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• W4385881710 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA binding proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks. Editor's evaluation This paper provides important evidence that the RNA binding protein SAM68 regulates endothelial cell migration through multiple mechanisms including localizing actin mRNA to focal adhesions and stimulating transcription of the fibronectin gene. The evidence is generally convincing, although the relative roles of transcription and RNA localization in SAM68 functions and the dynamics of RNA movement to adhesion sites remain unknown. The paper will be of interest to cell biologists investigating post-transcriptional regulatory mechanisms. https://doi.org/10.7554/eLife.85165.sa0 Decision letter eLife's review process Introduction Maintenance of tissue homeostasis requires reciprocal interactions between the extracellular matrix (ECM) and cells that organize their microenvironment. Endothelial cells play a central role in tissue homeostasis and they actively contribute to growth during development, tissue patterning, and regeneration processes in which the remodeling of blood vessel networks is highly dynamic (Ramasamy et al., 2015). Fine tuning of interactions between endothelial cells and their perivascular ECM is essential for vascular network formation and integrity (Marchand et al., 2019). These interactions occur at integrin adhesion site complexes or ‘adhesomes’, specialized mechanosensitive hubs for integration of extracellular stimuli and activation of cytoplasmic signaling pathways to control cell adaptive responses (Humphries et al., 2019). Large-scale proteomic analyses of integrin adhesomes have revealed the robust presence of RNA Binding Proteins (RBPs) in these macromolecular assemblies (Byron et al., 2015; Horton et al., 2016; Horton et al., 2015; Mardakheh et al., 2015) yet their precise functions remain to be fully understood. SAM68 (Src associated in mitosis) is an RBP present in endothelial cell adhesomes (Atkinson et al., 2018) whose activities have been associated to cell adhesion and migration (Huot et al., 2009a; Locatelli and Lange, 2011; Naro et al., 2022; Wu et al., 2015). SAM68 belongs to the STAR (signal transduction and activation of RNA) family of proteins that link intracellular signaling pathways to RNA processing. First described as a direct target of tyrosine phosphorylation by Src kinase during mitosis (Fumagalli et al., 1994; Taylor and Shalloway, 1994), SAM68 was subsequently shown to act as a scaffold protein following activation of diverse transmembrane receptors and intracellular signaling pathways (Sánchez-Jiménez and Sánchez-Margalet, 2013). A direct role in signal relay has been demonstrated for SAM68 in TNFα signaling following TNFR1 activation (Ramakrishnan and Baltimore, 2011), in modulation of the Wnt/β-catenin pathway (Benoit et al., 2017) and in Src signaling (Huot et al., 2009a; Richard et al., 2008). Moreover, SAM68 has been widely described as a regulator of alternative splicing. As all STAR family proteins, it contains a structural domain for binding of RNA composed of a KH RNA-binding module embedded within a conserved regulatory and signaling region (STAR domain). SAM68 has been shown to regulate the alternative splicing of CD44 in a signal-dependent manner (Matter et al., 2002) and the generation of large variants of tenascin-C (Moritz et al., 2008). More recently, SAM68 has been identified as regulator of a splicing program involved in migration of triple-negative breast cancer cells (Naro et al., 2022). In addition to its major role in alternative splicing, SAM68 impacts other RNA processing events such as transcription, RNA translation, and regulation of long noncoding RNA (Frisone et al., 2015). In light of the signal relay functions of SAM68 and its presence in integrin adhesion complexes, we set out to elucidate the role of SAM68 in endothelial cell-ECM interactions. We surveyed the spatio-temporal distribution of the RBP following integrin activation and analyzed its participation in integrin signaling and adhesion maturation. We show transient localization of SAM68 near nascent adhesion sites where it participates in cytoskeletal remodeling and local delivery of β-actin mRNA, which is known to contribute to adhesion site stabilization and growth. Moreover, we demonstrate that SAM68 orchestrates cell-matrix interactions by regulating the expression of key perivascular ECM components and assembly of the subendothelial matrix, a structure that provides important instructive signals for cell migration and morphogenesis. We propose that these coalescent activities of SAM68 play a key role in the adaptation of endothelial