Abstract
The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.
Highlights
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response
In Δ133p53 mRNA, the AUG3 initiation codon for Δ133p53 protein is located at nucleotide positions 279–281, while AUG4 codon for Δ160p53 isoform is located at positions 360–362 (Fig 1)
We proposed for the first time the secondary structure of the 50-terminal region of p53 mRNA that started from the P2 transcription initiation site (Δ133p53 mRNA) (Fig 1)
Summary
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. The protein is expressed as at least twelve protein isoforms, resulting from the usage of alternative promoters, downstream initiation codons or alternative splicing [1,2,3]. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located. This transcript encodes two p53 isoforms: Δ133p53 and Δ160p53.
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