Figure 1.

The fast-UTR MPRA. (A) The BTV plasmid includes a bidirectional tetracycline regulated promoter (biTet) that drives expression of enhanced green fluorescent protein (EGFP) and a reference protein (truncated low-affinity nerve growth factor receptor, ΔLNGFR). The EGFP reporter transgene includes a multiple cloning site (MCS) for insertion of 3′ UTR test sequences and a polyadenylation signal (pAS). Pools of 160-mer oligonucleotides containing 3′ UTR segments were inserted into BTV together with random octamer indexes used to identify each clone. Cells were transduced with BTV lentiviral libraries and massively parallel sequencing was used to measure 3′ UTR segment sequences in genomic DNA and mRNA isolated from cells. (B) Steady state mRNA levels were determined from clone read counts for mRNA samples before the addition of Dox. mRNA stability was estimated from mRNA read counts obtained before and 4 h after the addition of Dox to inhibit transcription. The blue line represents a 3′ UTR segment with an element that promotes rapid mRNA decay and the green line represents a sequence with an inactivating mutation of the destabilizing element that increases steady-state mRNA levels and reduces the decay rate.