cells to their extracellular environment. Results SAM68 regulates endothelial cell adhesion site formation and maturation To determine whether SAM68 is involved in the regulation of the adhesive phenotype of endothelial cells, we performed loss-of-function studies in primary cultured HUVECs by RNA interference. Transfection of two different siRNAs directed against human SAM68 transcripts efficiently decreased SAM68 protein levels (up to 90%), compared to those obsered in control siRNA-transfected cells (Figure 1—figure supplement 1). Following an overnight incubation on uncoated glass coverslips, subconfluent cells transfected with the control siRNA displayed large lamellipodial protrusions (Figure 1A) and prominent stress fibers (Figure 1B). In contrast, cells expressing SAM68-targeting siRNA were less spread and they assembled smaller edge ruffles. Quantitative morphological analyses revealed a significant decrease in the spreading index of SAM68-depleted cells, whereas the relative elongation of these cells was increased (Figure 1A). In addition to limited lamellipodial expansion in SAM68-depleted cells, stress fiber formation was affected as can be seen by a marked decrease in the number of stress fibers and a more isotropic arrangement of actin filaments, when viewed at high magnification (Figure 1B). Figure 1 with 2 supplements see all Download asset Open asset SAM68 regulates endothelial cell spreading, formation and maturation of adhesion sites. (A) siRNA-transfected cells plated overnight on uncoated glass coverslips were stained for F-actin for cell shape analysis (n=50 cells per condition; N=3). Scale bar=50 μm. Spreading Index is expressed as the ratio of occupied area (hatched area) to the cell surface (solid grey area) and elongation ratio is expressed as the ratio of the major to minor cell axis. (B) Magnification images of endothelial cells from the experimental setting described in (A). Sacle bars=20 μm and 10 μm (zoomed). Average fiber number was quantified using FIJI software (n=16 cells per condition; N=3). (C) Vinculin staining was performed on siRNA-transfected cells plated overnight on glass coverslips. Analysis of adhesion sites was performed using FIJI software by quantifying at least 15 cells per condition (N=3). Statistics: p-values: *<0.05 **<0.01; ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, B, C). Pearson’s chi-squared test was used for adhesion length distribution (C). Figure 1—source data 1 Quantification of cell morphology parameters. https://cdn.elifesciences.org/articles/85165/elife-85165-fig1-data1-v1.zip Download elife-85165-fig1-data1-v1.zip Figure 1—source data 2 Quantification of stress fiber numbers and lengths. https://cdn.elifesciences.org/articles/85165/elife-85165-fig1-data2-v1.zip Download elife-85165-fig1-data2-v1.zip Figure 1—source data 3 Quantification of vinculin-positive structure numbers, lengths and sizes. https://cdn.elifesciences.org/articles/85165/elife-85165-fig1-data3-v1.zip Download elife-85165-fig1-data3-v1.zip As cytoskeletal organization and cell spreading is intimately linked to the assembly and maturation of cell-substrate attachment sites, we next examined the number and size of integrin adhesions by immunostaining of vinculin, a core component of adhesion complexes, in siRNA-transfected cells. Following overnight plating, significant differences were observed in the abundance, size and distribution of vinculin-positive structures between control siRNA-transfected cells and SAM68-targeting siRNA-transfected cells (Figure 1C). Following SAM68 depletion, the average number of vinculin adhesions was reduced by threefold. Moreover, the average size of vinculin-positive adhesions was decreased by approximatively twofold and adhesions were less elongated (Figure 1C and Figure 1—figure supplement 2). Adhesion sites are dynamic subcellular structures that have been classified into three main types, depending on their composition, size and distribution (reviewed in Geiger et al., 2001). Nascent dot-like focal complexes form at the cell periphery and rapidly disassemble or mature into more elongated focal adhesions, as force is applied upon linkage of adhesion components to the actin cytoskeleton. Long fibrillar adhesions that arise by translocation of α5β1 integrins out of focal adhesions along growing FN fibers on the cell surface are sites of FN fibrillogenesis in mesenchymal cells. Analyisis of the size distribution of vinculin-containing adhesions revealed a decrease in the proportion of long adhesions (>2 µm) in SAM68-depleted cells compared to control cells, and an increase in the proportion of the smallest adhesions (<1 µm; Figure 1C). Whereas growth of integrin adhesions to greater than 1 µm is a hallmark