Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling
- Tina Ruggiero†1,
- Michele Trabucchi†1,
- Marco Ponassi1,
- Giorgio Corte1, 2,
- Ching-Yi Chen3,
- Latifa al-Haj4,
- Khalid SA Khabar4,
- Paola Briata†1 and
- Roberto Gherzi†1Email author
© Ruggiero et al; licensee BioMed Central Ltd. 2007
Received: 19 December 2006
Accepted: 16 April 2007
Published: 16 April 2007
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 β-catenin mRNA through an impairment of KSRP function.
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 αT3-1 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 β-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.
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.
Regulated mRNA decay is a key factor in determining the expression pattern of many genes including those encoding for cytokines, proto-oncogenes, cell cycle regulators, and growth factors . Adenylate-uridylate-rich elements (AREs), present in the 3'-untranslated region (3'UTR) of many inherently labile mRNAs, are the most widespread and best characterized destabilizing sequences [1, 2]. Impairment of the ARE-mediated mRNA decay results in abnormal cell proliferation and angiogenesis, leading to cancer insurgence and progression , as well as in inflammatory diseases such as Crohn-like inflammatory bowel disease and inflammatory arthritis .
The interaction of regulatory proteins, ARE-binding proteins (ARE-BPs), with their target labile mRNAs determines the half-life (t1/2) of these transcripts. Some ARE-BPs are decay-promoting factors (TTP, BRF1, KSRP) . Others, such as HuR, are stabilizing factors, whereas AUF1 mainly promotes decay although certain isoforms might be stabilizers of ARE-containing mRNAs [1, 5, 6]. According to the recently proposed recruitment model, destabilizing ARE-BPs, recruit the enzymatic degradation machinery to their target mRNAs [7–9].
We and others have recently reported that KSRP promotes rapid decay of several ARE-containing mRNAs both in vitro and in vivo and that extracellular stimuli regulate its activity [7, 10–13]. We have shown that activation of either Wnt/β-catenin pathway in αT3-1 cells  or MAPK p38 signaling in C2C12 myoblasts  selectively regulates the stability of specific sets of labile mRNAs targeting KSRP. More recently, we demonstrated that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) signaling activation induces stabilization and enhances the steady-state levels of β-catenin mRNA in pituitary αT3-1 cell line through phosphorylation and functional inactivation of KSRP . PI3K-AKT signaling exerts a central role in metabolism, cell survival, motility, transcription and cell-cycle progression [ and literature cited therein].
It has been recently suggested that control of mRNA decay is utilized by the cell to coordinate the expression of genes involved in specific processes leading to the notion of 'post-transcriptional operons' . This would allow multiple genes to be co-regulated by a similar array of RNA-binding proteins in response to certain stimuli. On this basis, we hypothesized that PI3K-AKT activation could regulate the expression of transcripts additional to β-catenin by targeting KSRP. According to this hypothesis, a subset of KSRP target transcripts should be stabilized in response to PI3K-AKT signaling.
To verify this hypothesis, we systematically searched, among the AU-rich transcriptome, for KSRP target transcripts whose expression was upregulated by PI3K-AKT signaling. We identified a set of labile mRNAs, stabilized upon either KSRP knock-down or PI3K-AKT activation, encoding signaling factors, RNA binding proteins, and a replication-independent histone. These proteins could play a role in the cascade of cellular events initiated by PI3K-AKT activation.
Identification of KSRP target transcripts upregulated in cells expressing constitutively active myrAKT1
Transcripts whose levels are increased by at least 2 fold in αT3-myrAKT1 when compared with mock-αT3. Transcripts identified as KSRP targets (see Table 3) are typed in bold.
CDP-diacylglycerol-inositol 3-phosphatidyltransferase (phosphatidylinositol synthase)
Catalyzes the biosynthesis of phosphatidylinositol.
SNAP91, synaptosomal-associated protein, 91 kDa homolog
Component of clathrin-coated vescicles.
Solute carrier organic anion transporter family, member 1C1
Mediates the Na(+)-independent high affinity transport of organic anions such as the thyroid hormones thyroxine (T4) and rT3.
Heterogeneous nuclear ribonucleoprotein F (hnRNPF)
RNA binding protein, splicing.
Fibroblast growth factor 5
Oncogene, can transform NIH 3T3 cells.
Tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase 2
Involved in the regulation of telomere length.
Fibroblast growth factor 19
Has a role in brain development, overexpressed in colon adenocarcinoma cell line.
