Engineering protein-protein devices for multilayered regulation of mRNA translation using orthogonal proteases in mammalian cells


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RNA-based synthetic circuits offer the opportunity to reprogram cellular functions avoiding risks of insertional mutagenesis and immunogenicity. In Ron Weiss’ lab at MIT, I was involved in a project about designing RNA-based synthetic circuits using RNA binding proteins L7Ae and MS2-cNOT7 which were successfully interconnected to create post-transcriptional circuits where they function both as inputs and outputs of the regulatory devices(1). 

Importantly, to assemble multilayered circuits for broad biomedical and pharmaceutical applications, it is necessary to develop foundational tools that can control RNA activity. I thus proposed a project to my PhD student about re-engineering proteins that modulate RNA expression. I had in mind to realize something that could be potentially encoded only on RNA, so we needed to avoid transcriptional regulation. We thus created protein-protein devicesby re-engineering both RNA-binding proteins and viral proteases such as TEV protease2.  

We first studied L7Ae structure identifying possible sites for the insertion of a short aminoacidic sequence recognized by TEV protease. We had two main objectives in choosing the sites: (i) TCS insertions should minimally affect L7Ae structure or RNA binding, and (ii) TEVp cleavage should render L7Ae non-functional. We thus chose three candidate insertion sites for TCS in loop regions away from the K-turn binding domain and closer to the center of the L7Ae sequence (Fig.1a). In the absence of TEVp (State 1 in Figure 1b) and similar to the behavior with wildtype L7Ae, the three L7Ae-CS repressors downregulate translation of the reporter gene. In the presence of TEVp (State 2 in Figure 1b), L7Ae activity is disrupted by protein cleavage, and the mRNA is de-repressed.

Figure 1. Engineering a TEVp responsive L7Ae.
Figure 1. Engineering a TEVp responsive L7Ae.(a) Structure of L7Ae binding to box C/D RNA that forms a K-turn motif. TCS insertion sites in L7Ae are shown in red for each site.(b) Schematics of L7Ae with inserted TCS translational regulation. In the absence of TEVp, L7Ae-CS binds and repress the K-turn motifs in the 5‘UTR of mRNA target (EGFP OFF-State 1). When TEVp is expressed, it cleaves the TCS, disrupting L7Ae structure and inhibiting its function (EGFP ON-State 2). (c) Flow cytometry analysis of three engineered L7Ae-CS tested in HEK 293 cells in the absence or presence of TEVp.

We observed 13 fold derepression of CS2 and 77 fold derepression of CS3, but no appreciable response to CS1 (Figure 1c). We created post-transcriptional regulatory circuits including protein sensor, a cascade and a switch demonstrating their functionality (Fig.2).

Figure 2. L7Ae-CS3-based cascade and two-state switch
Figure 2. L7Ae-CS3-based cascade and two-state switch. (a) The cascade includes both protein-protein regulation and post-transcriptional regulation by siRNA.(b) Simplified logic circuit and corresponding flow cytometry data. The cascade implements the two-input logic “siRNA-FF5 OR (NOT siRNA-FF4)” (top). Bar charts of the EBFP and EYFP circuit outputs as additional stages of the cascade and the inputs are added to the experiment, starting from just the reporter (Stage 0), and then adding L7Ae-CS3 (Stage 1), input siRNA-FF5 (Input 1) or TEVp-2A-EYFP (Stage 2), and siRNA-FF4 (Input 2).(c) Schematics of the L7Ae-CS3-based switch. TEVp-2A-EYFP includes two K-turn motifs in the 5’UTR and four target sites for siRNA-FF4 in the 3’UTR. L7Ae-CS3 mRNA harbors four siRNA-FF5 target sites in the 3’UTR. To monitor L7Ae-CS3 activity, we included EBFP reporter with two K-turn motifs in the 5’UTR. siRNA-FF4 and siRNA-FF5 are used to set and reset the state of the switch. (d) Flow cytometry data representing network behavior.

Next, we re-engineered the chimeric RNA binding protein MS2-cNOT7 including insertion sites for TEV and other homologous proteases (TVMV, SuMMV and TUMV), designing post-transcriptional cascades. 

Finally, we tested whether we could extend our approach by designing proteases that include a cleavage site for an orthogonal protease, enabling a novel protein-protein regulation system (Fig.3).

Figure 3. Protease-protease systems
Figure 3. Protease-protease systems. (a) Schematics of the three-stage signaling cascades. EGFP translation is repressed by MS2-CS-cNOT7 (Stage 1), while RBP activity is disrupted by a protease (Stage 2), which is itself engineered to include a cleavage site for an orthogonal protease. When Stage 2 protease activity is inhibited by an upstream protease (Stage 3), MS2-CS-cNOT7 is able to repress target mRNAs. (b) Corresponding flow cytometry data. We implemented this system for TUMV/TVMV_TUCS/MS2-TVCS-cNOT7 protein-protein regulation cascades as well as for TVMV/TEV_TVCS/MS2-TCS-cNOT7 and TEV/TUMV_tCS/MS2-TUCS-cNOT7

Our study lays the foundation for protein-driven synthetic circuits that enable construction of tunable RNA-encoded networks. We envision that our platform could also be connected to intracellular protease inputs (i.e. caspases) without requiring modifications to endogenous pathways to link changes of intracellular state to output regulation. On a broader level, using proteases upregulated in specific diseases such as cancer we could enable cell-type specific expression of immunomodulators and reprogram cancer cells and immune response.



1.        Wroblewska, L. et al.Mammalian synthetic circuits with RNA binding proteins delivered by RNA. Nature biotechnology33,839–841 (2015).

2.        Siciliano, V. et al.Engineering modular intracellular protein sensor-actuator devices. Nat. Commun.1–7 doi:10.1038/s41467-018-03984-5

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