Abstract
Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Because mutant cells with defects in PM to endoplasmic reticulum cholesterol trafficking can be isolated on the basis of resistance to amphotericin B, we conducted an amphotericin B loss-of-function screen in Chinese hamster ovary (CHO) cells using insertional mutagenesis to identify genes that regulate this trafficking mechanism. Mutant line A1 displayed reduced cholesteryl ester formation from PM-derived cholesterol and increased de novo cholesterol synthesis, indicating a deficiency in retrograde cholesterol transport. Genotypic analysis revealed that the A1 cell line contained one disrupted allele of the U60 small nucleolar RNA (snoRNA) host gene, resulting in haploinsufficiency of the box C/D snoRNA U60. Complementation and mutational studies revealed the U60 snoRNA to be the essential feature from this locus that affects cholesterol trafficking. Lack of alteration in predicted U60-mediated site-directed methylation of 28 S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Our study adds to a growing body of evidence for participation of small noncoding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.
Highlights
The mechanisms regulating internalization of plasma membrane cholesterol in mammalian cells are not well understood
We have identified a novel pathway linking a box C/D small nucleolar RNA (snoRNA) to cholesterol homeostasis
We show that haploinsufficiency of the U60 snoRNA resulted in decreased plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol trafficking, as measured by reduced PM cholesterol esterification and increased de novo cholesterol synthesis
Summary
The mechanisms regulating internalization of plasma membrane cholesterol in mammalian cells are not well understood. Results: A cell line haploinsufficient for U60 snoRNA expression exhibits impaired plasma membrane to ER cholesterol trafficking and increased de novo cholesterol synthesis. Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum is essential for cellular cholesterol homeostasis The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Following internalization via the LDL receptor, LDL particles are trafficked to a late endocytic compartment where esters are hydrolyzed by acid lipase to free cholesterol and subsequently released from endolysosomes via the concerted actions of the Niemann-Pick C1 (NPC1)/Niemann-Pick C2 (NPC2) proteins Most of this cholesterol is transported to the PM with a minor amount transported directly to the endoplasmic reticulum (ER), Golgi, and mitochondria [1,2,3]. We describe a CHO cell mutant in which a genetic disruption of the U60 small nucleolar RNA (snoRNA) host gene impairs PM to ER retrograde cholesterol transport
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