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Programmable Ligand-Controlled Riboregulators.

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DOI: 10.1002/anie.200502700
Gene Expression
Programmable Ligand-Controlled Riboregulators**
Ronald Micura*
antisense reagents · aptamers ·
gene expression · riboregulators · RNA
complex issue for all living organisms is gene expression, with hundreds
of genes to be coordinated simultaneously. To handle this challenge, nature
has ingeniously created a great diversity
of different tools and mechanisms for
control and regulation. In this context,
the importance of noncoding RNA
(ncRNA) has become well recognized
in recent years.[1] ncRNAs include cis- as
well as trans-acting RNA elements.
Representatives of the first class are
riboswitches, sequences in the 5’-untranslated regions (5’-UTRs) of mRNA
with structural-recognition elements for
specific metabolites. Binding of these
small molecules leads to alternative
structures that can modulate translation
initiation or transcription termination.[2]
Riboswitches have also been identified
to exhibit ribozyme activity, resulting in
cleavage of mRNA and downregulation
of the corresponding gene.[3] The class of
trans-acting ncRNA elements includes
microRNAs (miRNAs), which interact
with complementary sequences in
mRNA and the genome,[4] small interfering RNAs (siRNAs), which function
through the RNA interference (RNAi)
pathway,[5] and not least, small ribozymes[6] and antisense RNAs.[7]
The prevalence of RNA-based regulators across diverse organisms from
prokaryotes to humans has also stimulated scientists to engineer artificial
[*] Prof. Dr. R. Micura
Center for Molecular Biosciences CMBI
Institute of Organic Chemistry
University of Innsbruck
Innrain 52 a, 6020 Innsbruck (Austria)
Fax: (+ 43) 512-507-2892
[**] The author acknowledges the Austrian
Science Fund (FWF) for generous funding
and Claudia H?bartner for critical reading
of the manuscript.
riboregulator systems. Already in 1998,
it was demonstrated that the insertion of
a small-molecule aptamer (previously
selected in vitro) into the 5’-UTR of a
mRNA allowed its translation to be
repressed by ligand addition in mammalian cells.[8] More recently, in E. coli, a
combined cis/trans system was developed in which the cis-acting RNA elements inhibit translation, but interaction
of the trans-activating RNAs with the
cis-acting elements reinitiates translation.[9] Furthermore, a cis-acting ribozyme-based gene-regulation system that
responds to the presence of exogenous
small molecules has also been reported.[10]
A highly promising new development in the area of engineered riboregulators are so-called antiswitches, introduced by Bayer and Smolke in the
single-cell eukaryote Saccharomyces
cerevisiae.[11] An antiswitch is trans-acting and can, in principle, be designed to
regulate the expression of any target
transcript and to respond to any ligand.
It consists of an aptamer domain that
specifically recognizes the effector molecule (ligand) and an antisense domain
(Figure 1). Importantly, effector binding
induces a conformational change in the
antisense domain, which is then able to
interact with the target mRNA and
consequently modulate its translation.
Bayer and Smolke distinguish two
types of ligand-responsive riboregulators—an “off antiswitch” and an “on
antiswitch”. In the absence of ligand, the
“off antiswitch” RNA sequesters the
antisense sequence partition in an intramolecular manner within a doublestranded region and it is therefore not
available for base pairing with a target
mRNA. Once specific binding of the
ligand occurs at the aptamer domain,
the allosterically induced conformation-
2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
al rearrangement presents the antisense
sequence as a single strand and thus
allows intermolecular base pairing with
the target mRNA. Consequently, the
target gene is downregulated.
In contrast, the “on antiswitch” is
designed with a free single-stranded
antisense sequence to bind a target
mRNA in the absence of the effector
molecule. Upon effector binding at the
aptamer domain, the “on antiswitch”
undergoes a structural rearrangement
that causes the antisense sequence to
form an intramolecular stem loop, rendering the antiswitch RNA inactive.
Thus, the target gene is upregulated.
The antiswitch concept has been
demonstrated with the well-characterized theophylline aptamer[12] and a 15nucleotide antisense sequence, which
undergoes base pairing with the region
around the start codon of a target
mRNA that encodes the green fluorescent protein (GFP). The antiswitch
RNA to be expressed in S. cerevisiae
was cloned between two hammerhead
ribozymes known for efficient selfcleavage in vivo, thus guaranteeing the
creation of RNAs with defined 5’ and 3’
ends. A plasmid containing a yeastenhanced GFP was used to transform
the same cells.
Strikingly, these engineered riboregulators provide sharp transitions between gene-expression states over small
changes in effector concentration. This
binary on/off switch behavior most likely originates from competition between
the aptamer and the antisense sequences for base pairing with the same
internal sequence of the antiswitch.
The great flexibility of the novel
riboregulator framework was further
substantiated by additional antiswitch
variants. Strategically introduced point
mutations direct the switching threshAngew. Chem. Int. Ed. 2006, 45, 30 – 31
Figure 1. Programmable ligand-controlled riboregulators of eukaryotic gene expression. The so called antiswitches are trans-acting ncRNAs that
consist of an antisense domain and an aptamer domain. For the “off antiswitch” (left), the antisense sequence (red) is sequestered in a stem
loop. In the presence of ligand (theophylline; orange), an allosteric conformational change is induced, and the antisense sequence is liberated
and can interact with the target mRNA at the 5’-end, thus suppressing gene expression. For the “on antiswitch” (right), the antisense sequence is
free to bind to its target mRNA and gene expression is repressed. Upon ligand binding, the antiswitch is released from the target mRNA and gene
expression is permitted.
olds as a function of effector concentration. Moreover, Bayer and Smolke
replaced the aptamer domain for theophylline with that for tetracycline and
demonstrated a similar behavior with a
different effector. In addition, they also
showed that two antiswitches with different aptamer domains and different
antisense domains in the same cell
responded independently to different
ligands, each regulating a different
mRNA target.
The novel antiswitches represent a
highly flexible rational concept that
enables both positive and negative regulation of potentially any target gene in
a diverse range of organisms. The consideration of particularly tiny aptamer
domains might have an additional impact on the design of antiswitches. For
instance, Anderson and Mecozzi recently reduced the original theophylline
Angew. Chem. Int. Ed. 2006, 45, 30 – 31
aptamer to a 13-nucleotide RNA and
found that the high affinity and specificity were retained.[13] A combination of
these tiny aptamers and corresponding
antisense domains would result in antiswitch riboregulators of a size comparable to that of siRNAs, with a potentially
expanded scope of regulative applications in gene networks.
Published online: September 20, 2005
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[2] M. Mandal, R. R. Breaker, Nat. Rev.
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[5] “RNA Interference”: D. R. Engelke,
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[6] D. M. Lilley, Trends Biochem. Sci. 2003,
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[13] P. C. Anderson, S. Mecozzi, J. Am.
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2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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riboregulators, programmable, controller, ligand
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