Journal of Biomolecular
Screening
http://jbx.sagepub.com/
A Review of Methods to Monitor the Modulation of mRNA Stability : A Novel Approach to Drug Discovery
and Therapeutic Intervention
Dominique Cheneval, Tania Kastelic, Peter Fuerst and Christian N. Parker
J Biomol Screen 2010 15: 609 originally published online 10 May 2010
DOI: 10.1177/1087057110365897
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Review Article
A Review of Methods to Monitor the Modulation of mRNA
Stability: A Novel Approach to Drug Discovery
and Therapeutic Intervention
Dominique Cheneval1, Tania Kastelic1, Peter Fuerst2, and Christian N. Parker2
Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism
for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the
maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the
metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific
control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key
regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human
diseases, including cancer, inflammatory disease, and Alzheimer’s. These observations suggest that modulating the stability
of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several
compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and timeconsuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a
high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be
poorly “drugable.” This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on
assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and
issues related to this approach. (Journal of Biomolecular Screening 2010:609-622)
Key words: mRNA stability, ARE elements, assays, cell-based assays, reporter gene assays
T
INTRODUCTION
he physiological levels of proteins are tightly
controlled by various mechanisms. In addition to the
rate of mRNA production (transcription) and protein degradation, mRNA stability and the efficiency of translation are two
important mechanisms controlling protein levels. In fact, posttranscriptional control of gene expression is much more elaborate than was at first thought.1 The regulation of mRNA
turnover is an essential step in determining message abundance
and therefore gene expression. Recently, it has been proposed
that in eukaryotic cells, modulation of mRNA stability by cisacting sequence motifs coordinates the expression of functionally related genes creating “RNA operons.”2 Aberrant mRNA
1
Novation Pharmaceuticals, Inc., Vancouver, BC, Canada.
Novartis Pharma AG, Novartis Institute for BioMedical Research, Center for
Proteomic Chemistry, Basel, Switzerland.
2
Received Oct 6, 2009, and in revised form Dec 21, 2009. Accepted for publication Jan 10, 2010.
Journal of Biomolecular Screening 15(6); 2010
DOI: 10.1177/1087057110365897
turnover leads to altered protein levels, which can dramatically
modify cellular properties. For example, proto-oncogene or
growth factor overexpression is often associated with abnormal
cell proliferation and malignant transformation. Because message turnover is an important component of gene regulation, it
is not surprising to find that the mRNA stability of key growth
regulatory genes is tightly controlled. Starting in the 1980s, the
control of mRNA stability has been shown to be a significant
regulatory mechanism involved in the expression of inflammatory cytokines, growth factors, and certain proto-oncogenes.3-5
For reviews see Ross,6 Wilusz et al.,7 Malter,8 Audic and
Hartley,9 Hollams et al.,10 and Khabar.11
The regulation of mRNA stability is complex and can involve
elements in both the coding and the untranslated regions of an
mRNA transcript. Regulation of mRNA stability appears to
depend on the interaction between intrinsic, cis-acting elements
and trans-acting factors (Fig. 1). The cis-acting elements consist
of either highly conserved primary sequences12-15 or stable stemloop structures.16 The best characterized cis-element is the socalled Shaw-Kamen box, AUUUA motif, a minimal AU-rich
element (ARE) that is found in the 3′-untranslated region (3′UTR)
of approximately 5% to 8% of mammalian gene transcripts.17
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Cheneval et al.
5’ cap
7-me
GpppG
mRNA
Stabilized mRNA
CRD
3’ polyA tail
AUUUA
AU-bp
Stability control
elements can be
found 5’, 3’ and in
the coding region
AAAAAAAAAAA
polyA BP
Binding proteins / miRNA
e.g. HuR, AUF1, TTP, CRDBP, miRNA16
nucleolin YB-1
7-me GpppG
CRD-BP
HuR
polyA BP
AAAAA n
ARE
e.g. Stemloops, CRD , ARE
AUF-1
miRNA
BRF1
TTP
Fig. 1. Cis-acting elements and trans-acting factors governing
mRNA half-life. ARE, AU-rich elements; CRD, coding region
instability determinant.
