p38 MAPK Controls Prothrombin Expression by Regulated RNA 3′ End Processing

Molecular Cell

Article p38 MAPK Controls Prothrombin Expression by Regulated RNA 30 End Processing Sven Danckwardt,1,2 Anne-Susan Gantzert,1,2 Stephan Macher-Goeppinger,3,9 Hans Christian Probst,4,9 Marc Gentzel,5,8,9 Matthias Wilm,6,8 Hermann-Josef Gro¨ne,7 Peter Schirmacher,3 Matthias W. Hentze,2,8,* and Andreas E. Kulozik1,2,* 1Department

of Pediatric Oncology, Hematology, and Immunology, University of Heidelberg, Heidelberg, Germany Medicine Partnership Unit, European Molecular Biology Laboratory (EMBL) and University of Heidelberg, Heidelberg, Germany 3Institute of Pathology, University of Heidelberg, Heidelberg, Germany 4Institute for Immunology, University of Mainz, Mainz, Germany 5Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany 6Conway Institute of Biomolecular and Biomedical Research, University College, Dublin, Ireland 7Department of Cellular and Molecular Pathology, German Cancer Research Center, Heidelberg, Germany 8European Molecular Biology Laboratory (EMBL), Heidelberg, Germany 9These authors contributed equally to this work *Correspondence: [email protected] (M.W.H.), [email protected] (A.E.K.) DOI 10.1016/j.molcel.2010.12.032 2Molecular

SUMMARY

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 30 end processing components and phosphorylates the RNAbinding proteins FBP2 and FBP3, which inhibit 30 end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 30 UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 30 end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 30 end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications. INTRODUCTION The serine protease thrombin (blood coagulation factor 2, or F2) is a protein with multiple physiological roles in blood coagulation, the complement system, angiogenesis, and cell growth (Coughlin, 2000; Rittirsch et al., 2008). F2 also contributes to the pathogenesis of multiple sclerosis, thrombosis, and cancer (Chapman, 2006; Lane and Grant, 2000; Nierodzik and Karpatkin, 2006). Interestingly, F2 expression is induced in various acute and

chronic inflammatory processes, e.g., as alternative C5 convertase (Huber-Lang et al., 2006), in diabetes (Murakami et al., 2003), during HIV infection (Boven et al., 2003), in cerebral ischemia (Riek-Burchardt et al., 2002), in neurotrauma (Citron et al., 2000; VanLandingham et al., 2008), in Alzheimer’s disease (Yin et al., 2010), and others (Kim et al., 1998; Zoubine et al., 1996). Yet, little is known about the underlying cellular and molecular mechanisms that control F2 gene expression and their potential (patho-)physiological role in health and disease. Deregulated F2 expression has recently been shown to result from mutations that interfere with constitutively weak 30 end formation signals (Danckwardt et al., 2004; Gehring et al., 2001), highlighting the functional importance of a tight F2 gene expression control in hemostasis (Poort et al., 1996) and other processes (Gingrich et al., 2000; Palumbo et al., 2007; Xi et al., 2003). Interestingly, F2 30 end mRNA processing relies on an unorthodox architecture of processing signals with a highly conserved upstream sequence element (USE) that positively counterbalances the relatively inefficient 30 cleavage site. This architecture characterizes a recently defined class of genes with the potential to be regulated at the level of 30 end processing (Danckwardt et al., 2007). Many human genes have multiple polyadenylation sites (Tian et al., 2005) and are thought to be differentially polyadenylated (Barabino and Keller, 1999; Carninci et al., 2005; Danckwardt et al., 2008; Edwalds-Gilbert et al., 1997; Licatalosi et al., 2008; Lutz, 2008; Tian et al., 2005; Wang et al., 2008). Alternative polyadenylation can affect gene regulation by inclusion or elimination of regulatory sequence elements, such as miRNA target sites (Danckwardt et al., 2008; Mayr and Bartel, 2009; Sandberg et al., 2008). Recent reports suggest that 30 end processing can be globally regulated in response to external stimuli (Wilhelm et al., 2008), during the immune maturation process (Sandberg et al., 2008; Takagaki et al., 1996), during tumorigenesis to activate oncogenes in cancer cells (Mayr and Bartel, 2009), and during reprogramming of induced pluripotent stem cells (Ji and Tian, 2009). Yet, the involved signaling pathways and molecular mechanisms that regulate mRNA 30 end processing and their role in health and disease remained enigmatic.

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We now uncover a mechanism by which cellular stress and inflammatory pathways control 30 end processing and F2 expression. Molecularly, this regulation is achieved via an mRNP switch that connects the stress-dependent recruitment of canonical and noncanonical 30 end processing factors to the USE. Furthermore, we provide evidence for possible roles of this mechanism in vivo during the acute phase response and in the cancer microenvironment, highlighting its biological and medical relevance. RESULTS p38-Mediated Upregulation of USE-Containing mRNAs We previously identified enhanced 30 end processing of the F2 mRNA as a mechanism predisposing to human disease (Danckwardt et al., 2004; Gehring et al., 2001). Prototypically for a family of genes, low-efficiency F2 mRNA 30 end processing signals are balanced by a USE (Figure 1A); gain-of-function mutations of the low-efficiency signals (CG / CA) cause pathological increases in F2 levels. The proteins hnRNPI, U2AF65, and U2AF35 bind to the USE and specifically promote mRNA 30 end processing by recruiting the canonical 30 end processing machinery, consisting of the CPSF and CstF complexes (Danckwardt et al., 2007). Interestingly, the function of these proteins is independent of their known activity in the splicing process. More than 1700 human transcripts appear to contain these highly conserved USEs, including many genes with key roles in inflammation and tumorigenesis. Although USEs are currently only known for their importance in steady-state mRNA expression, we hypothesized that the unusual architecture of 30 end processing elements may have evolved to enable regulation of 30 end processing and thereby allow the dynamic adaptation of F2 expression in various (patho-)physiological conditions (see above). Noticing that F2 expression is increased in response to stress, we first tested whether anisomycin treatment, which globally induces stress- and inflammation-dependent cell responses (Mahadevan and Edwards, 1991; Wagner and Nebreda, 2009), affects the expression of USE-containing transcripts. This treatment of HUH7 hepatoma cells significantly upmodulates F2 mRNA levels and simultaneously augments the expression of other USE-containing mRNAs, such as BCL2L2, IVNS1, and ACTR3. By contrast, control mRNAs lacking USEs, such as ACTG1, HPRT1, CBFB, and MAP3K1, are unaffected by anisomycin treatment (Figure 1B). Furthermore, the induction of the F2, BCL2L2, IVNS1, and ACTR3 mRNAs appears to occur posttranscriptionally at the level of mRNA processing or stability, because the pre-mRNA abundance is not induced by anisomycin (Figure S1A). These specific changes in gene expression occur in the context of the activation of the ubiquitously expressed stress- and inflammation-dependent protein kinases JNK1, JNK2, and p38a and their signal transduction pathways (Figure S1B). We then explored the effect of these kinases on the abundance of USE-containing mRNAs in siRNA experiments. While efficient depletion of JNK1 and JNK2 with RNAi has little effect on the levels of the USE-containing F2, BCL2L2, IVNS1, and ACTR3 mRNAs, depletion of p38 MAPK significantly reduces their mRNA abundance (Figures 1C and S1C). Next, we aimed

