Sam68 protein is expressed at high levels in the nuclei of prostate cancer cells 1014. We immunoprecipitated endogenous Sam68 protein in nuclear extracts prepared from LNCaP cells. Western analysis confirmed that Sam68 protein was efficiently immunoprecipitated by rabbit antisera specific for Sam68, but not by normal rabbit IgG (Additional File 1). Further analysis of the immunoprecipitates using SDS-PAGE followed by Coomassie staining revealed a number of immunoprecipitated proteins, including a protein of ~68 kDa and a protein co-migrating with IgG heavy chain (Figure 1A; compare lanes 1 and 4), as well as a number of other distinct proteins. Peptide mass fingerprinting by MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time Of Flight) mass spectrometry was carried out on each of the immunoprecipitated proteins. The Mascot search engine was used to match peptides with statistically significant MOWSE (Molecular Weight Search) scores mapping across full-length protein sequences obtained from the Swiss-Prot database (Figure 1B to 1E and Table 1). We identified the ~68 kDa band as Sam68 (Figure 1B), and other co-immunoprecipitated proteins as the known Sam68-interacting proteins hnRNP A1 3 (Figure 1C), hnRNP G 15 (Figure 1D), and hnRNP A2/B1 16 (Figure 1C).

In addition to the three previously known Sam68-associated proteins, we also identified hnRNP L as a novel potential Sam68-interacting protein partner (Figure 1E). By Coomassie staining, the levels of co-precipitated hnRNP A1 and hnRNP G were similar to the level of immunoprecipitated Sam68, but there was considerably less co-immunoprecipitated hnRNP L (Figure 1A, lane 1). Hence, to independently confirm the novel Sam68-hnRNP L interaction, both Sam68 and hnRNP L proteins were separately immunoprecipitated in LNCaP cell nuclear extracts using their cognate antibodies prior to Western analysis (Figure 2A, left and right panels). hnRNP L protein was efficiently immunoprecipitated by a monoclonal antibody specific for hnRNP L, and also co-immunoprecipitated by the antisera to Sam68 (Figure 2A, left panel). Likewise, Sam68 protein was efficiently immunoprecipitated by its cognate antisera, and also co-immunoreprecipitated by the anti-hnRNP L antibody (Figure 2A, right panel). Similar to previous reports 17, we observed hnRNP L to migrate as a doublet, and both of these isoforms interacted with Sam68 (Figure 2, left panel). No or very weak immunoprecipitation was observed in the absence of either antibodies (Figure 2A, both panels, lane 4).

To further verify the novel Sam68-hnRNP L detected interaction, HEK293 cells were transiently transfected with expression constructs encoding FLAG-tagged Sam68 and GST-tagged hnRNP L proteins. Anti-FLAG M2 agarose was used to immunoprecipitate FLAG-tagged proteins from HEK293 cells, and co-immunoprecipitated GST fusion proteins were detected using antisera specific to GST. Consistent with the observed interactions of endogenous proteins, hnRNP L-GST was efficiently co-immunoprecipitated with Sam68-FLAG (Figure 2B, compare lanes 1 and 3), but could not be co-immunoprecipitated in cells transfected with an empty FLAG vector (Figure 2B, compare lanes 2 and 4).

Note that all immunoprecipitations of both endogenous (Figure 2A) and FLAG-tagged proteins (Figure 2B) were carried out in the presence of Benzonase nuclease to ensure that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids.

Sam68 is quantitatively associated with small nuclear protein complexes of a size compatible with either monomers or dimers 18. In order to investigate and compare the size ranges of any endogenous nuclear complexes containing hnRNP L protein, we analysed velocity gradient fractions of HEK293 cell nuclear extracts prepared either without pretreatment or after prior micrococcal nuclease (MNase) treatment to eliminate any nucleic acid dependent complexes (Figures 2C and 18). Similarly to previously published data for Sam68, following MNase treatment, hnRNP L (monomeric molecular weight ~70 kDa) protein was mainly present in small stable protein complexes (of less than 158 kDa) corresponding to the expected size of monomeric or dimeric molecular complexes (Figure 2C, upper panel). Also like Sam68, hnRNP L protein was quantitatively associated with nucleic acids since it migrated with much larger complexes without prior MNase digestion (Figure 2C, lower panel).

