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. 2008 Jun 20;320(5883):1643-7.
doi: 10.1126/science.1155390.

Proliferating cells express mRNAs with shortened 3' untranslated regions and fewer microRNA target sites

Rickard Sandberg  1 , Joel R Neilson, Arup Sarma, Phillip A Sharp, Christopher B Burge
Affiliations

Proliferating cells express mRNAs with shortened 3' untranslated regions and fewer microRNA target sites

Rickard Sandberg et al. Science. .

Abstract

Messenger RNA (mRNA) stability, localization, and translation are largely determined by sequences in the 3' untranslated region (3'UTR). We found a conserved increase in expression of mRNAs terminating at upstream polyadenylation sites after activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3'UTRs, is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues. Forced expression of full-length 3'UTRs conferred reduced protein expression. In some cases the reduction in protein expression could be reversed by deletion of predicted microRNA target sites in the variably included region. Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3'UTR-based regulatory capacity of mRNAs.

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Figures

Figure 1
Figure 1. Reduced relative expression of extended 3′ UTR regions 48h after T lymphocyte activation
(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.
Figure 2
Figure 2. Reduced expression of extended 3′ UTR regions is widespread, conserved to human, and correlated with proliferation
(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 (Rs = Pearson; Rp = Spearman correlations). Data in (A-D) are mean +/- SD of 2-33 replicates (Table S8).
Figure 3
Figure 3. Differential protein expression conferred by extended 3′ UTR regions and miRNA seed matches
(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.
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