Immunologically Distinct p53 Molecules Generated by Alternative Splicing

Abstract

Transfection of a functional cloned p53 gene into an L12 p53 nonproducer cell line efficiently reconstituted p53 expression. The p53 protein synthesized in these clones was indistinguishable from that occurring naturally in tumor cells. When a p53 cDNA clone was used instead, we observed that the L12-derived clones exhibited a distinct immunological profile. In the present experiments we compared the immunological epitopes of p53 proteins encoded by several full-length cDNA clones. Immunoprecipitation of p53 proteins generated by in vitro transcription and translation of the various cDNA clones indicated variations in the content of immunological epitopes. Basically, two p53 protein species were detected. Both species contained the same antigenic determinants except the PAb421-PAb122 site, which was present in proteins encoded by p53-M11 and pcD-p53, but not in the p53 protein encoded by the p53-M8 cDNA clone. Sequence analysis of the various cDNA clones indicated the existence of a 96-base-pair (bp) insert in clone p53-M8 as compared with clone p53-M11 or pCD-p53. The 96-bp insert contained a termination signal which caused the premature termination of the protein, leading to the generation of a p53 product 9 amino acids shorter than usual. The existence of this insert also accounted for the lack of the PAb421-PAb122 epitope which was mapped to the 3' end of the cDNA clone, following the 96-bp insert. This insert shared complete homology with the p53 intron 10 sequences mapping 96 bp upstream of the 5' acceptor splicing site of p53 exon 11. It was therefore concluded that the different cDNA clones represented p53 mRNA species which were generated by an alternative splicing mechanism. Differential hybridization of the mRNA population of transformed fibroblastic or lymphoid cells with either the 96-bp synthetic oligonucleotide or the p53-M11 cDNA indicated that the various mRNA species are expressed in vivo.

Comparative Genomics and Evolution of Alternative Splicing: The Pessimists' Science

“…Until the late 1990s, although several important cases of alternative splicing had been described, such as mammalian immunoglobulins 18 and T-cell receptors, 19 the p53 gene, 20 various growth factors, [21][22][23] the CD44 receptor involved in a variety of lymphocyte functions, 24 myofibrillar proteins 25 (tropomyosin, myosin, titin, etc. ), P-element transposase, 26 the sex differentiation pathway of Drosophila melanogaster 27 and, later, other insects, 28 and many others, alternative splicing was considered to be a relatively rare phenomenon occurring, for instance, in about 5% of human genes.…”

A comprehensive study of p53 protein

“…The alternative splicing discussion by Wolf and colleagues [144][145][146] produces immunologically different p53 molecules. In this publication, they provided the first experimental proof that variably spliced p53 exists in normal cells of many tissues and is not a transformationspecific event type.…”

“…A rat embryo fibroblast 2. The MDM-2 cosmid and the vector for this murine gene CV001 [143][144][145] that can bind to p53-R175H to reactivate p53-R175H. Hotspot mutations cause the encoding of mutant p53 proteins, which gain functions that promote the growth of cancer while losing the classical wild-type p53 capabilities.…”

Mutant mice lacking alternatively spliced p53 isoforms unveilAckr4as a male-specific prognostic factor in Myc-driven B-cell lymphomas

“…Like its human TP53 homolog, the murine Trp53 gene encodes multiple isoforms differing in their N– or C-termini (Arai et al, 1986; Marcel et al, 2011). Mouse models to evaluate the role of p53 isoforms differing in their N-terminus revealed that Δ40-p53 overexpression leads to accelerated ageing (Maier et al, 2004; Steffens Reinhardt et al, 2020).…”