Polypyrimidine-tract-binding protein (PTB): a role in alternative splicing of the Fibroblast Growth Factor Receptor (FGFR) 2 gene.
Session type: Poster / e-Poster / Silent Theatre session
PTB, implicated in a wide variety of cellular functions, negatively regulates splicing of the FGFR2 gene. FGFR2 undergoes alternative splicing in a tissue-specific manner giving rise to two receptor isoforms, each with different binding specificities. FGFR2-IIIb is expressed exclusively in epithelial cells and FGFR2-IIIc, mesenchymal cells. An isoform switch is thought to underlie epithelial mesenchymal transition, implicated in tumourigenesis. PTB, which has several homologues, is also expressed in a tissue specific manner and represses exon IIIb inclusion in mesenchymal cells by binding multiple elements flanking exon IIIb. In this study we profile the pattern of PTB expression in breast cancer cells.
RNA was extracted from breast cancer cells MDA-MB-436/468 differentially expressing FGFR2 isoforms using the RNeasy kit (Qiagen) and reverse transcribed (High Capacity kit, AB). A PCR product was generated using primers that flank the region of nPTB that undergoes alternative splicing, cloned into pPCR-Script (Stratagene) and the sequence from several clones (25 clones/ cell line) analyzed. Nuclear extracts prepared from MDA-MB-436/468 cells were subjected to 1 and 2D gel electrophoresis and followed by western blotting using an anti-PTB antibody (Santa Cruz).
Three regions of the nPTB cDNA underwent alternative splicing giving rise to eight different isoforms. nPTB7, the predominant isoform in MDA-MB-468 made up 48% of the clones. Results from the western blotting revealed a lower molecular weight protein (~33kDa).
nPTB7 undergoes alternative splicing to exclude a 34bp exon. This introduces a frameshift in the reading frame resulting in a premature stop codon and truncated protein lacking RNA recognition motifs. We hypothesise that since this protein lacks the motifs to bind RNA it acts to sequester other PTB proteins from binding elements adjacent to exon IIIb, allowing the splicing machinery access to splice sites and resulting in exon IIIb inclusion in epithelial tissues.