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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.


Simultaneous translational repression and activation of apoptotic proteins by synchronized ON/OFF switches.(a) Regulation of apoptosis by the synchronized Bcl-xL-ON and Bim-OFF switches. Cells that contain Kt-Bim and Kt-Sh-Bcl-xL were protected from Bim-induced apoptosis by expressing L7Ae (lane 3), whereas cells that contain dKt-Bim and dKt-Sh-Bcl-xL induced apoptosis (lane 6). The cells were analysed by using flow cytometry. Kt-ON/OFF switches are shown in red. The results are presented as the mean±s.d. of triplicate experiments. (b) Cell morphology analysis of Figure 6a by phase microscopy. Scale bars represent 200 μm.
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f6: Simultaneous translational repression and activation of apoptotic proteins by synchronized ON/OFF switches.(a) Regulation of apoptosis by the synchronized Bcl-xL-ON and Bim-OFF switches. Cells that contain Kt-Bim and Kt-Sh-Bcl-xL were protected from Bim-induced apoptosis by expressing L7Ae (lane 3), whereas cells that contain dKt-Bim and dKt-Sh-Bcl-xL induced apoptosis (lane 6). The cells were analysed by using flow cytometry. Kt-ON/OFF switches are shown in red. The results are presented as the mean±s.d. of triplicate experiments. (b) Cell morphology analysis of Figure 6a by phase microscopy. Scale bars represent 200 μm.

Mentions: The combined use of the ON/OFF systems was tested for controlling apoptotic pathways in human cells. The OFF and ON switches were designed to repress proapoptotic Bim (Bim-OFF) and to activate antiapoptotic Bcl-xL (Bcl-xL-ON), respectively (Fig. 1b). As expected, L7Ae simultaneously repressed Bim expression and activated Bcl-xL expression to successfully modulate apoptosis (Fig. 6a, lane 3; Fig. 6b, top left). We were able to unidirectionally control human cell fate by appropriately connecting the two systems to the existing signalling networks.


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 apoptotic proteins by synchronized ON/OFF switches.(a) Regulation of apoptosis by the synchronized Bcl-xL-ON and Bim-OFF switches. Cells that contain Kt-Bim and Kt-Sh-Bcl-xL were protected from Bim-induced apoptosis by expressing L7Ae (lane 3), whereas cells that contain dKt-Bim and dKt-Sh-Bcl-xL induced apoptosis (lane 6). The cells were analysed by using flow cytometry. Kt-ON/OFF switches are shown in red. The results are presented as the mean±s.d. of triplicate experiments. (b) Cell morphology analysis of Figure 6a by phase microscopy. Scale bars represent 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Simultaneous translational repression and activation of apoptotic proteins by synchronized ON/OFF switches.(a) Regulation of apoptosis by the synchronized Bcl-xL-ON and Bim-OFF switches. Cells that contain Kt-Bim and Kt-Sh-Bcl-xL were protected from Bim-induced apoptosis by expressing L7Ae (lane 3), whereas cells that contain dKt-Bim and dKt-Sh-Bcl-xL induced apoptosis (lane 6). The cells were analysed by using flow cytometry. Kt-ON/OFF switches are shown in red. The results are presented as the mean±s.d. of triplicate experiments. (b) Cell morphology analysis of Figure 6a by phase microscopy. Scale bars represent 200 μm.
Mentions: The combined use of the ON/OFF systems was tested for controlling apoptotic pathways in human cells. The OFF and ON switches were designed to repress proapoptotic Bim (Bim-OFF) and to activate antiapoptotic Bcl-xL (Bcl-xL-ON), respectively (Fig. 1b). As expected, L7Ae simultaneously repressed Bim expression and activated Bcl-xL expression to successfully modulate apoptosis (Fig. 6a, lane 3; Fig. 6b, top left). We were able to unidirectionally control human cell fate by appropriately connecting the two systems to the existing signalling networks.

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.