to study the mRNA decay kinetics upon p38 MAPK RNAi by using actinomycin D as a widely used and well-established experimental procedure to estimate the relative stability of endogenous mRNAs (Chen et al., 1995; Shyu et al., 1989). Because all of the USE-containing mRNAs studied here have a half-life in the range of hours, we used the PTEN mRNA as positive control. Although the sensitivity of measuring mRNA stabilities with a long half-life by using this approach is limited (Harrold et al., 1991), the inclusion of an appropriate control such as PTEN enables an assessment of the relative differences of endogenous mRNAs with these limitations. We could thus validate the actinomycin D approach used here for the USE-containing F2, BCL2L2, IVNS1, and ACTR3 mRNAs. Following p38 MAPK depletion, the relative stability of the PTEN mRNA (lacking a USE) decreased 2-fold, confirming the effect of p38 MAPK on the turnover of some mRNAs (Briata et al., 2005). By contrast, the relative decay kinetics of the endogenous USE-containing F2, BCL2L2, IVNS1, and ACTR3 mRNA, which have half-lives comparable to PTEN under normal conditions (Figure 1D, black line), are unaffected (Figure 1D). These data suggest that—within the sensitivity of this assay system—RNA processing, rather than the stability of the prototypic F2- and other USE-containing mRNAs, is regulated in a stress- and p38 MAPK-dependent manner. Proteomic Definition of the USE Ribonucleoprotein Complex To elucidate the mechanism of how USE-containing mRNAs respond to stress, we analyzed the composition of the USE-ribonucleoprotein (RNP) complex. USE affinity purifications followed by mass spectrometry identify more than 25 distinct proteins with specific USE-binding properties (Figures 2A and 2B, Table S1). We had previously identified three of these, U2AF65, U2AF35, and hnRNPI, to interact with USEs, recruit the canonical 30 end processing machinery, and specifically promote mRNA 30 end processing without affecting other processing steps (Danckwardt et al., 2007). This comprehensive analysis now reveals several AU-rich element (ARE)-binding proteins (BPs) that bind to the USE with high affinity and specificity (Figures 2 and S2). Interestingly, several ARE-BPs are known to respond to and integrate extracellular signals to regulate gene expression posttranscriptionally in response to environmental stimuli (Shyu and Wilkinson, 2000; Wilusz and Wilusz, 2004). We therefore hypothesized that these USE-binding proteins may relay cellular signals to the USE, and analyzed the RNP composition of a radioactively labeled 21-mer F2 USE-RNA, comparing nuclear extracts prepared from control or anisomycin-treated cells from which p38 MAPK had or had not been depleted by RNAi (Figure 3A). UV-crosslinking experiments and electromobility shift assays (EMSAs) show that anisomycin treatment changes the composition of the USE-RNP complex qualitatively and quantitatively (Figure 3A, USE x-link, lanes 1 and 3). This effect is also seen under native conditions in EMSAs, where the major USE-binding complex is diminished in extracts from anisomycin-treated cells (Figure 3A, USE EMSA, lanes 1 and 3). Most interestingly, the qualitative and quantitative changes of the USE-RNP complex induced by anisomycin (the ‘‘stress footprint’’) require p38

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Figure 1. p38 Regulates mRNA Processing of USE-Containing mRNAs (A) Schematic representation of the USE-containing (AUUAUUUUUGUGUUU) F2 30 end processing signal, including the suboptimal CG cleavage dinucleotide, the GU/U-rich downstream sequence element, and the poly (A) signal (AAUAAA). (B) Anisomycin (ANISO) treatment of HUH7 cells upmodulates the mRNA abundance of the USE-containing genes prothrombin (F2), B Cell lymphoma apoptosis regulator like 2 (BCL2L2), influenza virus NS1A binding protein (IVNS1), ARP3 actin-related protein 3 homolog B (ACTR3), but not of the mRNAs with a canonical 30 end processing signal, i.e., actin gamma 1 (ACTG1), hypoxanthine phosphoribosyltransferase 1 (HPRT1), corebinding factor beta (CBFB), mitogen-activated protein kinase kinase kinase 1 (MAP3K1) (bars show mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001), measured by qRT-PCR and normalized to GAPDH and DMSO-treated cells, respectively. (C) Endogenous mRNA abundance of USE-containing genes (F2, BCL2L2, IVNS1, ACTR3) after depletion of p38a, JNK1, and JNK2 by RNAi (Figure S1C) (bars show mean ± SD; *p < 0.05, **p < 0.01). (D) Decay kinetics of USE-containing genes (F2, BCL2L2, IVNS1, ACTR3) and control mRNAs (Jun, PTEN) after depletion of p38a by RNAi (mean ± SD; *p < 0.05). Further data are available in Figure S1.

p38 MAPK phosphorylates select RNAbinding proteins to control their RNA-binding activities (Briata et al., 2005). Next, we wanted to explore whether p38 MAPK activation results in phosphorylation of USE-binding effector proteins, and assayed for their differential phosphorylation in response to stress using 32 P-orthophosphate labeling experiments. Neither of the positive effector proteins that we tested (hnRNPI, U2AF35, U2AF65) are detectably altered (Figure 3B, right panel, lanes 15–20). In contrast, orthophosphate labeling reveals that the phosphorylation of FBP1, FBP2, and FBP3 is induced by anisomycin, whereas HUR, CUGBP, TIA, and TIAR do not display significant changes of their phosphorylation status (Figure 3B, lower panel, lanes 1–14, and S3A). Thus, while most of the USE-BPs do not appear to be differentially phosphorylated, FBP1, FBP2, and FBP3 are selectively hyperphosphorylated in response to anisomycin treatment via p38 MAPK (Figures 3B, S3A, and S3B).

