sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism

    sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRESdependent mechanism Michael M. Kunze, Fabienne Benz, Thilo F. Brauß, Sebastian Lampe, Julia E. Weigand, Johannes Braun, Florian M. Richter, Ilka Wittig, Bernhard Br¨une, Tobias Schmid PII: DOI: Reference:

S1874-9399(16)30095-5 doi: 10.1016/j.bbagrm.2016.05.005 BBAGRM 1034

To appear in:

BBA - Gene Regulatory Mechanisms

Received date: Revised date: Accepted date:

23 November 2015 15 April 2016 5 May 2016

Please cite this article as: Michael M. Kunze, Fabienne Benz, Thilo F. Brauß, Sebastian Lampe, Julia E. Weigand, Johannes Braun, Florian M. Richter, Ilka Wittig, Bernhard Br¨ une, Tobias Schmid, sST2 translation is regulated by FGF2 via an hnRNP A1mediated IRES-dependent mechanism, BBA - Gene Regulatory Mechanisms (2016), doi: 10.1016/j.bbagrm.2016.05.005

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IRES-dependent sST2 translation

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated

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IRES-dependent mechanism

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Michael M. Kunze 1, Fabienne Benz 1, Thilo F. Brauß 1, Sebastian Lampe 1, Julia E. Weigand , Johannes Braun 2, Florian M. Richter 3, Ilka Wittig 3, Bernhard Brüne 1, Tobias Schmid 1,*

Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590

Frankfurt, Germany; Darmstadt, Germany;

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Department of Biology, Technical University Darmstadt, 64287

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Functional Proteomics, SFB 815 Core Unit, Faculty of Medicine,

Corresponding author at: Institute of Biochemistry I, Faculty of Medicine, Goethe-University

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Goethe-University Frankfurt, 60590 Frankfurt, Germany

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Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.

IRES-dependent sST2 translation

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Running title:

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E-mail address: [email protected]

Key words:

IRES, hnRNP A1, ITAF, MEK/ERK, RNA-binding protein

Abbreviations:

5’UTR, 5’ untranslated region; CHX, cycloheximide; eIF, eukaryotic initiation

factor;

FGF2,

basic

fibroblast

growth

factor;

hnRNP,

heterogeneous nuclear ribonucleoprotein; IRES, internal ribosome entry site; ITAF, IRES trans-acting factors; sST2, soluble suppression of tumorigenicity 2

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ABSTRACT Translation is an energy-intensive process and tightly regulated. Generally, translation is

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initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor

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microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent

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translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES

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trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the

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IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5’UTR of sST2. Furthermore, we found that

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MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation.

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Determination of the sST2-5’UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5’UTR to be required for optimal IRES activity.

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Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5’UTR, acts as an ITAF, and thus controls the activity of the sST2IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic

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translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5’UTR contains an IRES element, which is activated by a MEK/ERKdependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2.

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1. INTRODUCTION Adaptation of cells to environmental signals requires changes in the expression of proteins.

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So far research predominantly characterized altered transcriptional profiles, largely

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neglecting post-transcriptional regulatory mechanisms. Yet, controlling mRNA stability and/or

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translation provides a means to rapidly modify the protein output [1]. Translation is a highly energy consuming process and therefore is tightly regulated [2]. As translation initiation is considered to be the rate limiting step in this process, it constitutes the prime target for

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translational regulation [3]. Eukaryotic translation initiation starts with binding of the eukaryotic initiation factor (eIF) 4E to the 5’ 7-methyl-guanosine cap of the mRNAs. Upon

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recruitment of additional initiation factors, including the RNA helicase eIF4A, the scaffolding protein eIF4G, and eventually the 43S preinitiation complex, this complex scans the 5’

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untranslated region (5’UTR) for a start codon [4]. Upon recognition of a start codon in the

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correct sequence context, the mature ribosome is formed, peptide synthesis is initiated, and translation proceeds to the elongation step [5].

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The PI3K-mTOR signaling cascade plays a central role in regulating global translation initiation. Specifically, mTOR inhibits sequestration of eIF4E by the 4E-binding proteins through inactivating hyperphosphorylation of the later [6]. mTOR further activates p70S6K by

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phosphorylation, which subsequently phosphorylates and activates the 40S ribosomal subunit [7], but also marks the translation inhibitor programmed cell death 4 for degradation [8]. Regulation of global, i.e. cap-dependent translation is important both under physiological (e.g. mitosis) as well as under stress conditions (e.g. hypoxia, genotoxic stress, nutrient deprivation). To ensure the continuous translation of selected mRNAs, for example under conditions when cap-dependent translation is inhibited, alternative translation mechanisms take over [9]. One of these alternative modes is internal ribosome entry site (IRES)-mediated translation. IRESs are elements in the 5’UTR that can recruit the ribosome capindependently. In line with the fact that IRESs allow for selective translation under otherwise translation-inhibitory environmental conditions, IRES elements are found in many mRNAs encoding for proteins contributing to various aspects of tumor development such as cell 3

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survival (e.g. Bcl-xL, cIAP1) [10, 11], proliferation (e.g. c-myc, Cyclin D1, FGF2) [12-14], and angiogenesis (e.g. VEGF, HIF-1α) [15, 16]. IRES-mediated translation depends on RNAbinding proteins, the so-called IRES trans-acting factors (ITAFs), which interact with specific

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sequence or structure elements within the 5’UTR of mRNAs and facilitate the assembly of initiation complexes independent of the cap-binding protein eIF4E [17, 18]. As ITAFs are

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critical for IRES-dependent translation, their activity emerges as an important regulatory principle for controlling translation of specific mRNAs [19].

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Both cap-dependent and -independent translation have been linked to all steps of tumor initiation and progression [20]. In line, various inflammatory mediators (e.g. IL-1β, IL-6) and

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growth factors (e.g. FGF2, IGF), which are commonly found in the tumor microenvironment, have been shown to impact translation in tumor cells [10, 21-23]. The present study aimed at characterizing the translational regulation of the IL-33 decoy

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receptor soluble suppression of tumorigenicity 2 (sST2). We provide evidence that sST2 translation is induced by the growth factor FGF2 -. We identified and verified an IRES in the

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5’UTR of sST2, which is bound and induced by the RBP hnRNP A1. Furthermore, we show that FGF2-mediated sST2-IRES activation requires intact MEK/ERK signaling to enhance

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cytoplasmic localization of hnRNP A1. 2. MATERIALS AND METHODS 2.1 Materials

All chemicals were purchased from Sigma-Aldrich, if not indicated otherwise. Rapamycin, PD98059, U0126, LY294002, and SB203580 came from Cell Signaling Technology. Antibodies were obtained from the following sources: anti-hnRNP A1 (NB100-672) from Novus Biologicals; anti-phospho-ERK (9101S) and anti-total-ERK (4696S) from Cell Signaling Technology; anti-nucleolin (sc-13057) from Santa Cruz Biotechnology; anti-H3 (07-690) from Merck Millipore; anti-HA (901501) from BioLegend; IRDyes 680LT and 800CW secondary antibodies (926-68022/ -68023/ -32212/ -32213) from Li-COR Biosciences GmbH;

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Alexa Fluor 546 (A-11030) from Thermo Fisher. Anti-PTB was a kind gift of Prof. Anne Willis [24].