Heterogeneous nuclear ribonucleoprotein A/B (hnRNPA/B)
RNA binding protein.
Microtubule-associated protein RP/EB family member 1
Component of the microtubule cytoskeleton.
Protocadherin beta 9
Calcium-dependent cell-adhesion protein.
H3 histone, family 3A (H3.3A)
Replacement histone, replication independent protein.
Thyroid hormone receptor interactor 4
Transcriptional coactivator of nuclear receptors.
ELL associated factor 2
Transcriptional transactivator of ELL and ELL2 elongation activities.
RUN and SH3 domain containing 1
Cytochrome c oxidase subunit VIIc
Component of cytochrome c oxidase.
Zinc finger protein 192
Notch homolog 3
Forms a transcriptional activator complex.
PHD finger protein 12
GNAS complex locus (Gsα)
Guanine nucleotide-binding protein.
Nascent-polypeptide-associated complex alpha polypeptide
Prevents inappropriate targeting of non-secretory polypeptides to the endoplasmic reticulum.
Protein phosphatase 1, catalytic subunit, beta isoform
Ser/Thr phosphatase, essential for cell division.
Sorbin and SH3 domain containing 1 (SORBIN)
Involved in insulin receptor signaling.
G protein modulator.
Immunoglobulin mu-binding protein 2
DNA binding protein.
ornithine decarboxylase antizyme 1
Destabilizes and promotes degradation of ornithine decarboxylase.
Involved in cytokinesis.
epidermal growth factor
Death-associated protein kinase 1
Pro-apoptotic calcium/calmodulin-dependent serine/threonine kinase.
Scaffolding protein within caveolar membranes. Interacts directly with G-protein alpha subunits and can functionally regulate their activity.
Dual specificity protein phosphatase 4
Regulates mitogenic signal transduction by dephosphorylating both Thr and Tyr residues on MAP kinases ERK1 and ERK2.
Brix domain-containing protein 2
Biogenesis of the 60S ribosomal subunit.
Catenin beta (CTNNB) *
Wnt signaling, cell transformation
Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform (PP2ACA)
Dephosphorylates several Ser/Thr kinases.
Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1)
RNA binding protein.
Transcripts whose levels are increased by at least 3 fold upon GST-KSRP chromatography when compared with control GST chromatography.
11 ARE pentamers
2 ARE pentamers
Guanine nucleotide -binding protein
Protein phosphatase 2A catalytic subunit alpha Isoform (PP2ACA)
6 ARE pentamers
Sorbin and SH3 domain containing 1 (SORBIN)
11 ARE pentamers
Histone 3.3A (H3.3A)
7 ARE pentamers
2 ARE pentamers
Prothymosin alpha (28)
1 ARE pentamer
4 ARE pentamers
ATP synthase mitochondrial F0 complex subunit G
4 ARE pentamers
3 ARE pentamers
2 ARE pentamers
Ecotropic viral integration site 5
15 ARE pentamers
Catenin beta (CTNN) *
KSRP associates with AUF1p45 and hnRNPA1 in the cytoplasm of αT3-1 cells
Molecular partners of KSRP identified by two-hybrid screening.
RNA binding protein
RNA RNA binding protein
ARE binding protein
Poly(A) binding protein
eIF2B beta subunit
We investigated whether either AUF1p45 or hnRNPA1 or both directly interacted with KSRP target transcripts. UV-crosslinking experiments failed to display high affinity interaction of these ARE-BPs with the KSRP target transcripts in vitro (data not shown). This finding suggest that AUF1p45 and hnRNPA1 are part of the KSRP-containing ribonucleoprotein complex but do not directly interact with KSRP targets.
KSRP knock-down in αT3-1 cells stabilizes KSRP target transcripts
To verify the relevance of KSRP in the decay control of its target transcripts, stable knock-down of KSRP using a short-hairpin vector was performed in αT3-1 cells (αT3-1-shKSRP; Figure 3A). KSRP knock-down led to two- to five-fold increase of the steady-state levels of KSRP target mRNAs in αT3-1-shKSRP when compared to mock-transfected cells (Figure 3B). No changes were seen with the control β2-MG RNA levels (Figure 3B). Next, using actinomycin D, we analyzed the t1/2 of the identified KSRP target mRNAs in both mock-αT3-1 and αT3-1-shKSRP cells. Results presented in Figure 3C showed that KSRP knock-down in αT3-1 cells strongly increased the t1/2 of all the transcripts (from less than 60 min. in mock-αT3-1 to more than 2 hours in αT3-1-shKSRP cells, see Additional file 6).