MAPK (Figure 3A, lanes 3 and 4). These experiments show that p38 MAPK influences the composition of the USE-RNP complex in response to anisomycin-induced stress.

Anisomycin Increases the Recruitment of Canonical and Noncanonical 30 End Processing Factors to the USE To directly monitor alterations of the binding of proteins to the USE in response to stress, we performed USE affinity purifications followed by immunoblotting with extracts from anisomycin-treated or untreated cells (Figure 4). Eluates from anisomycin-treated cells are enriched in

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Figure 2. USE RNP Complexes Include Components Involved in 30 End Processing and Splicing in Addition to ARE-BPs (A) Silver-stained SDS-PAGE polyacrylamide gel of protein samples (HeLa nuclear extract) derived from affinity purification with immobilized 30 biotinylated 21-mer RNA oligonucleotides, with the highly conserved F2 USE motif (USE, lanes 1, 4, and 7); with a mutated, functionally incompetent USE motif (Danckwardt et al., 2007) (USEmut, lanes 2, 5, and 8); or with an unrelated sequence (Unrel, lanes 3, 6, and 9). The left panel (lanes 1–3) shows protein samples eluted with up to 2000 mM NaCl (pooled fractions eluted with a NaCl concentration 150–2000 mM). The right panel shows fractionations of protein samples exclusively eluted under less stringent (‘‘low salt eluates,’’ NaCl concentrations up to 400 mM, lanes 4–6) or stringent (‘‘high salt eluates,’’ NaCl concentration 500–2000 mM, lanes 7–9) conditions, respectively. Silver-stained bands in lanes 4 and 7 were cut out and subjected to mass spectrometry (see Table S1). (B) Immunoblots of protein eluates derived from affinity purifications (Table S1). Lanes 1–9 correspond to samples as indicated in (A). Proteins in the top panel are implicated in USE-mediated mRNA 30 end processing (hnRNPI, U2AF65, U2AF35) (Danckwardt et al., 2007; Hall-Pogar et al., 2007; Millevoi et al., 2009; Moreira et al., 1998). Proteins in the middle panel either belong to the canonical 30 end processing machinery (CstF and CPSF subcomplexes) or to the multifunctional p54nrb/NonO-PSF complex implicated in facilitating transcription termination and 30 end processing. Proteins in the lower panel belong to the class of AU-rich element-binding proteins (AREBPs). Further data is available in Figure S2.

proteins in the 30–80 kDa range (Figure 4A, lanes 3 and 4), which is confirmed by UV-crosslinking experiments (Figure 4B, lanes 3 and 4). In immunoblots, the known USE-binding 30 end processing factors U2AF35, U2AF65, and hnRNPI are specifically enriched in eluates from stressed cells compared to control cells (Figure 4C, lanes 3 and 4). Consistent with our earlier results (Danckwardt et al., 2007), p54nrb/NonO, which has been implicated in facilitating transcription termination and 30 end processing, and the canonical 30 end processing machinery, represented here by the CPSF and CstF subcomplexes and the scaffold protein symplekin (Sullivan et al., 2009), co-purify with U2AF35, U2AF65, and hnRNPI in a stress-dependent fashion (Figure 4C,

lanes 3 and 4, middle and lower panel). Notably, while most of the USE-binding ARE-BPs identified by mass spectrometry (Figure 2) are also enriched in USE-mRNPs from stressed cells, FBP2 and FBP3 are specifically reduced under these conditions (Figures 4C, lanes 3 and 4, upper panel, and S3C). This correlates with their reduced binding affinity upon phosphorylation (Figures S3A and S3B). We also note that anisomycin treatment induces the total expression of immunoreactive CPSF 160, CPSF 30, CstF 77, CstF 64, CstF 50, and p54nrb/NonO (Figure 4C, lanes 1 and 2). These observations are highly consistent with earlier findings that extracellular stimuli (such as lipopolysaccharides) induce the expression of the 30 end processing machinery (Shell et al., 2005) and suggest that this induction facilitates the formation of functional 30 end processing complexes via the USE under stress conditions. Stress Induces Polyadenylation of USE-Containing mRNAs To define the mechanism by which anisomycin promotes RNA processing via the USE, we next performed in vitro cleavage and polyadenylation assays. USE-containing mRNA substrates are cleaved with similar efficiency in extracts from stressed

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Figure 3. p38 MAPK Signaling Modifies the USE RNP Composition and Hyperphosphorylates FBP1, FBP2, and FBP3 Contained in This Complex (A) Silver staining (top panel) shows integrity and equal loading of the nuclear extracts used for UV-crosslinking studies (USE X-link) and electromobility shift assays (EMSA). Immunoblots against pan-p38a and phosphop38a demonstrate depletion of p38a upon RNAi (lanes 2 and 4) and phosphorylation of p38 after anisomycin treatment (lane 3), respectively. USE X-linking and EMSA were carried out with a USE-containing 21-mer RNA oligonucleotide after incubation in HeLa nuclear extract of cells treated with (lanes 3 and 4) or without anisomycin (lanes 1 and 2) after siRNA-mediated depletion of p38a MAPK (lanes 2 and 4). Arrowheads highlight differentially bound protein (complexes). (B) 32P-orthophosphate labeling experiments with HeLa cells to determine the phosphorylation status of USEbinding proteins in response to anisomycin stress. After immunoprecipitation, the FLAG-tagged USE-binding proteins were separated by SDS gel electrophoresis, and gels were exposed to X-ray films (lanes 1–14, left, show ARE-binding proteins; lanes 15–20, right, show positive USE-effector proteins). Upper two panels show loading and purity of the immunopurified proteins (silver staining and western blot against FLAG peptide). The autoradiography in the lower panel shows metabolic labeling of the respective proteins after exposure to anisomycin. Phosphoimaging of the band intensities was used to quantify the fold change of phosphorylation (above background, shown is a representative image of two independent experiments; n.a. = not applicable; red asterisks highlight immunopurified proteins, black arrowheads highlight differentially phosphorylated proteins). For further quantification of the phosphorylation status and the functional role of p38 MAPK, see Figures S3A and S3B.

