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

The development of RNA-encoded regulatory circuits relying on RNA-binding proteins (RBPs) has enhanced the applicability and prospects of post-transcriptional synthetic network for reprogramming cellular functions. However, the construction of RNA-encoded multilayer networks is still limited by the availability of composable and orthogonal regulatory devices. Here, we report on control of mRNA translation with newly engineered RBPs regulated by viral proteases in mammalian cells. By combining post-transcriptional and post-translational control, we expand the operational landscape of RNA-encoded genetic circuits with a set of regulatory devices including: i) RBP-protease, ii) protease-RBP, iii) protease–protease, iv) protein sensor protease-RBP, and v) miRNA-protease/RBP interactions. The rational design of protease-regulated proteins provides a diverse toolbox for synthetic circuit regulation that enhances multi-input information processing-actuation of cellular responses. Our approach enables design of artificial circuits that can reprogram cellular function with potential benefits as research tools and for future in vivo therapeutics and biotechnological applications.

Populations of live, single cells were first determined based on forward and side scatter. Represented plots were established based on negative control (non-transfected) cells and cells transfected with EGFP, EBFP or mKate (transfection marker) only (not shown). EYFP is coupled to TEVp levels via a 2A self-cleaving peptide. EBFP is the functional output of L7Ae-CS3 activity. siRNAFF4 and siRNAFF5 are used to set the state of the switch (schematics is shown is Figure 2c).  (Fig 3b). Reporter EGFP includes 8x binding sites for MS2 in the 3'UTR, where MS2-TCS-cNOT7 binding induces mRNA degradation due to cNOT7 deadenylation activity. In the presence of TEVp, MS2-TCS-cNOT is cleaved and EGFP is derepressed.

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Supplementary Note 1: Engineering a novel protease-based protein-protein regulation system.
We hypothesized that re-engineering proteases to include protease cleavage sites could provide for novel protein-protein system. Towards this goal, we first designed TVMVp with three alternative insertion sites for the TUMVp cleavage site and tested TVMV-CS along with MS2-TVCS-cNOT7 (MS2-cNot7 with cleavage site responsive to TVMVp) which in turn represses EGFP translation. In this topology, TVMV-CS upregulates EGFP expression, whereas co-expression of TUMVp results in TVMV-CS inhibition and EGFP down-modulation by MS2-TVCS-cNOT7 (Figure 4a).
Our data show that TVMVp_TUCS2 expression interferes with MS2-TVCS-cNOT7 activity, while expression of TUMVp in turn inhibits TVMVp_TUCS2, resulting in decreased EGFP expression (Figure 4b). Conversely, we did not observe inhibition of MS2-TVCS-cNOT7 by TVMVp_TUCS1 and TVMVp_TUCS3, perhaps because the insertions alter the structure and function of the proteins (Supplementary Figure 11a,b).
TEVp and TVMVp share a high degree of structural similarity, the crystal structure is resolved and for both 3,4 . Importantly, the amino acid sequence flanking the insertion point of TVMVp-TUCS2 (residues 116-121 NFQQ-KS) is highly similar to TEVp (amino acid residues 114-119 NFQT-KS).