Overall these data indicate that KSRP interacts with a subset of mRNAs up-regulated in cells expressing constitutively active AKT1 and regulates their stability and steady-state levels in αT3-1 cells.
PI3K-AKT activation stabilizes KSRP target transcripts
Notably, both the t1/2 and steady-state levels of some of the KSRP targets (Table 2), as exemplified by PTMA, were not affected by PI3K-AKT activation although increased by KSRP knock-down in αT3-1 cells (Additional file 8).
Altogether, these data indicate that activation of PI3K-AKT signaling increased both the t1/2 and the steady-state levels of a subset of KSRP target transcripts.
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.
In conclusion, we have identified several KSRP target mRNAs that are overrepresented upon activation of PI3K-AKT signaling. The interaction of KSRP with these transcripts was validated in vitro and in intact cells. Importantly, both KSRP knock-down and PI3K-AKT activation were found to increase the stability and the steady-state levels of these target mRNAs. Our findings provide comprehensive and valuable insight into the KSRP-containing ribonucleoprotein complexes that govern gene expression at the posttranscriptional level.
Yeast two hybrid screening
A cDNA fragment encoding amino acids 47–711 of human KSRP was cloned into pDBLeu vector (Invitrogen) and used as the bait. MaV203 yeast cells containing pDBLeu-KSRP constructs were tested for self-activation and the concentration of 3-Amino-1,2,3,-Triazole required to inhibit the basal endogenous expression of HIS3 gene was determined. A e12.5 mouse embryo head cDNA library was prepared using the pEXP-AD502 vector according to manufacturer's (Invitrogen) instructions. pDBLeu-KSRP-containing MaV203 yeast cells were transfected with the library and selected according to the activation of the three reporter genes HIS3, URA3 and LacZ according to the manufacturer's (Invitrogen) protocol.
Isolation of KSRP-co-purifying RNAs
To isolate mRNAs co-purifying with KSRP, the SNAAP (isolation of s pecific n ucleic a cids a ssociated with p roteins) technique described by Trifillis et al.  was used with minor modifications. Briefly, both GST and GST-KSRP fusion protein were expressed in Escherichia coli BL21. Cells expressing either protein were resuspended in lysis buffer (20 mM HEPES, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1X Complete protease inhibitors (Roche)), disrupted by sonication, and insoluble material removed by centrifugation. To eliminate bacterial RNAs, the extract was treated with 200 U/ml micrococcal nuclease (GE Healthcare) in the presence of 1 mM CaCl2 at 30 C for 20 min, and the reaction was stopped with the addition of 5 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA). Approximately 4 mg of either GST or GST-KSRP were bound to 1 ml GST-beads in a total volume of 5 ml in RNA binding buffer (RBB; 10 mM Hepes, pH 7.6, 3.0 mM MgCl2, 100 mM KCl, 2 mM DTT, 5% glycerol, 0.5% Triton X-100, 1X Complete) at 4°C for 1 h. Unbound proteins were removed with ten 5-ml washes in RBB. The washed beads were resuspended in 5 ml of RBB containing 50 μg/ml heparin (Calbiochem) and 200 U/ml RNasin (Promega). Fifty mg of cytoplasmic S100 extracts from αT3-1 cells were precleared with 2 ml of glutathione Sepharose slurry (extensively washed in RBB) to remove background RNAs that bind to the glutathione Sepharose beads. Incubation of the precleared S100 extracts to the above described washed beads was carried out at 4°C for 1 h under rotation, followed by six 5-ml washes in RBB. The RNA was then extracted with phenol/chloroform (1:1) and chloroform, ethanol precipitated with GlycoBlue (Ambion), and washed with 70% EtOH. The dried RNA was resuspended in 50 μl DEPC-treated H2O.
Microarray hybridization and computational analysis of AREs
The glass microarrays contained cDNA probes representing more than 3000 ARE-cDNAs and control clones (their identities were obtained from AU-rich element-containing mRNA database ARED 3.0 ). The microarrays were hybridized with cDNA generated from total RNA (15 μg) and labeled with either Cy5 or Cy3 (control). The utilized hybridization protocol (Genisphere kit, Genisphere, Inc., Hatfield, PA) eliminated the possibility of signal contribution from genomic DNA . cDNA microarrays scanning, pre-processing, filtering of erroneous signals, and normalization were performed as described in .
