ELUCIDATING THE BIOLOGICAL PATHWAYS REGULATED BY AN RNA-BINDING PROTEIN IMPLICATED IN A NEURODEVELOPMENTAL DISORDER

Abstract

Neurodevelopmental disorders (NDDs) are complex conditions arising from genetic disruptions in pathways that regulate brain development, affecting over 3% of children worldwide. Among these, PUM1-Associated Developmental Disability, Ataxia, and Seizure (PADDAS) syndrome is linked to mutations in the PUM1 gene, which encodes a conserved RNA-binding protein involved in post-transcriptional gene regulation. Despite its clinical relevance, the molecular mechanisms by which PUM1 dysfunction contributes to disease pathology remain poorly understood. To investigate these mechanisms in vivo, this research utilizes the C.elegans ortholog puf-9 as a model system. Phenotypic characterization of the puf-9(ok1136) null allele revealed a significant reduction in fertility and defects in body size, providing a measurable framework for genetic analysis. A forward genetic suppressor and enhancer screen were performed using ethyl methanesulfonate (EMS) mutagenesis to identify modifiers of the puf-9(ok1136) phenotype. From this screen, 294 F2 lines were isolated, exhibiting a wide range of phenotypic variations, including both suppressors and enhancers of fertility and growth defects. From this population, 24 candidate strains were selected based on phenotypic criteria related to N2 wild-type control. To complement the genetic screen, transcriptomic profiling was conducted using RNA sequencing to identify genes regulated by puf-9 mutant strains. Comparative analysis between N2 and mutant strains revealed that 493 genes were upregulated in puf-9(ok1136) Day 1 adults relative to N2, while puf-9(syb2398) and puf-9(syb2404) showed minimal transcriptional changes. These findings are expected to provide insight into the downstream regulatory pathways controlled by puf-9. Together, this research establishes a framework for identifying genetic modifiers and regulatory pathways associated with puf-9 function. The integration of forward genetics with transcriptomic analysis provides a comprehensive approach to understanding conserved mechanisms underlying PUM1- associated NDDs. These findings may contribute to the identification of candidate genes and pathways relevant to human disease and inform future therapeutic strategies.