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2.2 Cell culture

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MCF7 cells came from LGC Standards GmbH. MCF7 cells with inducible hnRNP A1 k/d

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(MCF7 k/d) were generated by lentiviral transduction of MCF7 cells with pTRIPZ-hnRNP A1 shRNA clone V2THS_201776 (Thermo Fisher Scientific). A non-targeting sh control cell line was included to exclude doxycycline induced off-target effects. MCF7 cells were maintained

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in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate, and 2 mM L-glutamine.

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Additionally, media of MCF7 k/d cells contained 1 µg/mL puromycin and were supplemented with tetracycline-free FBS. Cells were kept at 37°C in a humidified atmosphere with 5% CO 2.

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Medium and supplements were purchased from Gibco (Life Technologies). FBS came from

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Capricorn, and tetracycline-free FBS from Clontech.

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2.3 Polysomal fractionation

7.5 x 106 MCF7 cells were seeded in a 15-cm dish 1 d prior to treatment of the cells, followed by polysomal fractionation. Briefly, after incubation with 100 µg/mL cycloheximide (CHX) for

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10 min at 37°C, cells were harvested in PBS/CHX (100 µg/mL) and lysed in 750 µL polysome buffer (140 mM KCl, 20 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.5% NP40, 0.5 mg/mL heparin, 1 mM DTT, 100 U/mL RNasin (Promega), 100 µg/mL CHX). After pelleting, the cytoplasmic lysates were layered onto 11-mL 10–50% continuous sucrose gradients. The gradients were centrifuged at 35,000 rpm for 2 h at 4°C without brake, using a SW40 rotor in a Beckman ultracentrifuge. Afterward, the gradients were collected in 1-mL fractions using a Gradient Station (Biocomp). Absorbance was measured at 254 nm. RNA was precipitated by addition of sodium acetate (3 M) and isopropanol. RNA was further purified using the Nucleospin RNA Kit (Macherey-Nagel) according to the manufacturer’s manual. 2.4 mRNA analysis

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To analyze mRNA expression levels, RNA was isolated from MCF7 cells using the PeqGold RNAPure Kit (PeqLab Biotechnology) according to the manufacturer’s instructions. For all PCR-based approaches (expression and polysomal fractionation), mRNA was reverse

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transcribed using the Maxima First Strand cDNA synthesis kit (Thermo Fisher Scientific). For quantitative analyses individual mRNAs were subsequently analyzed by real-time PCR using

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specific primers and the iQ SYBR Green Supermix (BioRad). The following primers were used: hnRNP A1 for: AAC GCT CAC GGA CTG TGT GGT AAT; hnRNP A1 rev: GTG GCC

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TTG CAT TCA TAG CTG CAT; GAPDH-fwd: TGC ACC ACC AAC TGC TTA GC; GAPDHrev: GGC ATG GAC TGT GGT CAT GAG; sST2-fwd: AGT CTA TGA GGA GGG ACC TAC

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A; sST2-rev: CAG AGG GGA TGA ACT TGG AGT; firefly for: GGT TCC ATC TGC CAG GTA TCA GG; firefly rev: CGT CTT CGT CCC AGT AAG CTA TG; renilla for: CGT GGA AAC CAT GTT GCC ATC AA; renilla rev: ACG GGA TTT CAC GAG GCC ATG ATA; phpRF

GAT CTT TCC GC.

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2.5 Protein analysis

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CDS for: GCC ACC ATG ACT TCG AAA GTT TAT GA; phpRF CDS rev: TTA CAC GGC

For the analysis of total protein changes, cells were lysed and snap-frozen in lysis buffer (50

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mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1x protease inhibitor mix (Roche)). For subcellular fractionation, cells were collected and incubated in buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1x protease inhibitor mix (Roche)) for 15 min. Then 10 µl NP40 (10%) were added to the sample followed by centrifugation to collect the nuclei. After transferring the supernatant (cytosolic proteins) to a fresh collection tube, the nuclei were washed two times with PBS, lysed with SDS-lysis buffer (6.65 M Urea, 10 mM Tris–HCl, 10% Glycerol, 1% SDS) and sonified. For total proteins and cytoplasmic fractions 50 µg and for nuclear fractions 30 µg protein were separated on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), transferred to nitrocellulose membranes, detected using specific antibodies and appropriate secondary antibodies, and visualized and quantified on an Odyssey infrared imaging system (Li-COR Biosciences GmbH).

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2.6 Plasmid construction Primers were designed to amplify products of 267 nt containing the human sST2-5’UTR (not including the authentic start codon) using RNA extracted from human MCF7 cells. The

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amplification product generated with phpRF-compatible primers (fwd: ACC TCG AAT CAC TAG AGT CTA TGA GGA GGG ACC; rev: TTG GCG TCT TCC ATG TCT GTT TAT CAA

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TCA AGA GCA) was inserted into the hairpin-containing bicistronic vector phpRF (kind gift of Prof. Anne Willis) [25] with Spe I and Nco I, resulting in phpR-sST2-F. For control purposes

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the sST2-5’UTR was also inserted into phpRF in reverse orientation (fwd: TTG GCG TCT TCC ATG AGT CTA TGA GGA GGG ACC; rev: ACC TCG AAT CAC TAG TCT GTT TAT

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CAA TCA AGA GCA) resulting in phpR-rev-sST2-F. The product generated with pcDNA3.1(+)-compatible primers (fwd: GTT TAA ACT TAA GCT AGT CTA TGA GGA GGG

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ACC; rev: AAA CGG GCC CTC TAG TCT GTT TAT CAA TCA AGA GCA) was inserted into

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pcDNA3.1(+) using Hind III and Xho I, resulting in pcDNA3.1(+)-sST2. The amplicon generated with pGL3-compatible primers (fwd: CTA GCC CGG GCT CGA AGT CTA TGA

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GGA GGG ACC; rev: CCG GAA TGC CAA GCT TCT GTT TAT CAA TCA AGA GCA) was inserted into pGL3-basic with Hind III and Xho I resulting in pGL3-sST2. In order to generate monocistronic control plasmids, PCR primers (ΔRL fwd: TTC TAG AGC TTA TCG ATA CCG

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T; ΔRL rev: AGT GGA CAC CTG TGG AGA GAA AG) were phosphorylated by T4 polynucleotide kinase treatment (37°C, 1 h) prior to amplification of phpRF or phpR-sST2-F by inverse PCR. After digestion with Dpn I for 2 h, the vectors were purified by agarose gel electrophoresis and ligated using T4 DNA ligase (16°C, 4 h) (Thermo Fisher Scientific) resulting in pF and p-sST2-F. To generate deletions of the sST2-5’UTR, PCR primers (Δ34100 for: GCT ACT CTT CCC AAC TCA GT; Δ34-100 rev: TTT CCA GTC TTT GTA GGT CC; Δ101-180 for: CAT CTG GAG TAA TCT CAA CA; Δ101-180 rev: CCT ATA TCT CAA CCA AAC TGA AAG; Δ181-237 for: TCG AGT CTA GAG GGC CC; Δ181-237 rev: AGT CAC TGG CAT ACG ACA G; Δ181-203 for: AGT TAC CAA TAC TTG CTC TTG ATT G; Δ204-220 for: CTT GAT TGA TAA ACA GAT CGA G; Δ204-220 rev: CGT TGT TGA GAT TAC TCC AG; Δ221-237 rev: AGC AAG TAT TGG TAA CTC GTT G) were used as described above to gain 7

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the appropriate constructs based on pcDNA3.1(+)-sST2. Subsequently, the deletions were transferred into phpRF with the In-fusion Cloning system (Clontech) using the following primers (phpRF for: ACC TCG AAT CAC TAG AGT CTA TGA GGA GGG ACC; phpRF rev:

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TTG GCG TCT TCC ATG TCT GTT TAT CAA TCA AGA GCA).

