UTR motif screen in PTBP1KO T cells

To systematically examine how PTBP1 regulates protein expression through 3’UTR motifs, we employed a massively parallel reporter assay (MPRA) with a library of 467 synthetic 3’UTRs fused to GFP, each containing six occurrences of a 6-7 nucleotide oligomer (Nicolet, 2025). After retroviral transduction of the MPRA library into activated CD8+ T cells, we deleted PTBP1 or used non-targeting RNPs as control. After 6 days of rest, we reactivated PTBP1 KO and control T cells for 16h with a-CD3/a-CD28, or left them nonactivated. The 15% top (GFPhi) and 15% bottom (GFPlo) GFP-expressing cells were FACS-sorted, and motif enrichment for high or low protein expression was determined by sequencing.