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Synthetic human cell fate regulation by protein-driven RNA switches.

Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T - Nat Commun (2011)

Bottom Line: Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways.A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression.The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. [2] International Cooperative Research Project, Japan Science and Technology Agency, 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. [3] The Hakubi Center, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

No MeSH data available.


Related in: MedlinePlus

Simultaneous translational repression and activation of two fluorescent proteins by synchronized ON/OFF switches.(a) Simultaneous translational activation and repression of EGFP and AsRed2 by synchronized ON/OFF switches. The fluorescence intensity of the transfected cells was analysed by flow cytometry. These dot plot data correspond to the fluorescent microscopic images in Figure 5c. (b) Flow cytometric analysis of the simultaneous regulation of EGFP-ON and AsRed2-OFF switches by L7Ae. The efficiencies of translation were normalized to control translational rates in the absence of pL7Ae (AsRed2-OFF) or in the presence of 0.3 μg of pL7Ae (EGFP-ON). The results are presented as the mean±s.d. of triplicate experiments. Green solid line with circles; EGFP relative intensity (Kt), green dashed line with triangles; EGFP relative intensity (dKt), red solid line with circles; AsRed2 relative intensity (Kt), red dashed line with triangles; AsRed2 relative intensity (dKt). (c) Merged fluorescent microscopic images of cells that contained EGFP-ON and AsRed2-OFF switches. A scale bar represents 200 μm.
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f5: Simultaneous translational repression and activation of two fluorescent proteins by synchronized ON/OFF switches.(a) Simultaneous translational activation and repression of EGFP and AsRed2 by synchronized ON/OFF switches. The fluorescence intensity of the transfected cells was analysed by flow cytometry. These dot plot data correspond to the fluorescent microscopic images in Figure 5c. (b) Flow cytometric analysis of the simultaneous regulation of EGFP-ON and AsRed2-OFF switches by L7Ae. The efficiencies of translation were normalized to control translational rates in the absence of pL7Ae (AsRed2-OFF) or in the presence of 0.3 μg of pL7Ae (EGFP-ON). The results are presented as the mean±s.d. of triplicate experiments. Green solid line with circles; EGFP relative intensity (Kt), green dashed line with triangles; EGFP relative intensity (dKt), red solid line with circles; AsRed2 relative intensity (Kt), red dashed line with triangles; AsRed2 relative intensity (dKt). (c) Merged fluorescent microscopic images of cells that contained EGFP-ON and AsRed2-OFF switches. A scale bar represents 200 μm.

Mentions: We next attempted to simultaneously repress the translation of one mRNA and activate expression of a second mRNA in human cells by using L7Ae. Plasmids encoding AsRed2, designed to serve as a repressible gene (AsRed2-OFF), and EGFP, designed to serve as an activating gene (EGFP-ON), were constructed. L7Ae expression simultaneously and selectively repressed the translation of AsRed2 and activated EGFP, which we confirmed by flow cytometry (Figs 5a,b) and fluorescence microscopy (Fig. 5c). The results indicate that a single protein simultaneously represses and activates translation of two different mRNAs in human cell.


Synthetic human cell fate regulation by protein-driven RNA switches.

Saito H, Fujita Y, Kashida S, Hayashi K, Inoue T - Nat Commun (2011)

Simultaneous translational repression and activation of two fluorescent proteins by synchronized ON/OFF switches.(a) Simultaneous translational activation and repression of EGFP and AsRed2 by synchronized ON/OFF switches. The fluorescence intensity of the transfected cells was analysed by flow cytometry. These dot plot data correspond to the fluorescent microscopic images in Figure 5c. (b) Flow cytometric analysis of the simultaneous regulation of EGFP-ON and AsRed2-OFF switches by L7Ae. The efficiencies of translation were normalized to control translational rates in the absence of pL7Ae (AsRed2-OFF) or in the presence of 0.3 μg of pL7Ae (EGFP-ON). The results are presented as the mean±s.d. of triplicate experiments. Green solid line with circles; EGFP relative intensity (Kt), green dashed line with triangles; EGFP relative intensity (dKt), red solid line with circles; AsRed2 relative intensity (Kt), red dashed line with triangles; AsRed2 relative intensity (dKt). (c) Merged fluorescent microscopic images of cells that contained EGFP-ON and AsRed2-OFF switches. A scale bar represents 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3105309&req=5

f5: Simultaneous translational repression and activation of two fluorescent proteins by synchronized ON/OFF switches.(a) Simultaneous translational activation and repression of EGFP and AsRed2 by synchronized ON/OFF switches. The fluorescence intensity of the transfected cells was analysed by flow cytometry. These dot plot data correspond to the fluorescent microscopic images in Figure 5c. (b) Flow cytometric analysis of the simultaneous regulation of EGFP-ON and AsRed2-OFF switches by L7Ae. The efficiencies of translation were normalized to control translational rates in the absence of pL7Ae (AsRed2-OFF) or in the presence of 0.3 μg of pL7Ae (EGFP-ON). The results are presented as the mean±s.d. of triplicate experiments. Green solid line with circles; EGFP relative intensity (Kt), green dashed line with triangles; EGFP relative intensity (dKt), red solid line with circles; AsRed2 relative intensity (Kt), red dashed line with triangles; AsRed2 relative intensity (dKt). (c) Merged fluorescent microscopic images of cells that contained EGFP-ON and AsRed2-OFF switches. A scale bar represents 200 μm.
Mentions: We next attempted to simultaneously repress the translation of one mRNA and activate expression of a second mRNA in human cells by using L7Ae. Plasmids encoding AsRed2, designed to serve as a repressible gene (AsRed2-OFF), and EGFP, designed to serve as an activating gene (EGFP-ON), were constructed. L7Ae expression simultaneously and selectively repressed the translation of AsRed2 and activated EGFP, which we confirmed by flow cytometry (Figs 5a,b) and fluorescence microscopy (Fig. 5c). The results indicate that a single protein simultaneously represses and activates translation of two different mRNAs in human cell.

Bottom Line: Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways.A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression.The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. [2] International Cooperative Research Project, Japan Science and Technology Agency, 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan. [3] The Hakubi Center, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan.

ABSTRACT
Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

No MeSH data available.


Related in: MedlinePlus