Post-translational enzyme activation in an animal via optimized conditional protein splicing

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Control over the timing, location and level of protein activity in vivo is crucial to understanding biological function1. Living systems are able to respond to external and internal stimuli rapidly and in a graded fashion by maintaining a pool of proteins whose activities are altered through post-translational modifications2. Here we show that the process of protein trans-splicing3 can be used to modulate enzymatic activity both in cultured cells and in Drosophila melanogaster. We used an optimized conditional protein splicing4 system to rapidly trigger the in vivo ligation of two inactive fragments of firefly luciferase in a tunable manner. This technique provides a means of controlling enzymatic function with greater speed and precision than with standard genetic techniques and is a useful tool for probing biological processes.

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Figure 1: Conditional protein splicing.
Figure 2: Splicing and activation of luciferase in cultured cells.
Figure 3: Optimization of the luciferase CPS system.
Figure 4: Activation of luciferase in living D. melanogaster.

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We would like to thank M. Hahn and M. Pratt for helpful comments on the manuscript, and Ariad Pharmaceuticals for providing compound AP21967. This work was supported by grants from the US National Institutes of Health to T.W.M. (EB001991 and GM072015) and M.W.Y. (NS053087), and by a Howard Hughes Medical Institute fellowship to E.C.S.

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Correspondence to Tom W Muir.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Attempts to splice at luciferase Arg437. (PDF 137 kb)

Supplementary Fig. 2

Splicing of luciferase437–551 with maltose-binding protein. (PDF 71 kb)

Supplementary Fig. 3

Dose-dependent splicing in response to rapamycin or rapamycin analog. (PDF 34 kb)

Supplementary Fig. 4

CPS-activated luciferase in mammalian cells. (PDF 29 kb)

Supplementary Fig. 5

Poor expression of C-lucFRB3. (PDF 96 kb)

Supplementary Fig. 6

Additional luciferase traces. (PDF 412 kb)

Supplementary Fig. 7

Generation of full-length luciferase through CPS in adult D. melanogaster. (PDF 93 kb)

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