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2.7 RNA affinity chromatography

For in vitro transcription, pcDNA3.1(+)-sST2 was linearized with Xho I. pDrive-hr-gapdh [22] was linearized with Not I. sST2-5’UTR (NM_003856.3) and partial human reverse gapdh

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were transcribed in vitro with the MEGAShortscript Transcription kit (Ambion) according to the manufacturer’s protocol. The transcript was biotinylated at the 5’ end with the 5’ EndTag

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Nucleic Acid Labeling System (Enzo Life Science) according to the manufacturer’s instructions. Biotinylated RNA (20 µg) was conjugated to streptavidin agarose beads in

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incubation buffer (10 mM Tris-HCl pH 7.4, 150 mM KCl, 0.5 mM DTT, 0.05% NP40, 100

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U/mL RNasin) for 2 h at 4°C with continuous rotation. Cytoplasmic protein lysates (1 mg) of MCF7 cells that were lysed in incubation buffer containing 0.5% NP40 were added to the

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beads and incubated for 1 h at 4°C followed by 15 min at room temperature. Beads were washed five times with incubation buffer, resuspended in 30 µL 4x Laemmli buffer, and

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boiled for 10 min. Eluted proteins were separated on 12% SDS-PAGE, transferred to nitrocellulose membranes, detected using specific antibodies and appropriate secondary antibodies, and visualized on an Odyssey infrared imaging system (Li-COR Biosciences GmbH). 2.8 In-line probing sST2-5’UTR was in vitro transcribed, dephosphorylated, and 5’ described [26]. For the in-line reaction 100 pM of the 5’

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P-labeled as previously

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P-labeled sST2-5’UTR were

incubated for 40 h at 25°C in in-line reaction buffer (10 mM Tris–HCl pH 7, 10 mM MgCl2, 100 mM KCl). To generate size markers 100 pM of the 5’

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P-labeled sST2-5’UTR were

subjected to alkaline hydroxylation (OH), i.e. incubated for 2 or 4 min at 96°C in Na2CO3 (pH 9), or incubated for 3 or 5 min at 55°C with RNase T1 (10 U/µL) (T1) at denaturing conditions

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to identify guanines. After the incubation, 10 µL urea (10 M) were added and the product was separated by polyacrylamide gel electrophoresis. To achieve a better resolution, each sample was separated on two polyacrylamide gels (8% and 10%). Gels were dried and

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analyzed using a Storm Phosphoimager (GE Healthcare).

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2.9 Reporter assays and transient transfection

MCF7 cells were transiently transfected with 500 ng plasmid DNA using Rotifect reagent (Roth) according to the manufacturer’s protocol. After 16 h, the medium was changed, and

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cells were stimulated. Following stimulation, cells were lysed, and firefly and renilla luciferase activities were determined using a Dual Luciferase kit assay (Promega) on a Mithras LB 940

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luminometer (Berthold). β-galactosidase activity was measured using a β-Galactosidase Enzyme Assay System (Promega) on a Apollo LB 911 luminometer (Berthold). Co-

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transfections were performed using 800 ng hnRNP A1 wt or hnRNP A1 NLS [27] (kindly

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provided by Prof. Danilo Perrotti) in combination with 200 ng phpR-sST2-F, or using 450 ng pGL3-basic, pGL3-sST2, or pGL3-SV40 in combination with 50 ng pRL-TK, or using 500 ng

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phpRF, phpR-sST2-F, pF, or p-sST2-F in combination with 200 ng SV40-β-Gal. For RNA transfection, phpRF or phpR-sST2-F plasmids were linearized with Bam HI, in vitro

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transcribed and capped using the mMESSAGE mMACHINE T7 Kit (Ambion) according to the manufacturer’s protocol and purified with the MEGAclear Kit (Ambion). RNA (500 ng) was transfected as described for DNA. Luciferase activities were measured 24 h posttransfection. To knock-down hnRNP A1 expression for analysis in reporter assays, MCF7 cells were co-transfected with 5 nM SMARTpool ON-TARGETplus hnRNP A1 siRNA (Dharmacon) or AllStarsNegative control siRNA (Qiagen) and 500 ng phpR-sST2-F using GenMute siRNA transfection reagent (SignaGen). 2.10 Immunofluorescence MCF7 cells were grown on chamber-slides (25 x 75 mm; Thermo Fisher). After treatment, cells were fixed in 4% paraformaldehyde for 20 min and rinsed with PBS. Cells were then permeabilized in 0.2% Triton-X 100-PBS for 15 min followed by blocking in 5% BSA (in 0.1% Triton-X 100-PBS) for 30 min and incubation with hnRNP A1 antibody (1:200 in 5% BSA9

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PBS) overnight. After rinsing with 0.1% Triton-X 100-PBS the secondary antibody (1:200 in 5% BSA-PBS) was added for 1 h. Nuclei were stained with Hoechst 33258 that was added directly into the secondary antibody solution for 10 min. Cells were rinsed with 0.1% Triton-X

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100-PBS, covered with Fluoromount G (SouthernBiotech) and mounted with a cover slip (24 x 60 mm; Thermo Fisher). Imaging was carried out using an LSM 510 laser scanning

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microscope (Carl Zeiss) and software with an x 40 oil-immersion objective. 2.11 Statistical analysis

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Data are presented as mean values ± SEM of at least three independent experiments. Statistical analyses were performed using Student’s t-test, or one-way ANOVA with a

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Tukey’s post-hoc test (Fig. 5C).

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3. RESULTS

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3.1 FGF2 enhances sST2 translation hnRNP A1-dependently We previously characterized translational changes in tumor cells in response to an

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inflammatory tumor microenvironment [22, 28] and identified hnRNP A1 as a potential ITAF. We further identified the decoy receptor for IL-33, sST2, as a candidate for which translation

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appeared to be induced by inflammatory conditions in an hnRNP A1-dependent manner. As the basic fibroblast growth factor (FGF2) was recently shown to induce hnRNP A1dependent translation of XIAP and Bcl-xL [10], and elevated serum levels of FGF2 are commonly detected in tumor patients [29], we initially determined the impact of FGF2 on the translation of sST2. To this end we treated MCF7 cells with FGF2 (10 ng/mL) for 4 h and subjected the resulting lysates to polysomal fractionation (Fig. 1A). As previously published [28], we normalized the relative distribution for each fraction of stimulated cells to control cells to determine stimulus-induced changes in mRNA distribution across the gradients. While the distribution of GAPDH mRNA was not responsive to FGF2, sST2 mRNA distribution shifted from the subpolysomal fractions (fractions 1-3) to the polysomal fractions (fractions 4-9) (Fig. 1B). At the same time FGF2 treatment enhanced sST2 mRNA levels

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(Fig. 1C), thus FGF2 markedly enhances both transcription and translation of sST2 in MCF7 cells. In order to test whether hnRNP A1 is involved in FGF2-induced translational upregulation of

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sST2, we stably introduced a tetracycline-inducible shRNA against hnRNP A1 into MCF7 cells (MCF7 k/d). As shown in Figure 1D, treatment of these cells with 2 µg/mL doxycycline

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(dox) for 72 h led to a robust knockdown of hnRNP A1 at mRNA (0.59 ± 0.09) and protein levels (0.33 ± 0.08). To assess the role of hnRNP A1 in FGF2-enhanced sST2 translation,

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hnRNP A1 depletion was induced with dox for 72 h, prior to the treatment with FGF2 (10 ng/mL) for 4 h. In line with our previous finding, sST2 mRNA distribution shifted to the

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polysomal fractions upon FGF2 treatment (Fig. 1E – black bars). However, if hnRNP A1 expression was reduced by inducible knockdown, this shift was markedly attenuated (Fig. 1E – white bars, see also Supplementary Fig. S1 for a detailed explanation of this data

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presentation). Similarly, the FGF2-enhanced polysomal distribution of sST2 mRNA was significantly reduced in hnRNP A1-depleted cells as compared to sh control cells (Fig. 1F).

