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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

Quantitative regulation of apoptosis by L7Ae-DsRedM protein derived from DNA integrated into genome.(a) Western blotting analysis of cell lysates 24 h after transfection with pKt-Bcl-xL-I-GFP (or pdKt-Bcl-xL-I-GFP) and pBim in the absence or presence of tetracycline (0–15 ng ml−1) using anti-Bcl-xL antibody. (b) Flow cytometric analysis of the same cells. Cells positive for EGFP and Pacific Blue were considered dead. Blue column, Kt; yellow column, dKt. The results are presented as the mean±s.d. of triplicate experiments.
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f7: Quantitative regulation of apoptosis by L7Ae-DsRedM protein derived from DNA integrated into genome.(a) Western blotting analysis of cell lysates 24 h after transfection with pKt-Bcl-xL-I-GFP (or pdKt-Bcl-xL-I-GFP) and pBim in the absence or presence of tetracycline (0–15 ng ml−1) using anti-Bcl-xL antibody. (b) Flow cytometric analysis of the same cells. Cells positive for EGFP and Pacific Blue were considered dead. Blue column, Kt; yellow column, dKt. The results are presented as the mean±s.d. of triplicate experiments.

Mentions: Finally, we investigated to see whether a protein of interest encoded in the genome quantitatively controls apoptosis pathways as a function of the protein expression. The insertion of L7Ae into the genome allows us to use L7Ae as a molecular tag for a protein of interest. As a pilot system, we attempted to quantitatively regulate apoptosis by controlling the expression level of an input protein (L7Ae-tagged red fluorescent protein: DsRed-L7Ae) encoded in the genome. A stable cell line (T-REx HeLa) was used for the conditional expression of DsRed-L7Ae in the presence of tetracycline. Western blotting analysis confirmed that the repression of Bcl-xL production depends quantitatively on the expression of DsRed-L7Ae (Fig. 7a). Moreover, apoptosis of the target cells was induced by the expression of DsRed-L7Ae in a dose-dependent manner (Fig. 7b). We were also able to repress apoptosis by controlling FADD expression by using DsRed-L7Ae expressed from the genome (Supplementary Fig. S7). Thus, it seems certain that a specific protein encoded in the genome can drive the translational regulatory systems to control cell fate.


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

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

Quantitative regulation of apoptosis by L7Ae-DsRedM protein derived from DNA integrated into genome.(a) Western blotting analysis of cell lysates 24 h after transfection with pKt-Bcl-xL-I-GFP (or pdKt-Bcl-xL-I-GFP) and pBim in the absence or presence of tetracycline (0–15 ng ml−1) using anti-Bcl-xL antibody. (b) Flow cytometric analysis of the same cells. Cells positive for EGFP and Pacific Blue were considered dead. Blue column, Kt; yellow column, dKt. The results are presented as the mean±s.d. of triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Quantitative regulation of apoptosis by L7Ae-DsRedM protein derived from DNA integrated into genome.(a) Western blotting analysis of cell lysates 24 h after transfection with pKt-Bcl-xL-I-GFP (or pdKt-Bcl-xL-I-GFP) and pBim in the absence or presence of tetracycline (0–15 ng ml−1) using anti-Bcl-xL antibody. (b) Flow cytometric analysis of the same cells. Cells positive for EGFP and Pacific Blue were considered dead. Blue column, Kt; yellow column, dKt. The results are presented as the mean±s.d. of triplicate experiments.
Mentions: Finally, we investigated to see whether a protein of interest encoded in the genome quantitatively controls apoptosis pathways as a function of the protein expression. The insertion of L7Ae into the genome allows us to use L7Ae as a molecular tag for a protein of interest. As a pilot system, we attempted to quantitatively regulate apoptosis by controlling the expression level of an input protein (L7Ae-tagged red fluorescent protein: DsRed-L7Ae) encoded in the genome. A stable cell line (T-REx HeLa) was used for the conditional expression of DsRed-L7Ae in the presence of tetracycline. Western blotting analysis confirmed that the repression of Bcl-xL production depends quantitatively on the expression of DsRed-L7Ae (Fig. 7a). Moreover, apoptosis of the target cells was induced by the expression of DsRed-L7Ae in a dose-dependent manner (Fig. 7b). We were also able to repress apoptosis by controlling FADD expression by using DsRed-L7Ae expressed from the genome (Supplementary Fig. S7). Thus, it seems certain that a specific protein encoded in the genome can drive the translational regulatory systems to control cell fate.

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