Prader-Willi Syndrome (PWS) is a genetic disease that results in abnormal hormone levels, developmental delay, intellectual disability, hypogonadism, and excessive appetite. The disease is caused by a de novo genetic deletion in chromosome 15. While many of the deleted genes have been identified, there is little known about their molecular function. There is evidence that a cluster of non-coding RNA genes in the deleted region known as the SNORD116 genes may be the most critical genes deleted in Prader-Willi Syndrome. It is unknown what the SNORD116 genes do at the molecular level, but recent evidence suggests they regulate the expression of other genes involved in the neuroendocrine system. Specifically, the SNORD116 gene is implicated in regulation of NHLH2, a transcription factor gene which plays a key role in development, hormonal regulation, and body weight. In this study we identify phylogenetically conserved regions of SNORD116 and predict interactions with its potential downstream RNA targets. We show that mouse Snord116 post-transcriptionally increases Nhlh2 RNA levels dependent on its 3'UTR and protects it from degradation within 45 minutes of its transcription. Additionally, a single nucleotide variant within Nhlh2 at the predicted Snord116 interaction site may disrupt Snord116's protective effect. This is the first observation of a molecular mechanism for Snord116, identifying its role in RNA stability, and leads us closer to understanding Prader-Willi Syndrome and finding a possible treatment. However, Snord116 in vitro knockdown or paternally inherited in vivo deletion fail to detect differential expression of Nhlh2, likely due to missing the key timepoint of Snord116 regulatory effects on Nhlh2 RNA soon after its transcriptional stimulation, and dependent on leptin signals. Furthermore, the hypothalamic mRNA expression profile of PWS mouse models fed a nutraceutical dietary supplement of conjugated linoleic acid reveals minimal overall changes, while the effect of diet may be stronger than genotype and potentially changes gene expression of metabolic molecular pathways. / Doctor of Philosophy / Prader-Willi Syndrome is a genetic disease that results in abnormal hormone levels, slow development, intellectual disability, gonad deficiency, and excessive appetite. The disease is caused by a genetic deletion in chromosome 15 that is almost always a spontaneous mutation not inherited from the parents. While many of the deleted genes have been identified, there is little known about what their molecular function is. There is evidence that a cluster of genes in the deleted region known as the SNORD116 genes may be the most critical genes deleted in Prader-Willi Syndrome. It is unknown what the SNORD116 genes do at the molecular level, but recent evidence suggests that it regulates other genes involved in the hormone system. Specifically, the SNORD116 gene is implicated to regulate the levels of NHLH2, a gene which plays a key role in development, hormonal regulation, and body weight. In this study we identify key regions of SNORD116 and predict interactions with its potential downstream targets. We show that SNORD116 increases NHLH2 levels and slows its degradation at the RNA transcript level. This is the first observation of a molecular mechanism for SNORD116 and leads us closer to understanding Prader-Willi Syndrome and finding a possible treatment. However, other mouse models of Snord116 deletion fail to find differences in Nhlh2. This is likely due to missing a brief key timepoint and hormonal signal when Nhlh2 is most subject to Snord116's effects. Furthermore, PWS mouse models fed a supplement intended for weight loss leads to mild overall gene expression changes in the hypothalamus, a brain region that regulates many hormonal signals including appetite and energy balance. The effect of diet may be stronger than genotype in this brain region, with diet potentially changing the activity of metabolic molecular pathways.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/112674 |
| Date | 27 May 2021 |
| Creators | Kocher, Matthew Afshin |
| Contributors | Graduate School, Good, Deborah J., Grange, Robert W., Thompson, Christopher, Kojima, Shihoko, Huang, Wenda |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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