(A) Scatter plot of common and extended UTR region expression of tandem APA genes in resting and stimulated (48h) T lymphocytes. Significantly increased expression of isoforms resulting from distal PAS (blue) or proximal PAS (red) (P < 0.02, 20% FDR) are colored. Inset: Direction of change (13 distal, 86 proximal; P < 2.3×10^-14, binomial test). (B) Relative expression of mutually exclusive 3′ exons in resting and stimulated (48h) T lymphocytes. Significant events (P < 0.0031; 5% FDR) and inset are as in (A). (C) Expression of common versus extended 3′ UTR regions in resting (0h, n = 1768) versus activated (48h, n = 1761) cells. Difference P < 6.6×10^-19, t-test. (D) Relative expression (activated/resting) of all tandem UTR genes and in the set of significant events from (A) after stimulation.

(A) TLI analysis of murine CD4⁺ T lymphocytes stimulated with anti-CD3/anti-28 beads (this study). (B) TLI values from human CD4⁺ T cells, B cells, and monocytes activated for 30 h with the indicated stimulus. (C) TLI values of cultured cancer cell lines and of matched normal tissues. (D) Correlation of TLI with proliferation index across a panel of 135 different tissues and samples (R_s = Pearson; R_p = Spearman correlations). Data in (A-D) are mean +/- SD of 2-33 replicates (Table S8).

(A) Luciferase activity in stimulated primary murine T lymphocytes transfected with reporter constructs encoding common and extended 3′ UTR isoforms of indicated genes (Cnn3, acidic calponin 3; E4300, putative phosphodiesterase; Gtf2e2, General transcription factor II E, polypeptide 2; Hip2, Huntingtin-interacting protein 2; Rab1, Ras-associated protein Rab1). (B) Mean expression +/- SD of common transcript expression and relative 3′ UTR expression of the Hip2 transcript at 48 h post-stimulation. (C) Immunoblot of Hip2 protein in resting and stimulated (24 hour and 48 hour) primary CD4⁺ T cells. Znf207 serves as a loading control. (D) Percentage restoration of luciferase activity in stimulated primary murine T lymphocytes transfected with the indicated reporter constructs (Fig. S9). (E) Mean number of conserved 3′ UTR miRNA seed matches in proximal and distal PAS isoforms of genes with tandem APA sites. (F) Cumulative density plot of mean relative expression of extended regions (log₂) for conserved targets (n = 64) and non-targets (n = 64) of activation and proliferation-associated miRNAs (mir-155, miR-17-92 cluster) at 48h post-stimulation. (G) Distribution of fraction of mRNA targets lost for each miRNA upon shift from distal to proximal PAS in genes with tandem UTRs that increase or decrease in expression 48h post-stimulation. Data in (A) and (D) are mean +/- SD of triplicate measurements and are representative of 3-4 independent experiments.