Sometimes highly impactful serendipitous discoveries are made when performing genetic loss-of-function studies that were initially focused on putative tumor suppressors or other hypotheses.

Researchers working at The University of Texas M.D. Anderson Cancer Center reported on the August 6, 2021, issue of Science on the surprising discovery that a long non-coding RNA (lncRNA) involved in cancer turns out to serve more fundamental functions required for phenylalanine hydroxylase (PAH) function, prevention of phenylketonuria (PKU) disease, and a healthy lifespan.

PKU is the most common inborn error of amino acid metabolism and if left untreated will lead to the accumulation of phenylalanine, deficiency of tyrosine, and associated deficiencies of neurotransmitters to ultimately cause brain damage, mental retardation, decreased lifespan, and more symptoms. The severity of symptoms range depending on the degree of loss of PAH activity, with more than 1000 genetic mutations in PAH having been identified, but for some PKU cases the PAH gene is perfectly wild-type. Disease etiology is poorly understood. Moreover, modern treatments do not always work, but the new research discovery provides both a new potential explanation for PKU and a promising potential pharmacologic approach.

The story of serendipitous discovery started when cancer researchers at M.D. Anderson decided to genetically delete the mouse orthologue (Pair) of the human HULC (hepatocellular carcinoma upregulated lncRNA) gene in mice. Pair and HULC encode for lncRNAs that are highly expressed in the adult liver of mice and humans. Therefore, initially the researchers were hypothesizing that HULC may be a tumor suppressor.

By focusing on lncRNA the researchers took a road less traveled. The vast majority of genetic research focuses on regions of genes that encode for amino acids in proteins in part because it is much easier to understand how loss of protein synthesis will cause a disease. However, it turns out that most genetic mutations are not present in the coding region. Rather 98% of mutations occur in noncoding RNAs. The investigators wanted to determine the functional importance of lncRNAs in biology.

They profiled expression status of lncRNAs in mouse liver, brain, kidney, lung and skin, both in the early life and adult stage. They assumed that the highly expressed lncRNAs in adult stage, but not in embryo, indicate the important biological functions in these organs. Later, they developed genetic mouse models to deplete potential candidates. Pair knockout mice exhibited a surprising phenotype resembling human PKU disease.

However, to their surprise, the mice did not develop cancer. Rather Pair-deficient mice died early and instead developed metabolic symptoms consistent with excessive phenylalanine in the blood, ultimately resembling the metabolic PKU disease.

On the basis of biochemical studies, University of Texas M.D. Anderson principal investigator Chunru Lin, Associate Professor of Molecular and Cellular Oncology, reached out to M.D. Anderson colleague Dr. Shuxing Zhang, an Associate Professor in the Department of Experimental Therapeutics, to perform structural and computational biology studies. Zhang's team independently demonstrated the molecular mechanism of the interactions between lncRNAs, PAIR or HULC, with PAH from a structural biology point of view.

Most surprisingly, biochemical studies determined that the Pair and HULC lncRNAs bind directly to the PAH protein to increase its activity in converting phenylalanine into tyrosine. It was the first time a lncRNA was shown to bind to the PAH enzyme to increase and control its activity.

As Zhang explained to BioWorld Science, "The situation is analogous to why astrophysicists describe the calculated mass of the universe as 'dark matter' -- we can see the stars, but most of the matter in the universe is not visible. Similarly, we see the coding region and can make clear unambiguous connections between mutations and loss of functions, but we are still learning about lncRNAs." LncRNAs are known to have important roles in regulating gene expression by interacting with messenger RNA, but lncRNAs could possibly work via directly interacting with proteins, directly interacting with substrates/small molecules, or even possess inherent activities.

Current standard treatments for PAH typically involve dietary recommendations, phenylalanine restriction, tyrosine supplementation, BH4, and/or PAH enzyme substitution. However, for some patients having normal PAH genetics or treatments remain ineffective. Fortunately, the new study reveals a new understanding for potential PKU pathogenesis and most significantly demonstrates that mimetics of HULC hold promise in restoring PAH activity for PKU patients.

Loss-of-function genetic mutations of the PAH gene are the most appreciated cause of PKU, but this latest study suggests that deficiency of noncoding RNAs or other PAH allosteric factors are likely to cause PKU as well. To date, nobody has screened for HULC mutations.

Zhang explained that this is exciting because this is the first time anyone has systematically studied at the molecular, cellular and animal model level to understand at the molecular level how PKU can develop when there is a normal wild-type PAH enzyme and no BH4 genetic errors either. For the first time ever, the researchers identified how a lncRNA can serve a requisite role in supporting PAH enzymatic activity.

To further investigate this approach as a possible treatment strategy, the researchers created a mouse model of PKU, carrying the most found mutation in PAH. The HULC mimic improved phenylalanine metabolism as evidenced by a sustained reduction in phenylalanine levels. Significantly synthetic HULC mimetics were able to partially restore enzymatic activity in 13 of 17 different most common PAH mutants seen in clinical PKU.

Zhang emphasized the critical point that this approach is based on endogenous HULC molecules. Therefore, this is unlike a small-molecule drug developed against a structure. He explained that every drug, depending on the dosage, has a toxicity after a certain dose, but the use of an endogenous RNA mimicking approach is likely to have an exceptionally good safety profile.