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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.


Using the ON system to control cell fate.(a) Targeting the Bcl-xL gene with shRNAs containing a loop (1), Kt (2, green) and dKt (3, green). The sequence in red is the complementary strand of a region of Bcl-xL mRNA. The cleavage sites of the Dicer enzyme are indicated by arrowheads. (b) The Dicer cleavage assay in vitro. (c) Western blotting analysis of Bcl-xL using the L7Ae-K-turn ON switch in cells. (d) Flow cytometric analysis of cell death performed 24 h after co-transfection with pAsRed2-L7Ae, pBcl-xL, pBim and the corresponding pshRNA. The data are presented as the mean±s.d. of triplicate experiments. (e) Analysis of the morphology of the cells in d by phase microscopy. A scale bar represents 200 μm.
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f4: Using the ON system to control cell fate.(a) Targeting the Bcl-xL gene with shRNAs containing a loop (1), Kt (2, green) and dKt (3, green). The sequence in red is the complementary strand of a region of Bcl-xL mRNA. The cleavage sites of the Dicer enzyme are indicated by arrowheads. (b) The Dicer cleavage assay in vitro. (c) Western blotting analysis of Bcl-xL using the L7Ae-K-turn ON switch in cells. (d) Flow cytometric analysis of cell death performed 24 h after co-transfection with pAsRed2-L7Ae, pBcl-xL, pBim and the corresponding pshRNA. The data are presented as the mean±s.d. of triplicate experiments. (e) Analysis of the morphology of the cells in d by phase microscopy. A scale bar represents 200 μm.

Mentions: To regulate cell fate, we prepared three shRNA-coding plasmids that targeted the Bcl-xL gene: pSh-Bcl-xL (positive control), pKt-Sh-Bcl-xL and pdKt-Sh-Bcl-xL (Fig. 4a). L7Ae selectively inhibited the activity of Dicer on Kt-Sh-Bcl-xL RNA in vitro, but not on dKt-Sh-Bcl-xL RNA (Fig. 4b). Western blotting showed that transfection of pKt-Sh-Bcl-xL or pdKt-Sh-Bcl-xL repressed expression of Bcl-xL in cells after 24 h as effectively as the positive control, pSh-Bcl-xL (Fig. 4c, lanes 7 and 8). Conversely, L7Ae protected the expression of Bcl-xL from repression by pKt-Sh-Bcl-xL selectively and effectively (Fig. 4c, lane 5). The results indicate that the interaction between L7Ae and Kt-shRNA inhibits RNAi activity, which results in translation of the protected target mRNA.


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

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

Using the ON system to control cell fate.(a) Targeting the Bcl-xL gene with shRNAs containing a loop (1), Kt (2, green) and dKt (3, green). The sequence in red is the complementary strand of a region of Bcl-xL mRNA. The cleavage sites of the Dicer enzyme are indicated by arrowheads. (b) The Dicer cleavage assay in vitro. (c) Western blotting analysis of Bcl-xL using the L7Ae-K-turn ON switch in cells. (d) Flow cytometric analysis of cell death performed 24 h after co-transfection with pAsRed2-L7Ae, pBcl-xL, pBim and the corresponding pshRNA. The data are presented as the mean±s.d. of triplicate experiments. (e) Analysis of the morphology of the cells in d by phase microscopy. A scale bar represents 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Using the ON system to control cell fate.(a) Targeting the Bcl-xL gene with shRNAs containing a loop (1), Kt (2, green) and dKt (3, green). The sequence in red is the complementary strand of a region of Bcl-xL mRNA. The cleavage sites of the Dicer enzyme are indicated by arrowheads. (b) The Dicer cleavage assay in vitro. (c) Western blotting analysis of Bcl-xL using the L7Ae-K-turn ON switch in cells. (d) Flow cytometric analysis of cell death performed 24 h after co-transfection with pAsRed2-L7Ae, pBcl-xL, pBim and the corresponding pshRNA. The data are presented as the mean±s.d. of triplicate experiments. (e) Analysis of the morphology of the cells in d by phase microscopy. A scale bar represents 200 μm.
Mentions: To regulate cell fate, we prepared three shRNA-coding plasmids that targeted the Bcl-xL gene: pSh-Bcl-xL (positive control), pKt-Sh-Bcl-xL and pdKt-Sh-Bcl-xL (Fig. 4a). L7Ae selectively inhibited the activity of Dicer on Kt-Sh-Bcl-xL RNA in vitro, but not on dKt-Sh-Bcl-xL RNA (Fig. 4b). Western blotting showed that transfection of pKt-Sh-Bcl-xL or pdKt-Sh-Bcl-xL repressed expression of Bcl-xL in cells after 24 h as effectively as the positive control, pSh-Bcl-xL (Fig. 4c, lanes 7 and 8). Conversely, L7Ae protected the expression of Bcl-xL from repression by pKt-Sh-Bcl-xL selectively and effectively (Fig. 4c, lane 5). The results indicate that the interaction between L7Ae and Kt-shRNA inhibits RNAi activity, which results in translation of the protected target mRNA.

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.