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These data indicate that the FGF2-induced translation of sST2 is mediated by hnRNP A1. 3.2 Identification and verification of an IRES element in the sST2-5’UTR

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We next aimed to explore potential mechanisms to explain the translational regulation of sST2. Initially, we blocked cap-dependent translation by treating MCF7 cells with the mTOR inhibitor rapamycin (50 nM) for 16 h, followed by polysomal fractionation. As expected for suppression of global translation, the absorbance at 254 nm, reflecting the RNA content, decreased in the later fractions of a polysome profile, whereas the 60S and 80S peaks increased in response to rapamycin (Fig. 2A). In line, GAPDH mRNA decreased in the polysomal and increased in the sub-polysomal fractions (Fig. 2B). Interestingly, sST2 mRNA did not decrease in the polysomal fractions, instead it further shifted from the sub-polysomal into the polysomal fractions (Fig. 2C). The observation that sST2 translation can be maintained under conditions of impaired cap-dependent translation indicates that sST2 might be translated cap-independently. Therefore, we next analyzed if sST2 might be translated IRES-dependently. To test if the 5’UTR of sST2 indeed contains an IRES element, we 11

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introduced the 5’UTR of sST2 intercistronically into the bicistronic reporter plasmid phpRF resulting in phpR-sST2-F (Fig. 3A). This vector expresses renilla luciferase cap-dependently, whereas firefly luciferase is only translated if a functional IRES element is present

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intercistronically. A hairpin structure upstream of the renilla open reading frame minimizes cap-dependent translation and, thus, also a read-through across the renilla termination

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codon. While MCF7 cells transiently transfected with either phpRF or phpR-sST2-F showed similar renilla luciferase activities, firefly activity dramatically increased in the presence of the

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sST2-5’UTR (27.15 ± 2.77) as compared to the empty vector (Fig. 3B), implying the presence of an IRES element. Introducing the sST2-5’UTR intercistronically in a reverse orientation

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significantly attenuated firefly luciferase activity while it did not affect renilla luciferase activity as compared to the correct orientation (Fig. 3C). To verify that the observed increase is not due to cryptic splicing and/or promoter activities inherent to the vector system, we next tested

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if the vectors produce mRNAs of the expected size. Therefore, we isolated mRNA of vector transfected cells, DNase-treated the mRNA (to exclude plasmid DNA contamination), reverse

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transcribed the mRNA, and performed a PCR, using primers binding to the 5’ end of renilla and the 3’ end of firefly ORF, resulting in the amplification of the whole transcript. The expected length of the transcripts was 2587 nt for phpRF and 2866 nt for phpR-sST2-F. In

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both cases only one product of the expected size was found (Fig. 3D), i.e. the product of the phpR-sST2-F was larger than the non-5’UTR containing vector product, suggesting that in both cases a full length transcript including both ORFs and the intercistronic region (with or without the sST2-5’UTR) is generated. To rule out cryptic promoter activities exerted by the sST2-5’UTR, we next inserted the sST2-5’UTR into the promoterless pGL3-basic vector. MCF7 cells transfected with pGL3-sST2 did not show enhanced luciferase activities as compared to pGL3-basic vector transfected cells, whereas the SV40-promoter containing pGL3-SV40 displayed high firefly luciferase activities (Fig. 3E). Thus, cryptic promoter activity due to the insertion of the sST2-5’UTR into the bicistronic vector appeared unlikely. Since phpRF contains a chimeric intron including a splice donor site in the renilla ORF, cryptic splicing events facilitated by an introduced splice acceptor site could result in the generation

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of monocistronic firefly mRNAs. To address this issue we analyzed the expression of firefly and renilla mRNAs in MCF7 cells transfected with phpRF or phpR-sST2-F. If monocistronic firefly ORF containing mRNAs should occur, the ratio of firefly/renilla mRNA would be

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expected to differ between phpRF and phpR-sST2-F transfected cells. As shown in Figure 3F, firefly/renilla mRNA ratios remained unaltered (0.98 ± 0.002), thus ruling out cryptic splice

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events. To unambiguously prove that neither cryptic promoter nor cryptic splicing events account for the proposed IRES activity, we in vitro transcribed and capped the bicistronic

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vectors and transfected the resulting, DNAse-treated, mRNAs into MCF7 cells. In line with the plasmid DNA transfections, renilla luciferase expression was not altered in hpR-sST2-F

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compared to hpRF transfected cells, whereas firefly luciferase expression was markedly elevated (15.64 ± 1.11) (Fig. 3G). Finally, to assess how the sST2-5’UTR affects capdependent translation as compared to IRES-dependent translation we removed the renilla

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cistron from the bicistronic vector to allow for expression of the firefly ORF in a cap-

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dependent manner. While the sST2-5’UTR increased the firefly activity compared to the

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empty vector in the bicistronic setting, presence of the 237 nucleotide long sST2-5’UTR did not enhance, but rather attenuated, firefly expression in the monocistronic context (Fig. 3H). Taken together, these results prove that the 5’UTR of sST2 contains features that facilitate

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IRES-dependent translation.

3.3 Structure and localization of the sST2-IRES Since IRES elements are commonly considered to be structured, we next aimed to determine the structure of the full-length sST2-5’UTR, to narrow down the localization of the identified IRES element. Therefore, we analyzed the structure of the 237 nt long 5’UTR by inline probing. Briefly, comparing signals in the in-line probing samples with those generated by alkaline hydrolysis (single nucleotide ladder, OH) or RNase T1 digestion (identification of guanines, T1), allowed for the determination of single stranded regions (Fig. 4A). Modeling the structure based on the cleavage pattern using VARNA [30] revealed that the complete sST2-5’UTR is highly structured. It contains three hairpins at its 5’ end (nt 1-71), followed by a double stranded linker region containing only a few bulges (nt 72-97). The middle region 13

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contains two large hairpins in a Y-shaped conformation and two small hairpins (nt 98-191). The last part of the 5’UTR (nt 192-237) directly preceding the start codon contains a central stemloop structure between nucleotides 203 and 221 flanked by two regions which base-pair

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with the region between nucleotides 70 and 100 (Fig. 4B). To narrow down the localization of the element eliciting the IRES activity, we first generated deletion constructs missing

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approximately one third of the 5’UTR each in the phpR-sST2-F IRES-reporter. Specifically, we either deleted nucleotides 34-100 (blue), nucleotides 101-180 (green), or nucleotides

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181-237 (red) (Fig. 4B), resulting in Δ34-100, Δ101-180, and Δ181-237, respectively, as indicated in Figure 4C. We transfected MCF7 cells with these constructs and determined

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IRES activities by the ratio of firefly to renilla luciferase activities. Neither Δ34-100 nor Δ101180 altered the IRES activity compared to the full-length 5’UTR-containing phpR-sST2-F. In contrast, Δ181-237 displayed a significantly reduced IRES activity (0.42 ± 0.02) (Fig. 4C). As

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the last 57 nucleotides (nt 181-237) of the sST2-5’UTR contained only one structural element, we next assessed if this stemloop structure (nt 203-220) might be responsible for

