Engineering synthetic ligand-responsive RNA devices for controlling the cell cycle
Kathy Y. Wei, Christina D. Smolke
Dept. of Bioengineering, Stanford University
Contact: kywei@stanford.edu, christina.smolke@stanford.edu
System Design
o
o
o
o
Goal: Engineer synthetic RNA devices for the reversible arrest of mammalian cell
populations in G0/1.
Previous methods: Currently available small molecule inhibitors of the cell cycle tend
to broadly disrupt cell function
Our method:
o A switchable RNA platform allows inducible arrest through specific endogenous
gene targets with potentially any small molecule effector by modular replacement
of the target or aptamer-based sensing region.
o This platform represents a general class of synthetic biology tools for modular,
dynamic, and multi-input control over endogenous protein levels in mammalian
cells.
Motivation:
• Tools for the control of mammalian programming is a relatively underdeveloped
area.
• We propose to use simple, general-purpose components (an RNA ribozyme or
hairpin coupled to an RNA aptamer) to control a complex phenomenon (such as
regulation of the cell cycle).
Applications:
• Increase heterologous protein production.
• Better control of signal processing in the self-renewal versus differentiation
decision.
• Increase the reliability of mammalian genetic integration techniques.
o
[1]
Methods
Optimize expression of cell cycle regulatory nodes
Cell cycle arrest measured by DNA staining
Tuning regulatory node expression via synthetic sequences, cleavable peptides, and IRES
A) DNA stain to determine phase
B) Measure by flow cytometry
25
promoter
P2A/IRES
protein
pA
20
Tuning the expression of key regulator p27 using syn21 and
IRES improved the degree of control over cell cycle arrest.
The syn21 sequence is 21 nt that is inserted prior to the
start codon and was identified to increase expression in
Drosophila [5]. Introducing a second copy of p27 should
further increase the amount of expression. While linking the
two proteins with a cleavable peptide (P2A) did not improve
the amount G0/1 arrest (possibly due to lack of cleaving and
thus mis-folding), linking with an IRES was effective for
increasing the amount of control over the cell cycle.
15
D%G0/1
A) Cell cycle phase is determined by the amount of DNA
staining using an intercalating dye such as propidium
iodide (PI).
B) Flow cytometry is used to measure the proportion of
cells in G0/1 by examining a histogram of cell frequency
vs. DNA staining. Measurements are normalized to a
control (either empty or fluorescent) to allow easy
comparison across experiments and reported as:
D%G0/1 = (%G0/1 sample) – (%G0/1 control)
syn21 protein
10
5
0
-5
[2]
-
[3]
p27
syn21p27
x2P2A
xIRES
syn21p27
syn21p27
Tuning expression of regulatory nodes
Key regulators control cell cycle signaling network
RNA switches that control G0/1 arrest
Cell cycle signaling network is controlled by a few key regulatory nodes
Mechanism of small molecule inducible RNA switches
S
The signaling network controlling cell progression from G1
to S phase is large and interwoven with other processes in
the cell. While our understanding of the network is
incomplete, we do know that a very small set of key protein
regulators act as nodes. The regulatory nodes act such that
changes in the level of that node results in measurable
population level changes in cell cycle progression. Some key
nodes are boxed.
G1
h
AAAn
Gene
expression off
Basal
%G0/1
OR
h
M
Ribozyme
inactive
Inducer
p16, p21, and p27 identified as key regulators in G0/1 arrest
5
2
mCherry cyclin D1 DP-1
Cdh1
hRb
p16
p21
p27
HSVTKpA
promoter
protein
pA
Cells were transfected with plasmids overexpressing
potential regulatory protein nodes to identify which are
capable of inhibiting progression of G0/1 to S. mCherry
served as a non-disruptive control and cyclin D1 served as a
negative control. The most promising key regulators are
p16, p21, and p27.
-4
16
14
12
10
8
6
4
2
0
-2
-4
0.75 mM inducer
10
OFF only
Regulatory node overexpression
-7
ON only
Switch A
promoter
25
CMV
20
15
10
5
0
p27
-5
p21
-
p16
p21
p27
p27
p21
p21
p16
Combinatorial regulatory node expression
p16
ueGFP
HSVTKpA
promoter protein
pA
promoter protein
pA
promoter protein
pA
Many of the regulatory proteins in the cell cycle signaling
pathways naturally work together or in parallel branches to
achieve their effect. Therefore, combining the expression of
many regulatory nodes has the potential to increase the
amount of arrest in G0/1 compared to expression of a single
node. In these particular combinations however, the effect
of using multiple nodes did not exceed that of
overexpressing a single key regulator.
-2
syn21 protein
P2A/IRES
0
0.2
protein
rz switch
0.4
mM inducer
0.6
0.8
pA
Future Work
Multi-input/multi-output RNA switch control
11
8
5
2
-1
[1] Morgan, David O. The Cell Cycle: Principles of Control. 1999-2007 New Science Press. [2] http://en.wikipedia.org/wiki/Hoechst_stain [3] Pozarowski, P (2004) Analysis of cell cycle by flow
cytometry. Methods Mol Biol. [4] http://www.cellsignal.com/common/content/content.jsp?id=pathways-cc-g1s [5] Pfeiffer, B.D., Truman, J.W. et al. (2012) Using translational enhancers to increase
transgene expression in Drosophila. PNAS. [6] Wei, K.Y., Chen Y.Y. et al. (2013). A yeast‐based rapid prototype platform for gene control elements in mammalian cells. Biotech & Bioeng.
This research was made with Government support under and awarded by DoD, Air Force Office of Scientific Research, National Defense Science and Engineering Graduate (NDSEG) Fellowship, 32 CFR
168a, Siebel Foundation.
4
RNA (rz) switches were inserted behind the optimized expression construct for the key cell cycle regulatory node p27 and integrated into cell lines.
“OFF only” is a cleaving, non-switching control and “ON only” is a non-cleaving, non-switching control. Both Switch A and Switch B induce arrest of
cells in G0/1 with the addition of a small molecule inducer. Both switches show small molecule depend control of cell cycle and function best at 0.75
mM induction (p-value < 0.05).
D%G2/M
30
7
1
Switch B
Optimized regulatory node with RNA switch
Combining proteins does not appear to have an additive effect
Switch A
Switch B
13
D%G0/1
8
ueGFP
D%G0/1
CMV
11
D%G0/1
High
%G0/1
0 mM inducer
14
D%G0/1
Gene
expression on
G2
Small molecule control of G0/1 arrest through RNA switches
17
-1
S
G1
AAAn
[4]
Ribozymes are RNA sequence capable of self-cleavage.
These can be made ligand-responsive by incorporating an
aptamer such that ligand binding to the aptamer effects
ribozyme cleavage. Specifically, this results in an inducible
switch where absence of ligand results in an active
ribozyme and low gene expression and presence of ligand
results in inactive ribozyme and high gene expression. For
more information, see [6].
M
Ribozyme
active
No inducer
G2
-
pMyt1a
Chk2
Mad2 GADD45
Chk1
Emi1
-4
-7
G2/M regulatory node overexpression
BubR1
Future work includes identifying regulatory nodes that are
key to arresting cells in other phases such as G2/M (shown
here) and S. Putting such nodes under RNA switches that
are sensitive to different inducers and combining with RNA
switches that alter G0/1 arrest will create a sophisticated
controller that can provide multiple different phenotypic
outputs given different combinations of small molecule
inputs.