Widespread Effects of Chemokine 3' Untranslated Regions on mRNA Degradation and Protein Production in Human Cells

Abstract

Chemokines are a large family of chemotactic cytokines that play critical roles in inflammation, development, and diseases. Chemokine expression is highly regulated during development and in response to environmental stimuli. The 3' untranslated regions (3'-UTRs) of mRNA are believed to be important in the control of chemokine gene expression. However, the regulatory effects of most chemokine 3'-UTRs have not been characterized previously. In this work, we systematically studied the effects of 43 CC and CXC chemokine 3'-UTRs on gene expression in eight human cell lines and two types of human primary cells. We found that chemokine 3'-UTRs had a wide spectrum of regulatory effects on mRNA abundance and protein production that were tightly correlated with the effects on mRNA stability. In general, 3'-UTRs had remarkably similar effects across all cell types studied. The presence of AU-rich elements, microRNA targets, and Pumilio binding sites were associated with chemokine 3'-UTR activity but did not fully account for all 3'-UTR activity detected using the reporter assay. Mutational analysis illustrated how specific cis-regulatory elements contributed to the regulatory effect of chemokine 3'-UTRs. These findings bring new insights into the mechanisms by which chemokine expression is regulated by 3'-UTRs.

FIGURE 1

Quantification of the effects of chemokine 3’-UTRs on EGFP protein production with a reporter assay. (A) BTV reporter. Bi-Tet: bi-directional tetracycline-regulated promoter driving both EGFP and LNGFR expression. The 3’-UTR test sequence was cloned downstream of the EGFP open reading frame. (B) Flow cytometric analysis of BEAS-2B cells transduced with a negative control without EGFP (BTVnoGFP) or BTV without a 3’-UTR test sequence. (C) Flow cytometric analysis of effects of 3’-UTR test sequences. CXCL8 ARE: 160-nt ARE-containing sequence from CXCL8 3’-UTR; let-7 wt: synthetic let-7 target; let-7 mut: mutant let-7 target; CCL3: full length 3’-UTR of CCL3 mRNA. The dotted lines show the threshold for gating of LNGFR positive cells used in the analysis. (D) Effects of 43 chemokine 3’-UTRs on EGFP protein expression in BEAS-2B cells. The suffixes “.1” and “.2” represent 3’-UTR variants arising from alternative polyadenylation; “.1” is the longer version. All values represent mean ± s.d.. *, p < 0.05.

FIGURE 2

Comparison of the effects of chemokine 3’-UTRs on EGFP protein in cell lines and primary cells. (A) Effects of chemokine 3’-UTRs in immortalized cell lines. Reporters were introduced into each of 8 cell lines: RD (rhabdomyosarcoma), MG-63 (osteosarcoma), DLD-1 (colon cancer), BEAS-2B (airway epithelial), Jurkat (T lymphoblast), WIDR (colorectal adenocarcinoma), HelaS3 (cervical carcinoma), and H292 (airway epithelial). The color scale represents reporter protein expression relative to the control reporter with no 3’-UTR test sequence (100%) in the same cell line. (B, C) Correlation of reporter protein levels in one cell type with mean reporter protein levels in the other seven cell lines. BEAS-2B cells (B) had the highest correlation and Jurkat cells (C) had the lowest. Each point represents one of the 43 chemokine 3’-UTRs tested. (D, E) Correlation of reporter protein levels in primary lung fibroblasts (D) or primary bronchial smooth muscle cells (E) with mean reporter protein levels in the eight cell lines. All 43 chemokine 3’-UTRs were analyzed in fibroblasts and a subset of 15 chemokine 3’-UTRs were analyzed in smooth muscle cells.

FIGURE 3

Effects of chemokine 3’-UTRs on steady state mRNA levels and mRNA stability. (A) Quantification of the steady state EGFP mRNA in BEAS-2B cells with qRT-PCR. Results were normalized by comparing with BTV reporter with no 3’-UTR test sequence (100%). Values represent mean ± s.d.. *, p < 0.05. (B) Effects of chemokine 3’-UTRs on steady state EGFP reporter mRNA versus protein levels (R² = 0.91, p < 0.0001). (C) CXCL8 3’-UTR effects on mRNA degradation. (D) Effects of chemokine 3’-UTRs on reporter mRNA half-lives versus steady state mRNA levels (R² = 0.75, p < 0.0001). (E) Effects of chemokine 3’-UTRs on reporter mRNA half-lives versus protein levels (R² = 0.75, p < 0.0001). The dotted lines are linear regression lines. CCL22 and CXCL9 3’-UTRs have lower levels of mRNA than other 3’-UTRs with similar mRNA half-lives.

FIGURE 4

Relationships between chemokine 3’-UTR activity and the presence of predicted cis-elements. Comparison of the effects chemokine 3’-UTRs containing or lacking AREs (A), miRNA targets (B), Pumilio binding sites (C) or any of these elements (D) on EGFP protein level in BEAS-2B cells. Each points represents one chemokine 3’-UTR; bars represent group medians. P values were calculated using the Mann-Whitney test. (E) Examples of three predicted Pumilio binding sites (WT) and mutations tested in the BTV reporter assay. Numbers denote motif positions within the 3’-UTR. (F) Effects of mutating predicted Pumilio binding sites on reporter protein expression in BEAS-2B cells. *, p < 0.05; **, p < 0.005 by Student's t-test.

FIGURE 5

A let-7 target and an ARE in the CCL3 3’-UTR co-regulate gene expression. (A) CCL3 3’-UTR contains a conserved let-7 target and an ARE. The vertical bars represent conservation scores from PhyloP. The let-7 target is predicted by TargetScan; the ARE (based on ARED) contains a typical ARE motif (solid underline) and two non-typical ARE motifs (broken underline). (B) Mutagenesis of the let-7 target and the ARE resulted in the reduction of inhibitory activity of CCL3 3’-UTR in BEAS-2B cells. WT: CCL3 3’-UTR; ARE-del, deletion of the ARE in CCL3 3’-UTR; Let7-scr, scrambled seed sequence binding site in CCL3 3’-UTR; ARE-del&Let7-scr: mutation of both the ARE and the let-7 target. (C) The let-7 inhibitor abrogated the inhibitory effect of the let-7 target in CCL3 3’-UTR. Lipo: negative control transfection with lipofectamine; AntiLet7, let-7 inhibitor. (D) Scrambling the let-7 target seed sequence in CCL3 3’-UTR stabilized mRNA. **, p < 0.005; ns, no significant difference by one-way ANOVA and Dunnett’s test. All values represent mean ± s.d.