Here we report that KSRP controls the half-life and the steady-state levels of a set of unanticipated labile mRNAs in αT3-1 cells. The expression and the stability of the majority of these KSRP target transcripts is increased upon activation of PI3K-AKT signaling. Furthermore, we show that KSRP forms a ribonucleoprotein complex together with its target transcripts and the RNA binding protein hnRNPA1.
Recently, we have shown that activation of PI3K-AKT pathway induces KSRP-controlled regulation of β-catenin mRNA in αT3-1 cells . We hypothesized that PI3K-AKT activation could prolong the t1/2 of ARE-containing mRNAs additional to β-catenin by targeting KSRP.
In order to identify transcripts whose t1/2 and steady-state levels are controlled by KSRP and respond to PI3K-AKT activation, we performed a comparative analysis of the AU-rich transcriptome of αT3-1 cells focusing our attention onto KSRP target transcripts which are overrepresented in cells expressing a constitutively active AKT1. Microarray-based methods have been successfully used to study global patterns of transcript decay and comprehensively identify targets of RNA-binding proteins thus providing unique insights into gene expression programs [17, 20–25]. We identified a set of mRNAs that interact with KSRP and whose t1/2 and steady-state levels are consistently increased by either KSRP knock-down or PI3K-AKT activation. Among these transcripts, three encode RNA binding proteins mainly implicated in pre-mRNA splicing events (hnRNPA1, hnRNPF, and hnRNPA/B), three encode signaling molecules (Gsα, PP2ACA, SORBIN), and one encodes the replication-independent histone H3.3A. To our best knowledge, none of these transcripts has been yet reported to undergo posttranscriptional control of its expression through regulation of mRNA decay rates.
Our present data (see Additional file 2) together with our previous observations [7, 10–12] indicate that KSRP interacts with a rather broad array of ARE-like sequences. The criteria used by KSRP to recognize its RNA targets remain still unknown, and to date there are no reports that provide an explanation for its target recognition at the molecular level. Our unpublished structural studies on KSRP domains (M.F. Garcia-Mayoral et al., submitted) show a modularity of the interaction between K-homology (KH) domains 3 and 4 that can increase the adaptability to different RNA sequences/structures thus providing a possible explanation for the ability of KSRP to recognize highly heterogeneous RNA targets. These data indicate that KH3 and KH4 can adapt to different AU-rich sequences within the ARE without being limited by a rigid, pre-existing protein-protein interaction (M.F. Garcia-Mayoral et al., submitted). This provides the protein with a flexible recognition unit than can adapt to different RNA sequences and can mediate interactions in the structural environment of different 3'UTRs.
PI3K-AKT signaling has been reported to cause phosphorylation and activation of the SR-family members of splicing factors [26, 27]. Interestingly, hnRNPA1 has been shown to antagonize the splicing activity of SR proteins . Increased expression of hnRNPA1 could be viewed as a mean by which PI3K-AKT signaling finely modulates select splicing events. Intriguingly, hnRNPA1 transcript interacts with KSRP in the context of a complex that includes hnRNPA1 protein, thus suggesting the existence of an auto-regulatory loop.
GNAS gene encodes the Gsα that is required for hormone-stimulated cAMP generation . Recently, Chen et al. demonstrated that GNAS gene deletion causes increased insulin sensitivity targeting AKT . Our data allow the hypothesis that AKT could, in turn, regulate Gsα expression and activity operating a negative feed back-control on insulin responsiveness.
Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, whose minimal component is a well conserved catalytic subunit [reviewed in ]. PP2A plays a prominent role in cell cycle regulation, cell morphology and development . We have recently shown that PI3K-AKT activation increases the expression of β-catenin by prolonging its mRNA t(1/2) through functional inactivation of KSRP. Intriguingly, PP2A can de-phosphorylate β-catenin thus preventing its degradation and, therefore, it has been proposed as an activator of β-catenin signaling [32, 33]. Therefore PI3K-AKT, inducing stabilization of the two KSRP target transcripts β-catenin and PP2ACA, could enhance the cellular levels of β-catenin protein operating a combinatorial positive control. On the other hand, either inhibition or disruption of PP2A complexes leads to AKT activation . Therefore, it is possible that PP2ACA mRNA stabilization and enhanced expression could operate a negative feed-back on the effects of either exaggerate or inappropriate PI3K-AKT signaling activation . A potential model for KSRP-mediated control of PI3K-AKT/β-catenin signaling is presented in Additional file 9.
SORBS1, the human gene that encodes SORBIN, was mapped to the locus which is a candidate region for insulin resistance found in Pima Indians . CAP, the mouse homologue of SORBIN, is a cytoskeletal adaptor protein involved in modulating adhesion-mediated signaling events that lead to cell migration . It has been shown that stable cell lines overexpressing CAP exhibit a reduced growth rate . Recently, Katsanakis and Pillay showed that AKT phosphorylates the APS protein, a key factor in the signaling events that involve CAP . Our data support the existence of an additional point of cross-talk between PI3K-AKT signaling and the SORBIN/CAP pathway in insulin signaling.
Variations in the expression of histone H3.3A, a cell cycle-independent replacement histone, during differentiation of murine erithroleukemia cells, has been hypothesized to depend on post-transcriptional regulatory events . Although histone H3.3A expression regulation has not been reported to be controlled by PI3K-AKT signaling, it has been correlated to cell transformation and differentiation [40, 41].
Further investigations will be necessary to elucidate the functional role, if any, of the coordinated decay control of the identified transcripts by PI3K-AKT signaling under different physiological and pathological conditions.
Our data indicate that KSRP interacts with AUF1p45 and hnRNPA1 in the cytoplasm of αT3-1 cells. Only one of the PI3K-AKT-regulated KSRP targets, hnRNPA/B, is very weakly immunoprecipitated by anti-AUF1 antibody (Figure 1C and Additional file 5). Conversely, anti-hnRNPA1 antibody efficiently immunoprecipitates KSRP target transcripts (Figure 1C). Both AUF1p45 and hnRNPA1 bind very weakly to the same RNAs in vitro (data not shown). hnRNPA1 has been implicated in many aspects of mRNA maturation, transport, turnover and in telomere and telomerase regulation [42, 43]. Hamilton et al.  reported that hnRNPA1 interacts with ARE-containing mRNAs and suggested a role for this factor in ARE-mediated decay. Our findings allow to hypothesize that AUF1p45 and hnRNPA1 play some, yet unidentified, regulatory role in the ribonucleoprotein complex that includes KSRP and its target transcripts. We can hypothesize that, in response to certain stimuli, the two KSRP-interacting ARE-BPs could acquire high affinity binding for target mRNAs thus either potentiating or terminating the decay-promoting activity of KSRP on the same transcripts.
The mRNA stability promoting factor HuR interacts with KSRP target transcripts both in vitro (data not shown) and in intact cells (Figure 1C). We have previously reported that the balanced interaction of KSRP and HuR to common sets of transcripts could allow a fine tuning of mRNA decay regulation upon specific stimuli [10, 11]. Similar results were obtained by Linker et al. . Our present data further support the idea that complex interactions in the ARE-BP network are required to ensure accurate regulation of the t1/2 of select transcripts.