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the observed IRES activity. Surprisingly, deletion of the stemloop only (Δ204-220) did not attenuate the IRES activity. Similarly, deletion of the last 16 nucleotides of the 5’UTR (Δ221237) did not affect IRES activity. In contrast, removal of nucleotides 181 to 203 (Δ181-203)

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sufficed to mimic the loss of IRES activity compared to the full length 5’UTR as observed upon deletion of the entire last 57 nucleotides (Δ181-237) (Fig. 4D). These data indicate that nucleotides 181-203 of the 5’UTR are required for optimal sST2-IRES activity. 3.4 FGF2 enhances sST2-IRES activity hnRNP A1-dependently Based on our observations that FGF2 induces sST2 translation hnRNP A1-dependently and that the sST2-5’UTR harbors an IRES, we next tested the sST2-IRES for its responsiveness to FGF2. To this end, MCF7 cells were transfected with phpR-sST2-F and treated with FGF2 (10 ng/mL) for 24 h. FGF2 significantly enhanced sST2-IRES activity, i.e. the ratio of firefly to renilla luciferase activity, (1.67 ± 0.06) as compared to Ctr (Fig. 5A). To determine the hnRNP A1-dependency of this effect, we transiently knocked down hnRNP A1 using specific siRNAs (Fig. 5B). Depletion of hnRNP A1 significantly reduced the FGF2-induced sST214

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IRES activation (Fig. 5C). These data support the concept that hnRNP A1 is critically involved in this process. As nuclear-cytoplasmic translocation of hnRNP A1 was previously shown to be crucial for its activity [10, 31], we next analyzed the localization of hnRNP A1.

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As shown in Figure 5D, FGF2 treatment induced a two-fold increase in cytoplasmic hnRNP A1 within 60 min of treatment. To further support the role of hnRNP A1 in the regulation of

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the sST2-IRES activity, we next co-transfected MCF7 cells with either an empty vector (EV), a wildtype hnRNP A1 overexpression vector (A1 WT), or a vector overexpressing hnRNP A1

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fused to a nuclear localization signal (A1 NLS) in combination with phpR-sST2-F and analyzed the sST2-IRES activity. Overexpression of wildtype hnRNP A1 significantly

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increased the sST2-IRES activity (1.61 ± 0.03), whereas overexpression of the nuclearrestricted hnRNP A1 (see Supplementary Fig. S2 for verification of the nuclear restricted localization) decreased IRES activity (0.44 ± 0.08) as compared to control (EV) cells (Fig.

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5E). These results underline the importance of hnRNP A1 as a putative ITAF for IRESdependent regulation of sST2. Finally, to verify a direct interaction of hnRNP A1 with the

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sST2-IRES, we performed RNA-affinity chromatography with the 5’UTR of sST2. For this purpose we biotinylated in vitro transcribed sST2-5’UTR at the 5’ end, coupled the labeled transcript to streptavidin-agarose beads, and incubated it with whole-cell lysates of MCF7

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cells. After elution, interacting proteins were electrophoretically separated and detected by western analysis. In line with the prediction that hnRNP A1 acts as an ITAF for the sST2IRES, hnRNP A1 was efficiently immunoprecipitated with the sST2-5’UTR, in contrast to the negative control RNA (GAP) (Fig. 6A). Interestingly, polypyrimidine-tract binding protein (PTB), which was shown to contribute to the regulation of various IRESs [32, 33], also bound to the sST2-5’UTR, but not to the control. Both proteins were not found when immunoprecipitation was carried out in the absence of RNA. To validate the specificity of hnRNP A1 binding to the sST2-5’UTR, we added increasing amounts of in vitro transcribed, non-biotinylated sST2-5’UTR to the above-described RNA-affinity purification approach. Addition of the unlabeled sST2-5’UTR efficiently competed with its biotinylated counterpart for binding to hnRNP A1, i.e. equal amounts of unlabeled sST2 already reduced the amount

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of hnRNP A1 in the immunoprecipitation, whereas competition with 10-fold higher amounts of unlabeled sST2-5’UTR virtually abolished the interaction with biotin-labeled sST2-5’UTR (Fig. 6B). These data indicate that hnRNP A1, in concert with other RBPs such as PTB,

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directly interacts with the 5’UTR of sST2 and critically contributes to the FGF2-induced sST2-

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IRES activation.

3.5 FGF2-induced MEK/ERK signaling contributes to sST2-translation and -IRES activation

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To assess which signaling cascades contribute to the FGF2-induced sST2-IRES activation, we transfected MCF7 cells with phpR-sST2-F followed by stimulation with FGF2 (10 ng/mL)

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in combination with the MEK inhibitors PD98059 (50 µM) or U0126 (10 µM), the PI3K inhibitor LY294002 (10 µM), or the p38-MAPK inhibitor SB203580 (10 µM). While inhibition of

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PI3K and p38-MAPK signaling had no effect, both MEK inhibitors significantly attenuated

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FGF2-induced sST2-IRES activity (PD98059: 0.41 ± 0.03; U0126: 0.86 ± 0.05) (Fig. 7A). Western analysis verified that FGF2 strongly activated the MEK/ERK signaling cascade and

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that PD98059 reduced the activation at the concentrations used (Fig. 7B). To assess if the IRES-inhibitory effect might be due to changes in hnRNP A1 nuclear-cytoplasmic distribution,

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we determined hnRNP A1 localization in response to FGF2 (10 ng/mL) in the presence or absence of PD98059 (50 µM). As shown in Fig. 7C, FGF2 increased cytoplasmic hnRNP A1 (middle panels) as compared to control cell (upper panels), which was blocked by inhibition of MEK/ERK signaling (lower panels). Finally, we aimed to determine if the observed MEK/ERK-dependency of the FGF2-induced sST2-IRES activation is also reflected at the level of total sST2 translation. Therefore, we treated MCF7 cells with FGF2 (10 ng/mL) with or without PD98059 (50 µM) for 4 h and determined the polysomal distribution of sST2 mRNA of FGF2/PD98059 co-treated cells (FGF2+PD) relative to FGF2-only treated cells (FGF2). As shown in Figure 7D, MEK inhibition significantly reduced FGF2-dependent polysomal association of sST2 mRNA. In summary, FGF2-activated MEK/ERK signaling induces nuclear-cytoplasmic translocation of hnRNP A1, thus contributing to enhanced sST2-IRES activity, which results in an increased sST2 translation efficiency. 16

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4. DISCUSSION In the present study, we identify a novel IRES in the 5’UTR of sST2, which provides an

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alternative mode of translation regulation for sST2. We further establish that the IRES-

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dependent translation is induced by the growth factor FGF2 dependent on the RNA-binding

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protein hnRNP A1. Specifically, we show that hnRNP A1 binds to the 5’UTR of sST2, translocates to the cytoplasm in response to FGF2-stimulation in a MEK/ERK-dependent manner, and acts as a positive regulatory ITAF for the translation of sST2.

