Investigating the functions of the RNA-binding protein Pumilio: Sleep regulation, gut homeostasis, and therapeutic targeting.

Abstract

The precise control of gene expression via post-transcriptional regulation is fundamental to all biological processes. Central to this system are RNA-binding proteins (RBPs), including the highly conserved Pumilio/PUF family, which typically act as post-transcriptional repressors by binding target mRNAs to promote their degradation and inhibit their translation. This work leverages the <em>Drosophila melanogaster</em> model system to investigate the function of the conserved RBP Pumilio (Pum) from three perspectives: organismal behavior, cellular homeostasis, and translational applications.<br /> <br /> First, at the organismal level, we uncover a peripheral-to-brain signaling axis regulating behavior. We demonstrate that conditional knockdown of <em>pum </em>in two distinct peripheral tissues, the intestinal stem cells (ISCs) and the endocrine <em>corpus allatum</em> (CA), independently and significantly accelerates the onset of nighttime sleep. This behavioral change is associated with widespread transcriptional alterations in the brain, including a robust upregulation of stress-response genes such as <em>Hsp83</em>.<br /> <br /> Second, at the cellular level, we investigated Pum's role in intestinal stem cell homeostasis within the adult <em>Drosophila </em>posterior midgut. We find that <em>pum </em>knockdown in the ISC lineage results in a sex-specific disruption of cellular balance, observed only in males. This phenotype is characterized by an accumulation of esg-positive ISCs and a concomitant loss of differentiated Prospero-positive enteroendocrine (EE) cells. These findings suggest that Pumilio is required in males to maintain the proper balance between stem cells and their differentiated progeny.<br /> <br /> Finally, addressing the oncogenic role of human Pumilio homologs, we developed a structure-based computational drug discovery workflow to identify novel inhibitors. Targeting the conserved RNA-binding pocket, we virtually screened over 5 million compounds to yield 32 lead candidates. While initial<em> in vitro</em> validation did not yield an active hit, this work establishes a robust computational pipeline for future screening. Taken together, this work provides a multi-faceted analysis of Pumilio, illuminating its diverse and critical functions in regulating complex physiology, from cellular differentiation in the gut to systemic control of sleep behavior, and establishes a foundation for its therapeutic targeting in human disease.