several
molecules that had no effect on translation and others
that inhibited translation in a G-quadruplex-independent
manner. For example, the well-studied G-quadruplex
ligand TMPyP4 showed translational inhibition without
probably because of its poor selectivity for G-quadruplex
versus other nucleic acids structures (118,119). However,
we also identified molecules that displayed selective trans-
lational inhibition depending on the presence of the RNA
G-quadruplex. Among these, a pyridine-2,6-bis-quinolino-
dicarboxamide derivative (RR82, Figure 3) reduced the
translational efficiency of the NRAS 50-UTR by ?50%
at 1.25mM concentration; whereas under the same condi-
tions the control still exhibited ?80% translation effi-
ciency. We then tested structural variants of this
molecule and found that a para-fluorophenyl substituent
at pyridine C4 (RR110, Figure 3) considerably improved
G-quadruplex selectivity. At 10mM concentration, RR110
had no effect on the translational efficiency of the control
mRNA, but inhibited that NRAS 50-UTR translation by
?40%. This inhibitory effect was retained in the presence
of large excess of double-stranded DNA or hairpin RNA
competitors, and it also compared favorably with studies
based on in vitro selected RNA aptamers (115,116). Using
hydrogen-deuterium exchange followed by1H-NMR, we
demonstrated that RR110 stabilizes the NRAS RNA
G-quadruplex and we performed an mRNA stability
assay to show that the small molecule did not exert its
effect by altering the mRNA degradation rate (117). In
addition, we also showed that an RNA G-quadruplex
motif that did not display any intrinsic translational in-
hibitory activity could become a translational inhibitory
element upon binding to a G-quadruplex ligand, suggest-
ing that a G-quadruplex-binding small molecule can
trigger a translational effect (117). Collectively, these
in the50-UTRs of
4734Nucleic Acids Research, 2012,Vol.40, No. 11
observations support a mechanism whereby stabilization
of the NRAS RNA G-quadruplex affects initiation of the
translation process and provided proof-of-concept that a
small molecule G-quadruplex ligand is able to modulate
translation via selectively binding to a 50-UTR RNA
In a subsequent study, Gomez et al. (89) evaluated the
potency of three bisquinolinium compounds (360A,
PhenDC3 and PhenDC6, Figure 3) to bind the TRF2
RNA G-quadruplex and to alter translation in vitro.
These molecules were selected on the basis of previous
studies showing their high efficacy to bind and stabilize
DNA G-quadruplexes (120,121). The potential of these
moleculesto bindand stabilize
G-quadruplex was first assessed in a FRET melting com-
petition assay using a doubly labeled oligonucleotide
that mimics the human telomeric DNA G-quadruplex.
For all compounds, ligand-induced stabilization of the
DNA G-quadruplex structure was strongly decreased
in the presence of an excess of the TRF2 RNA
G-quadruplex-forming sequence, but was not affected by
a 26-bp DNA duplex or a TRF2 mutated RNA sequence
that is unable to form a G-quadruplex. These results
indicate that the three compounds are able to selectively
bind the TRF2 RNA G-quadruplex. Using an in vitro
coupled transcription-translation assay, the authors went
on to demonstrate that the binding of the ligands to the
TRF2 RNA G-quadruplex correlates with inhibition of
mRNA translation. For example, at 3mM concentration,
the most potent compound in this assay, PhenDC3
(Figure 3), inhibited GFP protein expression from a
50-UTR TRF2 reporter plasmid by about 4-fold; whereas
it did not affect the GFP expression from a control
plasmid containing G-quadruplex-disrupting mutations.
Furthermore, mRNA level analyses showed that protein
synthesis inhibition was not due to a transcriptional
effect, but rather arose from ligands interacting with
RNA G-quadruplex to interfere with the translation
In a following study, Hartig and co-workers (122) used
the same three bisquinolinium compounds to investigate
whether these molecules could be used to target a 50-UTR
RNA G-quadruplex in a cellular context. In this study,
fluorescence intercalator displacement (FID) and CD
melting experiments were used to demonstrate the
binding to and stabilization of rationally designed RNA
G-quadruplex-forming sequences of the form (G3U1–3)4.
FID experiments revealed high association constants to
RNA G-quadruplexes, in the 107–108M?1range, for the
three compounds. HEK293 cells were transfected with
luciferase gene under control of 50-UTRs containing dif-
ferent G-quadruplex sequences of the form (G3U1–3)4–6,
before treating cells with increasing concentrations of
the compounds. In all cases, a dose-dependent reduction
in luciferase expression was observed as compared to un-
treated cells. The most notable effect was a 86% decrease
in luciferase expression for a (G3U)6containing plasmid
when treated with 10mM of PhenDC3 compound
(Figure 3). In contrast, similar treatments showed little
effect on the expression of plasmids containing G-rich
control sequences unable to fold into G-quadruplex struc-
tures. G-quadruplex ligands did not affect mRNA abun-
dance of the investigated reporter genes. Taken together,
these results indicate that the bisquinolinium molecules
can inhibit protein expression in a RNA G-quadruplex-
dependent fashion within a cellular context.