AREs have been classified in a number of different ways depending on their different sequence arrangements.18 In contrast, isolated
AUUUA sequences may have other regulatory functions, for
example, in translation and mRNA localization.19,20 According to
some reports, to function as an instability determinant, the AUUUA
motifs must be arranged in tandem, forming at least one
UUAUUUAU/AU/A element.21,22
A number of trans-acting regulatory factors, including several mRNA binding proteins and some miRNAs, play a role in
regulating mRNA stability. A number of these regulatory factors are ARE binding proteins; their binding to mRNA has been
show to either stabilize or destabilize their RNA targets dependent on cellular conditions.23 Based on their patterns of activity,
these proteins have been tentatively assigned as stabilizers,24,25
destabilizers,26-28 or nucleocytoplasmic transporters of AUUUAcontaining mRNAs.29 The proto-oncogene c-fos mRNA, for
instance, has a half-life of approximately 30 min, and its instability is modulated by sequences within its 3′UTR. Replacement
of the 3′UTR from c-fos with the 3′UTR sequences from
β-globin, which has a half-life of approximately 24 h, produces
a stable chimeric transcript. Conversely, fusion of the c-fos
3′UTR to β-globin confers instability.30
A number of signal transduction processes have been associated with changes to mRNA stability. Such changes in mRNA
stability appear to be due to alterations in the affinity of RNA
binding proteins for their cognate mRNA (see Fig. 2). For
example, interleukin-2 (IL-2) mRNA in T cells is inherently
unstable due to the presence of AREs in the 3′UTR. Upon T
cell activation, IL-2 mRNA is stabilized via the c-Jun aminoterminal kinase (JNK) pathway.31 It has been proposed that a
JNK-activated stabilization factor acts through a JNKresponsive element (JRE) by the binding of two JRE binding
proteins, nucleolin and YB-1.32 Other cytokines, which also
contain AU-motifs, such as interleukin-6 (IL-6) and interleukin-8 (IL-8), are not affected by the JNK pathway.33 Instead,
the cytokine-induced stabilization of these transcripts requires
the activation of MEKK1, MKK6, or MK2 of the p38 mitogenactivated protein kinase (MAPK) pathway.34 For example,
the p38-MAPK/MK2 kinase cascade inhibits tristetraprolin
Destabilized mRNA
Fig. 2. Examples of control mechanisms influencing mRNA stability. YB-1, Y-box binding protein 1; CRD-BP, coding region instability
determinant binding protein; TTP, tristetraprolin; AUF1, AU-binding
protein (see references 31-44).
(TTP)–mediated degradation of ARE-containing transcripts
such as tumor necrosis factor α (TNFα) mRNA, thereby leading to TNFα mRNA stability and contributing to increased
TNFα production.35 This pathway is also implicated for protooncogenes such as bcl-2 and c-myc.36 In fact, Stoecklin et al.37
clearly established the ARE as an anti-oncogenic target, showing the importance of mRNA stabilization in oncogenesis and
that tumor suppression can be achieved by interfering with
mRNA turnover.
The heat shock–ubiquitin–proteasome pathway appears to
be associated with AUF1-mediated mRNA regulation in
another ARE-containing transcript, the granulocyte macrophage colony stimulating factor (GM-CSF).38 AU-motifdependent mRNA decay can thus be transiently inhibited by the
activation of the JNK and p38 MAP-kinases pathways. Once
the activation signal has disappeared, the AU-motif ensures
rapid return to a low level of basal expression. Thus, AU-binding
proteins act as mediators between the signaling cascade and the
mRNA degradation machinery.
Recently, it has become apparent that, at least in the case of
AU-mediated mRNA stability, a third player in the form of
microRNA (miRNA) is at work modulating mRNA half-life.
Jing et al.39 have reported that miR16 is involved in regulating
TNFα mRNA stability through interaction with RNA-induced
silencing complex (RISC) and TTP. The effect of miR16 is to
allow sequence-specific targeting of the RISC complex to the
ARE. On the other hand, the TNFα ARE was also reported to
enhance translation upon serum starvation, an effect that was
coupled to two microRNP-related proteins (FXR1 and AGO2),40
thus connecting two posttranscriptional regulatory mechanisms
and perhaps explaining why under some experimental conditions, mRNA degradation appears to be linked to ongoing
translation. This is further corroborated by the fact that AUF1
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Methods to Monitor the Modulation of mRNA Stability
has been reported to be involved in regulating translation but
not mRNA stability via the AU-motifs of bcl-2 mRNA.41
miRNAs also appear to be associated with, and interact
indirectly through, AGO2 and Dicer with AU-binding proteins to prevent translational initiation and induce mRNA
degradation.42 Furthermore, decapping and deadenylation
are promoted by the GW182 protein, which is also involved
in miRNA-mediated gene silencing. This protein is localized to Processing Bodies (P-Bodies), bringing together
Argonaute (AGO)–containing RISCs and mRNA decay
enzymes. miRNAs have also been implicated in directing
rapid deadenylation of mRNA.43,44 These examples demonstrate that there is growing evidence that posttranslational
regulation of gene expression is highly complex, allowing a
coordinated regulation by influencing mRNA stability and
translational efficiency.
In various disease states, mRNA half-life and the levels of
disease-related factors are altered due to aberrant mRNA stabilization. A number of human pathologies associated with
altered mRNA stability have recently been reviewed in the
context of ARE elements by Khabar.11 Other pathologies associated with RNA processing have also been identified. For
example, cystic fibrosis and muscular dystrophy can both be
caused by premature termination codons present in their coding
sequences, which leads to nonsense-mediated decay (NMD) of
their mRNAs.45,46 A number of examples of disease-relevant
genes that are regulated at the level of mRNA stability are
shown in Table 1.
