STAU1 and circHIPK3 finely regulate the translation of BRCA1 mRNA in RD cells

Circular RNAs (circRNAs) are a class of long non-coding RNAs composed of single- stranded RNA molecules that form a covalently closed structure without free ends. Their deregulation has been implicated in various pathological conditions, such as cancer. CircRNAs can influence gene expression in several ways, by interacting with DNA, RNA, and proteins. Our lab has identified a functional interaction between circHIPK3, a circRNA upregulated in rhabdomyosarcoma (RMS), and BRCA1 mRNA, where circHIPK3 binding displaces FMRP, a translational repressor, enhancing BRCA1 protein expression. BRCA1 is essential for DNA repair via homologous recombination, and its upregulation may contribute to resistance to genotoxic therapies. Considering the relevance that circRNA-mRNA interactions seem to exert in cancer cells, the aim of my project is to characterize the role of proteins that potentially bind these RNA- RNA duplexes in RD cells. In particular, we focused on STAU1, a double-stranded RNA- binding protein upregulated in RMS cells. In a circHIPK3 pulldown experiment under STAU1 knockdown (KD) condition, BRCA1 mRNA showed increased binding to circHIPK3, suggesting that STAU1 promotes dissociation of the duplex. However, BRCA1 protein levels decreased, while mRNA levels remained unchanged. This implies that STAU1 acts downstream of circHIPK3, and that the effect of circHIPK3 on BRCA1 mRNA becomes inhibitory for translation in the absence of STAU1. Polysome profiling confirmed that STAU1 KD causes a shift of BRCA1 mRNA from heavy polysomes fractions to light polysomes, implying an effect of STAU1 in the processivity of BRCA1 mRNA translation. Additionally, CLIP experiments demonstrated direct binding of STAU1 to BRCA1 mRNA. Since STAU1 is predicted to interact with many RNA helicases, we hypothesize it recruits helicases to unwind inhibitory RNA structures in BRCA1 mRNA. Supporting this, DHX9 KD, an RNA helicase predicted to bind STAU1, phenocopied the translational defect of STAU1 KD. Together, these findings highlight a complex regulatory mechanism involving circHIPK3, STAU1, and BRCA1 mRNA, and suggest that STAU1 contributes both to RNA duplex remodeling and to translation processivity, thus shedding light on novel aspects of post-transcriptional gene expression regulation in cancer.