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Since its discovery in the early 1990s [34] sST2 was shown to be involved in various inflammation-associated diseases, including asthma, fibroproliferative diseases, rheumatoid

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arthritis, sepsis, cardiovascular diseases, and cancer [35, 36]. As sST2 acts as a decoy receptor for IL-33, it has been primarily associated with inhibition of IL-33-induced pro-

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inflammatory signals [36]. With respect to its function in cancer, sST2 was recently shown as

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a negative prognostic marker in ER-positive breast cancer [37] and in hepatocellular carcinoma [38]. Yet, surprisingly little is known about the regulation of sST2. Our finding that

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sST2 translation is enhanced by the growth factor FGF2 therefore provides a first clue towards the regulation of sST2 levels. Interestingly, high serum levels of sST2 have recently been shown to positively correlate with elevated levels of vascular endothelial growth factor

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(VEGF) and platelet-derived growth factor-C (PDGF-C) in breast cancer patients [39]. In general, enhanced levels of growth factors are commonly found in the tumor microenvironment, which is shaped by tumor cells in concert with interacting stromal cells [40]. Especially, infiltrating immune cells strongly impact tumor progression not only by providing growth promoting stimuli, but also by generating an inflammatory environment, which contributes to various aspects of tumorigenesis [41]. Here, we show that sST2 translation is induced by FGF2, a growth factor which is highly expressed by tumor associated macrophages and mast cells, which are typically found in inflammatory tumor microenvironments [42]. Similarly, we recently described enhanced IRES-dependent translation of Egr2 and Cyp24A1 in response to inflammatory, pro-tumorigenic environments as provided by activated macrophages or IL-1β [22, 28]. IRESs have been initially identified 17

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in viruses [43, 44]. Yet, while the secondary structure of many viral IRESs and their regulation are well understood, the exact structural conformation underlying the regulation of eukaryotic IRESs largely remains to be characterized [45]. Nevertheless, cap-independent

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translation via IRES elements appears to be enriched in mRNAs encoding for oncogenic proteins [46]. Considering that preliminary in silico predictions [47] suggested the sST2-

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5’UTR to be highly structured (ΔG = -56.9 kcal/mol), which is expected to prevent efficient ribosomal scanning [48], the presence of an IRES appeared possible. It is important to note

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that the free energy necessary for unfolding of the entire 5’UTR does not necessarily reflect the energetic hurdles that have to be overcome for local unwinding, which occurs during

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scanning [49]. Yet, the observation that the 5’UTR attenuated firefly luciferase activity when inserted into a monocistronic vector, supports the concept that the sST2-5’UTR per se provides a translation restricting environment. The strong induction of the firefly luciferase

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activity upon insertion of the same sequence into a bicistronic system, indicates that

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alternative modes of translation might be favored by the same 5’UTR. Thus, based on

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additional circumstantial evidence (e.g. enhanced translation under translation inhibitory conditions – Fig. 2) as well as multiple crucial control assays (most importantly the transfection of the mRNA of the bicistronic vector – Fig. 3G) [50], we feel confident that the

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5’UTR of sST2 indeed contains an IRES. We verified the structure of the sST2-5’UTR, and initially determined the last 57 nucleotides (nt 181-237) of the 5’UTR to be required for optimal IRES activity. The use of additional deletion constructs, which still showed maximal IRES activity, ensured that the reduction of the IRES activity was not only due to decreased length of the 5’UTR. Strikingly, while the 57 nucleotides that proved to be essential for the sST2-IRES activity contained a stemloop structure similar to the stemloops of domains I and II of the FGF2-IRES [51], selective removal of this stemloop did not diminish the IRES activity. Furthermore, deletion of the last 17 nucleotides (Δ221-238) of the sST2-5’UTR did not significantly attenuate the IRES activity, thus implying that the structural integrity close to the translation initiating start codon is not critical for efficient ribosome recruitment and translational activity in this case, whereas the region in close vicinity to the start codon was

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recently shown to be crucial for efficient IRES-dependent translation of XIAP appeared [52]. Removal of 23 nucleotides (Δ181-203) right before the above-mentioned stemloop structure on the other hand sufficed to attenuate the IRES activity similar to the reduction observed

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upon deletion of the last 57 nucleotides (Δ181-237). Yet, while IRESs as short as 22 nt have been characterized in the past [53], in silico predictions suggest that deletion of either

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nucleotides 181-203 or nucleotides 181-237 rather than removing a specific structural element contribute to major structural reorganizations of the entire 5’UTR. In line many

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eukaryotic IRESs have been reported to be 150-300 nucleotides in length [54]. Future analyses of the exact secondary structures using chemical (e.g. NMIA, DMS) or enzymatic

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(e.g. RNases S1, T1, V1) probing [55] of the various deletion mutants are needed to determine those structures within the 5’UTR, which constitute the functional elements of the sST2-IRES. In line with the concept that larger structural features are required for the sST2-

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IRES activity, the minimally deleted segment (Δ181-203) does not contain a consensus hnRNP A1 binding site [56]. Yet, hnRNP A1 was previously shown to also interact with larger

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mRNA structures not only with specific sequence motifs [57]. Furthermore, while we determined the secondary structure of the sST2-5’UTR in a protein-free context, it will be interesting to see how the structure changes upon binding of one or more ITAFs. Along these

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lines, Mitchell and co-workers have shown that the structure of the Apaf-1-IRES changes upon the binding of the ITAF Unr, which provides the basis for concomitant binding of PTB, and subsequent recruitment of the translation machinery [58]. In addition to the herein characterized hnRNP A1, we also found PTB, an RBP reported to interact with many cellular IRESs [59], to bind to the 5’UTR of sST2. Similarly, hnRNP A1, which is well characterized as a splicing regulatory RNA-binding protein, appears to be involved in the regulation of a number of IRESs [10, 12, 13, 22]. Our data further indicate that FGF2 induces cytoplasmic translocation of hnRNP A1 and enhances sST2-IRES activity. This observation corroborates an earlier report that FGF2-mediated nuclear-cytoplasmic shuttling of hnRNP A1 contributes to the nuclear export of the hnRNP A1 target mRNAs Bcl-XL and XIAP, but sumoylation of hnRNP A1 and concurrent release of its mRNA targets in the cytoplasm was required to

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allow for activation of the respective IRESs [10]. The observation that overexpression of wildtype hnRNP A1 sufficed to enhance sST2-IRES activity, whereas, overexpression of nuclear-restricted hnRNP A1 [27] did not induce sST2-IRES activity supports the importance

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of the cytoplasmic localization of hnRNP A1 in our setting as well. Along the same lines, cytoplasmic accumulation of hnRNP A1 in response to stress conditions, such as osmotic

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shock or UVC irradiation, was shown to attenuate rather than activate IRES-dependent translation of anti-apoptotic mRNAs Bcl-XL, XIAP, and Apaf-1 [60-62]. Yet, hnRNP A1 was

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also suggested as a positive regulatory ITAF for IL-6-induced IRES-dependent translation of c-myc, where again nuclear-cytoplasmic shuttling of hnRNP A1 appeared crucial [21]. The

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IRES-regulatory properties of hnRNP A1 as well as its subcellular distribution are affected by a multitude of signaling cascades. Our observation that intact MEK/ERK signaling is required for FGF2-induced cytoplasmic localization of hnRNP A1, activation of the sST2-IRES, and

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enhanced translation of sST2, is in accordance with a recent study that convincingly showed that FGF2-induced nuclear export of hnRNP A1 occurs in a MEK/ERK-S6K2-dependent

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manner to enhance Bcl-XL and XIAP IRES activities [10]. While p38 MAPK inhibition did not affect FGF2-induced sST2-IRES activity, p38 MAPK and its downstream effector MAP kinase‐interacting kinase 1 (MNK1) were previously shown to contribute to cytoplasmic