314 mRNAs enriched by at least 1.8-fold in KSRP-bound RNA samples (KSRP targets) and 314 mRNAs that were not enriched in KSRP-bound RNA samples (non-KSRP targets) were extracted from the microarray data. The sequences of the 3' UTR of both groups were used as input for the MotifSampler algorithm. The MotifSampler algorithms finds over-represented motifs in sequence regions using Gibbs sampling that has been successfully applied for both promoter and unstranslated regions . This strategy has been applied previously .
Murine αT3-1 pituitary cells and rat HIRc-B fibroblasts were cultured in DMEM plus 10% FBS. αT3-1 cell transfections were performed using Lipofectamine Plus (Invitrogen), G418 (Invitrogen) was used at 500 μg/ml for selection. Cell pools of transfectants were used for experiments. Both mock-αT3-1 and αT3-1-myrAKT1 cells were starved in DMEM plus 0.5% FBS for 16 hrs prior to experiments. HIRc-B cells were starved in DMEM plus 0.1% FBS for 16 hrs prior to experiments or further treatments.
shRNA-mediated KSRP knock-down
pSUPER-Puro-shKSRP was previously described . αT3-1 cells were transfected using Lipofectamine Plus (Invitrogen). Transfectant pools were selected with 0.3 μg/ml puromycin (Sigma).
Recombinant proteins and antibodies
Affinity-purified human KSRP, expressed using the Baculovirus system, was described in Briata et al. . cDNA fragments encoding the entire coding sequence of human hnRNPA1, the entire coding sequence of murine AUF1p45, and nt. 202–2136 of human KSRP were cloned into the pGEX6 to generate GST-A1, GST-AUF1p45, and GST-KSRP respectively. E. Coli-expressed GST-A1 protein was digested by Prescission protease (GE Healthcare) according to manufacturer's instructions. Anti-KSRP rabbit polyclonal antibody was previously  described. Anti-AUF1 and anti-hnRNP-A1 monoclonal antibodies were a kind gift from Dr. Gideon Dreyfuss. Anti-TTP (rabbit polyclonal H-120) was from Santa Cruz. Anti-α-tubulin, and anti HuR (3A2) monoclonal antibodies were from Sigma and Santa Cruz, respectively.
RNA in vitro degradation and UV crosslinking
Immunoprecipitation of ribonucleoprotein complexes
Ribonucleoprotein complexes were immunoprecipitated from αT3-1 cell lysates as previously described . Total RNA, extracted from either immunocomplexes or total cell lysates (input) was subjected to RT-PCR reactions. Primers are listed in Additional file 10.
In vitro kinase assays
Kinase assays were performed using AKT kinase activity immunoprecipitated from cell lysated and histone H2 (Roche) as the substrate. [γ-32P]ATP (3000 Ci/mmol) was from GE Healthcare.
Cells under different culture conditions were treated with 5 μg/ml actinomycin D, harvested at the indicated times, and total RNA was isolated using RNeasy mini kit (Qiagen) and treated with DNAseI (Promega) according to manufacturer's instructions. cDNA first strand was obtained with Transcriptor Reverse Transcriptase (Roche) using 250 ng of total RNA and oligo-dT primer. PCR reactions were performed using the sequence-specific primers listed in Table 2 of the Additional Data. β2-microglobulin was used as an internal control for normalizing transcripts levels measured by RT-PCR. To optimize RT-PCR, preliminary dose-response experiments were performed to determine the range of first strand cDNA concentrations at which PCR amplification was linear for each target molecule essentially as reported in Briata et al. . For each species of RNA analyzed, the amount of RT-PCR product (measured as densitometric units) was plotted against the input of first strand cDNA.
ARE binding protein
myristylated form of AKT1
Protein phosphatase 2A
The authors thank Ms. Hana Abulleef for her bioinformatics assistance. We are indebted with Dr. G. Dreyfuss for antibodies. M.T. present address is: George Palade Laboratories, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0648 USA. This work has been partly supported by grants from Associazione Italiana Ricerca sul Cancro, AIRC (to RG), Fondazione Telethon (GGP04012, to PB), and FISM (to GC). TR was supported by a fellowship from FIRC, MT was supported by an American Italian Cancer Foundation fellowship. PB is recipient of a Senior Scholar Consultancy from the American Italian Cancer Foundation
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