Engineering dynamic cell cycle control with synthetic small molecule-responsive RNA devices

Abstract

Background: The cell cycle plays a key role in human health and disease, including development and cancer. The ability to easily and reversibly control the mammalian cell cycle could mean improved cellular reprogramming, better tools for studying cancer, more efficient gene therapy, and improved heterologous protein production for medical or industrial applications.

Results: We engineered RNA-based control devices to provide specific and modular control of gene expression in response to exogenous inputs in living cells. Specifically, we identified key regulatory nodes that arrest U2-OS cells in the G0/1 or G2/M phases of the cycle. We then optimized the most promising key regulators and showed that, when these optimized regulators are placed under the control of a ribozyme switch, we can inducibly and reversibly arrest up to ~80 % of a cellular population in a chosen phase of the cell cycle. Characterization of the reliability of the final cell cycle controllers revealed that the G0/1 control device functions reproducibly over multiple experiments over several weeks.

Conclusions: To our knowledge, this is the first time synthetic RNA devices have been used to control the mammalian cell cycle. This RNA platform represents a general class of synthetic biology tools for modular, dynamic, and multi-output control over mammalian cells.

Keywords: RNA engineering; Synthetic biology; cell cycle; cellular devices.

Fig. 1

Screen to identify key regulatory nodes that produce cell cycle arrest in G0/1 and G2/M. a Schematic of the progression through the phases of cell cycle and a simplified representation of the identified key node function in cell cycle regulation. b, c Potential regulatory node proteins were overexpressed and the resulting cell populations were assayed for changes in the percentage of cells that were in G0/1 phase (b) or G2/M phase (c) relative to a negative control (i.e., control plasmid that does not alter cell cycle progression). *, p < 0.05. Cells were transiently co-transfected with 3–3.75 μg of the plasmids encoding the expression of these candidate proteins and 1.13–1.5 μg of a plasmid encoding a GFP reporter. Error bars represent standard deviation across biological triplicates

Fig. 2

Impact of experimental parameters on the activity of regulatory nodes for cell cycle control. a Impact of combinatorial expression of key regulatory nodes on cell cycle arrest in G0/1. U2-OS cells were transiently co-transfected with plasmids encoding overexpression of combinations of one, two, and three key node proteins. The resulting arrest in G0/1 of the cell population was assayed via DNA staining and flow cytometry. b Impact of p16 on cell cycle arrest in G0/1 of different cell lines. HeLa, HEK293, or U2-OS cells were transiently transfected with a plasmid encoding p16 or a control and the percentage of the cell population in G0/1 was measured. c Impact of the arrangement of expression cassettes encoding regulatory nodes on cell cycle arrest in G0/1. U2-OS cells were transiently transfected with a single plasmid containing two expression cassettes, encoding a transfection marker and a regulatory node (p16 or p27) in tandem, or with two plasmids separately encoding the transfection marker and the regulatory node. The resulting arrest in G0/1 of the cell population was assayed. *, p < 0.05. Error bars represent standard deviation across biological triplicates

Fig. 3

Small molecule-responsive ribozyme switches control arrest of cells in G2/M. a Schematic of the mechanism by which ribozyme switches mediate small molecule-dependent transition of cells into a G2/M arrest state, rather than the normal G2/M state, by regulating expression of CCNB1m. b A set of theophylline-responsive switches and a non-switch control (wild-type sTRSV hammerhead ribozyme; OFF control) were inserted in the 3′ UTR of CCNB1m, stably integrated into U2-OS T-Rex Flp-In cells, and tested with 0 or 1 mM theophylline (theo) for their ability to arrest cells in G2/M. c Characterization of gene-regulatory activity of switch th-A measured as reporter protein activity as a function of theophylline concentration. HEK293 cells were transiently transfected with a plasmid encoding switch th-A in the 3′ UTR of a mCherry reporter and induced with varying levels of theophylline. Mean fluorescence of the population was measured by flow cytometry. d Characterization of gene-regulatory activity of switch th-A measured as CCNB1m transcript levels in the presence and absence of theophylline. Cell lines harboring the stably integrated OFF control and switch th-A in the 3’UTR of the CCNB1m expression cassette were grown in 0 or 1 mM theophylline and CCNB1m transcript levels relative to that of a housekeeping gene (ACTB) were measured by qRT-PCR. *, p < 0.05, ****, p < 1E-4. Error bars represent standard deviation across biological triplicates for (b) and duplicates for (c) and (d)

Fig. 4

Small molecule responsive ribozyme switches for titratable and dynamic control over cell cycle. a Schematic of the progression through the phases of cell cycle (grey and white) with the introduction of the synthetic G0/1 cell cycle control system (blue) and synthetic G2/M cell cycle control system (orange). b Theophylline responsiveness of the G0/1 cell cycle control system (integrated p27-switch controller). Cell lines harboring the control system or a negative control were induced with a range of theophylline (theo) concentrations and assayed for the percentage of cells in G0/1 via DNA staining and flow cytometry. c Theophylline responsiveness of the G2/M cell cycle control system (integrated CCNB1m-switch controller). Cell lines harboring the control system or a negative control were induced with a range of theophylline (theo) concentrations and assayed for the percentage of cells in G2/M via DNA staining and flow cytometry. d The response of the G0/1 cell cycle control system to changes in theophylline concentration over time. Cell lines harboring the G0/1 control system (switch th-A) or a negative control (OFF control) were induced with 0 or 1 mM theophylline. After 3 days, samples were assayed for arrest in G0/1 by flow cytometry (induce) and re-seeded with 0 mM theophylline. After an additional 3 days, samples were assayed for G0/1 arrest (remove). e The response of the G2/M cell cycle control system to changes in theophylline concentration over time. Cell lines harboring the G2/M control system (switch th-A) or a negative control (OFF control) were induced with 0 or 1 mM theophylline. After 3 days, samples were assayed for arrest in G2/M by flow cytometry (induce) and re-seeded with 0 mM theophylline. After an additional 3 days, samples were assayed for G2/M arrest (remove). *, p < 0.05. Error bars represent standard deviation across triplicates

Fig. 5

Reliability of cell cycle control systems over time. a Performance of the cell cycle controller for G0/1 (integrated p27-switch controller) over time. Cells harboring the G0/1 cell cycle controller (p27 switch th-A) or a negative control (p27 OFF control) were induced with 0 or 1 mM theophylline (theo) and the percentage of the cell population in G0/1 was measured via DNA staining and flow cytometry after creation of the cell lines (left of hash marks) and at 1 and 3 weeks after cell lines were frozen and revived (right of hash marks). b Performance of the cell cycle controller for G2/M (integrated CCNB1m-switch controller) over time. Cells harboring the G2/M cell cycle controller (CCNB1m switch th-A) or a negative control (CCNB1m OFF control) were induced with 0 or 1 mM theophylline and the percentage of the cell population in G2/M was measured via DNA staining and flow cytometry at 1, 2, 3 and 6 weeks after creation of the cell lines