As the “governor of the cell”, p53 gathers different cues provided to it by the cellular environment, assimilates them through its regulatory structures and domains and makes well-pondered judgments. And when one regulatory pathway fails to trigger – e.g. due to mutation – p53 can often still be activated via an alternate mechanism. This happens for example with p53 mRNA at the level of translation due to the function of specific RNA structures called IRES (Internal Ribosome Entry Site). Different signals can thus activate different p53 mRNA and protein variants (p53 isoforms) that will in turn pass the appropriate verdicts to the cell.
Several different types of cellular stresses/deprivations shut-down the translation machinery to avoid unnecessary energy expenditures and the formation of abnormal products. p53 mRNA can compensate for that by harboring internal ribosome entry sites (IRESs) that allow for translation to be initiated without the need for the canonical translation initiation apparatus. IRESs are structures in the RNA that facilitate the binding to 40S subunits of the translation initiation complex without the need for a 5’cap-structure or the cap-binding complex eIF4F. Two IRESs have been described in p53 mRNA, one in the 5´ untranslated region (5’UTR) and another in the first most 5’ 117 nucleotides of the coding region (5’CR), upstream of the initiation codon for the Δ40p53 isoform lacking the first 39 amino-acids of the full-length (FL) p53 protein. As expected from their locations, 5’UTR-IRES mainly regulates the translation of FLp53 whereas 5’CR-IRES regulates Δ40p53 translation. The structures of these IRESs have been estimated by three different laboratories using different assays. There were small differences among the studies, but the main elements are consistent: two stem-loop (SL) structures. The larger one (SL1) includes the 5’UTR and encompasses the initiation codon for FLp53 (AUG1) and the first nucleotides of the coding sequence and the smaller one (SL2) locates downstream between the two initiation codons. The region between SL2 and AUG40 (the initiation codon for Δ40p53) is involved in the folding of the 5’UTR. In vitro, AUG1 is translated mostly 5’cap-dependently while AUG40 is largely 5’cap-independent. While Δ40p53 seems to be translated primarily 5’cap-independently, FLp53 can be expressed by both cap-dependent and independent mechanisms (the latter, for example, following DNA damage). The function of these two p53 IRESs is critical for the activation of the p53 FL and Δ40p53 protein isoforms and helps dictate stimulus-specific outcomes in the cell.
p53 protein isoforms
p53 is the most commonly mutated gene in cancer. However, p53 mutation status fails to accurately predict cancer formation, aggressiveness or recurrence. This is possibly because mutations in p53 regulate the levels and activity of a collection of different protein isoforms with different roles in the cell. Understanding the roles and regulation of these isoforms in normal and cancer cells may help define more clinically relevant biomarkers for cancer.
Recent studies have started to elucidate the immense diversity of p53 isoform functions in different tissues, disease and species and it soon became clear that the full-length (FL) p53 only manifests a small fraction of the p53 gene’s capacities. Shorter p53 isoforms have been shown to be involved in a variety of cellular and organismal physiological processes and pathologies as diverse as: cell cycle progression, cell motility, cell death, senescence, tubulogenesis, 3D structure formation, DNA repair, glucose homeostasis, mitochondrial function, epigenetics, stem cell induction and potency, differentiation, organ and body development, life span, aging, premature death, neurodegeneration, cognitive decline, synaptic impairment, inflammation, immune response, cancer, tissue injury, Alzheimer, amyotrophic lateral sclerosis, diabetes and bacterial and viral infections. Several of the isoforms are well conserved among species and studies have been performed using human cells, mouse, Mongolian gerbil, drosophila, zebrafish and white shrimp. Importantly, many of the isoforms’ functions are also conserved among the species, suggesting an early co-evolution of FL and shorter p53 isoforms, both harboring equally important roles in cell fate decisions. In fact, for Drosophila melanogaster (common fruit fly), the shorter isoform (Dp53A) is the most abundant and loss-of-function mutants indicate that it is the primary mediator of pro-apoptotic gene transcription after ionizing radiation and not FL p53 (Dp53B).
The exact number of p53 isoforms existent is yet unclear and difficult to determine because their expression is dependent on tissue and cell type, developmental stage and pathological condition. In humans the isoforms are originated through different usages of alternative promoters, splice sites and translation initiation codons. The second promoter (P2) is located between intron 1 and exon 5 and originates a shorter p53 mRNA . Both the P1 and P2 p53 nuclearRNAs can then undergo alternative splicing. Alternative splicing has been observed at introns 2 , 5 , 6, 9 and between exons 7 and 9. Lastly, fully spliced p53 mRNAs can be translated from different initiation sites: AUG1, AUG40, AUG133 and AUG160.
The most studied shorter p53 isoforms are ∆39 and ∆132 N-terminally truncated proteins, named ∆40p53 and ∆133p53, respectively. ∆40p53 lacks the first transactivation domain (TAD1) of FL p53, while ∆133p53 lacks both TAD1 and TAD2, the proline-rich domain and part of the DNA-binding domain. ∆40p53is expressed from AUG40 in the P1 p53 mRNA, mostly via cap-independent translation, due to the presence of an IRES structure (5’CR-IRES) upstream of its initiation codon. Several types of cellular stress, such as DNA damage, ionizing radiation, ER stress, glucose deprivation, oncogene-induced senescence and Alzheimer disease-associated APP intracellular domain (AICD) levels, were shown to activate 5’CR-IRES and induce ∆40p53 expression. ∆40p53 then acts together with FL p53 in tetramers to regulate the transcription of genes involved in cell-cycle arrest (p21Cip1, 14-3-3sigma), apoptosis (Bax), senescence (p21Cip1), microtubule stability (Dyrk1A, GSK3ß, Cdk5, p35 and p39), regeneration, mitochondrial function (MARS2, carnitine, acetyl coA, ATP and Krebs cycle intermediates) and aging (IGFBP-3). ∆40p53 can also be induced by retention of intron 2, which possesses an in frame stop codon leading to premature translation termination of FL p53 and initiation of ∆40p53 at AUG40.
∆133p53 and ∆160p53 isoforms are translated from the first and second initiation codons present in the shorter P2 p53 mRNA: AUG133 and AUG160, respectively. Unlike the p53 isoforms that contain at least one TAD and favor tumor suppression (e.g. FL p53, ∆40p53, p53ß), shorter p53 isoforms lacking both TADs (∆133p53, ∆160p53) tend to possess contrasting pro-oncogenic functions such as the capacity to induce proliferation or inhibit apoptosis. More unique attributes include still: enhanced migration/invasion, the formation of invasive structures and disrupted mammary tissue architecture. ∆133p53 was furthermore shown to play a role in angiogenesis, stem cell induction and potency, DNA double-strand break repair, replicative senescence and also, together with ∆160p53, response to bacterial infection. ∆133p53 isoforms act by interfering with the transcriptional activity of the p53 tetramer (and in such way repressing, for example, p21Cip1, miR34a, PAI-1, IGFBP7, which help the induction of pluripotent stem cells) as well as by directly associating with target proteins, such as the small GTPase RhoB, a pro-apoptotic protein.