G-quadruplex structures in TP53 intron 3: role in alternative splicing and in production of p53 mRNA isoforms.

The tumor suppressor gene TP53, encoding p53, is expressed as several transcripts. The fully spliced p53 (FSp53) transcript encodes the canonical p53 protein. The alternatively spliced p53I2 transcript retains intron 2 and encodes Δ40p53 (or ΔNp53), an isoform lacking first 39 N-terminal residues corresponding to the main transactivation domain. We demonstrate the formation of G-quadruplex structures (G4) in a GC-rich region of intron 3 that modulates the splicing of intron 2. First, we show the formation of G4 in synthetic RNAs encompassing intron 3 sequences by ultraviolet melting, thermal difference spectra and circular dichroism spectroscopy. These observations are confirmed by detection of G4-induced reverse transcriptase elongation stops in synthetic RNA of intron 3. In this region, p53 pre-messenger RNA (mRNA) contains a succession of short exons (exons 2 and 3) and introns (introns 2 and 4) covering a total of 333 bp. Site-directed mutagenesis of G-tracts putatively involved in G4 formation decreased by ~30% the excision of intron 2 in a green fluorescent protein-reporter splicing assay. Moreover, treatment of lymphoblastoid cells with 360A, a synthetic ligand that binds to single-strand G4 structures, increases the formation of FSp53 mRNA and decreases p53I2 mRNA expression. These results indicate that G4 structures in intron 3 regulate the splicing of intron 2, leading to differential expression of transcripts encoding distinct p53 isoforms.