Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with a known genetic etiology but a complex epigenetic basis. PWS is an imprinted disorder, meaning that genes expressed only on the paternal but not the maternal chromosome 15 are responsible. Furthermore, unlike genetic mutations that affect protein-coding genes, the smallest genetic deletions causing PWS only affect noncoding transcripts of RNA. At the heart of the minimally deleted region in PWS are two types of noncoding RNAs. First, the HBII-85/SNORD116 snoRNAs localize to the nucleolus, a subnuclear structure that enlarges in mature neurons and serves as a cellular factory for ribosomes. Second, the host gene surrounding the snoRNA components surprisingly stays in the nucleus and forms a large RNA cloud-like structure that increases in size with neuronal maturity. While most of the focus in the PWS field has been on understanding the function of the snoRNA components, the host snoRNA-lncRNA may be of equal if not greater importance to understanding and treating PWS. In this proposal, we seek to test the hypothesis that the host snoRNAlncRNA directly alters the structure of the PWS genetic locus by forming an RNA:DNA structure called an R-loop during neurodevelopment, creating a highly active nuclear domain important for neuronal maturity. The approaches include novel imaging methods to observe RNA and DNA structural changes in individual neuronal nuclei in mouse and human brain, novel mouse-human chromosome 15 hybrid cell lines, and novel molecular R-loop detection methods. Results from these investigations are expected to improve understanding of the epigenetic basis of PWS and may enable epigenetic therapy for PWS by reactivating inactive transcripts through noncoding RNA-based therapies.