and nonstressed cells (Figure S5A); however, polyadenylation is strongly enhanced by 3- to 4-fold in extracts from stressed cells (Figure 5A, left panel, compare lanes 5–8 with 1–4). RNA substrates in which the USE was specifically replaced by a (nonfunctional [Danckwardt et al., 2007]) unrelated sequence context, which is incapable of binding the positive effector or

other proteins identified here (Figure 2A, lane 3), do not display any stress-dependent increases of polyadenylation efficiency (Figure 5A, right panel, compare lanes 9–12 with 13–16), thus demonstrating the specificity of the effect and showing their USE dependence. We next explored the role of the differentially phosphorylated FBPs in these reactions and hypothesized that 30 end processing efficiency is inhibited by hypophosphorylated FBP2 and/ or FBP3 in nonstressed cells. We first added FBP2 or FBP3 isolated from control (DMSOtreated) cells to in vitro polyadenylation reactions (Figure 5B, lanes 9–12) and observed that neither of these two proteins individually interferes with active 30 end processing of USE-containing substrates in extracts from anisomycin-treated cells (Figure 5B, compare lane 8 with 9–12). Interestingly, however, combined addition of FBP2 and FBP3 antagonizes stress-induced 30 end processing in a dose-dependent manner (Figure 5B, compare lane 8 with 13 and 14). This effect is specific, because it is not detected with equal amounts of hyperphosphorylated FBP2 and FBP3 obtained from cells

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Figure 4. Stress Upmodulates the Expression of Components of the 30 End Processing Machinery and Promotes the Formation of a Stimulatory USE-RNP Complex (A) Affinity purification of proteins binding to the F2 USE under conditions before and after activation of p38 signaling by anisomycin. The silver stain demonstrates the load or integrity of whole-cell lysates obtained from HeLa cells treated with DMSO and anisomycin (‘‘input,’’ lanes 1 and 2, respectively), which were used as starting material (Figure S4) for affinity purifications. Lanes 3 and 4 show proteins enriched in F2 USE-affinity purifications (‘‘eluates’’). (B) USE 21-mer RNA oligonucleotide X-linking with wholecell lysates (lanes 1 and 2) and with eluates obtained from affinity purifications (lanes 3 and 4). (C) Immunoblotting of whole-cell lysates (lanes 1 and 2) and eluates obtained from affinity purifications (lanes 3 and 4; lanes 5 and 6 represent dilutions of the stress eluates shown in lane 4) with antibodies as indicated (silver stain in (A) shows loading). Proteins in the first group belong to the class of AU-rich element-binding proteins (ARE-BPs) with high USE binding affinity (Figures 2A and 2B), which show a stress- and p38 MAPK-dependent induction of phosphorylation and loss of USE-binding (Figures 3B, 4C, and S3A, S3B). Proteins in the second group are implicated in USE-mediated mRNA 30 end processing (Danckwardt et al., 2007; Hall-Pogar et al., 2007; Millevoi et al., 2009; Moreira et al., 1998). Proteins in the third group belong to either the canonical 30 end processing machinery (CstF and CPSF subcomplexes, the scaffold protein symplekin) or to the multifunctional p54nrb/ NonO-PSF complex. Tubulin (fourth group) was used as a loading control. The bar diagram at the bottom shows the fold change of protein abundance (mean ± SD) in the F2 USE RNP upon stress induction (lane 4), compared to mock treated cells (lane 3) (n R4 experiments; *p < 0.05; log 2 values are shown).

treated with anisomycin (Figure 5B, compare lanes 13 and 14 with 15 and 16). To further control these experiments, we performed in vitro polyadenylation reactions with recombinant poly(A) polymerase (PAP) in the presence or absence of FBP2 and FBP3. Recombinant PAP efficiently polyadenylates USE-containing RNA templates (Figure S5B, lane 2), which is not affected by the addition of hypophosphorylated FBP2 and FBP3 either during or after the reaction (Figure S5B, lanes 3 and 4). These data show that FBP2 and FBP3 do not affect the processivity of PAP per se; furthermore, they do not induce deadenylation of the polyadenylated products. While hypophosphorylated FBP2 and FBP3 from unstressed cells inhibit polyadenylation in a dose-dependent manner (Figures 5B, lanes 13 and 14, and S5B, lanes 6–8), their

addition after the reaction does not perturb polyadenylation (Figure S5B, compare lanes 6 with 9 and 10). We thus conclude that FBP2 and FBP3 modulate mRNA 30 end processing efficiency in a USE-dependent manner without affecting PAP processivity or deadenylation. Stress upmodulates the expression of the canonical 30 end processing machinery and induces the hyperphosphorylation of FBP2 and FBP3 via p38 MAPK, which in turn interferes with their RNA-binding properties and/or induces remodeling of the USE-dependent mRNP. Lack of interference by FBP2 and FBP3 allows for effective loading of the USE with positive 30 end processing factors and concomitantly corecruited canonical 30 end processing components. This reorganization of the USE-RNP effects more efficient 30 end processing of USE-containing mRNAs in response to environmental cues such as stress (Figure 6I). In line with this model, the combined depletion of FBP2 and FBP3 by RNAi reduces their antagonistic function at the USE and suffices to significantly upmodulate USE-containing mRNAs (such as F2, BCL2L2, IVNS1, ACTR3B, OTUD7B, KIN, ZCCHC8,