Within the nuclei of cancer cells, Sam68 protein exhibits a general nucleoplasmic distribution but is also concentrated within distinct peri-nucleolar structures called SNBs 13. We compared the localization of Sam68 and hnRNP L proteins in cancer cell lines using indirect immunofluorescence (representative images are shown in Figure 3A and 3B, and Additional File 2). Confocal microscopy demonstrated that the endogenous Sam68 and hnRNP L proteins directly overlapped within SNBs in 100% of all cells examined. In addition to SNBs, further large pools of hnRNP L and Sam68 proteins were seen in the general nucleoplasm, and in the case of hnRNP L, towards the nuclear periphery in Saos-2 cells (the position of representative SNBs are indicated by arrows in Figures 3A, B and Additional File 2: note also a large population of both Sam68 and hnRNP L proteins outside SNBs). In parallel experiments, using the same combination of secondary antisera, no co-localisation was observed between Sam68 protein (identified using specific rabbit antisera) and SC35-containing splicing speckles (identified using a specific mouse monoclonal antibody) (Figure 3C). The distribution of Sam68 and hnRNP L proteins was examined in three different cell lines (LNCaP, Saos-2 and HeLa) and found to be very similar suggesting that the hnRNP L association with SNBs may be ubiquitous (Figure 3A, B, and Additional File 2). Furthermore, the other identified Sam68-associated proteins (hnRNP A1, A2/B1, and G) identified in our proteomic screen exhibited diffuse subnuclear distributions and did not localise within SNBs (Additional File 2 and 19).

Only a small number of molecules have been identified as SNB components, and each of these share a role in cell signalling and/or RNA processing. hnRNP L has a closely-related homolog within the cell called hnRNP LL (hnRNP L-like), which also regulates signal-dependent splicing. We carried out further experiments to test if hnRNP LL protein would be also found within SNBs. Surprisingly, these experiments indicated that hnRNP LL protein exhibits a nucleoplasmic distribution outside SNBs (Figure 3D and Additional File 2). The observed hnRNP LL distribution was punctate and somewhat reminiscent to that of splicing speckles. We compared the localisation of hnRNP LL protein with SC35, but found that the speckled distribution of hnRNP LL is in fact distinct from the SC35-containing splicing speckles (Figure 3E).

To verify these localization findings, HeLa cells were transiently transfected with expression vectors encoding hnRNP L-GST, hnRNP LL-GST, or GST alone. We then detected the localisation of the ectopically expressed proteins using antisera to detect the GST tag, and compared this distribution with that of endogenous Sam68 protein (Figure 4). Consistent with the data described above, hnRNP L-GST but not hnRNP LL-GST, was found within SNBs (Figure 4A and 4B). The GST moiety had no effect on protein localization, since GST alone was detected both within the nuclei and cytoplasm of transfected cells, and not within SNBs (Figure 4C).

The frequency of cells in a population containing SNBs is cell line-dependent. To test whether the association of Sam68 and hnRNP L was dependent on the presence of SNBs, we performed side-by-side immunoprecipitations in whole cell extracts obtained from LNCaP and NIH3T3 cells (only ~5.5% of NIH3T3 cells have SNBs) 13. Sam68 protein was efficiently immunoprecipitated by its cognate antisera from both these cell lines (Figure 5A and 5B, left panels). Consistent with the interaction of Sam68 and hnRNP L also occurring outside of SNBs, virtually equal levels of hnRNP L protein was co-immunoprecipitated by the antisera to Sam68 from NIH3T3 cells as LNCaP cells (Figure 5A and 5B, right panels). Notice that neither Sam68 nor hnRNP L proteins were immunoprecipitated by normal rabbit IgG or beads alone. All immunoprecipitations were carried out in the presence of Benzonase nuclease to confirm that the detected associations were mediated by protein-protein interactions and not bridged by nucleic acids.

The above experiments indicate that Sam68 and hnRNP L proteins interact and are both present within SNBs. Although SNBs contain some splicing regulators and RNA, they do not detectably contain key spliceosome components 13. Hence SNBs are not thought to be sites for active pre-mRNA splicing, which takes place in the general nucleoplasm 20. Both Sam68 and hnRNP L proteins are independently implicated in the regulation of distinct alternative splicing events during which exon inclusion can be either activated or repressed depending on the target transcript 421. Although active splicing of Sam68 and hnRNP L target pre-mRNAs is unlikely to take place within SNBs, we also detected significant nucleoplasmic populations of both Sam68 and hnRNP L proteins outside SNBs, and protein interactions between Sam68 and hnRNP L occurred equally well in a cell line which exhibits a low frequency of SNBs. These observations raise the question as to whether the Sam68-hnRNP L interaction might have a co-operative effect on pre-mRNA splicing in the general nucleoplasm.

