Fig. 3

Engineering protease-dependent MS2-cNOT7 proteins. a Schematics of the cascade. MS2 is fused to cNOT7 via the TEVp cleavage site (MS2-TCS-cNOT7). MS2 binds its cognate sequences in the 3’UTR of target EGFP mRNA, resulting in RNA de-adenylation by cNOT7. When TEVp is co-expressed, MS2-TCS-cNOT7 is cleaved and the two domains are separated, rescuing EGFP translation. b Flow cytometry data of the protease cascade. MS2-TCS-cNOT7 shows repression similar to its wild-type counterpart MS2-cNOT7 (Supplementary Figure 10). EGFP translation recovers in the presence of TEVp. NC: negative control (no repression). Data represent geometric mean and standard deviation of EGFP normalized by transfection marker mKate for n = 3 replicates. ru relative units. Significant changes determined by unpaired t-test are indicated with asterisks *p-value < 0.05, n = 3 replicates. c Proteases and cognate cleavage site sequences used to create additional regulatory MS2-CS-cNOT7 proteins. d Analysis of MS2-CS-cNOT7 variants responsive to SuMMVp, TVMVp, TUMVp in the absence (Repressed) or presence (Derepressed) of the protease. Cleavage sites include either a Serine or a Glycine in P’1 with reported high affinity toward the associated proteases. The proteases are ordered from most efficient to least in terms of derepression of EGFP (TUMV > TVMV > SuMMV). Data represent geometric mean and standard deviation of EGFP normalized by transfection marker mKate for n = 3 replicates. NC: negative control (no repression). ru relative units. e Proteases orthogonality. Regulatory effect is shown between all combination of proteases and MS2-CS-cNOT7, for cleavage sites with Serine in P1 (left) and with Glycine in P1 (right). Data represent geometric mean of EGFP normalized by transfection marker mKate for n = 3 replicates. Data collected 48 h post transfection