Feedback Control of Protein Expression in Mammalian Cells by Tunable Synthetic Translational Inhibition

Feedback regulation plays a crucial role in dynamic gene expression in nature, but synthetic translational feedback systems have yet to be demonstrated. Here we use an RNA/protein interaction-based synthetic translational switch to create a feedback system that tightly controls the expression of proteins of interest in mammalian cells. Feedback is mediated by modified ribosomal L7Ae proteins, which bind a set of RNA motifs with a range of affinities. We designed these motifs into L7Ae-encoding mRNA. Newly translated L7Ae binds its own mRNA, inhibiting further translation. This inhibition tightly feedback-regulates the concentration of L7Ae and any fusion partner of interest. A mathematical model predicts system behavior as a function of RNA/protein affinity. We further demonstrate that the L7Ae protein can simultaneously and tunably regulate the expression of multiple proteins of interest by binding RNA control motifs built into each mRNA, allowing control over the coordinated expression of protein networks

Association of the candidate polymorphisms with the risk of aspirin induced enteropathy.

<p>OR, odds ratio. <i>p</i> Values, odds ratio (OR) and 95% confidence interval (CI) were obtained by Mantel-Haenszel statistics.</p

Association between various relating factors and aspirin induced enteropathy.

<p>The unadjusted odds ratio (OR) and 95% confidence interval (CI) were obtained by Mantel-Haenszel statistics and the adjusted OR and 95%CI by multiple logistic regression analysis after adjustment for the other factors. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001. </p

Allele and genotype frequencies of the candidate genes.

<p><i>p</i> Values by using the chi-square test; a, Hardy-Weinberg equilibrium (HWE) of allele frequencies at individual loci was assessed by comparing the observed and expected genotype frequencies.</p

Relative expression levels of ABC transporter family in HCC4006ER cells compared with HCC406.

<p>Relative expression levels of ABC transporter family in HCC4006ER cells compared with HCC406.</p

Effect of siRNA-mediated knockdown for CDH1 in HCC4006ER cells.

<p>(A) Total cell lysates were harvested 72 hours after reverse-transfection of negative control siRNA (siNC) or validated siRNAs for CDH1 which encodes E-cadherin mixed with Lipofectamine RNAiMAX. (B-D) Tumor cells were reverse-transfected at the same time plating into 96-wells and then incubated for 24 hours. They were incubated with various concentrations of docetaxel, paclitaxel and vinorelbine for additional 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay.</p

Expression of proteins which were reported to be associated with sensitivity to anti-microtubule agents.

<p>(A) Protein expression was evaluated by western blot analysis. Expression values of class Ⅲ beta-tubulin (TUBB3) relative to beta-actin were determined using Just TLC software. (B) Representative images of HCC4006 and HCC4006ER cells immunohistochemically stained with antibodies to ATP-binding cassette subfamily B, member 1 (ABCB1).</p

The characteristics of EGFR-TKI sensitive cell lines and their resistant clones.

<p><b>Abbreviations: EMT</b>, epithelial mesenchymal transition.</p><p>The characteristics of EGFR-TKI sensitive cell lines and their resistant clones.</p

IC50 values for cytotoxic agents in EGFR-TKI sensitive and their resistant clones.

<p><b>Abbreviations: CDDP</b>, cisplatin; <b>GEM</b>, gemcitabine; <b>DOC</b>, docetaxel; <b>PTX</b>, paclitaxel; <b>VNR</b>, vinorelbine.</p><p>IC50 values for cytotoxic agents in EGFR-TKI sensitive and their resistant clones.</p

Additional effects of entinostat in HCC4006 and HCC4006ER cells.

<p>(A-C) Tumor cells were incubated with various concentrations of docetaxel (D), paclitaxel (P) and vinorelbine (V) and with or without 1μM entinostat (E) for 72 hours. Percent growth relative to DMSO-treated controls was evaluated by Cell Counting Kit-8 assay. (D) Total cell lysates were harvested after addition of 1μM entinostat.</p