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. 2008 Jul 11:9:37.
doi: 10.1186/1471-2121-9-37.

The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response

Mattias K Andersson  1 , Anders Ståhlberg, Yvonne Arvidsson, Anita Olofsson, Henrik Semb, Göran Stenman, Ola Nilsson, Pierre Aman
Affiliations

The multifunctional FUS, EWS and TAF15 proto-oncoproteins show cell type-specific expression patterns and involvement in cell spreading and stress response

Mattias K Andersson et al. BMC Cell Biol. .

Abstract

Background: FUS, EWS and TAF15 are structurally similar multifunctional proteins that were first discovered upon characterization of fusion oncogenes in human sarcomas and leukemias. The proteins belong to the FET (previously TET) family of RNA-binding proteins and are implicated in central cellular processes such as regulation of gene expression, maintenance of genomic integrity and mRNA/microRNA processing. In the present study, we investigated the expression and cellular localization of FET proteins in multiple human tissues and cell types.

Results: FUS, EWS and TAF15 were expressed in both distinct and overlapping patterns in human tissues. The three proteins showed almost ubiquitous nuclear expression and FUS and TAF15 were in addition present in the cytoplasm of most cell types. Cytoplasmic EWS was more rarely detected and seen mainly in secretory cell types. Furthermore, FET expression was downregulated in differentiating human embryonic stem cells, during induced differentiation of neuroblastoma cells and absent in terminally differentiated melanocytes and cardiac muscle cells. The FET proteins were targeted to stress granules induced by heat shock and oxidative stress and FUS required its RNA-binding domain for this translocation. Furthermore, FUS and TAF15 were detected in spreading initiation centers of adhering cells.

Conclusion: Our results point to cell-specific expression patterns and functions of the FET proteins rather than the housekeeping roles inferred from earlier studies. The localization of FET proteins to stress granules suggests activities in translational regulation during stress conditions. Roles in central processes such as stress response, translational control and adhesion may explain the FET proteins frequent involvement in human cancer.

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Figures

Figure 1
Figure 1
EWS expression in salivary gland. EWS shows cytoplasmic expression in ductal and serous cells but is undetectable in the cytoplasm of mucous cells. DC – ductal cells, MC – mucous cells, SC – serous cells.
Figure 2
Figure 2
FET protein localization in cultured human cells. (a) Actively proliferating cells stained with antibodies for FET proteins. FUS and TAF15 show both nuclear and cytoplasmic localization while EWS is found solely in nuclei. Scale bars indicate 10 μm. (b) HT-1080 cells stably expressing FET-GFP proteins show nuclear localization of all three proteins and in addition cytoplasmic localization for FUS-GFP and TAF15-GFP. Scale bars indicate 10 μm. (c) Western blots showing FET-GFP proteins of expected sizes and specificity of FET antibodies used. Wells contain the following lysates: FUS-GFP clone 1 (1), EWS-GFP clone 1 (2), TAF15-GFP clone 1 (3), GFP clone 1 (4), HT-1080 (5), FUS-GFP clone 2 (6), EWS-GFP clone 2 (7), TAF15-GFP clone 2 (8), GFP clone 2 (9). No crossreactivity is seen between different FET antibodies. Endogenous FET proteins correspond to the lower bands seen in all lanes and tagged FET proteins to upper bands. GFP adds approximately 27 kDa to the total size of the respective protein. FUS-GFP and EWS-GFP are expressed at slightly augmented levels compared with their endogenous counterparts while TAF15-GFP is expressed at much higher levels than wild type TAF15.
Figure 3
Figure 3
FET proteins localize to stress granules. Images show FET proteins (green) and immunostaining for the stress granule marker TIA-1 (red). Nuclei are visualized by DAPI staining (blue) in the bottom image of each column (a) Overexpression of FET-GFP proteins cause stress granule formation in transiently transfected HT-1080 cells. (b) HT-1080 cells stably expressing FET-GFP proteins show localization of tagged proteins to stress granules upon arsenite treatment. (c) Endogenous FET proteins localize to stress granules in response to oxidative stress in HeLa cells. (d) Stable transfectants of FUSA-GFP and GFP show similar minor signal in granules upon arsenite treatment. Scale bars indicate 5 μm.
Figure 4
Figure 4
FUS and TAF15 localize to spreading initiation centers. F470 and HT-1080 cells co-stained with FET antibodies and focal adhesion/SIC markers Vinculin, FAK and RACK1. Arrowheads indicate areas of cytoplasmic overlap and nuclei are shown in blue by DAPI staining in the merge images. Bars indicate 5 μm.
Figure 5
Figure 5
Heterogenous FET protein expression in human tissues. Brown staining indicates FET expression while blue staining shows negatively staining nuclei. (a) Individual epithelial cells of esophagus show large heterogeneity in FET expression levels. (b) FET expression is elevated in proliferating endometrium (upper panel) compared to differentiated secretory endometrium (lower panel).
Figure 6
Figure 6
FET expression is reduced during neuronal differentiation of SH-SY5Y neuroblastoma cells. Cells were treated with 1 μM of all-trans retinoic acid and lysed at different time points. Relative quantification (RQ) values were obtained by normalizing FET expression against beta actin expression in each sample and by further comparing RA treated with untreated cells at each time point. Data from one of two independent experiments yielding similar results.
Figure 7
Figure 7
FET gene expression is attenuated in differentiating human embryonic stem cells. (a) A colony of spontaneously differentiating hESCs. Inner and peripheral parts of the colonies were harvested based on cell morphology. (b) Relative expression of selected mRNAs in samples from inner or peripheral parts of colonies analyzed by quantitative real-time PCR with ACTB as endogenous control. POU5F1 – pluripotency marker,SOX2 – ectodermal marker, VIM – mesodermal marker,CCNA2 – proliferation marker. The day 12 measurement consists of one sample taken from inseparable, mixed cell populations. (c) Undifferentiated hESCs show positive FET protein staining. Cells were stained with primary antibodies for FET proteins and visualized with Cy3-conjugated secondary antibodies (red). The merge images additionally show DAPI staining of nuclei (blue).
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