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distribution of hnRNP A1, enhancing the IRES-dependent translation of various mRNAs [21, 63, 64]. Yet, MNK1-mediated phosphorylation of hnRNP A1 was also shown to inhibit the capacity of the later to interact with TNFα mRNA thereby enhancing its translation without influencing hnRNP A1 localization [31]. Using a broad kinase screen Courteau et al. recently verified the role of p38 MAPK in regulating hnRNP A1 cytoplasmic localization and subsequent Bcl-XL–IRES activation in response to hypertonic stress. Interestingly, they further identified hexokinase 2 to be also necessary for the osmotic shock induced hnRNP A1 translocation [65]. The observation that the inhibition of PI3K signaling, which was shown to impact the ITAF function of hnRNP A1 in the past [66, 67], did not affect FGF2-induced sST2-IRES activation further supports the notion that FGF2 induces IRES-dependent translation of sST2 largely via MEK/ERK-mediated regulation of the hnRNP A1 activity. While

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we provide ample evidence for the regulatory mechanism of the sST2-IRES, it is important to keep in mind that the MEK/ERK cascade also controls cap-dependent translation at various levels. For example, MEK/ERK and p38 MAPK both activate MNK1, which in turn

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phosphorylates the cap-binding protein eIF4E thereby facilitating recruitment of the ribosomes [68]. In addition, ERK-dependent activation of p90 ribosomal S6 kinase (RSK)

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enhances phosphorylation of ribosomal protein S6 which promotes its recruitment to the 7methyl-guanosine cap complex [69].

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Taken together, we present evidence that sST2 can be regulated translationally in a MEK/ERK-dependent manner by FGF2. Moreover, we identify and characterize the 5’UTR of

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sST2 to contain an IRES, which is bound and regulated by hnRNP A1. Off note, since hnRNP A1 was shown to positively regulate IRES-dependent translation of FGF2 [70] and FGF2 on the other hand induces the IRES activating properties of hnRNP A1, this constitutes

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a translation-regulatory and -regulated positive feedback loop. As sST2 correlates with poor prognosis of tumor patients, our findings might help to establish sST2, or rather IRES-

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dependent translation of sST2, as a novel therapeutic target. Translation is already wellestablished as a target for tumor therapies, yet so far most approaches focused on the inhibition of global, cap-dependent translation [71]. Only very recently, IRES-dependent

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translation was recognized as a potentially more specific means of intervention [72]. Considering that IRESs appear to be enriched in oncogenes, it might be envisioned that not only selected IRESs could be targeted but rather cohorts of IRES-dependently regulated transcripts could be inhibited, e.g. by aiming at regulatory ITAFs, as new therapeutic options [73].

ACKNOWLEDGEMENTS This work was supported by grants from the DFG (GRK1172, SCHM2663/3, SFB815)

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in response to endoplasmic reticulum stress. Biochem J, 449: p. 543-53. 65. Courteau, L., J. Crasto, G. Hassanzadeh, S.D. Baird, J. Hodgins, U. Liwak-Muir, G.

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69. Roux, P.P., D. Shahbazian, H. Vu, M.K. Holz, M.S. Cohen, J. Taunton, N. Sonenberg, and J. Blenis (2007), RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem, 282: p.

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growth factor 2 mRNA. J Biol Chem, 280: p. 4144-53.

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Targeting the translation machinery in cancer. Nat Rev Drug Discov, 14: p. 261-78. 72. Vaklavas, C., Z. Meng, H. Choi, W.E. Grizzle, K.R. Zinn, and S.W. Blume (2015), Small molecule inhibitors of IRES-mediated translation. Cancer Biol Ther, 16: p. 1471-85.

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FIGURE LEGENDS Fig. 1. sST2 is translationally upregulated by FGF2. (A) Representative profile of MCF7

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cell lysates measured at 254 nm during polysomal fractionation. (B) MCF7 cells were treated

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with FGF2 (10 ng/mL) or vehicle control (Ctr) for 4 h and analyzed by polysomal

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fractionation. RNA was isolated from single fractions and the mRNA abundance of GAPDH and sST2 mRNAs was analyzed by RT-qPCR. The distribution of the respective mRNAs across the gradient was determined relative to the total RNA extracted from the gradients.

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Changes of GAPDH (left panel) and sST2 (right panel) mRNA distribution induced by FGF2 were normalized to Ctr. Data are presented as means ± SEM (n > 3, * p<0.05, ** p<0.01, ***

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p<0.001). (C) Changes of sST2 mRNA levels were analyzed by RT-qPCR using the total RNA extracted from the gradients from (B). Data are presented as means ± SEM (n > 3, *

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p<0.05). (D) MCF7 cells with an inducible hnRNP A1 knockdown were generated (MCF7

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k/d). hnRNP A1 knockdown was induced with doxycycline (2 µg/mL) for 72 h before the hnRNP A1 knockdown (+dox) was verified at the mRNA level (normalized to GAPDH mRNA

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and relative to -dox) by RT-qPCR analysis (left panel) or at the protein level by western analysis (right panel). Data are presented as means ± SEM (n > 3, ** p<0.01) and blots are

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representative for at least 3 independent experiments. (E) MCF7 k/d cells were grown for 72 h in the presence or absence of doxycycline (2 µg/mL). Subsequently, cells were stimulated with FGF2 (10 ng/mL) or vehicle control (Ctr), subjected to polysomal fractionation, and sST2 mRNA distribution was determined relative to Ctr-treated cells for hnRNP A1-depleted cells (+dox, white bars) and control cells (-dox, black bars). Data are presented as means ± SEM (n > 3, * p<0.05). (F) MCF7 cells expressing a control shRNA (sh Ctr) or MCF7 k/d cells (sh A1) were grown for 72 h in the presence of doxycycline (2 µg/mL). Subsequently, cells were stimulated with FGF2 (10 ng/mL) or vehicle control (Ctr), subjected to polysomal fractionation, and sST2 mRNA distribution was determined relative to Ctr-treated cells for hnRNP A1-depleted cells (sh A1, white bars) and sh control cells (sh Ctr, black bars). Data are presented as means ± SEM (n > 3, * p<0.05, ** p<0.01).

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Fig. 2. sST2 can be translated cap-independently. MCF7 cells were treated with rapamycin (50 nM) for 4 h and subjected to polysomal fractionation. (A) Representative UV profile across the gradients under control conditions (black line) and after rapamycin

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treatment (grey line). RNA of single fractions was collected and GAPDH (B) and sST2 (C) mRNA distribution changes were analyzed as described before. Data are presented as

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means ± SEM (n > 3).

Fig. 3. sST2 contains an IRES element. (A) Schematic representation of the bicistronic

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control (phpRF) and sST2-5’UTR-containing (phpR-sST2-F) luciferase vectors used for the reporter assays. (B) Bicistronic reporter vectors phpRF (white bars) and phpR-sST2-F (black

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bars) were transfected into MCF7 cells. 48 h after transfection firefly and renilla luciferase activities were measured. Data are presented as means ± SEM relative to phpRF (n > 3, ***

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p<0.001). (C) MCF7 cells were transfected with phpR-sST2-F (white bars) or phpR-rev-

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sST2-F (black bars), which contains the sST2-5’UTR in reverse orientation. 48 h after transfection firefly and renilla luciferase activities were measured. Data are presented as

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means ± SEM relative to phpRF (n > 3, *** p<0.001). (D) RNA isolated from MCF7 cells transfected with the bicistronic reporter vectors was DNase treated and reverse transcribed.

