Limits...
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.


Regulation of the apoptosis pathway using the OFF system.(a) Schematic illustration of the ON/OFF states of the switch. In cells that do not express L7Ae, translated Bcl-xL protein binds to Bim to prevent apoptosis (ON state). L7Ae represses translation of Bcl-xL, and the apoptosis signal from Bim can proceed (OFF state). (b) Western blotting analysis of Bcl-xL expression in HeLa cells co-transfected with pL7Ae and p(d)Kt-Bcl-xL-I-GFP. Bcl-xL and L7Ae were detected using anti-Bcl-xL and anti-myc antibodies, respectively. (c) Flow cytometric analysis performed 24 h after co-transfection with pBim and the plasmids described in b. The data are presented as the mean±s.d. of triplicate experiments. (d) Phase microscopic images of the cells analysed in c. A scale bar represents 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105309&req=5

f2: Regulation of the apoptosis pathway using the OFF system.(a) Schematic illustration of the ON/OFF states of the switch. In cells that do not express L7Ae, translated Bcl-xL protein binds to Bim to prevent apoptosis (ON state). L7Ae represses translation of Bcl-xL, and the apoptosis signal from Bim can proceed (OFF state). (b) Western blotting analysis of Bcl-xL expression in HeLa cells co-transfected with pL7Ae and p(d)Kt-Bcl-xL-I-GFP. Bcl-xL and L7Ae were detected using anti-Bcl-xL and anti-myc antibodies, respectively. (c) Flow cytometric analysis performed 24 h after co-transfection with pBim and the plasmids described in b. The data are presented as the mean±s.d. of triplicate experiments. (d) Phase microscopic images of the cells analysed in c. A scale bar represents 200 μm.

Mentions: The OFF system was applied for controlling human cell fate by modulating the translation of apoptosis regulatory proteins (Fig. 1b). The mitochondrial-dependent apoptosis pathways were used to control intrinsic apoptosis pathways (Fig. 1b, Bcl-xL/Bim-dependent pathways) because the balance between proapoptotic (that is, Bim) and antiapoptotic (that is, Bcl-xL) Bcl-2 family proteins determines cell fate24. Namely, the control of apoptosis pathways by gene regulation requires strict expression balance between pro- and antiapoptotic proteins25. For instance, the leaky expression of a proapoptotic gene makes it difficult to suppress apoptosis, as it amplifies apoptosis signals by positive feedback mechanism2627. To control apoptosis by L7Ae, we constructed the following three plasmids: pKt-Bcl-xL-I-GFP (an antiapoptotic agent), pdKt-Bcl-xL-I-GFP (a negative control plasmid containing a defective Kt) and a plasmid encoding Bim (pBim, a proapoptotic agent) (Fig. 2a, Supplementary Fig. S2). Western blot analysis showed that L7Ae expressed in HeLa cells efficiently represses the expression of Bcl-xL from pKt-Bcl-xL-I-GFP (Fig. 2b, lane 5), but has no effect on the defective Kt mutant, pdKt-Bcl-xL-I-GFP (Fig. 2b, lane 7). Flow cytometric analysis confirmed that expression of Bcl-xL from pKt- or pdKt-Bcl-xL-I-GFP efficiently repressed Bim-induced apoptosis in cells (Fig. 2c, lane 2 versus lane 3 or 5). In addition, L7Ae triggered an apoptosis-inducing signal in the corresponding cells by promoting the translational regulation of Bcl-xL. Expression of L7Ae induced apoptosis specifically in cells containing both pBim and pKt-Bcl-xL-I-GFP (Fig. 2c, lane 4; Supplementary Fig. S3). An analysis of cell morphology confirmed that L7Ae induced apoptosis by repressing Bcl-xL expression in target cells (Fig. 2d, left, middle picture), whereas the normal phenotype was observed for cells containing the control plasmid (Fig. 2d, right, middle picture). Thus, L7Ae expressed in cells containing Kt-Bcl-xL mRNA induces apoptosis by repressing the expression of Bcl-xL.