Therapeutic approaches for the modulation of mRNA stability may occur at a number of different levels. The possibility
of directly altering protein-RNA interaction, although conceptually the simplest way to alter mRNA stability, seems the
most unlikely mechanism by which a small molecule would be
able to influence such a system. The reason for this is that
protein-protein interactions have proven difficult to inhibit by
small molecules because of the tendency for such interactions
to occur over large surfaces of the proteins with generally
hydrophobic and van der Waals interactions. It might be
expected that similar interactions will drive RNA-protein
interactions.47 There are a number of examples of small molecules that alter the interaction of proteins with an RNA molecule by altering the conformation of the RNA such that the
protein of interest can no longer recognize the RNA molecule.
Examples of small molecules binding RNA and altering its
biological activity include tRNA and rRNA with aminoglycosides,48 small-molecule interaction with iron response elements
(IRE) sequences altering structure,49 and studies from the
Dervan group50 with molecules able to interact with helical
nucleic acid sequences. However, we believe that the most
likely mechanism for small molecules to modulate the stability
of specific mRNAs is by altering the activity of proteins
that interact with target mRNA or by interfering with signal
Table 1. Examples of Disease Targets for Which Evidence
Exists Showing That Their Transcripts Are Regulated at the
Level of mRNA Stability and May Represent Valid
Drug Targets
Therapeutic Area
mRNA Target
Inflammation/transplantation
IL-1β
TNFα
IL-8
E-selectin, ICAM,
VCAM
MIP2α (Gro-β)
GM-CSF
CCL4 (MIP1β)
CCL3 (MIP1α)
CCL2 (MCP-1)
CCL8
IL-10RA
IL-23A
IL-2
IL-6
uPAR
iNOS
TCR
bcl-2
c-myc
VEGF
DNMT1
K-Ras
MDR1
β-catenin
IL-3
APP
Hsp70
β-adrenergic receptor
PTP1B
SCD1
GLUT1
CCR5
Oncology
Neurodegeneration/regeneration
Cardiovascular diseases
Diabetes/obesity
Infectious diseases
Reference
63
96
95
63
95
95
95
95
95
95
95
95
31
63
97
98
99
89
100
101
102
103
104
105
106
107
108
109
110
111
112
113
transduction pathways governing mRNA stability or translatability. A number of such compounds that have been described
having such an effect will be discussed.
The primary aim of this review is to discuss the methods
available for detecting mRNA levels and thus screening for
such molecules.
Current Screening Methods to Monitor
mRNA Stability
A number of methods are available for the detection and
quantification of mRNA present in a cell sample. Traditional
methods such as Northern blots, RNase protection, or slot blots
have the disadvantage that they only measure either one or only
a limited number of transcripts at a time and are very difficult
to automate. Newer techniques for analysis of gene expression
include: (a) unbiased, open systems such as serial analysis of
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Cheneval et al.
gene expression (SAGE),51 differential display (DD) analysis,52
RNA arbitrarily primer (RAP)–PCR,53 restriction endonucleolytic analysis of differentially expressed sequences (READS),54
amplified restriction fragment-length polymorphism (AFLP),55
total gene expression analysis (TOGA),56 and the quantitative
multiplex RT-PCR method, Standardized RT-PCR (StaRTPCR)52; and (b) focused, closed systems such as high-density
cDNA filter hybridization analysis: (HDFCA), suppression
subtractive hybridization (SSH), differential screening (DS),
cDNA arrays, or oligonucleotide chips and tissue microarrays,
methods that have been reviewed previously.52
Although closed systems are excellent for the initial screening of a large number of gene transcripts, the value of the
information generated is biased because the techniques are
generally limited to an often arbitrarily chosen set of known
sequences. Only an open system or platform has the potential
to evaluate the expression patterns of tens of thousands of
genes that have not yet been cloned or partially sequenced in a
quantitative and unbiased manner.
Low-throughput methods
Methods to measure abundance of specific mRNA sequences
are essential prerequisites to monitor stability of a specific
transcript. The first method used for quantifying mRNA levels
was with Northern blots. In such experiments, the level of an
RNA transcript could be quantified either when the RNA is in
a steady state or after the inhibition of RNA synthesis, by
actinomycin D or alpha-amanitin, to measure the half-life of
specific mRNAs.
The development of reverse transcription–PCR (RT-PCR) has
generally become accepted as the new “gold standard” for measuring the relative abundance of RNA transcripts, with the availability of kits, automated PCR machines, and the ability to
multiplex, allowing the convenient normalization of transcript
numbers to internal controls (usually a housekeeping gene such
as GAPDH or absolute quantification using an amplicon).