Since ectopically expressed Sam68 protein stimulates splicing inclusion of CD44 variable exon v5 7, and TJP1 exon 20 inclusion is repressed by hnRNP L 21, we examined the effect of both Sam68 and hnRNP L protein expression on these known target exons (Figure 6). We firstly monitored splicing in HEK293 cells transfected with a minigene containing CD44 variable exon v5 together with expression constructs encoding Sam68, hnRNP L or both (Figure 6A). Ectopic expression of hnRNP L protein stimulated exon v5 inclusion albeit with a lower potency than Sam68 (Figure 6B, compare lane 1 with lanes 2 and 3). We carried out similar experiments with a minigene containing TJP1 exon 20 (Figure 6C). Due to the low levels of expression of TJP1 exon 20, it was not easy to draw clear conclusions from this experiment, however ectopic expression of either hnRNP L or Sam68 proteins alone appeared to repress exon 20 inclusion (Figure 6D, compare lane 1 with lanes 2 and 3). Together, these experiments indicate that both Sam68 and hnRNP L proteins can regulate the same pre-mRNA splicing events in the same direction, with hnRNP L activating CD44 exon v5 inclusion like Sam68, and Sam68 repressing TJP1 exon 20 inclusion like hnRNP L. Importantly in either case, however, co-expression of hnRNP L and Sam68 proteins did not have a significant additive effect on either TJP1 exon 20 skipping or CD44 exon v5 inclusion (Figures 6B and 6D, compare lanes 2, 3 and 4). Hence it seems unlikely that the general nucleoplasmic populations of hnRNP L and Sam68 proteins co-operate in splicing control of these shared target exons, despite their observed interaction, localisations, and ability to regulate these target exons by themselves.

LNCaP cells were cultured in RPMI-1640 (PAA Laboratories) supplemented with 10% foetal calf serum (FCS) (PAA Laboratories). HEK293, HeLa, NIH3T3, and Saos-2, and cells were cultured in Dulbecco's Modified Eagle Media (D-MEM) with glutamax-1 (PAA Laboratories) with 10% FCS. Plasmid transfections were performed as previously described 18 using Genejammer (Stratagene) according to manufacturer's instructions.

The following antibodies were used: normal rabbit IgG sc-2027 and anti-Sam68 sc-333 rabbit antisera (Santa Cruz Biotechnology); anti-hnRNP A1 9H10, anti-hnRNP L 4D11, anti-SC35 SC-35, and anti-β-actin AC15 mouse monoclonal antibodies (Sigma); anti-hnRNP A2/B1 DP3B2 mouse monoclonal antibody (Abcam); anti-hnRNP LL rabbit antisera (Aviva Systems Biology); anti-GST (glutathione S-transferase) goat antisera (GE Healthcare).

The following plasmids have been described previously: FLAG-Sam68 10; pGFP3-Sam68 31; pETv5 32 (from Stefan Stamm, University of Kentucky, USA). The following plasmids were made using standard cloning procedures, the details of which are available on request: pcDNA-GST, pcDNA-hnRNP L-GST, and pcDNA-hnRNP LL-GST encode full-length GST, hnRNP L 33, and hnRNP LL with a C-terminal GST tag, respectively. pcDNA-hnRNP G encodes full-length hnRNP G. pcDNA3-TJP1-wWT contains exon 19, 20 and 21 of TJP1 with intervening intronic sequence.

Immunoprecipitated proteins were resolved on NuPAGE Novex 10% Bis-Tris gels (Invitrogen) and stained with Coomassie blue (GE Healthcare). Excised bands were digested with trypsin, and identified by peptide mass fingerprinting. Mass spectra were obtained using the Ultraflex II MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometer (Bruker Daltonics) in standard MALDI-TOF mass spectrometry (MS) mode. The peak lists were searched against the non-redundant Swiss-Prot protein sequence database using the Mascot 34 search engine version 2.2 (Matrix Science). Probability-based MOWSE (Molecular Weight Search) 35 scores greater than 55 were considered as significant (p < 0.05).

LNCaP cell nuclear extracts were obtained using the CelLytic NuCLEAR Extraction Kit (Sigma) according to manufacturer's instructions. Benzonase nuclease (Sigma) was used at 100 units/ml where indicated. Immunoprecipitations were performed using Dynabeads Protein A (Invitrogen) or anti-FLAG M2 agarose (Sigma) according to manufacturers' instructions. Where indicated, antibodies were cross-linked to Dynabeads Protein A according to manufacturer's instructions. Recovered material was resolved by SDS-PAGE and subjected to Western analysis as previously described 36.

Sucrose gradients were run using HEK293 cell nuclear extract, pre-treated or not with micrococcal nuclease (MNase), and recovered material resolved by SDS-PAGE and subjected to Western analysis as previously described 18.

LNCaP, HeLa, and Saos-2 cells were grown and transfected on glass coverslips (VWR International), and stained with primary and secondary antibodies as previously described 18. All images were captured using the LSM510 (Zeiss) or SP2 MP (Leica) confocal microscopes and associated software.

HEK293 cells were grown in 6-well plates (Asahi Techno Glass), and transfected with DNA as detailed in figure legends. RNA was extracted using TRIzol (Invitrogen) and RT-PCR was performed using the One-Step RT-PCR kit (Qiagen) as previously described 31. Densitometric band quantification was performed as previously described 37. All experiments shown are the mean of at least three independent experiments ± standard error.