The Box C D Snorna U60 Regulates Intracellular Cholesterol Trafficking

Mobilization of plasma membrane (PM) cholesterol to the endoplasmic reticulum (ER) is essential for cellular cholesterol homeostasis. The mechanisms regulating this retrograde, intermembrane cholesterol transfer are not well understood. Since mutant cells with defects in PM to ER 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 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 28S rRNA in the A1 mutant suggests that the U60 snoRNA modulates cholesterol trafficking by a mechanism that is independent of this canonical function. Additional studies were conducted to isolate and identify direct RNA targets of the U60 snoRNA and to define cellular pathways that are affected by U60 expression. This study adds to a growing body of evidence for participation of small non-coding RNAs in cholesterol homeostasis and is the first to implicate a snoRNA in this cellular function.