Although Northern blots and RT-PCR allow the abundance of
specific transcripts to be measured, they do not allow a global
assessment of the effect of a treatment on mRNA levels in a cell.
Methods that allow a global assessment of transcript abundance
include array methods where oligonucleotides or cDNAs are
immobilized on a solid support, and SAGE. Array methods
require amplification and labeling of the transcripts, allowing
relative/comparative assessments of transcript abundance. Array
methods, however, retain some bias as they depend on design,
number of genes selected, controls, and gene sequences. SAGE
offers a completely unbiased analysis of the entire genome but is
very work intensive, time-consuming, and expensive.
The strength of these methods lies in their ability to monitor,
in a comprehensive manner, the changes in mRNA abundance
to as wide a number of transcripts as possible. Such a transcriptome-wide analysis then allows assessment of the global effects
of a treatment on a cell, be it by agents that stimulate the cells
such as growth factors, antigens, stress, or low molecular
weight compounds. Technology development in the field of
“deep sequencing” means that it is now possible to use such
technology for the sequencing and even quantification of a
comprehensive portion of the mRNA transcriptome.57 Recently,
a method to allow comparison of the effects of treatments on
the transcriptome to be made has been published.58
Higher throughput assay methods
Biochemical approaches. Such biochemical systems must
all include: (a) in vitro transcription of the target mRNA; (b) a
cell-free extract that provides the proteins necessary for translation, an important factor controlling mRNA stability, as well as
other factors required for mRNA degradation or stabilization;
and (c) a means to measure the levels of RNA, even those in a
specific conformation. Such biochemical methods allow faster
and thus higher throughput analysis using standard biochemical readouts used in high-throughput screening (HTS) labs
(such as fluorescence polarization [FP] or time-resolved fluorescence resonance energy transfer [TR-FRET]). An example
of a cell-free system was recently described by Meisner et al.59
Here we describe a minimal primary consensus sequence
together with the conformation-determining flanking sequence
of an mRNA stability region by first defining a single-stranded
mRNA accessibility sequence. By elongating the oligonucleotide up- and downstream of the core, they reduced the accessibility of the RNA binding site by modulating the secondary
structure of the binding motif without changing the core
sequence. It has been possible to define “opener” and “closer”
oligonucleotides for manipulating the accessibility of the
mRNA binding protein HuR. These oligonucleotides result in a
change in conformation, either allowing or preventing the protein from binding, thereby mimicking the events in mRNA
stability regulation. The cell-free system thus described could
potentially also be used to screen for small molecular weight
compounds that mimic the effect of these openers or closers,
allowing one to target a large number of disease-relevant genes
by using a common assay format.
The throughput of such biochemical assays is usually not
limited, as the assays can readily be miniaturized to at least
384- or 1536-well formats such that reader capacity becomes
the limiting step (with throughput of approximately 200, 1536well plates per day being possible). The real limiting factor
with such assays is the provision of assay reagents and the
stability of RNA as a reagent (due to the ubiquitous nature of
RNases in the environment).
Cell-based approaches. The most common way to determine if mRNA sequences present in a transcript are responsible
for conferring mRNA stability is to create chimeric gene
sequences, commonly fused to a stable gene such as β-globin,
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Methods to Monitor the Modulation of mRNA Stability
and to then monitor transcript levels after inhibition of transcription.22 The abundance of the RNA transcripts is then commonly monitored by Northern blots60 or by RT-PCR61 following
the inhibition of transcription. However, such analysis of RNA
levels is not suitable for rapid HTS.
A number of reporter gene-based assays have been developed
to address this limitation and can be used for the discovery of
novel compounds affecting mRNA stability/translatability. One
of them is described by Kastelic and Cheneval.62 The objective
of this assay system is to exploit earlier findings that it is possible
to modulate mRNA levels with low molecular weight compounds.63 Because AREs and other mRNA stability elements
appear in a number of mRNAs known to be relevant in several
human diseases, it allows this approach to be expanded into a
platform technology that exploits the presence of stability determining elements. This approach was also recently suggested by
Benjamin et al.64 and shown to be suitable for screening.
The throughput of such assay systems in a well-equipped
HTS lab, even using a workstation approach, in our experience,
is limited to approximately 200 plates per day per full-time
equivalent (FTE), 3 times a week. The use of frozen cells65 can
increase the number of times a week an assay can be run.
Miniaturization to 1536-well formats is usually possible, and
so the bottleneck for performing screening campaigns with this
assay format is often that of cell line generation.
These systems allow screening for compounds that modulate reporter gene expression through mRNA sequence motifs
in a high-throughput format. The stability (half-life) of a target
mRNA sequence motif in the assay is monitored through the
expression and activity of a reporter gene. The reporter construct, having been designed to include stability sequences of
the target mRNA present in appropriate regions of the reporter
construct, is stably transfected into a disease-relevant cell line.
