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

Abstract

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.

The folding of 5′-UTR human G-quadruplexes possessing a long central loop

“…The existence of RNA G-quadruplexes in human cells was recently confirmed using a structure-specific antibody (Biffi et al 2013). A significant number of studies have linked G-quadruplexes to important biological processes, including mRNA splicing, polyadenylation, translation repression, and localization (Shafer and Smirnov 2000;Perreault 2010, 2013;Marcel et al 2011;Bugaut and Balasubramanian 2012;Millevoi et al 2012), thus rendering them interesting potential therapeutic targets (Patel et al 2007;Collie and Parkinson 2011;Marcel et al 2011;McLuckie et al 2013).…”

A Study of TP53 RNA Splicing Illustrates Pitfalls of RNA-seq Methodology

“…1B). Another mechanism by which D40TP53 transcripts are generated depends on G-quadraplex structures in intron 3, which affect the splicing of intron 2 (36). Finally, alternative splicing between exon 9 and exons 9B/10 generates three alternative 3 0 ends for TP53 transcripts (TP53a, b, and g; ref.…”

An RNA G-quadruplex in the 3' UTR of the proto-oncogene PIM1 represses translation

“…[1][2][3][4][5][6][7][8][9][10][11] In addition, RNA G-quadruplex structures can also stimulate mitochondrial transcription termination 12 and regulate alternative splicing in TP53 intron 3. 13 In addition, a G-quadruplex close to a cleavage sites in the 3' UTR of insulinlike growth factor II mRNA has been proposed and its structure formation has been demonstrated in vitro.…”