mRNA degradation plays a significant role in the program of gene expression regulated by phosphatidylinositol 3-kinase signaling

Abstract

Control of gene expression by the phosphatidylinositol (PI) 3-kinase/Akt pathway plays an important role in mammalian cell proliferation and survival, and numerous transcription factors and genes regulated by PI 3-kinase signaling have been identified. Because steady-state levels of mRNA are regulated by degradation as well as transcription, we have investigated the importance of mRNA degradation in controlling gene expression downstream of PI 3-kinase. We previously performed global expression analyses that identified a set of approximately 50 genes that were downregulated following inhibition of PI 3-kinase in proliferating T98G cells. By blocking transcription with actinomycin D, we found that almost 40% of these genes were regulated via effects of PI 3-kinase on mRNA stability. Analyses of β-globin-3' untranslated region (UTR) fusion transcripts indicated that sequences within 3' UTRs were the primary determinants of rapid mRNA decay. Small interfering RNA (siRNA) experiments further showed that knockdown of BRF1 or KSRP, both ARE binding proteins (ARE-BPs) regulated by Akt, stabilized the mRNAs of a majority of the downregulated genes but that knockdown of ARE-BPs that are not regulated by PI 3-kinase did not affect degradation of these mRNAs. These results show that PI 3-kinase regulation of mRNA stability, predominantly mediated by BRF1, plays a major role in regulating gene expression.

FIG. 1.

Inhibition of PI 3-kinase increases mRNA degradation. Proliferating T98G cells were treated with actinomycin D (ActD) in either the presence (dashed lines) or the absence (solid lines) of the PI 3-kinase inhibitor LY294002 (A and B) or PI-103 (C). Total RNA was isolated after 1, 2, 4, or 6 h, and the mRNA levels of 48 genes were determined by real-time RT-PCR. Inhibition of PI 3-kinase with LY294002 increased the mRNA degradation of 18 genes (A). The results for six representative genes for which degradation was not affected by inhibition of PI 3-kinase with LY294002 are shown in panel B. PI 3-kinase inhibition using PI-103 increased mRNA degradation, with the results for six representative genes shown in panel C. Data are presented as the percentages of mRNA relative to the level for a DMSO vehicle control at each time point and are the means of results from two (B and C) or three (A) independent experiments ± standard errors (SE).

FIG. 2.

Correlation between mRNA half-lives and effects of PI 3-kinase on mRNA degradation. The half-lives of mRNAs that were <10 h (A) or <5 h (B) following treatment with actinomycin D and LY294002 (LY) were compared to the ratio of mRNA levels in cells treated for 4 h with actinomycin D with and without LY294002, as calculated from the data in Fig. 1. For panel A, correlation coefficient r² = 0.3, P = 0.0001, and n = 39. For panel B, correlation coefficient r² = 0.4, P = 0.0001, and n = 29.

FIG. 3.

mRNA degradation is induced by the 3′ UTRs of genes downregulated upon PI 3-kinase inhibition. HeLa Tet-off cells were transfected for 24 h with either a β-globin reporter gene (solid line) or a β-globin-3′ UTR fusion transcript (dashed lines). Transcription was stopped by the addition of doxycycline. Total RNA was harvested after the indicated times and the levels of β-globin transcript determined by real-time RT-PCR. The 3′ UTRs either induced rapid decay of the β-globin reporter gene, with half-lives of <1 h (A), induced moderate decay, with half-lives of 2 to 2.5 h (B), or had no effect (C). Data represent the percentages of mRNA relative to the control level and are representative of 2 to 6 experiments (means ± SE). Shown in panel D is the correlation between mRNA half-lives following treatment with actinomycin D plus LY294002 (Fig. 1) and half-lives of 3′ UTR fusion transcripts. Correlation coefficient r² = 0.4, P = 0.009, and n = 14.

FIG. 4.

Stabilization of mRNA degradation by knockdown of BRF1 or KSRP. Proliferating T98G cells were transfected for 72 h with a nonspecific control siRNA, BRF1 siRNA, or KSRP siRNA. Immunoblots were used to measure knockdown of BRF1 and KSRP (A). mRNA degradation was measured by real-time RT-PCR following treatment with either DMSO or LY294002 for the indicated times (B). Data are presented as the percentages of mRNA compared to the level for the vehicle control at each time point.

FIG. 5.

Global effects of BRF1 and KSRP knockdown on mRNA degradation following PI 3-kinase inhibition. Proliferating T98G cells were transfected for 72 h with BRF1 siRNA (A), KSRP siRNA (B), or a nonspecific control siRNA. Cells were treated with DMSO or LY294002 for 4 h, after which levels of 48 downregulated mRNAs were measured by real-time RT-PCR. Data are presented only for those mRNAs that were significantly affected by either BRF1 or KSRP knockdown (P < 0.05) and represent the percentages of mRNA compared to the level for the DMSO vehicle control (means of results from four [A] or three [B] independent experiments ± SE).

FIG. 6.

Knockdown of either AUF1 or HuR does not affect PI 3-kinase-regulated mRNA degradation. Proliferating T98G cells were transfected for 72 h with AUF1 siRNA, HuR siRNA, or a nonspecific control siRNA. Immunoblot analysis was used to measure knockdown of AUF1 and HuR (A). Cells were treated with DMSO or LY294002 for 4 h, after which levels of 48 downregulated mRNAs were measured by real-time RT-PCR (B). Data represent the percentages of mRNA compared to the level for the DMSO vehicle control and are the means of results from five (nonspecific control), three (AUF1), or two (HuR) independent experiments ± SE.

FIG. 7.

Correlations between mRNA stabilization by BRF1 siRNA, mRNA half-lives, and PI 3-kinase-regulated mRNA degradation. The mRNA stabilization resulting from BRF1 knockdown (the ratio of mRNA levels in cells transfected with BRF1 siRNA compared to the level for control siRNA) (Fig. 5) was compared to the mRNA half-lives following treatment with actinomycin D plus LY294002 (LY) (Fig. 1) (r² = 0.13; P = 0.01; n = 39) (A), the half-lives of the β-globin-3′ UTR fusion transcripts (Fig. 3) (r² = 0.3; P = 0.02; n = 14) (B), or the ratio of mRNA levels in cells treated for 4 h with actinomycin D with or without LY294002 (Fig. 1) (r² = 0.2; P = 0.005; n = 39) (C).

FIG. 8.

Relationship between genes regulated by PI 3-kinase and ARE-BPs. Shown is the distribution of the 48 mRNAs downregulated upon PI 3-kinase inhibition according to whether their mRNA degradation increased upon PI 3-kinase inhibition (PI3K-regulated) or was stabilized by KSRP and/or BRF1 knockdown.