of adhesion maturation (Doyle et al., 2022), nearly all adhesion sites in SAM68-depleted cells remained shorter than 0.75 µm. The finding that SAM68-depleted cells display fewer, smaller and less elongated adhesion sites, together with the reduced number of actin stress fibers assembled in these cells, suggested that their spreading defect stems from a defect in the formation or maturation of integrin adhesions. SAM68 transiently localizes to leading edges of spreading cells To explore how SAM68 contributes to the stability and growth of endothelial cell adhesion sites, we first examined the localization of the protein in untransfected endothelial cells. SAM68 has previously been shown to relocate near the plasma membrane following fibroblast attachment (Huot et al., 2009a). More recently, it was found by mass spectrometry to associate with β3 integrin-based adhesion complexes in endothelial cells plated on FN for 90 min (Atkinson et al., 2018). Therefore, we determined SAM68 localization in endothelial cells at early times after plating on FN-coated coverslips. Total internal reflection fluorescence (TIRF) microscopy was used to selectively detect SAM68 near the membrane-coverslip interface (internal depth of ~150 nm) and to minimize the fluorescence signal from nuclear SAM68. Twenty minutes after seeding, SAM68 was present in dot-like structures at the basal surface of cells. At this early time of adhesion, the majority of the submembraneous SAM68 puncta were concentrated at the edge of spreading cells in, or adjacent to, sites of active cortical actin assembly (Figure 2A). Staining of peripheral SAM68 partially overlapped with that of cortactin, a well-known regulator of cortical actin polymerization and substrate of Src kinase (similar to SAM68). Anti-phosphotyrosine staining was also strongest at the periphery of spreading cells, in close proximity to SAM68 puncta (Figure 2A). Figure 2 with 1 supplement see all Download asset Open asset SAM68 localization in spreading cells. (A) HUVECs plated on FN-coated coverslips for 20 min were stained for SAM68, F-actin, cortactin and phospho-tyrosine. Scale bars=10 μm. Dotted squares depict enlarged areas (10 μm wide) shown in the same panel. (B) Labelling of SAM68 and FAK was performed on HUVECs plated on FN-coated coverslips for the indicated times; dotted squares depict enlarged areas shown in the same panel. Live-cell imaging of eGFP-SAM68-expressing HUVEC spreading on a FN-coated substrate between 20 and 45 min revealed that SAM68-containing particles in the peripheral submembraneous compartment were extremely motile (Figure 2—video 1). This time-dependent relocalization of SAM68 during cell spreading and focal adhesion formation is illustrated in Figure 2B. After 20 min of adhesion, SAM68 dots formed a peripheral ring that partially overlapped with FAK-containing nascent adhesions at cell edges. By 45 min, SAM68-positive puncta became more diffuse as FAK-labeled adhesions became larger and more elongated. After 120 min of cell spreading and adhesion maturation, SAM68 puncta were distributed across the entire basal cell surface and no longer co-localized with FAK-positive focal adhesions. These results regarding the transient localization of SAM68 at the cell periphery, together with our observed effects of SAM68 depletion on cell spreading and adhesion formation, strongly suggest that SAM68 regulates an initial step of adhesion stabilization and maturation. SAM68 locally regulates adhesion site signaling In light of the dynamic and transient association of SAM68 with nascent adhesions at the cell periphery during spreading on ligand-coated coverslips, this experimental setting was not ideal for investigating early recruitment and local functions of the molecule. Therefore, we employed an alternative system for this purpose in which FN-coated beads are deposited onto fully spread endothelial cells following an overnight incubation to generate ectopic integrin adhesion sites at the apical surface of cells, as described (Atkinson et al., 2018; Chicurel et al., 1998) and schematized in Figure 3A. Twenty minutes after addition of beads, the formation of an actin-rich cup containing vinculin was observed at sites of contact with FN-coated beads (Figure 3B). Co-staining of vinculin and SAM68 at the apical surface of cells, as shown in the optical sections of Figure 3B, illustrates that SAM68 is recruited to these ectopic nascent adhesion sites where it may contribute to some early steps of adhesion complex formation and actin remodeling. Figure 3 with 1 supplement see all Download asset Open asset SAM68 regulates integrin signaling and RNA composition at adhesion sites. (A) Scheme of the experimental procedure used to induce and image