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Figure 5. FBP2 and FBP3 Regulate Polyadenylation and Gene Expression in a Stress-Dependent and USE-Dependent Way (A) In vitro polyadenylation assays (left and right panel, respectively) carried out on RNA substrates with a functional USE (USE substrate, lanes 1–4 and 5–8) or on RNA substrates in which the USE was replaced by an unrelated sequence (unrel. substrate, see schematic drawing above, and lanes 9–12 and 13–16) in nuclear extracts (NE) obtained from cells treated with (NE ‘ANISO’; lanes 6–8 and 14 and 15) or without anisomycin (NE ‘DMSO’; lanes 2–4 and 10–12). Black arrowheads indicate the size of input precursor RNA substrates. Polyadenylated RNA products of heterogeneous size are indicated with an open bracket. A representative result obtained with at least four independent extract preparations is shown; the quantification shows the fold change of polyadenylation efficiency after stress induction (lanes 7, 8, and 16), compared to mock controls (lanes 3, 4, and 12) after normalization (polyadenylation efficiency in lane 3, 4, and 12 is set 1.0) (USE substrate; n = 4, *p < 0.05; unrel. substrate; n = 3). (B) In vitro polyadenylation assays (as performed in panel (A) on USE-containing substrates with or without coincubation of FBP2 and/or FBP3 obtained from FLAG-immunoselection of transiently transfected cells treated with anisomycin (FBP2A or FBP3A, hyperphosphorylated) or DMSO (FBP2D or FBP3D, hypophosphorylated), respectively (see also Figures 3B and S2). FLAG-immunoblot (lower panel) shows the amount of FBP2 and/or FBP3 used in the respective reactions (A) representative result obtained with at least four independent extract preparations is shown; the quantification shows the fold change of polyadenylation inhibition by FBP2 and/or FBP3 after stress induction (lanes 6–16) compared to mock control (lane 8) after normalization (polyadenylation efficiency in lane 8 is set 1.0, *p < 0.05). Further data is available in Figure S5. (C) Endogenous F2, BCL2, IVNS1, ACTR3B, cellular zinc finger anti-NF-kappa-B protein (OTUD7B), antigenic determinant of recA protein homolog (KIN), zinc finger CCHC domain-containing protein 8 (ZCCHC8), programmed cell death 10 (PDCD10), sterol-C4-methyl oxidase-like (SC4MOL), platelet-derived growth

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PDCD10, SC4MOL, and PDGFRA) in living cells, even under normal (nonstressed) conditions (Figure 5C). In contrast, 7 of the 9 controls (either with a USE located at a nonfunctional position far upstream of a potential poly(A) signal or lacking a USE completely) were unaffected by FBP2/FBP3 depletion. We also note that stress coinduced the expression of the canonical (and noncanonical) 30 end processing factors during stress (see above). This induction may thus help to further amplify enhanced 30 end processing of USE-containing substrates during stress (see below). In Vivo Models for Inflammation and Tumor Invasion Suggest Important Roles of USE-Mediated Control The critical function of F2 in hemostasis, innate immunity, and cancer cell biology (Coughlin, 2000; Nierodzik and Karpatkin, 2006; Rittirsch et al., 2008) suggested that USE-mediated regulation may play a particular role in the regulated (patho-)physiology of F2 gene expression in vivo (Boven et al., 2003; Citron et al., 2000; Dihanich et al., 1991; Huber-Lang et al., 2006; Kim et al., 1998; Murakami et al., 2003; Riek-Burchardt et al., 2002; VanLandingham et al., 2008; Yin et al., 2010; Zoubine et al., 1996). First, we examined whether experimental septicemia upmodulates the F2 mRNA. We injected immunocompetent C57BL/ 6 mice with lipopolysaccharide (LPS) to induce a systemic inflammatory response (Figure 6A). The inflammatory increase of TNFa 4 hr after LPS injection—both LPS and TNFa activate p38 MAPK (Raingeaud et al., 1995)—is paralleled by a marked increase of F2 mRNA, but not of the pre-mRNA (Figure 6B). Furthermore, the F2 induction closely mirrors the extent of inflammation, as is indicated by the induction of fibrinogen b, during experimental septicemia (Figure S6B). Moreover, LPS treatment also induces the mRNAs for 30 end processing factors in liver samples, i.e., CPSF 160, CstF 64, CstF 50, and p54nrb/ NonO (Figure S6C), which corresponds to the changes observed in anisomycin-stressed cells (compare Figure 4C, lanes 1 and 2, Figure S6C). By contrast, the mRNA that encodes coagulation factor 10 (F10), which is also expressed in the liver but does not contain a USE, is not induced under the same conditions (Figures S6A and S6B), indicating that the upmodulation of the 30 end processing machinery per se is not sufficient to induce mRNAs (nonspecifically) in vivo. Finally, F2 protein expression is seen to increase in liver sections (Figure 6C) and immunoblots (Figure 6D). In view of the data shown in Figures 1D, 4C , 5A , 5B, 6B, and S6C, we suggest that these data reflect the upmodulation of 30 end processing efficiency in a USE-dependent manner. To further probe whether this in vivo response likely reflects a USE-dependent switch, we also studied USE mRNP composition in crosslinking experiments with whole-cell liver homogenates obtained from LPS-treated and untreated animals (Figure 6E). LPS treatment triggers both quantitative and qualitative changes of the F2 USE mRNP (Figure 6E, compare lanes 1–3), reminiscent of the changes observed in stressed cells (Figure 3A). Furthermore, the endogenous murine F2 mRNA is

