We previously identified over 50 genes that were downregulated at least 1.8-fold in response to inhibition of PI 3-kinase in proliferating T98G cells (55
), and the present study has demonstrated that ARE-mediated mRNA degradation plays a key role in regulating their expression. By blocking transcription, we determined that the degradation of almost 40% of these downregulated mRNAs increased when PI 3-kinase was inhibited. Notably, the half-lives of mRNAs were highly correlated with the effect of PI 3-kinase inhibition on the rate of degradation, such that the degradation of mRNAs with short half-lives was most likely to be regulated by PI 3-kinase. In fact, of the 18 genes that exhibited PI 3-kinase-regulated mRNA degradation, only 3 had half-lives that were greater than 3 h. Therefore, our results suggest that PI 3-kinase regulates the mRNA stability of many of its target genes, the majority of which have mRNAs that are rapidly degraded.
Rapid and selective degradation of many mRNAs is mediated by AREs within their 3′ UTRs (14
), and statistical analysis of the genes that were downregulated following inhibition of PI 3-kinase indicated that their 3′ UTRs were enriched in sequences defined as common ARE motifs. Using a β-globin
reporter gene fused to the 3′ UTRs of PI 3-kinase-regulated mRNAs, we found that there was a strong correlation between the half-lives of the β-globin
-3′ UTR transcripts and the half-lives of intact mRNAs in actinomycin D-treated cells, indicating that degradation of these mRNAs was primarily mediated by sequences within their 3′ UTRs. PI 3-kinase signaling has previously been shown to stabilize ARE-containing transcripts, including interleukin 3
, as a result of phosphorylation of the ARE-BPs KSRP and BRF1 by Akt (29
). We therefore performed siRNA experiments against BRF1 and KSRP and found that the mRNAs of almost 70% of the downregulated genes were stabilized upon knockdown of either BRF1 or KSRP. BRF1 was the dominant factor in controlling mRNA stability downstream of PI 3-kinase signaling, with 31 out of 48 mRNAs stabilized by knockdown of BRF1. In contrast, knockdown of KSRP stabilized 11 mRNAs, with 9 of these also stabilized by knockdown of BRF1. We also showed that knockdown of ARE-BPs that have not been shown to be regulated by PI 3-kinase did not affect mRNA levels following PI 3-kinase inhibition, further illustrating the significance of BRF1 and KSRP in mediating mRNA degradation downstream of PI 3-kinase. Importantly, 16 out of the 18 genes that exhibited PI 3-kinase-regulated mRNA degradation were stabilized by BRF1 knockdown, indicating that BRF1 was the major factor involved in controlling mRNA stability downstream of PI 3-kinase signaling.
Other global studies have previously identified mRNAs that are targeted for degradation by KSRP. Ruggiero et al. compared mRNAs that were bound by KSRP and those that were upregulated by overexpression of Akt and identified a set of eight mRNAs that both were targets of KSRP and were induced by Akt overexpression (49
). In a similar manner, Winzen et al. identified a set of 100 mRNAs that were induced in response to stimulation with the inflammatory cytokine interleukin 1 and were both bound by KSRP and stabilized by KSRP knockdown (60
). Of the 11 mRNAs that we found to be stabilized by knockdown of KRSP following inhibition of PI 3-kinase, 9 had previously been identified by Winzen et al. as bound and/or stabilized by KSRP knockdown (60
), while CCL2
are newly identified targets of KSRP.
