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. 2007 Apr 16:8:28.
doi: 10.1186/1471-2199-8-28.

Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

Tina Ruggiero  1 , Michele Trabucchi, Marco Ponassi, Giorgio Corte, Ching-Yi Chen, Latifa al-Haj, Khalid S A Khabar, Paola Briata, Roberto Gherzi
Affiliations

Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling

Tina Ruggiero et al. BMC Mol Biol. .

Abstract

Background: KSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile beta-catenin mRNA through an impairment of KSRP function.

Results: Aim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary alphaT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to beta-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation.

Conclusion: Our study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway.

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Figures

Figure 1
Figure 1
KSRP associates with a set of unstable mRNAs overrepresented in myrAKT1-αT3-1 cells. (A) In vitro RNA degradation assays using S100 extracts from αT3-1 cells. Internally 32P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with extracts for the indicated times and their decay analyzed as described in Methods. (B) The interaction between 32P-labeled RNAs (indicated on the right) and recombinant purified KSRP (30–300 nM) was evaluated by UV-crosslinking. (C) Immunoprecipitation of ribonucleoprotein complexes containing different KSRP target mRNAs. The proteins were immunoprecipitated from αT3-1 cell extracts using the indicated antibodies. RNA was extracted from the immune complexes and analyzed by RT-PCR as described in Methods.
Figure 2
Figure 2
KSRP associates with AUF1p45 and hnRNPA1 in cytoplasmic extracts of aT3-1 cells. (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1.
Figure 3
Figure 3
KSRP is required for rapid degradation of a set of unstabletranscripts. (A) Immunoblot analysis of total extracts from either mock-αT3-1 (empty pSUPER-Puro vector-transfected) or αT3-1-shKSRP (pSUPER-Puro-shKSRP-transfected) cells using affinity-purified anti-KSRP and anti-α-tubulin antibodies. (B) Expression of a set of KSRP-interacting mRNAs and β2-MG (as a control), monitored by RT-PCR, in either mock-αT3-1, or αT3-1-shKSRP cells. (C) Semi quantitative RT-PCR analysis of both labile KSRP-associated mRNAs and β2-MG mRNA in either mock-αT3-1 (black lines), or αT3-1-shKSRP cells (red lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 6.
Figure 4
Figure 4
PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression. (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[32P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file 7.
Figure 5
Figure 5
Insulin stabilizes a set of KSRP-interacting mRNAs. (A) Serum-starved HIRc-B cells were treated for 1 h with either PBS (control) or insulin (10-6 M). Total extracts were immunoprecipitated with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[32P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) In vitro RNA degradation assays using S100 extracts from either control or insulin (10-6 M)-treated HIRc-B cells. Internally 32P-labeled, capped RNA substrates (see Additional file 4 for sequences) were incubated with S100 extracts for the indicated times and their decay analyzed as described in Methods.
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