Molecular Mechanism of long Noncoding RNAs in Phenylketonuria

Research Summary The functional roles and molecular mechanisms of non-coding RNAs (lncRNA) in human genetic disease serve as promising new frontiers for establishing the foundation of future research directions and therapeutic avenues for disorders that currently lack sufficient treatment options. The inborn metabolic disorder phenylketonuria (PKU) has been considered to be caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in disruptions to its enzymatic activity and consequent accumulation of phenylalanine in the blood. Our preliminary data demonstrated that the human lncRNA HULC is functionally conserved with mouse lncRNA Pair. PKU patients harbor genomic variants of the HULC gene. Knockout of Pair leads to hypo-pigmentation, growth retardation, progressive neurological symptoms, and seizures, which faithfully models human PKU. Deletion of HULC in primary human hepatocytes leads to elevated cellular Phenylalanine, suggesting the functional importance of HULC/Pair in PKU. We defined the RNA motifs of both HULC and Pair that are integral to establishing lncRNA-enzyme interactions and designed peptide-tagged lncRNA mimics that restored the enzymatic activity of PAH. Administration of HULC mimics in PKU mouse model successfully alleviated excess phenylalanine levels in serum, providing mechanistic insight into the basis of inherited genetic diseases. The proposed study seeks to establish long non-coding RNAs as important players in the initiation and progression of inborn metabolic disorders and develop a lncRNA-based innovative approach for determining new strategies to tackle the molecular basis of PKU. The central hypothesis is that the genetic mutation and deletion of lncRNA results in the enzymatic deficiency of PAH, which could be alleviated through targeting therapies to enhance the protein stability and enzymatic activity of PAH. Using iPS-differentiated hepatocytes derived from PKU patients, we will first demonstrate the biological significance of HULC in the development and progression of PKU by addressing the impact of common HULC mutations and assessing the correlations between mutations or deletions of HULC and PKU symptoms. We will then elucidate the molecular mechanisms of lncRNA-mediated regulation of PAH enzymatic activity. Finally, we will use the administration of liver-enriched lncRNA mimics and small molecule agonists/antagonists to establish their mechanistic role in restoring impaired enzymatic function. The aforementioned research aims will not only elucidate the roles of lncRNAs in metabolic disorders, but also illustrate the potential therapeutic value of lncRNA mimics and small molecule inhibitor-based medicine in treating inborn genetic diseases. We anticipate that the results of this study will shift the current research and clinical paradigm to incorporate the physiological and pathological aspects of non-coding genes and pave the way for the development of future lncRNA-based medicines.