Control of alternative RNA splicing and gene expression by eukaryotic riboswitches

104

124

Riboswitches are cis-acting elements that regulate gene expression by affecting transcriptional termination or translational initiation in response to binding of a metabolite. A typical riboswitch is made of an upstream aptamer domain and a downstream expression platform. Both domains participate in the folding and structural rearrangement in the absence or presence of its cognate metabolite. RNA polymerase pausing is a fundamental property of transcription that can influence RNA folding. Here we show that pausing plays an important role in the folding and conformational rearrangement of the Escherichia coli btuB riboswitch during transcription by the E. coli RNA polymerase. This riboswitch consists of an approximately 200 nucleotide, coenzyme B12 binding aptamer domain and an approximately 40 nucleotide expression platform that controls the ribosome access for translational initiation. We found that transcriptional pauses at strategic locations facilitate folding and structural rearrangement of the full-length riboswitch, but have minimal effect on the folding of the isolated aptamer domain. Pausing at these regulatory sites blocks the formation of alternate structures and plays a chaperoning role that couples folding of the aptamer domain and the expression platform. Pausing at strategic locations may be a general mechanism for coordinated folding and conformational rearrangements of riboswitch structures that underlie their response to environmental cues.gene regulation | translational control | metabolism R NA structures fulfill important roles in the regulation of gene expression including the control of translational initiation, transcriptional termination, and alternative splicing (1-5). In many cases, an RNA conformational change is crucial to gene regulation since one RNA sequence may adopt alternate structures. Regulation of gene expression is achieved when an input domain senses varying cellular conditions, resulting in shifting the balance between two or more structural states.A prime example of gene regulation by RNA conformational changes in bacteria is riboswitch control. Riboswitches are cisacting elements that regulate gene expression in response to the concentration of an intracellular metabolite. The coenzyme B12 riboswitch is located in the 5′ untranslated region of the btuB gene which encodes an outer membrane transporter for B12 (6) (Fig. 1A). The btuB riboswitch consists of two domains: an upstream coenzyme B12 binding aptamer and a downstream expression platform. When the cellular concentration of coenzyme B12 is high, this metabolite binds to the aptamer, reconfiguring the expression platform to form a structure in which the ribosome binding site (RBS) is base paired and inaccessible. When the concentration of coenzyme B12 is low, the mRNA forms an alternate structure which allows for translation (6, 7).RNA folding and conformational switching occurs during transcription in vivo. Folding during transcription is particularly important for riboswitch regulation (8). Although bacterial RNA ...

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confidence: 99%

Transcriptional pausing coordinates folding of the aptamer domain and the expression platform of a riboswitch

Perdrizet

Artsimovitch

Furman

et al. 2012

104

124

“…Translational riboswitches usually function via sequestration of the ribosome-binding site (Shine-Dalgarno (SD) sequence), thereby blocking translation initiation in a metabolitedependent manner. The increasing understanding of the functional principles of bacterial riboswitches and the possibility to produce novel aptamers by in vitro evolution of RNA molecules has facilitated attempts to design synthetic switches (10-12), which potentially can provide versatile tools for genetic engineering and synthetic biology applications.While, over the past years, numerous riboswitches have been discovered in prokaryotes (13) and also a few in eukaryotes including plants (14,15), no riboswitches are known in organellar (plastid and mitochondrial) genomes. Plastids (chloroplasts) and mitochondria are derived from formerly free-living bacteria and have retained a largely prokaryotic gene expression machinery.…”

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confidence: 99%

“…While, over the past years, numerous riboswitches have been discovered in prokaryotes (13) and also a few in eukaryotes including plants (14,15), no riboswitches are known in organellar (plastid and mitochondrial) genomes. Plastids (chloroplasts) and mitochondria are derived from formerly free-living bacteria and have retained a largely prokaryotic gene expression machinery.…”

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confidence: 99%

Inducible gene expression from the plastid genome by a synthetic riboswitch

Verhounig

Karcher

Bock

2010

120

Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology.chloroplast | plastid transformation | translational regulation R iboswitches are natural RNA sensors that mediate control of gene expression via their capacity to bind small molecules (metabolites). They fold into RNA secondary structures whose conformation switches between an "on" state and an "off" state in response to ligand binding (reviewed, e.g., in refs. 1-4). Riboswitches can be divided into two distinct structural domains: an aptamer and an expression platform. The aptamer domain binds the metabolite and this triggers conformational changes in the expression platform, which either permit or prevent gene expression. Depending on the nature of the response to metabolite binding, "on" switches and "off" switches are distinguished. In bacteria, riboswitches reside mainly in the 5 0 untranslated regions (UTRs) of mRNAs and an emerging theme is that anabolic genes are mainly controlled by "off" switches (5-7), whereas catabolic genes are controlled by "on" switches (8), thus providing an efficient mechanism of feedback control of metabolic pathways. In bacteria, most riboswitches act at the transcriptional level [e.g., by resolution of rho-independent transcription termination hairpins (8, 9)], but a few translational switches have also been identified (5, 7). Translational riboswitches usually function via sequestration of the ribosome-binding site (Shine-Dalgarno (SD) sequence), thereby blocking translation initiation in a metabolitedependent manner. The increasing understanding of the functional principles of bacterial riboswitches and the possibility to produce novel aptamers by in vitro evolution of RNA molecules has facilitated attempts to design synthetic switches (10-12), which potentially can provide versatile tools for genetic engineering and synthetic biology applications.While, over the past years, numerous riboswitches have been discovered in prokaryotes (13) and also a few in eukaryotes including plants (14,15), ...

“…The bacterial riboswitches normally operate through the binding of a metabolite to an aptamer domain, which, through allosteric effects, changes the conformation of an adjacent expression platform resulting in either the premature termination of transcription, inhibition of translation initiation, or mRNA self-cleavage (3)(4)(5)(6). In eukaryotes, riboswitches present within introns have also been shown to affect alternative splicing pathways (7). These simple protein-free mechanisms make the engineering of natural or synthetic riboswitches an attractive strategy for developing new small-molecule responsive regulatory systems (8).…”

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confidence: 99%

Reengineering orthogonally selective riboswitches

Dixon

Duncan²,

Geerlings³

et al. 2010

151

163

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependent, orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes.