A Novel BAT3 Sequence Generated by Alternative RNA Splicing of Exon 11B Displays Cell Type-Specific Expression and Impacts on Subcellular Localization

Background: The human lymphocyte antigen (HLA) encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3. Methodology/Principal Findings: By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and D24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved

The Limonoids TS3 and Rubescin E Induce Apoptosis in Human Hepatoma Cell Lines and Interfere with NF-κB Signaling.

Hepatocellular carcinoma (HCC) is extremely resistant towards pharmacological therapy. To date, the multi-kinase inhibitor Sorafenib is the only available therapeutic agent with the potential to prolong patient survival. Using the human hepatoma cell lines HepG2 and Huh7, we analyzed anti-cancer activities of 6 purified havanensin type limonoids isolated from the traditional African medicinal plant Trichilia rubescens Oliv. Our results show that two of the compounds, TR4 (TS3) and TR9 (Rubescin E) reduced hepatoma cell viability, but not primary hepatocyte viability, at TC50s of 5 to 10 μM. These were significantly lower than the TC50s for Sorafenib, the histone deacetylase inhibitor SAHA or 5-Fluoruracil. In comparison, TR3 (Rubescin D), a limonoid isolated in parallel and structurally highly similar to TR4 and TR9, did not interfere with hepatoma cell viability. Both, TR4 and TR9, but not TR3, induced apoptosis in hepatoma cells and interfered with NF-κB activation. TR4 as well as TR9 significantly supported anti-cancer activities of Sorafenib. In summary, the limonoids TR4 and TR9 exhibit anti-cancer activities and support Sorafenib effects in vitro, having the potential to support future HCC therapy

Impact of leptomycin B treatment on the subcellular localization of transfected BAT3 splice variants and of endogenous BAT3.

<p><b>A.</b> HeLa cells transfected with BAT3 Δ11B,24 were cultured on coverslips in the presence (lower panel) or absence (upper panel) of leptomycin B (LMB) for 2 h. Cells were subsequently stained with V5 mAb and inspected with standard immunofluorescence microscopy (second panel). Left panel shows DAPI stained nuclei, third panel merging of images and right panel displays phase contrast images. Scale bars = 10 µm. <b>B.</b> Raji cells were cultured for 2 h in the presence (lower panel) or absence (upper panel) of LMB and then plated on coverslips. Cells were subsequently stained with the polyclonal anti-BAT3 serum and with ISCR3 mAb (HLA-DR) for evaluation by immunofluorescence microscopy. Left panel shows DAPI staining, second panel staining for BAT3, third panel staining for HLA-DR and images were merged in the right panel. Scale bars = 5 µm.</p

Frequency of spliced exons in MelJuSo cells.

*<p>A total number of 100 clones was analyzed.</p

Subcellular localization of endogenous BAT3 in % of cells.

<p>Subcellular localization of endogenous BAT3 in % of cells.</p

Tissue specific expression of <i>BAT3</i> splice variants.

<p>Tissue specific expression of <i>BAT3</i> splice variants.</p

Subcellular localization of BAT3 variants in transfected HeLa cells.

<p>Cells were transfected with BAT3 splice variants and stained after 24 hours with a monoclonal V5 antibody for evaluation by immunofluorescence microscopy. Left panel displays DAPI staining, second panel BAT3 staining, third panel merging of images and right panel shows corresponding phase contrast images. The displayed transfected cells show examples for nuclear (BAT3 full-length, upper panel), for nuclear and cytosolic (BAT3 Δ11B, middle panel) and for enhanced cytosolic staining (BAT3 Δ11B, 24, lower panel). Scale bars = 10 µm.</p

Subcellular localization of endogenous BAT3 in four cell types.

<p>HeLa cells (Adenocarcinoma), the human melanoma cell line MelJuSo, the B lymphoma Raji and monocyte-derived dendritic cells (moDCs) were plated on coverslips and stained for BAT3 using a polyclonal serum against a C-terminal peptide (middle lane). Cell nuclei (left lane) were visualized with DAPI (A) or 7AAD (B). Merged images are shown in the right lane. <b>A.</b> Immunofluorescence staining was evaluated with a standard fluorescence microscope and <b>B.</b> by confocal microscopy. Scale bars = 10 µm. <b>C.</b> Nuclear and cytosolic staining of endogenous BAT3 in Raji cells was evaluated in 10 single cells using ImageJ. MFI, mean fluorescence intensity per region of interest <b>D.</b> Western blot analysis of subcellular fractions from Raji and HeLa cells. Nuclei (N) and cytoplasm (C) were separated by SDS-PAGE and immunoblotted for BAT3, GADPH (cytosolic marker) and histone H3 (nuclear marker).</p

Detection of exon sequences in BAT3 cDNA clones by PCR.

<p><b>A.</b> Exon-intron-structure of the <i>BAT3</i> gene. Exon sequences are indicated as arrows. The start codon for translation in exon 2 is indicated. Potentially spliced exons are numbered. Exon 1 and 7 exist in different fragment lengths, 247 bp or 271 bp for exon 1 (NCBI) and 236, 254 or 278 bp for exon 7 (NCBI, ENSEMBL). Exons highlighted in grey could be deleted without changing the reading frame. The novel exon 11B with a length of 108 bp is labeled by arrow. Exon 11B also can be deleted without a change of the reading frame of the adjacent exon sequences. <b>B.</b> Schematic presentation of BAT3 cDNA. Exon boundaries are indicated and protein-encoding exons are numbered from 2 to 25. Potentially spliced exons are highlighted in grey. The position of complementary primer pairs (a to d) for forward (f) and reverse (r) PCR are indicated by arrows. <b>C.</b> Primers indicated in B were used to characterize BAT3 cDNA clones. Digestion of two BAT3 cDNAs is shown for example. Lane M contains size standards with bp indicated on the left. Lanes a: PCR products of clones 1 and 2 exhibit a band of 280 bp (no exon 11B) or of 380 bp (with exon 11B). Lanes b: Both clones generate a PCR product of 660 bp, indicating the presence of exon 9. Lanes c: The sizes of PCR fragments from clone 1 and clone 2 are 800 bp or 950 bp, respectively. This size is consistent with the presence or absence of exon 24. Lanes d: The presence of exon 5 is verified in clones 1 and 2 by a PCR product of 400 bp.</p