Abstract
p53 isoforms remain poorly characterized despite recent evidence that they possess distinct functions in response to different cellular stresses. In Drosophila , four p53 isoforms are expressed from a single locus, but their individual roles under different stress conditions are not yet fully understood, partly due to limited tools for isoform-specific analysis. To address this gap, we developed and validated a set of isoform-specific qPCR primers that accurately distinguish all endogenous Drosophila p53 transcripts. Using this approach, we examined how each isoform responds to nucleolar stress, a mechanism triggered in humans by deregulation of ribosome biogenesis, often accompanied by increased p53 and apoptosis. In Drosophila , knockdown of Nucleolar complex protein 1 ( NOC1 ) impairs rRNA maturation and ribosomal assembly, triggering nucleolar stress and leading to upregulation of p53 level. Using our isoform-specific qPCR primers, we found that p53B and the previously uncharacterized p53C isoforms are selectively upregulated in response to NOC1 knockdown, while p53A expression remains largely unaffected. This differential regulation underscores a stress-specific pattern of isoform activation distinct from the response typically observed under genotoxic stress conditions, where p53A predominates. These findings suggest the existence of isoform-specific roles in nucleolar stress signaling. Furthermore, our data support the possibility of autoregulatory interactions among p53 isoforms, whereby the stress-induced upregulation of one isoform may influence the transcriptional activity or stability of others. Notably, we also identified a previously unrecognized and evolutionarily conserved transcriptional response of p53 to nucleolar stress, which is detectable in both Drosophila and human systems. Together, these results highlight the significance of dissecting p53 isoform-specific functions to advance our understanding of cellular stress responses and their conservation across species.