Because the aim of this system is to find compounds affecting
mRNA stability, or indeed translation, through stability regulatory motifs, a control is built into the same cell. The control, a
second identical expression plasmid that expresses a different
reporter gene but does not contain any mRNA stability motifs,
is simultaneously introduced into the cell. Thus, control and
motif-specific measurements can be carried out at the same
time in the same cell. This enables specific selection of compounds with effects dependent only on the motif introduced.
Other groups66-70 have described similar systems with the
aim of facilitating compound characterization. For example,
the GEMS (Gene Expression Modulation by Small Molecules)
system has been described by PTC Therapeutics.71
Paillusson et al.72 have developed a reporter system that
mimics nonsense-mediated mRNA decay by constructing a T
cell receptor minigene in which the green fluorescent protein–
open reading frame (GFP-ORF) was inserted such that the stop
codon acts as a premature stop codon (PTC). The authors then
show that eliminating essential NMD factors resulted in an
increase in GFP fluorescence, so that the system faithfully
reflects NMD. The system, however, does not rule out other
effects such as enhanced transcription or translation, or inhibition of general mRNA turnover.
Boelz et al.73 describe a cell-based chemiluminescence
reporter system to monitor NMD. Using a dual-luciferase
approach, the authors introduced a PTC into the 3′ region of a
renilla luciferase/β-globin fusion gene. Co-transfection of this
construct with a control firefly luciferase expression vector
allows for normalization and overcomes some of the drawbacks of using co-reporters such as CAT, β-gal, and GFP, which
have different chemistry, handling, or instrumentation needs.
Furthermore, this study showed that the luminescence measured faithfully reflected mRNA levels.
The Quanti Gene™ nucleic acid quantification kit as used
by Warrior et al.74 employs a branched DNA (bDNA) technology to measure mRNA directly from cells. Unlike PCR, which
employs target amplification, this method relies on signal
amplification for detection. Using a “sandwich” nucleic acid
hybridization procedure, direct measurement of mRNA from
crude cell lysates is possible. Target-specific capture extenders
and label extenders are added to cell lysates, which allow the
tethering of the target mRNA to the assay plate and subsequent
hybridization of the bDNA amplifier. The branches of the DNA
amplifier are then labeled with alkaline phosphatase, providing
the amplification step for the assay. This approach has the
advantage that it is easy and fast to develop compared to
reporter gene assays, which optimally require stably transfected cell lines, result in more physiologically relevant data,
and can be used with any cell type, including primary cell lines.
However, the assay itself is a multistep, complex, and nonhomogenous process with a lower throughput than for standard
reporter gene assays. In the experience of the authors, such
assay formats are limited to 384-well plate formats (because of
the need for liquid transfer of the cell lysate to the capture
plate). Because of the multiple assay steps, the throughput is
limited to processing one hundred to two hundred 384-well
plates a day, for one researcher. As with reporter gene assays,
the need to seed and treat cells in the plates limits the number
of days such an assay can be run to 3 to 4 days a week.
Martel et al.75 have taken RNA analysis one step further by
introducing the multiplexed Array Plate mRNA assay. In this
approach, cells grown and subjected to compound treatment in
96-well plates were subjected to an in situ nuclease protection
assay against a set of predefined targets. Processed cell lysates
were then transferred to a microplate that contained a predefined oligonucleotide array that separated and immobilized the
assay probes. Quantitative detection of array-bound probes was
by enzyme-mediated chemiluminescence. In this way, effects
of compounds on the mRNA levels of several genes can be
determined. Although this represents a step up compared to
reporter gene assays with respect to higher content, the method
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remains biased to the choice of the predefined targets and, with
multiple assay steps, will remain a challenge to automate for
true HTS.
DNA microarray assays provide a further step in the direction of transcriptome-wide quantitative analysis of mRNA
levels and the understanding of gene expression. Canales
et al.76 compared microarray results with other quantitative
gene expression platforms. The methods used were TaqMan, an
assay using RT-quantitative real-time PCR,77-79 which has also
been used to validate microarray data earlier,80-82 and standardized (Sta)RT-PCR, as described by Willey et al.,83 which is a
competitive PCR-based method that measures the amount of a
PCR end-product. After conversion of mRNA to cDNA using
reverse transcriptase and either random or gene-specific oligonucleotides, hexamers, or oligo dT, the cDNA is added to
standardized mixtures of competitive templates and dispensed
into microplate wells containing gene-specific primers. The
end-products are separated and quantified by high-throughput
microfluidic electrophoresis. This method has also been used to
validate microarray data84 and the QuantiGene System
described above by Warrior et al.74 They observed a high correlation between quantitative gene expression values and
microarray results, with only few differences among all platforms. The microarray systems were less sensitive, and detection levels varied only for lower expressed genes. Overall,
there was an excellent correlation between each method tested,
and it was concluded that the microarray data were validated by
alternative quantitative gene expression platforms, supporting
the use of microarray platforms for the quantitative characterization of gene expression.
