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First published online 2 January 2007
doi: 10.1242/jcs.03344

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Alternative RNA splicing complexes containing the scaffold attachment factor SAFB2

¹ Institute of Human Genetics, University of Newcastle, International Centre for Life, Central Parkway, Newcastle, NE1 3BZ, UK
² IGBMC, Department of Gene Expression and Neurogenesis, Illkirch, F-67400, France
³ Department of Inserm U596, Illkirch, F-67400, France
⁴ Department of CNRS UMR7104, Illkirch, F-67400, France
⁵ Université Louis Pasteur, Strasbourg, F-67000, France

View larger version (49K): [in this window] [in a new window]   Fig. 1. Structure and potential protein interaction partners of scaffold attachment factor, SAFB1 and SAFB2. (A) Cartoon of SAFB1 and SAFB2 proteins, showing the position of conserved domains, the epitopes used to raise the antisera and corresponding heterologous peptides used in mock pre-absorption of the antisera. (B) Summary of known protein interaction partners for these proteins (mainly based on SAFB1), grouped by family according to their role in transcription (left hand side) or RNA processing (right hand side). Protein families in each group implicated in intracellular signalling are on a hatched background; proteins tested in this study are asterisked, and proteins identified as stable components of the core SAFB1/SAFB2 complex by immunoprecipitation are in bold. See text for details.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Monospecific antisera to SAFB1/SAFB2. Cell lysates from HEK293 cells transfected with plasmids encoding SAFB1-GFP, SAFB2-GFP and GFP alone were resolved by SDS-PAGE and immunoblotting using -SAFB1 (lanes 1-3) and -SAFB2 (lanes 4-6). The position of the 175 kDa marker is shown.

View larger version (54K): [in this window] [in a new window]   Fig. 3. The nuclear distribution of SAFB2 is distinct from that of SAFB1. (A,B). Nuclear distribution of SAFB2 and SAFB1 in HeLa cells. The distribution of SAFB1 protein directly co-localises with the protein detected by monoclonal antibody 6F7, but is distinct from SAFB2. (C) Both SAFB1 and SAFB2 have a much more general subnuclear distribution pattern in HEK293 cells compared with HeLa cells. (D) In adult human testis SAFB2 is specifically upregulated within the nuclei of Sertoli cells (arrowed) whereas SAFB1 expression is negligible in these cells. The images are pseudocoloured, and merged images are shown at the right of the panel. Any overlapping nuclear signal appears yellow. Bars, 10 µm. SC, Sertoli cell; Spg, spermatogonium; Spc, spermatocyte; Rtd, round spermatid.

View larger version (18K): [in this window] [in a new window]   Fig. 4. A GFP-SAFB2 fusion protein inhibits splicing of a Tra2 variable exon. (A) In vivo splicing activity of SAFB1- and SAFB2-GFP fusion proteins after co-transfection with a minigene containing a Tra2-dependent variable exon. Splicing activity was monitored using RT-PCR and agarose gel electrophoresis. (B) Quantitative results from three independent experiments.

View larger version (51K): [in this window] [in a new window]   Fig. 5. SAFB2 protein is a component of higher molecular mass nuclear complexes, which contain a core set of protein interaction partners. Nuclear extracts were fractionated either (A) directly on a sucrose gradient, or (B) after pre-treatment with micrococcal nuclease to remove endogenous RNA. Fraction 20 was from the top of the gradient, and fraction 1 from the bottom. The pelleted material is in lane P. The migration of individual proteins was monitored by SDS-PAGE and immunoblotting. The mobility of size markers on the gradients is shown. (C,D) Both -SAFB1 and -SAFB2 efficiently immunoprecipitate their cognate proteins, and SAFB1 and SAFB2 proteins reciprocally co-immunoprecipitate each other. (E-H) Antisera to both proteins also co-immunoprecipitate (E) Sam68 and (F) hnRNP G. No co-immunoprecipitation was observed with (G) PRP19, or (H) the large subunit of RNA polymerase II containing the carboxyl-terminal domain (CTD).

View larger version (24K): [in this window] [in a new window]   Fig. 6. The stable protein interactions between SAFB1/SAFB2 and Sam68/T-STAR are primarily mediated through their glutamate/arginine (ER)-rich regions. (A) Detection of protein interactions between SAFB1 and SAFB2 with T-STAR in vivo by immunoprecipitation. HEK293 cells were transfected with a construct encoding a T-STAR-FLAG fusion protein, and immunoprecipitated with affinity purified -SAFB1 and -SAFB2 antisera. Immunoprecipitated proteins were detected by probing with the FLAG antibody. (B) The ER-rich regions of SAFB1 and SAFB2 primarily mediate the protein interactions with T-STAR and Sam68, although a weaker interaction was identified with the glycine-rich region. SAFB1 and SAFB2 interact with the arginine and glycine-(RG) and tyrosine-rich (YD) containing C-terminal domains of Sam68 and T-STAR. Protein interactions were assayed by directed yeast two hybrid assays. The organisation of the interacting proteins are shown as cartoons, with the regions described in the text indicated, and the approximate extent of regions used in the assays illustrated underneath by double arrows. The strength of protein interaction in a filter lift assay is based on the rate at which colonies turned blue, and is indicated as strong (+++); medium (++); weak (+) and negative (–).

View larger version (71K): [in this window] [in a new window]   Fig. 7. SAFB2 quantitatively co-localises with its stable interaction partner Sam68. (A) Co-localisation of SAFB2 and Sam68 in HeLa cells. (B) Co-localisation of SAFB2 and the carboxyl-terminal domain of RNA polymerase II in HeLa cells. (C) Co-localisation of SAFB1 and the oestrogen receptor ER in MCF7 cells. Bar, 10 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 8. SR and SR-related splicing regulators are present in parallel but SAFB-independent nuclear protein complexes. (A,B). Sucrose gradient fractionation was as described in Fig. 5, again without treatment (A) and after treatment with micrococcal nuclease (B). The migration of individual proteins was monitored by immunoblotting. The mobility of size markers on the gradients is shown. (C,D) Under identical conditions to Fig. 5, no co-immunoprecipitation was observed (C) between SAFB2 and Tra2, while (D) antibodies to Tra2 efficiently immunoprecipitated Sam68. (E-H) No co-immunoprecipitation was observed between SAFB proteins and (E) hnRNP A1; (F) SR proteins identified with the pan-SR family monoclonal antibody 10H3, (G) ASF/SF2 or (H) 9G8; (I) PTB.

View larger version (79K): [in this window] [in a new window]   Fig. 9. SAFB2 and other RNA splicing regulators are quantitatively released into soluble nuclear extracts. To follow the extraction of the different proteins, 1/5000 of several fractions [cytoplasmic fraction (Cyto), pellet 1 (P1), pellet 2 (P2), final nuclear extract (NE)] were analysed by SDS-PAGE followed by Western-blotting using specific antibodies against SAFB1, SAFB2, Sam68, hnRNPG, Tra2 and 9G8. Where there are additional aberrantly migrating proteins, the position of the expected protein band is shown with an asterisk.

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