artificial adhesion sites in contact with FN-coated beads. (B) Labelling of SAM68 and vinculin was performed 20 min after seeding cells on FN-coated beads. Dotted squares in top panels depict enlarged z-projections shown in the middle panel. Orthogonal views are shown in bottom panels. Scale bars=5 μm. (C) Immunolabeling of α5β1 and activated FAK (pFAK-Y397) were performed 20 min after deposition of FN-coated beads onto siCTRL- or siSAM68-transfected cells and pFAK-Y397 foci were quantified (n=at least 8 beads per condition, N=3). (D) siSAM68-transfected cells were transduced with lentiviral constructions encoding SAM68 WT and mutants shown in the left panel of the figure. Immunolabeling of activated FAK (pFAK-Y397) was performed 20 min after deposition of FN-coated beads onto cells and pFAK-Y397 foci were quantified (n=at least 5 beads per condition, N=4). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used for (C, D). Figure 3—source data 1 Quantification of pFAK397 foci from Figure 3C. https://cdn.elifesciences.org/articles/85165/elife-85165-fig3-data1-v1.zip Download elife-85165-fig3-data1-v1.zip Figure 3—source data 2 Quantification of pFAK397 foci from Figure 3D. https://cdn.elifesciences.org/articles/85165/elife-85165-fig3-data2-v1.zip Download elife-85165-fig3-data2-v1.zip To investigate the function of SAM68 at adhesion sites, we next examined the impact of SAM68 depletion on ectopic adhesion site formation and on integrin-dependent signal transduction initiated at these structures. We first controlled that SAM68-targeted siRNA efficiently diminished SAM68 recruitment to FN-coated beads (Figure 3—figure supplement 1). Next, as a readout of integrin signalling, we performed immunolabeling of FAK autophosphorylated on the Src-family kinase binding site tyrosine 397 (pFAK-Y397). pFAK-Y397 is known to be present in both nascent and growing focal adhesions following integrin activation and clustering. As shown in Figure 3C, depletion of SAM68 in endothelial cells reduced FAK signaling at ectopic adhesions, as determined by the decreased number of pFAK-Y397-positive foci at the interface of cells with FN-coated beads. Thereafter, we set out to determine whether the observed effects of SAM68 at adhesion sites could be attributed to its scaffolding activity via interaction with SH3 domain proteins, notably Src (Taylor and Shalloway, 1994), or to RNA-processing activities of the protein. To this end, we generated lentiviral constructs encoding wild type SAM68 (SAM68 WT) or the functional mutants depicted in Figure 3D. A proline to alanine substitution of residue 358 in the ‘P5’ SH3 binding domain of SAM68 (SAM68 P358A) has previously been shown to impair Src binding to this domain (Asbach et al., 2012), whereas deletion of residues 157–256 (SAM68 ΔKH) results in a full deletion of the SAM68 RNA binding domain and impairment of its binding to RNAs (Lin et al., 1997). Endothelial cells were transfected with an siRNA directed against the 3’UTR of the endogenous SAM68 transcript and then transduced to express either WT or mutant SAM68 coding sequences in a rescue experiment. As shown in Figure 3D, expression of SAM68 P358A in endothelial cells depleted for endogenous SAM68 increased the number of pFAK-Y397 foci around beads. In contrast, expression of the RNA binding-defective mutant SAM68 ΔKH drastically decreased integrin signaling, as illustrated by reduced pFAK-Y397 staining at ectopic adhesion sites, and phenocopied the adhesion formation defects observed upon SAM68 depletion. Altogether, these results indicate that both scaffolding and RNA binding activities of SAM68 are required to modulate integrin signaling. SAM68 locally delivers mRNA to nascent adhesion sites The maturation of integrin adhesions is a finely regulated process which involves not only recruitment and scaffolding of adhesome complex components, but also linkage of the growing adhesions to polymerzing actin networks for force transmission. Interestingly, it has been shown that β-actin mRNA localization at focal adhesions contributes to adhesion stability (Katz et al., 2012). Importantly, β-actin mRNA is a bona fide RNA target of SAM68 (Itoh et al., 2002; Klein et al., 2013) and SAM68 has recently been identified as belonging to the RBP proteome recruited onto β-actin mRNA (Mukherjee et al., 2019). Moreover, the dot-like pattern of SAM68 staining at adhesions is reminiscent of ribonucleoprotein (RNP) particles. Thus, using the ectopic adhesion assay we futher explored potential RNA binding functions of SAM68 at endothelial cell adhesion sites. To do so, we first tested whether β-actin mRNA is recruited to ectopic integrin adhesions upon cell binding to FN-coated beads. As depicted in Figure 4A, RNA smFISH