In addition to the above-described studies using
‘drug-like’ small molecules, Ito et al. (123) have recently
demonstrated the use of a short (9-nt long) G-rich RNA to
Figure 3. Chemical structures of synthetic molecules that have been demonstrated to exert selective RNA G-quadruplex mediated translation
Nucleic Acids Research, 2012,Vol.40, No. 114735
inhibit translation of a EGFP reporter mRNA in living
cells throughthe formation
G-quadruplex in its 50-UTR.
Whereas all these studies have exploited plasmid-based
reporter assays, there is one report were an observed
decrease in Aurora A protein level that accompanied
a M-phase cell cycle arrest in cells treated with an
hypothesized to arise through stabilization of an endogen-
ous 50-UTR G-quadruplex in the mRNA sequence of the
Aurora A gene (124,125). However, no direct evidence for
that has yet been provided, and further studies are
required to confirm this possibility.
Although all the examples discussed so far suggest a
mechanism by which small molecule stabilization of a
50-UTR RNA G-quadruplex structure inhibits translation,
there is also an example where a G-quadruplex ligand has
been shown to increase the translation efficiency of
a 50-UTR RNA G-quadruplex-containing mRNA. In
this study, Ofer et al. (126) showed that the cationic por-
phyrin TMPyP4 affected the electrophoretic mobility in
non-denaturing agarose gel of an RNA construct contain-
ing a 50-UTR r(CGG)33derived from the FMR1 mRNA,
which they previously proposed to fold into intramolecu-
lar G-quadruplex (110). In contrast, gel migration of a
presence of potassium ions was required to observe an
electophoretic mobility shift. Based on these results
and onthe previous observation
destabilized bimolecular (CGG)n tetraplex structures
(127), the authors proposed that the small molecule
slowed electrophoretic migration through G-quadruplex
unfolding (126). When assessed in functional assays,
both by itself and synergistically with the CBF-A and
hnRNP A2 proteins, TMPyP4 increased translation effi-
ciency of a 50-UTR r(CGG)99luciferase reporter mRNA
in vitro in rabbit reticulocyte lysates and in cellulo in
HEK293 tissue cultured cells. Since TMPyP4, but not its
positional isomers TMPyP2 or TMPyP3, which were both
previously shown to be unable to unfold (CGG)n
G-quadruplexes (127), enhanced translation efficiency,
these results suggested that the elevated translation effi-
ciency of the 50-UTR r(CGG)99reporter mRNA was a
consequence of the ability of TMPyP4 to destabilize the
translational blocking 50-UTR G-quadruplex structure.
Even though G-quadruplex formation by the r(CGG)n
repeat element is still a subject of debate (128), and the
selectivity of TMPyP4 for G-quadruplex is limited
(118,119), these findings offer an alternative perspective
on RNA G-quadruplex targeting by small molecule that
warrant further investigation.
Finally, very recently, while this manuscript was under
review, Xodo and co-workers (129) have reported on an
alkyl derivative of TMPyP4 (TMpyP4-C14), which effi-
ciently enter cells and preferentially localize into the cyto-
plasm. In this study, the authors demonstrated that
TMPyP4-C14 binds to G-quadruplex structures in the
50-UTR of KRAS mRNA and, upon photoactivation, se-
lectively induces mRNA degradation, resulting in a about
90% downexpression of KRAS protein in pancreatic
CONCLUSION, CHALLENGES AND PERSPECTIVES
A great deal of scientific research has been carried out
during the past 15–20 years to study the roles of RNA
structures on gene expression regulation at the transla-
tional level (68–72,80–83). These studies have mainly
concentrated on structural arrangements based on stem–
demonstrated that an RNA G-quartet-based structure in
an IRES motif within the 50-UTR of a human FGF2 gene
transcript had a role in cap-independent translation initi-
ation. It is <5 years ago since we reported that a naturally
occurring intramolecular RNA G-quadruplex structure in
the 50-UTR of the human NRAS transcript inhibits trans-
lation (73). Furthermore a computational search of
50-UTRs human transcripts for RNA PQSs led us to pos-
tulate that translational regulatory G-quadruplex struc-
tures in the mRNA 50-UTRs may be common (73).
Subsequently there have been many publications on add-
itional genes that have reinforced our initial observation
on the NRAS RNA G-quadruplex (Table 1). In addition
to mRNA 50-UTRs, RNA PQSs have also been computa-
tionally identified in 30-UTRs, and in pre-mRNAs near
splicing, transcription termination and polyadenylation
sites, indicating that RNA G-quadruplexes might also
regulate other stages of RNA metabolism (42,43,74).
Indeed, several studies published within the past 4 years
have provided experimental
G-quadruplex structures in introns affect the splicing
and expression patterns of Bcl-xL, FMR1 and TP53
G-quadruplex formation has recently been shown to
control human mitochondrial transcription termination
and mRNA localization in cortical neurites (133,134).
