Direct and specific chemical control of eukaryotic translation with a synthetic RNA–protein interaction

Goldfless, Stephen J.; Belmont, Brian J.; Paz, Alexandra de; Liu, Jessica F.; Niles, Jacquin C.

doi:10.1093/nar/gks028

Cited by 38 publications

(53 citation statements)

References 38 publications

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“…5C and Table 2). These dissociation constants were similar to those previously measured for the isolated aptamer [24], [25]. Notably, no binding was observed in the absence of a functional TetR aptamer (Fig.…”

Section: Resultssupporting

confidence: 89%

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Inducible Control of Subcellular RNA Localization Using a Synthetic Protein-RNA Aptamer Interaction

Belmont

Niles

2012

PLoS ONE

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Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis–elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs), which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically–inducible RNA–protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i) the Tet Repressor protein (TetR) genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii) a target transcript containing genetically encoded TetR–binding RNA aptamers. TetR–fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

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Section: Resultssupporting

confidence: 89%

“…2C and [25]]. Therefore, we tested whether using a highly structured 5′UTR RNA element that can decrease the efficiency with which vYFPΔ_ 5–1.2 is translated would improve its localization.…”

Section: Resultsmentioning

confidence: 99%

Inducible Control of Subcellular RNA Localization Using a Synthetic Protein-RNA Aptamer Interaction

Belmont

Niles

2012

PLoS ONE

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“…Natural small molecule-RNA interactions operate as transcript-embedded riboswitches that fine-tune ligand-inducible transcription and translation of target genes 12 , whereas natural ribonucleoprotein (RNP) complexes provide structural assemblies enabling translational repression (MS2 stem-loop-MS2 coat protein 13 ), ribosome scaffolding (C/Dbox-L7Ae protein 14 ), antitermination control (nutR-boxB-N-peptide 15 ), RNAi 16 and RNA-guided splicing 17 . Many natural RNPs have served as blueprints for the design of synthetic protein-controlled RNA-based gene switches that inhibit translation when engineered into the 5′ UTR of eukaryotic mRNAs [18][19][20] , modulate splicing upon detection of disease markers 21 and use NF-κB to control short hairpin RNA processing 22 . A new level of synthetic translation control was put into practice by transcript-embedded hammerhead ribozymes (HHRs), which directly cleave mammalian mRNAs and irreversibly inhibit translation 23 . HHRs consist of three stem-loop structures that form a three-way junction surrounding the catalytic core 24 .…”

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confidence: 99%

A general design strategy for protein-responsive riboswitches in mammalian cells

et al. 2014

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RNAs are ideal for the design of gene switches that can monitor and program cellular behavior because of their high modularity and predictable structure-function relationship. We have assembled an expression platform with an embedded modular ribozyme scaffold that correlates self-cleavage activity of designer ribozymes with transgene translation in bacteria and mammalian cells. A design approach devised to screen ribozyme libraries in bacteria and validate variants with functional tertiary stem-loop structures in mammalian cells resulted in a designer ribozyme with a protein-binding nutR-boxB stem II and a selected matching stem I. In a mammalian expression context, this designer ribozyme exhibited dose-dependent translation control by the N-peptide, had rapid induction kinetics and could be combined with classic small molecule-responsive transcription control modalities to construct complex, programmable genetic circuits.

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“…[2a] There are many natural and engineered versions of gene switches that respond to an enormous variety of chemical input signals (e.g.a ntibiotics, [19] metabolites, [20] and other disease-related signals [21] ), biological signals (proteins [22] and nucleic acids [23] ), and even physical signals (light [24] and temperature; [25] Table 2). [2a] There are many natural and engineered versions of gene switches that respond to an enormous variety of chemical input signals (e.g.a ntibiotics, [19] metabolites, [20] and other disease-related signals [21] ), biological signals (proteins [22] and nucleic acids [23] ), and even physical signals (light [24] and temperature; [25] Table 2).…”

Section: Gene Switchesmentioning

confidence: 99%

Synthetic Biology—The Synthesis of Biology

2017

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Synthetic biology concerns the engineering of man-made living biomachines from standardized components that can perform predefined functions in a (self-)controlled manner. Different research strategies and interdisciplinary efforts are pursued to implement engineering principles to biology. The "top-down" strategy exploits nature's incredible diversity of existing, natural parts to construct synthetic compositions of genetic, metabolic, or signaling networks with predictable and controllable properties. This mainly application-driven approach results in living factories that produce drugs, biofuels, biomaterials, and fine chemicals, and results in living pills that are based on engineered cells with the capacity to autonomously detect and treat disease states in vivo. In contrast, the "bottom-up" strategy seeks to be independent of existing living systems by designing biological systems from scratch and synthesizing artificial biological entities not found in nature. This more knowledge-driven approach investigates the reconstruction of minimal biological systems that are capable of performing basic biological phenomena, such as self-organization, self-replication, and self-sustainability. Moreover, the syntheses of artificial biological units, such as synthetic nucleotides or amino acids, and their implementation into polymers inside living cells currently set the boundaries between natural and artificial biological systems. In particular, the in vitro design, synthesis, and transfer of complete genomes into host cells point to the future of synthetic biology: the creation of designer cells with tailored desirable properties for biomedicine and biotechnology.

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Direct and specific chemical control of eukaryotic translation with a synthetic RNA–protein interaction

Cited by 38 publications

References 38 publications

Inducible Control of Subcellular RNA Localization Using a Synthetic Protein-RNA Aptamer Interaction

Inducible Control of Subcellular RNA Localization Using a Synthetic Protein-RNA Aptamer Interaction

A general design strategy for protein-responsive riboswitches in mammalian cells

Synthetic Biology—The Synthesis of Biology

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