Although Akt has also been shown to inhibit the activity of BRF1, which led to the stabilization of a reporter gene containing the 3′ UTR of interleukin 3 (4
), other genes whose mRNA degradation is regulated by the PI 3-kinase pathway via BRF1 have not been identified prior to this study. Furthermore, only a few BRF1 target genes have been determined, although its family member TTP has been much more extensively studied (2
). Of the 31 mRNAs that we found to be stabilized by BRF1 knockdown, only BIRC3/c-IAP2
has previously been reported to be regulated by BRF1 (38
), although TTP has been shown to regulate CCL2
) and CCND1
Although there was a significant enrichment of sequences defined as ARE motifs within the 3′ UTRs of genes downregulated by PI 3-kinase inhibition, the presence of these motifs alone is not sufficient to predict ARE-mediated decay and may reflect the need for additional sequence features to make a functional ARE. Using multiple variations of the motif, including the pentamer, nonamers, conserved heptamer, variations on the heptamer, and total number of all AREs, we did not find any significant correlation between the number of ARE motifs and the half-lives of the downregulated mRNAs. Although the nonamer was present in 30% of the rapidly degraded mRNAs (half-lives of <3 h), it was also present in 10% of the stable mRNAs (half-lives of >3 h). Furthermore, both the pentamers and the heptamers were found in 85% and 70% of the mRNAs in both groups. Thus, although many short-lived mRNAs contain an ARE motif, these elements are also present in more stable mRNAs. Conversely, many rapidly degraded mRNAs do not have an ARE sequence. Several global studies have investigated the relationship between mRNA decay rates and the presence of typical ARE motifs, which have also determined that the presence of an ARE motif does not predict a short mRNA half-life (25
). Our results and those obtained by others demonstrate that these sequence elements are not an accurate indicator of rapidly degraded mRNAs, suggesting that other signals are also responsible for degradation. Of note, a functional ARE may also include a U-rich region, an overall U-rich content, or multiple motifs (3
). Therefore, simply identifying the presence of an ARE motif or motifs does not reflect the complexity of regulation of these elements and does not recognize other sequence features that may be necessary to mediate mRNA degradation.
Additionally, we investigated the relationship between the presence of an ARE-BP binding motif and mRNA degradation regulated by an ARE-BP. Because it has previously been shown that the multiple domains of KSRP allow it to bind to a broad range of motifs as a result of each domain recognizing different sequences (26
), we focused on mRNAs regulated by BRF1. The optimal binding sequence for TTP family members, which includes BRF1, has been identified as the UUAUUUAUU nonamer (5
). TTP can also bind to variations of this sequence, namely, a UAUUUAU heptamer (36
), where the internal U sequence can consist of either two or four Us with minimal loss of affinity (8
). Of the mRNAs stabilized by BRF1 knockdown, 19% contained a nonamer and 58% contained some variation of the heptamer; however, we did not find that any of these motifs were sufficient to predict BRF1 regulation. There was no strong correlation between AREs and amount of stabilization, and in fact, several mRNAs that were stabilized by BRF1 knockdown did not contain any of these AREs. Conversely, some mRNAs that did contain a motif were not affected by knockdown of BRF1. These results are similar to what has been observed in studies of TTP, in which only 33 out of 250 mRNAs stabilized in TTP−/−
cells were found to have at least two of the heptamer sequences (36
), and in a separate study, where fewer than half of TTP-bound mRNAs contained a consensus nonamer (54
). Overall, this suggests that the sequence to which these ARE-BPs bind may be more flexible than previously thought or that these proteins bind indirectly to the mRNA via interactions with other proteins. Indeed, it has previously been shown that not only can many mRNAs bind to multiple ARE-BPs but there can be functional cooperation between the ARE-BPs, with either synergistic or antagonistic effects (3
). Moreover, mRNA stability can be influenced by sequences flanking the ARE (42
), by mRNA structure (24
), or by other elements, such as miRNAs, further illustrating the complexity involved in regulating mRNA decay.
Previous studies of the control of gene expression by PI 3-kinase signaling have focused primarily on transcriptional regulation. Our present results indicate that regulation of mRNA stability, predominantly mediated by BRF1, also constitutes a significant component of the overall program of gene expression controlled by PI 3-kinase in proliferating cells. Since it is well known that PI 3-kinase signaling is a critical regulator of cell survival and proliferation, these results imply a significant role for regulation of mRNA decay in these vital processes.