revealed punctuate β-actin mRNA staining around beads. Interestingly, β-actin mRNA particles partially overlapped with those containing SAM68, suggesting that they could be co-localized in the same RNA-protein assemblies. Next we evaluated the implication of SAM68 in the local delivery of β-actin mRNA to nascent apical adhesions. Indeed, upon SAM68 depletion the number of β-actin mRNA foci around beads specifically decreased, as compared to control cells, with no change of foci density in the cytoplasm (Figure 4B). Figure 4 Download asset Open asset SAM68 is involved local delivery of β-actin mRNA at adhesion sites. (A) smRNA FISH and SAM68 stainings were performed 20 min after deposition of FN-coated beads. Scale bars top image=10 μm, enlarged area 5 μm. Arrowheads point to overlapping signals between β-actin mRNA and SAM68 protein. (B) smRNA FISH of β-actin performed on cells 20 min after addition of FN-coated beads to cultures of siCTRL- or siSAM68-transfected cells (n=at least 12 beads per condition, N=3). Scale bars = left 10 μm, enlarged area 5 μm. (C) smiRNA FISH staining of β-actin performed on cells 20 min after deposition of FN-coated beads onto endothelial cells transfected with CTRL or blocking oligonucleotides (#SBE1 and #SBE2, as indicated) (n=at least 12 beads per condition, N=3). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used. Figure 4—source data 1 Quantification of pFAK397 foci from Figure 4B. https://cdn.elifesciences.org/articles/85165/elife-85165-fig4-data1-v1.zip Download elife-85165-fig4-data1-v1.zip Figure 4—source data 2 Quantification of pFAK397 foci from Figure 4C. https://cdn.elifesciences.org/articles/85165/elife-85165-fig4-data2-v1.zip Download elife-85165-fig4-data2-v1.zip To test whether the β-actin mRNA localizing activity of SAM68 is direct, we used antisense blocking oligonucleotides (chimeric 2′-O-methyl DNA oligos) antisense to the Sam68-binding sequence of β-actin mRNA (Itoh et al., 2002), previously shown to disrupt binding between SAM68 its β-actin mRNA cargo in dendrites (Klein et al., 2013). In endothelial cells, transfection of the blocking oligonucleotides #SBE1 and #SBE2, indicated in Figure 4C, resulted in a decrease in the recruitment of β-actin mRNA particles around beads, as compared to their recruitment in control (scrambled) oligonucleotide-transected cells. Importantly, there was no change of foci density in the cytoplasm. These results confirm the involvement of SAM68 in β-actin mRNA delivery to adhesion sites and indicate that β-actin transcript recruitment is likely caused by direct binding of SAM68 to the 3′ UTR of β-actin mRNA. SAM68 modulates FN synthesis and fibrillogenesis In endothelial cells, stabilized focal adhesions elongate and mature into fibrillar adhesions spanning the ventral cell surface. The accumulation of fibrillar adhesions can be visualized by immunostaining of integrin α5β1 (Figure 5A). Consistent with the observed role of SAM68 in adhesion maturation, we detected a significant decrease in the number and length of fibrillar adhesions in SAM68-depleted cells, as shown in Figure 5A. Figure 5 with 2 supplements see all Download asset Open asset SAM68 is involved in FN assembly and expression. (A) Immunofluorescence staining of α5β1 integrin was performed to identify and quantify fibrillar adhesion in siRNA transfected cells plated overnight on glass coverslips (n=at least 15; N=3). (B) Immunofluorescence staining of FN was performed on siRNA transfected cells plated on glass coverslips and quantification on whole-coverslip scans is expressed as the ratio of FN-stained area to the number of cells (N=8). Representative 40x field views. (C) Representative high magnification images of FN staining are shown with areas between the dotted lines selected for fluorescence intensity profiles. (D) Western blot analysis of cell-associated FN in siRNA transfected cells with densitometric quantification indicated below (N=3). (E) qPCR analysis of total FN (tFN) and Extra Domain-containing isoform expression in siRNA transfected cells using the indicated qPCR primer pairs (N=7). (F) Measurements of Luciferase activity driven by the FN1 promoter when SAM68 is overexpressed (N=5). (G) DNA fragments located in the FN1 promoter were quantified by qPCR in anti-SAM68 or IgG immunoprecipitated complexes (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, B, D, E, F). Statistical analysis of fold enrichment in (G) was performed with R using pairwise t-test with p-values adjusted using ‘Bonferroni correction’. Figure 5—source data 1 Quantification of endothelial fibrillar adhesions. https://cdn.elifesciences.org/articles/85165/elife-85165-fig5-data1-v1.zip Download elife-85165-fig5-data1-v1.zip Figure 5—source data 