Length variants of the 5′ untranslated region of p53 mRNA and their impact on the efficiency of translation initiation of p53 and its N-truncated isoform ΔNp53

Abstract

Recently, we have determined the secondary structure of the 5'-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5' direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5'-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5'-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.

The role of the 5' terminal region of p53 mRNA in the p53 gene expression

“…Translation of proteins with alternative initiation codons may take place by re-initiation, by leaky scanning by the ribosome or by the IRES: all these possibilities have been discussed with regard to the synthesis of Δ40p53 (Błaszczyk & Ciesiołka, 2011). Importantly, it has been demonstrated that the longer 5' terminus of p53 mRNA strongly reduces the synthesis of the p53 and Δ40p53 proteins (Górska et al, 2013). This is consistent with the observations that the longer 5'UTR results in lower productivity of the cap-dependent translation system (Vassilenko et al, 2011).…”

“…Additionally, it has been demonstrated that the coding sequence is necessary for proper folding of the 5' terminus of the P1-initiated transcript (Błaszczyk & Ciesiołka, 2011). In the P0-initiated p53 mRNA three small hairpins were identified in the P0-P1 region (Górska et al, 2013). In vitro and in vivo experiments have shown that the P0-P1 region influences p53 translation efficiency (Strudwick et al, 2003;Górska et al, 2013).…”

“…In the P0-initiated p53 mRNA three small hairpins were identified in the P0-P1 region (Górska et al, 2013). In vitro and in vivo experiments have shown that the P0-P1 region influences p53 translation efficiency (Strudwick et al, 2003;Górska et al, 2013). Interestingly, it has been shown that two There are three promoter sites located in exon 1: P0, P1 and P1'.…”

“…Translation initiation codons, AUG1 for full-length p53 and AUG2 for Δ40p53, are marked with frames. The 5' terminal region of p53 mRNA in the p53 gene expression characteristic hairpin elements, G56-C169 and U180-A218, are preserved in the P0-initiated transcript (Górska et al, 2013). In the P1-initiated mRNA both translation initiation codons, AUG1 and AUG2, are located in the helix-bulge junction and the A residues are unpaired.…”

“…The structural environment of AUG1 is likely to limit the p53 translation efficiency (Błaszczyk & Ciesiołka, 2011;Kozak 2005). Structure probing of the 5' terminus of the P1-initiated mRNA with intron 2 retained, has shown that the large hairpin containing the AUG1 codon is also preserved in this transcript (Górska et al, 2013). Thus, the presence of G56-C169 hairpin in which the AUG1 codon is embedded in various p53 mRNA transcripts strongly indicates an important role of this motif in the p53 gene expression.…”