Application to other targets such as miRNA
Genetic screens to identify genes modulated by miRNA
activity have also been reported and are conceptually similar to
the some of the assay systems reported for monitoring mRNA
stability. By introducing the miRNA binding site from the UTR
of the targeted gene into a suitable reporter gene, it is possible
to monitor the effect of miRNAs whether they act at the level
of mRNA stability or to alter the level of translational efficiency.85,86 In fact, the availability of such assay systems has
already allowed the description of small molecules interacting
to modulate the activity of miRNAs.87,88
Tool Compounds Described in the
Literature
A number of compounds have already been described as
modulating mRNA stability and thus can act as possible control
compounds for developing assays to monitor this process.
Possibly the first example of such an effect was the fungal
metabolite radicicol analog A (RAA) that was shown to
induce a rapid degradation of IL-1β, TNFα, IL-6, and Cox-2
transcripts in stimulated THP-1 cells mediated by the presence
of AREs in the target mRNA’s 3′UTR.63 This discovery demonstrated that mRNA stability can be influenced with small
molecular weight compounds. Other molecules also reported to
influence mRNA stability include paclitaxel,89 cyclosporin A,90
thalidomide,91 and glucocorticoids92 (see Table 2). Recent
reports describe how kinase inhibitors of Chk2 can also alter
the stability of Sirt1 mRNA.93 Such control compounds can
thus be used during assay development to either optimize the
assay signal window or, to at least monitor the selectivity of a
given assay.
Specificity of the assay
Compound specificity is a key consideration when identifying molecules that can modulate a specific target mRNA stability. In addressing this subject, however, it is important to
distinguish between two different issues: (1) the specificity of
the cellular regulatory mechanism that governs modulation of
a target mRNA (thus leading to construction of an assay) and
(2) the inherent specificity of compounds identified through
such an assay (which is an issue for the pharmaceutical development of any compound).
Regulation of mRNA stability is also cell type specific.
Certain mRNAs are unstable in one cell type but not in another.
For example, cell-specific posttranscriptional regulation of
CFTR (cystic fibrosis transmembrane conductance regulator)
mRNA has been reported.94 In a 3′UTR-dependent manner,
TNFα decreased CFTR mRNA in human HT-29 colon cells but
not in pulmonary Calu-3 cells, confirming that in this example,
regulation of mRNA stability is cell type specific. Therefore, as
mRNA regulation is cell type specific, it is possible for small
molecular weight compounds to act differently on the same
transcript depending on the cell type in which it is expressed.
Specificity of the compounds
Examples of molecules able to selectively modulate specific
mRNAs exist. Mak et al.95 reported RAA to be highly selective
for altering the stability of mRNAs from a family of cytokines.
In researching the mechanism of action of RAA, a SAGE
analysis showed that of approximately 18,000 genes reviewed,
only 34 were downregulated by RAA, and of these, only 11, all
having an ARE in their 3′UTR, were destabilized when validated by RT-PCR. All 11 of these confirmed mRNAs were
related proinflammatory chemokines. Of note is the observation that other mRNAs, also containing AUUUA motifs, were
not affected. This suggests that RAA was highly selective for
the related mRNAs and was able to discriminate between transcripts having a similar instability motif.
Further evidence that specificity of mRNA destabilizing
compounds identified is dependent on the class of compound,
as well as the cell type or disease model being studied, has been
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Table 2. Compounds Known to Influence mRNA Stability
Pharmacological compound
Target mRNA
Aspirin
O
Mechanism
Reference
Increase in COX2 mRNA stability
p38 activation
Mifflin et al.114
Decrease in IL-3 mRNA stability
3′UTR/FKBP12/calcineurin
Nair et al.90
Decrease in IL-3 mRNA stability
3′UTR/FKBP12/calcineurin
Banholzer et al.106
Decrease in IL-3 mRNA stability
3′UTR/FKBP12/calcineurin
Ing115
Stabilized in low-density lipoprotein
receptor mRNA
ERK-dependent signaling pathway through
3′UTR
Kong et al.,116 Abidi
et al.117
Decrease in bcl-2 mRNA stability
Downregulation or inactivation of nucleolin
(bcl-2 mRNA destabilizing mRNA binding
protein)
Otake et al.118
Decrease in bcl-2 mRNA stability
Downregulation or inactivation of nucleolin
(bcl-2 mRNA destabilizing mRNA binding
protein)
Otake et al.118
Decrease in bcl-2 mRNA stability
Downregulation or inactivation of nucleolin
(bcl-2 mRNA destabilizing mRNA binding
protein)
Otake et al.,119
Bandyopadhyay et al.89
Increase in eNOS mRNA stability
ROCK inhibitor
Rikitake et al.120
OH
O
O
CH3
Cyclosporin A
H 3C
CH 3
H3C
HO
O
CH 3
H3C
N
N
H 3C
CH 3
N
H
CH 3
CH 3
N
O
N
H 3C
H
N
O
CH 3
O
O
CH 3
CH 3
O
N
CH 3
H
N
N
CH 3 O
CH 3
O
H 3C
O
CH 3
H3C
O
CH 3
CH 3
H
N
N
CH 3
O
CH 3
Rapamycin
O
O
N
O
HO
O
OH
O
O
O
O
O
O
FK 506 (tacrolimus)
HO
MeO
O
O
N
O
HO
O
OH
O
O
OMe OMe
Berberine
O
O
N
MeO
+
OMe
All-trans retinoic acid
O
OH
Okadaic acid
C H3
OH
O
O
HO
H3 C
OH
O
O
C H2
O
O
OH
O
C H3
O
OH
C H3
Taxol
O
O
O
OH
H3C
H
O
O
N
H
O
OH
O
H
O
OH
O
O
CH3
O
Hydroxyfasudil
H
N
N
O
S
O
NH
HCl
O
(continued)
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Cheneval et al.
