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snoRNA U17 as a Novel Regulator of Cholesterol Homeostasis
Date of Award
Doctor of Philosophy (PhD)
Cholesterol is an essential cellular lipid that is a regulator of membrane organization and fluidity, and is an obligate precursor for synthesis of steroid hormones, oxysterols and bile acids. Cellular cholesterol homeostasis is tightly regulated through a network of transcriptional and non-transcriptional pathways. To further delineate intracellular cholesterol trafficking pathways, we initiated a loss-of-function genetic screen in Chinese hamster ovary cells (CHO), selecting for CHO mutants with altered cholesterol trafficking.
Here we characterize one of the isolated mutants in which the locus encoding small nucleolar RNA hosting gene 3 (SNHG3) is disrupted. Altered cholesterol trafficking phenotype is not due to reduced expression of the SNHG3 mRNA, but rather results from haploinsufficiency of the H/ACA small nucleolar RNA (snoRNA) U17, a small non-coding RNA produced from the hosting gene SNHG3 introns. Previously identified function of U17 snoRNA is to mediate 18S rRNA processing, yet cells haploinsufficient for U17 show normal maturation and amount of 18S rRNA, indicating that altered cholesterol trafficking is not caused by defects in rRNA processing.
Through expression profiling, we identify that mRNA of mitochondrial adapter protein HUMMR is a target that is negatively regulated by U17 snoRNA. HUMMR mRNA is up-regulated U17 snoRNA-deficient cells and down-regulated in U17 snoRNA-overexpression cells. Up-regulation of HUMMR itself is sufficient to alter cholesterol trafficking, suggesting HUMMR functions downstream of U17 snoRNA-initiated regulation of cholesterol homeostasis. We show that up-regulation of HUMMR protein in U17 snoRNA-deficient cells promotes formation of ER-mitochondrial contacts, decreasing esterification of cholesterol and facilitating cholesterol trafficking to mitochondria, which increases the synthesis of 27-hydroxy cholesterol and pregnenolone in mitochondria. We also show that levels of U17 snoRNA and HUMMR are developmentally regulated in steroidogenic tissues. As HUMMR level increases in maturing ovarian tissue, expression of U17 snoRNA gradually decreases. This suggests negative regulation of HUMMR by U17 snoRNA in vivo that may be critical for regulation of steroidogenesis.
Together, this dissertation describes U17 snoRNA as a new class of non-coding RNA that modulates cholesterol trafficking by regulating downstream coding genes. U17 snoRNA and HUMMR regulate mitochondrial synthesis of steroids, and are developmentally regulated in steroidogenic tissues, suggesting that the U17 snoRNA-HUMMR pathway may serve a previously unrecognized, physiological role in gonadal tissue maturation and steroidogenesis.
Chair and Committee
Daniel S Ory
Justin Fay, Scott Matkovich, Jean Schaffer, Tim Schedl, Paul Schlesinger
Jinn, Sarah, "snoRNA U17 as a Novel Regulator of Cholesterol Homeostasis" (2014). Arts & Sciences Electronic Theses and Dissertations. 346.