MicroRNA-directed control of complex mRNA stability patterns across cell types

Limiting expression to target cell types or states is a longstanding goal in gene therapy, which could be met by sensing endogenous microRNA. However, an unclear association between microRNA expression and activity currently hampers such an approach. Here, we probe this relationship by measuring the stability of synthetic microRNA-responsive 3'UTRs across 10 cell lines in a library format. By systematically addressing biases in microRNA expression data and confounding factors such as microRNA crosstalk, we demonstrate that a straightforward model can quantitatively predict the stability of reporters containing full microRNA target sites purely from expression data. We use this model to design constructs with previously unattainable response patterns across our cell lines. We then generalize this approach to primary tissues and cells and show that mRNA can be engineered to be differentially active between rested and exhausted T cell states. The rules we derive for microRNA expression data selection and processing should apply to microRNA-responsive devices for any environment with available expression data. Overall design: Libraries were prepared using four different protocols. All protocols start with plasmid libraries containing reporters with microRNA target sequences in their 3'UTR or 5' UTR, which were also sequenced. 1) 3' or 5' UTR libraries were transiently transfected into cell lines for n=2 replicates. After two days, library RNA was extracted, reverse transcribed, and sequenced. 2) Plasmid libraries were transfected with helper plasmids for murine stem cell virus (MSCV) into HEK293T. MSCV particles were purified and their RNA sequenced. MSCV particles were used to transduce either P2C2 cells or primary mouse CD8 T cells. T cells were expanded according to various protocols, then their library RNA and/or library constructs integrated into gDNA were extracted and sequenced. 3) Plasmid library was transiently transfected into K562 cells using electroporation. 48 hours later, Actinomycin D was added. Samples were extracted at 0 min, 30 min, 1h, 2h, 4h, 8h, 12h, and 24h after the addition of ActD. Sample library RNA was sequenced. 4) Plasmid library was transiently transfected into HEK293T or HeLa cells. 24 hours later, samples were lysed in the presence of cycloheximide and underwent a polysome profiling workflow. Individual polysome fractions were sequenced as separate libraries.