Whereas several computational algorithms exist that
can reasonably predict G-quadruplex formation within
DNA, such a tool for predicting RNA G-quadruplex
more accurately would be beneficial. The detailed rules
governing RNA G-quadruplex
starting to be discovered. Given the high propensity of
single-stranded RNA to fold into secondary structures
G-quadruplex formation in RNA may be complex. The
sequence and the local structural context could of particu-
Beaudoin and Perreault (93) have provided evidence that
the presence of cytosine tracks within a putative
G-quadruplex-forming sequence could be detrimental to
G-quadruplex formation, presumably because they favor
the formation of competing stem structures. In addition, a
recent report by Patel and co-workers (67) has exemplified
the importance of sequence context by showing the for-
mation of a RNA G-quadruplex structure with an unpre-
cedented fold, which contains a mixed-junction quartet
that connects between the G-quadruplex and a flanking
duplex stem. It is noteworthy that while several soft-
ware packages exist to computationally predict RNA
4736 Nucleic Acids Research, 2012,Vol.40, No. 11
secondary structures, none of them takes account of RNA
G-quadruplex formation. Advancements in the prediction
of RNA G-quadruplex formation would enhance future
research in this field.
While evidence is emerging to suggest critical roles
for RNA G-quadruplexes in biological processes, it is
striking that none of the high-resolution crystallographic
or NMR structures of naturally occurring complex RNA
molecules, such as tRNAs, rRNAs or ribozymes, reported
so far have revealed incident G-quartet structures. In a
recent bioinformatics search of the model plant species
A. thaliana, RNA PQSs were shown to be strongly
underrepresented in non-coding RNA molecules, such as
tRNAs, rRNAs and sno RNAs (75). These absences may
suggest that RNA G-quadruplexes are unsuitable as
long-lived architectural elements. Perhaps their roles are
confined to be transient and regulatory? In light of the
recent findings presented here, RNA G-quadruplex struc-
tures in the 50-UTRs of mRNAs seem to represent key
elements in translational regulation. A simple view
would be that the presence of a RNA G-rich sequence
with the required thermodynamic parameters is sufficient
to form a stable G-quadruplex structure that affects
translation. However, RNA G-quadruplex formation
within the context of a 50-UTR is likely to be in equilib-
rium with alternative secondary/tertiary
possibly involving cellular co-factors, and more complex
mechanisms for translational regulation by 50-UTR RNA
G-quadruplexes could be involved (93). If this view is
correct, an important and experimentally challenging
goal will be to demonstrate where and when 50-UTR
RNA G-quadruplexes naturally exist within cellular
mRNAs. For example, they may only be present at very
defined points of the cell cycle or in particular cell states.
Further evidence is needed to help us better understand
whether such structures are merely incidental or tightly
The discovery of naturally occurring proteins with pref-
erential RNA G-quadruplex recognition properties goes
some way towards helping address the existence and
function of naturally occurring RNA G-quadruplexes.
Immunoprecipation followed by genome-wide microarray
analysis studies of the RNA-binding FMPR protein and
the RHAU helicase have provided some evidence for
RNA G-quadruplex in cellular mRNAs (38,58). The
discovery of more RNA G-quadruplex-binding pro-
teins will be a major step to further elucidate the occur-
G-quadruplexes. It should also be noted that more
genome-wide scale based on enzymatic degradation of
RNA populations have not yet taken account of RNA
G-quadruplexes nor have they considered the cellular
context of RNA structure bound to proteins in vivo
(135–137). The application and refinement of such
approaches and other methods, including SHAPE and
CLIP-SEQ (138,139), could shed light on the existence
and nature of RNA G-quadruplexes in cells.
There is now a growing body of evidence that has
established a link between deregulation of translational
control and disruption of normal cell behavior in human
diseases, especially cancers (140–142). Due to their intri-
cate roles in translational regulation, RNA cis-regulatory
elements located in the 50-UTRs are considered to be
active players in translational regulation breakdowns
(71,72). Despite the recent elucidation of translational
control by 50-UTR RNA G-quadruplexes and the identi-
fication of RNA G-quadruplex in a large number of genes,
including numerous proto-oncogenes, no explicit link
between RNA G-quadruplex formation/disruption and
cancer development has yet been established; future
studies may provide further insights.
G-quadruplex binding ligands can selectively target
RNA G-quadruplexes and derail translation initiation
opens up a new and attractive avenue in RNA-directed
drug design. Clearly, more research will be needed to
rigorously validate RNA G-quadruplexes as drug targets
for therapeutics applications and explore how selective
ligands can be for a given RNA G-quadruplex. Part of
the upcoming challenge will be to better understand the
mechanistic effects and selectivity factors on endogenous
mRNAs in the all complexity of a cellular and, ultimately,
an in vivo environment. However, it is clear that the RNA
G-quadruplex motif represents a structurally attractive
scaffold for small molecule targeting and given the
promising early insights into their functional effects, this
represents an attractive and fertile area for future research.
that small molecule
We thank Dr David Tannahill for critical reading of this
manuscript and Dr Pierre Murat for assistance with
G-quadruplex is funded by the Biotechnology and
Biological Sciences Research Council of the UK and
Cancer Research UK. Funding for open access charge:
Cancer Research UK.
in Balasubramanianlaboratoryon RNA
Conflict of interest statement. None declared.
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