specifically reduced in coimmunoprecipitation (coIP) assays with antibodies directed against FBP2 and FBP3 and enriched in coIPs with antibodies directed against the stimulatory hnRNPI protein, which is rate-limiting for USE-mediated 30 end processing of the F2 (Danckwardt et al., 2007) and also other mRNAs (Danckwardt et al., 2007; Hall-Pogar et al., 2007; Millevoi et al., 2009; Moreira et al., 1998), using liver lysates from LPS-treated animals (4 hr and 9 hr after LPS injection) (Figure 6F, compare lane 4 with 5 and 6). In light of the data shown in Figures 1D, 4C, 5A, 5B, and S6C, these results indicate that, upon induction of a systemic inflammatory response, hnRNPI is more abundantly loaded onto the USE of the murine F2 mRNA in the liver to form a complex that promotes F2 mRNA 30 end processing, whereas the inhibitory FBP2 and FBP3 interact less efficiently with the F2 mRNA. Interestingly, these findings closely mirror the stress-mediated remodeling of the F2 USE RNP upon p38 MAPK induction in anisomycin-treated cells (Figure S6D). Local inflammatory processes represent an integral part of the body’s response to various malignancies, involving protumorigenic cytokines, such as TNFa and others (Lin and Karin, 2007; Mantovani et al., 2008). p38 MAPK integrates multiple inflammatory stimuli and environmental stresses, such as TNFa or reactive oxygen species (Raingeaud et al., 1995), and deregulated p38 MAPK signaling is associated with cancers in humans and mice (Gilbert and Hemann, 2010; Wagner and Nebreda, 2009). Therefore, we finally used human tumor sections as an additional model to explore both the functional importance of deregulated p38 MAPK signaling and possible in vivo implications of our findings, without extrinsically inducing an inflammatory response. We obtained human liver specimens from patients with metastatic colon carcinoma. F2 mRNA and protein expression are notably increased at the tumor invasion front in the nontumorous liver tissue. This contrasts with the neighboring normal liver tissue and the inner parts of the tumor metastases (Figures 6G and 6H). Furthermore, the upmodulation of the F2 mRNA in the tumor microenvironment correlates with a local inflammatory induction of TRADD mRNA and downstream markers of p38 MAPK activation and FBP2 phosphorylation (MYO1B mRNA; see Figures 6H and S6E and Briata et al., 2005). Finally, F2 overexpression results in an induction of F2mediated receptor signaling (reflected by the upmodulation of V-SRC and ARHGEF2 mRNA as downstream surrogate markers) in the tumor microenvironment and induces the expression of Cathepsin D and Angiopoietin2 mRNAs, which are implicated in mediating the effect of F2 on tumor angiogenesis, growth, and metastasis (Figure 6H) (Hu et al., 2008; Nierodzik and Karpatkin, 2006). DISCUSSION Physiological gene expression requires intricate and precise transcriptional and posttranscriptional regulatory networks (Pawlicki and Steitz, 2010; Proudfoot et al., 2002). Numerous pathways linking external stimuli to transcriptional regulation

factor receptor alpha (PDGFRA), AGXT2, USP47, ABAT, NAALAD2, HPRT, blood coagulation factor 10 (F10), CBFB, MAP3K1, and ACTG1 mRNA following control RNAi (white bars) and following combined FBP2 and FBP3 RNAi (black bar) in Hep 3B cells (mean of n > 5 independent experiments ± SD ; *p < 0.05, **p < 0.01, n.s. = not significant).

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Figure 6. Inflammatory Stimuli Modify F2 USE-RNP Complex Formation In Vivo and Increase F2 Expression in a Model of Experimental Septicemia and in the Tumor Microenvironment of Human Liver Metastases (A) Serum TNF-a kinetics (mean ± SD) in C57BL/6 mice injected with 80 mg LPS (*p < 0.05). (B) F2 mRNA (upper diagram) and pre-mRNA (lower diagram) in liver specimens obtained from LPS-treated C57BL/6 mice (mean ± SD; **p < 0.01). The analysis of the F10 mRNA that does not contain a USE served as negative control (Figure S6A). (C) Prothrombin/thrombin immunohistochemistry on paraffin-embedded liver sections of mice treated with or without LPS (24 hr after injection). (D) Immunoblot for prothrombin/thrombin of liver lysates of LPS-treated and untreated mice (the diagram integrates the abundance of both prothrombin and thrombin and reflects the mean of at least three animals; **p < 0.01). (E) F2 USE-RNP complex analysis with a 21-mer F2 USE-RNA oligonucleotide and liver lysates obtained from LPS-treated animals (0 hr, 4 hr, and 9 hr after LPS injection). The top panel shows the load after PAGE blue staining and the bottom panel the USE X-link. (F) RNA-protein interaction study based on in vivo crosslink RNP-coIPs with (whole-cell) liver extracts of LPS-treated animals (0 hr, 4 hr, and 9 hr after LPS injection) and antibodies directed against FBP2, FBP3, or hnRNPI, respectively. The F2 mRNA contained in the immunoprecipitates (lanes 4–6) was analyzed by RT-PCR after reversal of the crosslink (the bar diagram shows mean ± range of two independent coIP reactions after normalization against 0 hr values; log2 values are shown). (G) F2 immunohistochemistry of peripheral liver tissue and a liver metastasis of a colon carcinoma (representative immunohistochemistry from 2 out of 3 analyzed patient samples). Cells from regions A and B were microdissected and subjected to RNA analysis shown in (H). (H) F2, TRADD, MYO1B, V-SRC, ARHGEF2, Angiopoietin 2, and Cathepsin D RT-PCR mRNA expression analysis in tissue samples corresponding to regions A and B (tumor-adjacent [black bars ± SD] and peripheral normal liver [white bars ± SD] tissue) obtained with palm laser capture microdissection (after normalization against GAPDH; n.d. = not detectable).