2 Western blot uncropped membranes. https://cdn.elifesciences.org/articles/85165/elife-85165-fig5-data2-v1.zip Download elife-85165-fig5-data2-v1.zip Figure 5—source data 3 Quantification of FN1 mRNA levels. https://cdn.elifesciences.org/articles/85165/elife-85165-fig5-data3-v1.zip Download elife-85165-fig5-data3-v1.zip Figure 5—source data 4 Quantification of FN1 promotor reporter activity. https://cdn.elifesciences.org/articles/85165/elife-85165-fig5-data4-v1.zip Download elife-85165-fig5-data4-v1.zip Figure 5—source data 5 Quantification of SAM68 protein recruitment onto the FN1 promotor. https://cdn.elifesciences.org/articles/85165/elife-85165-fig5-data5-v1.zip Download elife-85165-fig5-data5-v1.zip Fibrillar adhesions are sites of FN fibrillogenesis. Thus, the sparsity of fibrillar adhesions in SAM68-depleted cells prompted us to investigate the ability of these cells to deposit a FN matrix. As illustrated by immunostaining of FN in endothelial cells plated on uncoated glass coverslips (Figure 5B), depletion of SAM68 markedly perturbed FN deposition. SAM68 knock down affected not only the amount of FN deposited beneath cells, but also perturbed the organization of the assembled protein. Thus, FN associated to SAM68-deficient cell monolayers was mostly present in the form of aggregates or thick cables, as opposed to the more homogeneous thin fibrillar networks assembled by control cells, as seen in intensity profiles of FN labeling along the lines indicated in Figure 5C. We did not observe differential retention of FN in the cytoplasm of SAM68-depleted cells. Rather, FN staining was strictly fibrillar (ECM-associated) in both control and SAM68-depleted cells and the intensity profile baseline values were similarly low, indicating that misregulation of FN deposition does not result from altered secretion of the molecule. FN assembly by endothelial cells was previously shown to be tightly coupled to autocrine production of the protein (Cseh et al., 2010). Therefore, we examined the possible role of SAM68 in the regulation of FN expression. Indeed, western blot analysis of total cell lysates revealed an approximately 50% decrease in the levels of cell-/matrix-associated FN in SAM68-depleted cells, as compared to control cells (Figure 5D). SAM68 depletion also led to a decrease in soluble FN in cell conditioned medium (Figure 5—figure supplement 1). As these results clearly indicated that SAM68 modulates production of the protein, we next evaluated the effect of SAM68 depletion on FN transcript levels (Figure 5E). Cellular FN differs from plasma FN by the inclusion of one or both ‘Extra Domains’ EDB and EDA (namely oncofetal FN isoforms) by alternative splicing. Therefore, we designed PCR primer pairs to specifically detect total FN transcripts (tFN) or FN transcripts containing sequences encoding the EDB- and/or EDA domains, which have been shown to differentially affect FN fibrillogenesis and endothelial cell behavior (Cseh et al., 2010; Efthymiou et al., 2021). Upon depletion of SAM68 in endothelial cells, a robust downregulation of total and Extra Domain-containing isoforms of FN was observed. Global downregulation of all FN transcripts in siSAM8-depleted cells suggested that SAM68 might affect FN1 gene transcription. To test this, we generated a reporter construct (pFN) containing a 3.5 kb sequence encompassing the Src signaling-responsive FN1 gene promoter region (Dean et al., 1987) upstream of the firefly luciferase coding sequence. SAM68-regulated promoter activity was examined by co-transfecting the pFN construct with increasing concentrations of a plasmid encoding SAM68 (pSAM68), or an empty vector (pcDNA3) control. Experiments were performed in HEK293 cells, since endothelial cells are poorly transfectable. In addition to their high transfection efficiency, HEK293 cells display nearly undetectable expression of FN and they are unable to assemble the molecule (even upon FN overexpression see Efthymiou et al., 2021). Luciferase measurements showed that increasing the amount of pSAM68 transfected in these cells augmented FN1-driven luciferase activity, compared to that induced by pcDNA3 transfection (Figure 5F). Notably, transfection of as little as 25 ng of pSAM68 yielded a 1.5-fold increase in luciferase activity, compared to cells co-transfected with the control plasmid, attesting to the involvement of SAM68 in FN1 promotor regulation. We confirmed the involvement of SAM68 in FN1 gene transcription in endothelial cells and characterized the recruitme" @default.
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• W4385881710 title "Author response: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly" @default.
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