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PCR was performed using specific primers amplifying full-length RF or R-sST2-F mRNAs. PCR products were visualized via agarose gel electrophoresis and ethidium bromide staining. Data are representative for at least 3 independent experiments. (E) MCF7 cells were co-transfected with the indicated reporter vectors and pRL-TK plasmid. 48 h after transfection firefly activity was measured and normalized to renilla activity. Data are presented as means ± SEM relative to pGL3-basic (n > 3). (F) RNA isolated from MCF7 cells transfected with the bicistronic reporter vectors was DNase treated and reverse transcribed. The amount of firefly mRNA was analyzed by qPCR and normalized to renilla mRNA. Data are presented as means ± SEM relative to phpRF (n = 3). (G) In vitro transcribed mRNA (hpRF and hpR-sST2-F) of the bicistronic reporter constructs (phpRF and phpR-sST2-F, respectively) was transfected into MCF7 cells. 48 h after transfection firefly and renilla activities were measured. Data are presented as means ± SEM relative to hpRF (n > 3, ** 31

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p<0.01). (H) MCF7 cells were co-transfected with an empty (EV) or sST2-5’UTR containing bicistronic (phpRF) or monocistronic (pF) vector, in combination with a SV40-β-Gal plasmid as indicated. 48 h after transfection firefly luciferase and β-galactosidase activities were

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measured. Data are presented as means ± SEM relative to EV (n > 3, * p<0.05, ** p<0.01).

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Fig. 4. Nucleotides 181 to 203 of the sST2-5’UTR are required for optimal sST2-IRES activity. (A) In vitro transcribed sST2-5’UTR was used for in-line probing. Autoradiogram of an 8% (left panel) and 10% (right panel) denaturing (8 M urea) polyacrylamide gel of the in-

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line probing reaction (IL) (lanes 6+7) and the non-reacted control (NR) (lane 1). Size markers were generated by alkaline hydrolysis (OH) of in vitro transcribed sST2-5’UTR for 2 (lane 2)

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or 4 min (lane 3) and by RNase T1 digestion (T1) of in vitro transcribed sST2-5’UTR for 3 (lanes 4+5) or 5 min (lanes 8+9). Numbers on the right indicate the position of the guanines

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within the sequence. Vertical bars indicate single stranded regions. (B) Structure model of

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the sST2-5’UTR created with VARNA according to the in-line probing [30]. Probing signals are indicated by black dots, and regions deleted in subsequent assay are indicated in blue

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(Δ34-100), green (Δ101-180), and red (Δ181-237). (C) MCF7 cells were transfected with phpR-sST2-F containing the full-length 5’UTR (1-237) or deletion mutants as indicated. After 48 h IRES activity was determined as ratio of firefly to renilla activity and is given relative to

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the full-length 5’UTR (1-237) containing vector. Data are presented as means ± SEM (n > 3, *** p<0.001). (D) MCF7 cells were transfected with phpR-sST2-F containing the full-length 5’UTR (1-237) or deletion mutants as indicated. After 48 h IRES activity was determined as ratio of firefly to renilla activity and is given relative to the full-length 5’UTR (1-237) containing vector. Data are presented as means ± SEM (n > 3, ** p<0.01, *** p<0.001). Fig. 5. FGF2 induces sST2-IRES activity hnRNP A1-dependently. (A) MCF7 cells were transfected with the reporter vector phpR-sST2-F. 24 h after transfection cells were treated with FGF2 (10 ng/mL) or vehicle control (Ctr) for 24 h and then assayed for sST2-IRES activity as described above. Data are given relative to Ctr and are presented as means ± SEM (n > 3, *** p<0.001). (B) MCF7 cells were co-transfected with hnRNP A1 siRNA (5 nM) (si A1) or control siRNA (si Ctr). After 48 h hnRNP A1 knockdown was verified on mRNA 32

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level by RT-qPCR (left panel) and on protein level by western analysis (right panel). Data are presented as means ± SEM relative to si Ctr (n > 3, *** p<0.001) and blots are representative for at least 3 independent experiments. (C) MCF7 cells were co-transfected with the reporter

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vector phpR-sST2-F and hnRNP A1 siRNA (5 nM) (si A1) or control siRNA (si Ctr). 24 h after transfection cells were treated with FGF2 (10 ng/mL) or vehicle control (Ctr) for 24 h and

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then assayed for sST2-IRES activity as described above. Data are presented as means ± SEM relative to si Ctr (n > 3, ** p<0.01, *** p<0.001, ## p<0.01). (D) MCF7 cells were treated

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with FGF2 (10 ng/mL) for the indicated times. Then cytoplasmic and nuclear lysates were fractionated, subjected to western analysis and analyzed using the indicated antibodies.

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Blots are representative for at least 3 independent experiments. (E) MCF7 cells were cotransfected with phpR-sST2-F and either pcDNA3.1(+) (EV), a wildtype hnRNP A1 overexpression vector (A1 WT), or a vector overexpressing hnRNP A1 fused to a nuclear

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localization signal (A1 NLS). 48 h after transfection sST2-IRES activity was measured as described above and is given relative to EV. Data are presented as means ± SEM (n > 3, *

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p<0.05, ** p<0.01).

Fig. 6. hnRNP A1 binds to the sST2-5’UTR. (A) Whole cell lysates of MCF7 cells were

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incubated with in vitro transcribed, biotinylated sST2-5’UTR or human reverse GAPDH (GAP), or without RNA (-RNA) before immunoprecipitation using streptavidin agarose beads. (B) Whole cell lysates of MCF7 cells were incubated with in vitro transcribed, biotinylated sST2-5’UTR (Ctr) and unlabeled sST2-5’UTR at equal amounts (1x) or ten-fold excess (10x) before immunoprecipitation using streptavidin agarose beads. Bound proteins were subjected to western analysis and probed with the indicated antibodies and analyzed by densitometry. Blots are representative for at least 3 independent experiments. Data are presented as means ± SEM (n = 3, ** p<0.01). Fig. 7. Intact MEK/ERK signaling is required for FGF2-induced sST2-IRES activation. (A) MCF7 cells were transfected with the reporter vector phpR-sST2-F. 24 h after transfection cells were treated with vehicle control (Ctr) or FGF2 (10 ng/mL) alone or in combination with PD98059 (50 µM), U0126 (10 µM), LY294002 (10 µM), or SB203580 (10 33

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µM) for 24 h. Then sST2-IRES activity was measured as described above and is given relative to Ctr. Data are presented as means ± SEM (n > 3, ** p<0.01, *** p<0.001). (B) MCF7 cells were treated with vehicle control or FGF2 (10 ng/mL) alone or in combination

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with PD98059 (50 µM) for the indicated time. Whole cell lysates were subjected to western analysis and probed with the indicated antibodies. Blots are representative for at least 3

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independent experiments. (C) MCF7 cells were pre-incubated with PD98059 (50 µM) or vehicle control for 1 h prior to treatment with or without FGF2 (10 ng/mL) for 30 min. The

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subcellular localization of hnRNP A1 was analyzed by immunofluorescence detection of hnRNP A1 and Hoechst staining on a laser scanning microscope. Pictures are representative

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for at least 3 independent experiments. (D) MCF7 cells were treated with FGF2 (10 ng/mL) alone or in combination with PD98059 (50 µM) for 4 h and subjected to polysomal fractionation. sST2 mRNA distribution of cells treated with FGF2 + PD98059 was normalized

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to FGF2 only treated cells. Data are presented as means ± SEM (n > 3, * p<0.05).

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HIGHLIGHTS FGF2 induces sST2 translation

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sST2-5’UTR contains an FGF2-responsive IRES element

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23 nucleotides within the sST2-5’UTR are required for optimal IRES activity

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Intact MEK/ERK signaling is required for FGF2-induced sST2-IRES activity

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hnRNP A1 binds to and activates the sST2-IRES

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