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

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

Regulation of the apoptosis pathway using the OFF system.(a) Schematic illustration of the ON/OFF states of the switch. In cells that do not express L7Ae, translated Bcl-xL protein binds to Bim to prevent apoptosis (ON state). L7Ae represses translation of Bcl-xL, and the apoptosis signal from Bim can proceed (OFF state). (b) Western blotting analysis of Bcl-xL expression in HeLa cells co-transfected with pL7Ae and p(d)Kt-Bcl-xL-I-GFP. Bcl-xL and L7Ae were detected using anti-Bcl-xL and anti-myc antibodies, respectively. (c) Flow cytometric analysis performed 24 h after co-transfection with pBim and the plasmids described in b. The data are presented as the mean±s.d. of triplicate experiments. (d) Phase microscopic images of the cells analysed in c. A scale bar represents 200 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Regulation of the apoptosis pathway using the OFF system.(a) Schematic illustration of the ON/OFF states of the switch. In cells that do not express L7Ae, translated Bcl-xL protein binds to Bim to prevent apoptosis (ON state). L7Ae represses translation of Bcl-xL, and the apoptosis signal from Bim can proceed (OFF state). (b) Western blotting analysis of Bcl-xL expression in HeLa cells co-transfected with pL7Ae and p(d)Kt-Bcl-xL-I-GFP. Bcl-xL and L7Ae were detected using anti-Bcl-xL and anti-myc antibodies, respectively. (c) Flow cytometric analysis performed 24 h after co-transfection with pBim and the plasmids described in b. The data are presented as the mean±s.d. of triplicate experiments. (d) Phase microscopic images of the cells analysed in c. A scale bar represents 200 μm.
Mentions: The OFF system was applied for controlling human cell fate by modulating the translation of apoptosis regulatory proteins (Fig. 1b). The mitochondrial-dependent apoptosis pathways were used to control intrinsic apoptosis pathways (Fig. 1b, Bcl-xL/Bim-dependent pathways) because the balance between proapoptotic (that is, Bim) and antiapoptotic (that is, Bcl-xL) Bcl-2 family proteins determines cell fate24. Namely, the control of apoptosis pathways by gene regulation requires strict expression balance between pro- and antiapoptotic proteins25. For instance, the leaky expression of a proapoptotic gene makes it difficult to suppress apoptosis, as it amplifies apoptosis signals by positive feedback mechanism2627. To control apoptosis by L7Ae, we constructed the following three plasmids: pKt-Bcl-xL-I-GFP (an antiapoptotic agent), pdKt-Bcl-xL-I-GFP (a negative control plasmid containing a defective Kt) and a plasmid encoding Bim (pBim, a proapoptotic agent) (Fig. 2a, Supplementary Fig. S2). Western blot analysis showed that L7Ae expressed in HeLa cells efficiently represses the expression of Bcl-xL from pKt-Bcl-xL-I-GFP (Fig. 2b, lane 5), but has no effect on the defective Kt mutant, pdKt-Bcl-xL-I-GFP (Fig. 2b, lane 7). Flow cytometric analysis confirmed that expression of Bcl-xL from pKt- or pdKt-Bcl-xL-I-GFP efficiently repressed Bim-induced apoptosis in cells (Fig. 2c, lane 2 versus lane 3 or 5). In addition, L7Ae triggered an apoptosis-inducing signal in the corresponding cells by promoting the translational regulation of Bcl-xL. Expression of L7Ae induced apoptosis specifically in cells containing both pBim and pKt-Bcl-xL-I-GFP (Fig. 2c, lane 4; Supplementary Fig. S3). An analysis of cell morphology confirmed that L7Ae induced apoptosis by repressing Bcl-xL expression in target cells (Fig. 2d, left, middle picture), whereas the normal phenotype was observed for cells containing the control plasmid (Fig. 2d, right, middle picture). Thus, L7Ae expressed in cells containing Kt-Bcl-xL mRNA induces apoptosis by repressing the expression of Bcl-xL.

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.