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have been characterized, whereas much less is known about the regulation of RNA processing by external cues. The importance of the tight regulation of mRNA 30 end formation is highlighted by thrombophilia from even subtle (1.5- to 1.7-fold) aberrations of this process (Danckwardt et al., 2004; Gehring et al., 2001). This report uncovers a regulatory pathway and mechanism at the level of 30 end processing and provides evidence for its pathophysiologic relevance in vivo. Specifically, we show that activation of p38 MAPK upmodulates the canonical 30 end processing machinery and directly induces the phosphorylation of the RNAbinding proteins FBP2 and FBP3, which bind to the highly conserved USE. Their USE-binding appears to interfere with the binding of stimulatory 30 end processing factors, such as hnRNPI, U2AF35, and U2AF65 (Danckwardt et al., 2007; HallPogar et al., 2007; Millevoi et al., 2009; Moreira et al., 1998), and thus limits 30 end processing efficiency. Following phosphorylation, USE-binding of FBP2 and FBP3 is reduced, facilitating USE access of stimulatory factors and, ultimately, the expression of the mRNA and protein of this recently discovered class of USE-containing genes (Figure 6I), by affecting the polyadenylation but not the cleavage step of 30 end processing. A similar specificity of regulation affecting these two steps of 30 end processing individually has recently also been reported for other factors (Xiang et al., 2010). These results predict that this mechanism of USE control could also affect alternative polyadenylation by switching the strengths of alternative polyadenylation sites, although this possibility remains to be explored in the future. Interestingly, the pre-mRNA levels of USE-containing mRNAs do not change significantly under stress conditions (Figure S1A). This observation is fully consistent with our model (and even its predictions) and all current concepts of general gene expression, whereby a cleaved precursor mRNA that is not yet processed further, i.e., polyadenylated, faces two alternative fates: either polyadenylation, and subsequent transport into the cytoplasm, or degradation in the nucleus (Kazerouninia et al., 2010). Thus, in case of upregulated polyadenylation, more of the pre-mRNA will be matured and exported, adding to the cytoplasmic pool of that mRNA. As a consequence, the net amount of premRNA (in the nucleus) should remain the same, while the amount of mRNA in the cytoplasm increases, which we observe here both in cells and in animals (Figures 1B, 6B, and S1A). The importance of inflammation as a driver of pathology is no longer confined to autoimmune and infectious diseases. Rather, inflammation is increasingly linked to diseases such as coronary artery disease, obesity, and cancer (Lin and Karin, 2007). Understanding regulatory networks underlying inflammatory responses has therefore become topical for both the elucidation of disease mechanisms and the quest for novel therapeutic strategies. Here, we identify p38 MAPK signaling as an upstream effector of a mechanism to control the expression of a class of mRNAs that encode proteins with pivotal roles in tumor progression and also tumor suppression (Danckwardt et al., 2007).

Increased procoagulatory activities are frequently detected during septicemia (Levi and Ten Cate, 1999; Rittirsch et al., 2008) and in cancer patients, sometimes even representing a forewarning of occult malignancy (referred to as Trousseau’s syndrome). A growing body of evidence indicates that this syndrome is not a mere paraneoplastic effect, but the result of mechanisms that provide a selective advantage to cancer cells (Boccaccio and Comoglio, 2005; Joyce and Pollard, 2009; Kuderer et al., 2009; Schulman and Lindmarker, 2000). In line with these findings, hyperactivation of blood coagulation is associated with more rapid tumor progression (Miller et al., 2004; Nierodzik and Karpatkin, 2006). Strikingly, the prevalence of prothrombotic mutations of F2 30 end processing, such as F2 20210 G > A (Danckwardt et al., 2004; Gehring et al., 2001), is higher in some cancer patients compared to controls (Blom et al., 2005). Conversely, impaired blood coagulation reduces the incidence of cancer (Schulman and Lindmarker, 2000) and inhibits the invasive growth of tumor cells and metastasis in patients treated with anticoagulants (Petralia et al., 2005) and in mice defective for F2 (Palumbo et al., 2007). We propose that these clinical observations are linked to the findings reported here. The acquisition of invasive/metastatic potential through protease expression is an essential event in tumor progression (Joyce and Pollard, 2009). The upmodulation of the serine protease thrombin in malignancies may thus actively promote tumor invasion, metastasis, and tumor angiogenesis (Nierodzik and Karpatkin, 2006). Moreover, F2 receptors are frequently upmodulated in highly metastatic human malignancies (Even-Ram et al., 1998; Finak et al., 2008). The induction of F2 in the tumor microenvironment shown here could therefore possibly represent a missing link in explaining how these receptors are activated to further aggravate tumor progression. In summary, the findings reported here decipher a mechanism that controls gene expression through regulated mRNA 30 end processing. This mechanism may also explain the enigmatic functional relationship between activation of blood coagulation and tumor growth, metastasis, and outcome (Blom et al., 2005; Boccaccio and Comoglio, 2005; Joyce and Pollard, 2009; Kuderer et al., 2009; Miller et al., 2004; Schulman and Lindmarker, 2000). Therefore, the results motivate testing of inhibitors of the signaling pathways that control regulated 30 end processing to attenuate inflammation-associated deregulation of blood coagulation in septicemia, the role of thrombin in the pathogenesis of inflammatory brain diseases (Boven et al., 2003; Chapman, 2006; Yin et al., 2010), and the progression of malignancies (Nierodzik and Karpatkin, 2006; Palumbo et al., 2007). EXPERIMENTAL PROCEDURES Stress Experiments HUH-7 and HeLa cells were treated with 10–20 mg/ml anisomycin (Calbiochem, Merck KGaA, Darmstadt, Germany) or DMSO (Sigma, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) as solvent control for indicated time periods (1–4 hr).

(I) Proposed model for a mechanism that enables the regulation of mRNA 30 end processing in response to extracellular stimuli, p38 MAPK activation and a USEdependent RNP-switch, as exemplified for the F2 mRNA (+ denotes a stimulatory interaction, [ indicates a stress-dependent upmodulation of CPSF and CstF complexes in response to p38 MAPK activation). For further details, see text and discussion.

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Cell Culture and (siRNA) Transfection, Protein and RNA Analysis Cell culture and (siRNA) transfection, protein and RNA analysis, and RNAprotein interaction studies were carried out as previously described (Danckwardt et al., 2007) (see Supplemental Information).

Affinity Purification and Mass Spectrometry Affinity purifications and mass spectrometry were performed as previously described (Danckwardt et al., 2007).

Metabolic Labeling HeLa cells were transfected with constructs encoding FLAG-tagged candidate proteins and then treated with anisomycin or DMSO in the presence of 32 P-orthophosphate. After harvest and FLAG immunoprecipitation (see Supplemental Information), the respective proteins were separated by SDS gel electrophoresis.