Table 2. (continued)
Pharmacological compound
Target mRNA
Y27632
N
Mechanism
Reference
Increase in eNOS mRNA stability
ROCK inhibitor
Rikitake et al.120
Decrease in tumor necrosis factor α
(TNFα) mRNA stability
3′UTR mediated
Moreira et al.,91 Fujita
et al.,121 Zhu et al.,122
Rawlins et al.123
TNFα
Induction of tristetraprolin
Smoak and Cidlowski124
APP
5’UTR mediated
Tucker et al.125
APP
5’UTR mediated
Tucker et al.125 APP
5’UTR mediated
Tucker et al.125 O
N
H
2HCl
CH 3
H
NH 2
Thalidomide
O
O
H
N
N
O
H
O
Glucocorticoids
OH
O
HO
OH
F
O
Paroxetine
F
O
O
O
N HCl
H
Erythromycin
O
OH
OH
OH
H
O
O
O
OMe
O
O
NMe2
O
OH
O
N-acetyl cysteine
O
HS
OH
O
HN
RAA
Decrease in IL-β and IL-6 mRNA stability 3′UTR mediated
OH
O
O
Kastelic et al.,63 Mak
et al.95
OH
O
O
O
OH
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Journal of Biomolecular Screening 15(6); 2010
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Methods to Monitor the Modulation of mRNA Stability
provided by Nair et al.90 Another example is cyclosporin A,
which has been reported to destabilize interleukin-3 (IL-3)
mRNA in an autocrine tumor cell line by acting through the
AUUUA instability motif in its mRNA. However, it shows no
effect on destabilizing IL-1β mRNA in monocytes that also has
the AUUUA motifs. RAA, on the other hand, destabilized
IL-1β mRNA in monocytes acting through AUUUA but had no
effect on IL-3 mRNA in mast cells or macrophages (unpublished results). This further demonstrates that low molecular
weight compounds from different classes, although affecting
mRNA stability through the same minimal stability regulatory
mechanism, are dependent on the compound class as well as
the cell type or disease. Clearly, different compounds affect the
regulation of mRNA stability differently, and the choice of cell
line for assay construction is important for a given target and
should be disease relevant and express the target of interest—
points crucial for ensuring the identification of disease-relevant
and selective compounds using an mRNA stability screen.
Considerations for Assay Design
Assay construction involves the introduction of a reporter
gene construct containing mRNA stability control elements
(SCE) of the selected target into an appropriate cell line.
Construction of each assay thus should make use of the unique
SCE, its configuration, and the use of a relevant cell type, so
that each assay is specific and unique, with respect to the
genome, for each mRNA to be targeted.
To capture the complexity of mRNA modulation, cell-based
assays should be developed in a cell line relevant for the disease of interest as cell type, cellular stimulation, the presence
or absence of mRNA binding proteins, miRNA, and other factors influence mRNA profiles. Such cell-based assays have
clear advantages above cell-free assay systems. A significant
advantage of such functional assays is that they can identify
any molecule that has a desired effect on the target of interest
irrespective of mechanism of action.
However, to assess the specificity of hits coming from an
initial screen against a given target in a given cell line, these
hits should undergo further evaluation, including comparison
to a co-transfected control plasmid, transfection of the target
into multiple pertinent cell lines for cross-comparison, and
transfection of multiple target constructs into one cell line for
cross-comparison and determination of selectivity. The latter
two comparison steps, together with the initial screening assay,
result in compounds that are selective for one target and do not
affect other targets or cells not relevant to the target of interest.
Selected compounds should then undergo cytotoxic assays,
measurement of their effect on endogenous mRNA levels,
protein-level expression, and DNA microarray to assess potential genome-wide effects on mRNA levels and provide further
assurance on specificity.