Phospholabeling In addition to the metabolic labeling experiments (above), the determination of the phosphorylation kinetics of FBP1, FBP2, and FBP3 was confirmed with phosphospecific stains (Pro-Q, Invitrogen, Carlsbad, California).

In Vitro Phosphorylation Studies To investigate whether a-p38 MAPK directly phosphorylates FBP1, FBP2, and FBP3, in vitro phosphorylation studies were carried out with activated a-p38 (Upstate/Millipore) in the presence of [g-32P]ATP. The reactions were then loaded onto SDS-polyacrylamide gels and visualized by exposure to X-ray films. For phosphospecific RNA-protein interaction studies, kinase assays were carried out with or without (non-radioactive) ATP (with FLAG-FBP1, -FBP2, and -FBP3), followed by radioactive USE-specific crosslinking assays as previously described (Danckwardt et al., 2007).

RNA-Protein Interaction Studies In Vivo For in vivo RNA-protein interaction studies, coIPs were carried out with cell and tissue lysates after formaldehyde crosslinking (see Supplemental Information). The immunoprecipitated RNAs were then analyzed by RT-PCR.

Laser Microdissection and Tissue RNA Extraction Quantitative gene expression analysis in archival formalin-fixed and paraffinembedded tumor tissue was performed after laser mircrodissection and RNA extraction with RT-PCR (see Supplemental Information).

In Vitro Cleavage and Polyadenylation Assays In vitro cleavage and polyadenylation assays were performed with nuclear extracts prepared from cells treated with or without anisomycin (see Supplemental Information) (Danckwardt et al., 2007).

Immunohistochemistry Immunohistochemistry experiments were carried out on formalin-fixed and paraffin-embedded tissue sections according to standard protocols. Antithrombin antibody (K-20; sc-16972; Santa Cruz) was used in dilutions of 1:150–1:500.

Mouse Treatment C57BL/6 mice (males) were intravenously injected with LPS as indicated (S. typhimurium [L6511], Sigma, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). To obtain 4% formaldehyde-fixed liver tissues (for immunohistochemistries and for in vivo crosslinking RNA-protein interaction studies) and snap-frozen liver samples (for protein analysis and RNA analysis), mice were sacrificed at the indicated time points. See Supplemental Information for further experimental procedures.

SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, one table, Supplemental Experimental Procedures, and additional references, and can be found with this article at doi:10.1016/j.molcel.2010.12.032. ACKNOWLEDGMENTS We are grateful to T. Heise, J. Patton, A. Krainer, B. Blencowe, C. and J. Lutz, H.-J. Chung, D. Levens, A.-B. Shyu, J. Izaquierdo, J. Valca´rcel, M. Sattler, A. Lamond, V. Lohmann, B. Jockusch for providing plasmids, cell lines, and antibodies. We thank the Tissue Bank of the National Center for Tumor Diseases (NCT), Heidelberg, Germany, for providing patient samples. We thank P. Ivanov, N. Gehring, S. Breit, G. Neu-Yilik, O. Reddy Bandapalli, and the team of the Molecular Medicine Partnership Unit for advice and discussions. This work was funded by the Young Investigator Award fellowship from the University of Heidelberg (to S.D. and to S.M.G.), by grants from the Deutsche Forschungsgemeinschaft (to S.D. and to A.E.K.), the Fritz-Thyssen Stiftung (to A.E.K.), the DFG Forschergruppe (FOR 426: ‘‘Complex RNA–protein interactions in the maturation and function of eukaryotic mRNA’’), and funds from the Lautenschla¨ger Research Prize (to A.E.K. and M.W.H.). Received: August 25, 2010 Revised: November 10, 2010 Accepted: December 23, 2010 Published: February 3, 2011 REFERENCES Barabino, S.M., and Keller, W. (1999). Last but not least: regulated poly(A) tail formation. Cell 99, 9–11. Blom, J.W., Doggen, C.J., Osanto, S., and Rosendaal, F.R. (2005). Malignancies, prothrombotic mutations, and the risk of venous thrombosis. JAMA 293, 715–722. Boccaccio, C., and Comoglio, P.M. (2005). A functional role for hemostasis in early cancer development. Cancer Res. 65, 8579–8582. Boven, L.A., Vergnolle, N., Henry, S.D., Silva, C., Imai, Y., Holden, J., Warren, K., Hollenberg, M.D., and Power, C. (2003). Up-regulation of proteinase-activated receptor 1 expression in astrocytes during HIV encephalitis. J. Immunol. 170, 2638–2646. Briata, P., Forcales, S.V., Ponassi, M., Corte, G., Chen, C.Y., Karin, M., Puri, P.L., and Gherzi, R. (2005). p38-dependent phosphorylation of the mRNA decay-promoting factor KSRP controls the stability of select myogenic transcripts. Mol. Cell 20, 891–903. Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.C., Maeda, N., Oyama, R., Ravasi, T., Lenhard, B., Wells, C., et al; FANTOM Consortium; RIKEN Genome Exploration Research Group and Genome Science Group (Genome Network Project Core Group). (2005). The transcriptional landscape of the mammalian genome. Science 309, 1559–1563. Chapman, J. (2006). Thrombin in inflammatory brain diseases. Autoimmun. Rev. 5, 528–531. Chen, C.Y., Xu, N., and Shyu, A.B. (1995). mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colonystimulating factor transcripts: different deadenylation kinetics and uncoupling from translation. Mol. Cell. Biol. 15, 5777–5788. Citron, B.A., Smirnova, I.V., Arnold, P.M., and Festoff, B.W. (2000). Upregulation of neurotoxic serine proteases, prothrombin, and protease-activated receptor 1 early after spinal cord injury. J. Neurotrauma 17, 1191–1203. Coughlin, S.R. (2000). Thrombin signalling and protease-activated receptors. Nature 407, 258–264. Danckwardt, S., Gehring, N.H., Neu-Yilik, G., Hundsdoerfer, P., Pforsich, M., Frede, U., Hentze, M.W., and Kulozik, A.E. (2004). The prothrombin 30 end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations. Blood 104, 428–435.

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