Cell-based mRNA stability assays have the advantage that
they are able to identify compounds that modulate mRNA levels through a number of possible mechanisms. These could
include direct interaction of the compound with the mRNA,
interference of the compound with protein-mRNA interaction,
and/or influencing processes that govern the modulation of
mRNA stability such as signal transduction, enzymatic mRNA
degradation, or cotranslational mRNA degradation. Although
all “effective” regulatory domains engineered into mRNA stability assays are unique, further steps should be taken to ensure
that selected compounds will be specific for the intended
mRNA target. A built-in control allows compounds that do not
work through the SCE to be identified and eliminated as false
positives. Only compounds specifically influencing the reporter
containing the SCE are considered because the activity of the
compound is dependent on the presence of the SCE introduced.
The use of counterscreens will further confirm specificity
and identify compounds that are highly selective for the mRNA
of interest, but have no activity against other mRNAs screened.
Hits identified with one mRNA stability screen are counterscreened against other mRNA stability assays. In this way, the
identification of compounds that may affect targets other than
the one of interest is minimized. As more screening campaigns
are run using a given compound library, compounds repeatedly
identified as active, and therefore not specific, can be eliminated from future screens. In this way, identification of highly
selective compounds becomes faster as the information generated for each compound in a library becomes cumulative.
The outcome of a primary screen and counterscreens can be
displayed by so-called heat maps, which compare the activities
of a compound on all assays. Data from these heat maps can
identify compound hits that show activity against only one
specific SCE. A schematic hypothetical heat map (Fig. 3)
shows 3 mRNA stability assays screened against the same
library. The fold difference either stimulated (green) or inhibited (red) of the target assay relative to the control assay is
shown for each compound. Therefore, compounds that uniquely
downregulate the signal can be identified specifically for
the mRNA regulatory domain (assay A, for example, has specific hits with compounds 17, 18, and n-2). To maximize information obtained from a screening campaign, a cytotoxicity
assay can be carried out concurrently with the mRNA stability
assay.
Conclusion
Selectivity and specificity is a critical issue when targeting
mRNA. However, a wealth of evidence exists showing that it is
possible to identify small molecules that can specifically modulate the stability of selective mRNA transcripts. Specificity
concerns are common for all compounds, not just those related
to mRNA stabilization. Concern with respect to off-target
Journal of Biomolecular Screening 15(6); 2010 www.sbsonline.org 617
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Cheneval et al.
Therapeutics Inc.), and other companies are selling reagents
that allow the effect of compounds on the stability of specific mRNAs to be monitored (e.g., SwitchGear Genomics
[www.switchgeargenomics.com]). In addition, companies
have started to sell reporter constructs using the presence of
ARE elements to destabilize the reporter RNA so as to make
the reporter gene more responsive to the chosen inducer
used in these reporter constructs (e.g., Promega and New
England Biolabs). Although these are obviously an interesting application of these RNA stability motifs, they do introduce the possibility that reporter constructs designed to
monitor effects on a given promoter will now also detect the
influence of compounds or miRNAs that modulate mRNA
through these ARE elements.
In conclusion, there are now a number of methods for
monitoring mRNA levels, some of which are suitable for HTS.
The availability of compounds reported to alter mRNA metabolism will also aid in the development of assays to identify treatments capable of altering mRNA levels.
Assay
cmpd#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
A
B
C
n-4
n-3
n-2
n-1
n
inhibition
References
stimulation
Fig. 3. Comparison of compounds screened against 3 different
mRNA stability motifs target A, B, or C in different cell lines. The heat
map readout shows compounds that either inhibit (red) or stimulate
(green) the target. Compounds that show stimulation regardless of the
mRNA motif (e.g., compound 1) would be of no further interest.
Similarly, compounds that show inhibition regardless of the motif
(e.g., compound 4) would also be of no further interest. n is the number of compounds screened.
effects is common for all compounds no matter how they are
discovered, and thus effects may be inherent to the structure of
the molecule rather than any mRNA modulating action. This is
a general pharmaceutical development issue. Even though the
precise molecular target and mechanism of action may be
known, unpredicted side effects may still occur. Of greater
concern may be “on-target” side-effects if the molecular target
plays multiple roles. These potential side effects can be evaluated early on through standard parallel screens that can be
incorporated into lead selection. The fact that compounds of
known pharmacological relevance have been shown to affect
the stability of selective mRNAs suggests that even compounds
targeting other aspect of cell physiology might have effects on
transcript stability.
For these reasons, a number of different companies have
started discovery efforts to identify compounds modulating
mRNA stability (e.g., Novation Pharmaceuticals and PTC
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622 www.sbsonline.org
Address correspondence to:
Christian N. Parker
Novartis Institute for BioMedical Research
Center for Proteomic Chemistry
Novartis Pharma AG
CH-4056 Basel, Switzerland
E-mail: [email protected]
Journal of Biomolecular Screening 15(6); 2010
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