title: Box C/D small nucleolar RNA (snoRNA) U60 regulates intracellular cholesterol trafficking.
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.
authors.affiliation: From the Diabetic Cardiovascular Disease Center, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: Katrina A
authors.initials: KA
authors.lastname: Brandis
authors.forename: Sarah
authors.initials: S
authors.lastname: Gale
authors.forename: Sarah
authors.initials: S
authors.lastname: Jinn
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Langmade
authors.forename: Nicole
authors.initials: N
authors.lastname: Dudley-Rucker
authors.forename: Hui
authors.initials: H
authors.lastname: Jiang
authors.forename: Rohini
authors.initials: R
authors.lastname: Sidhu
authors.forename: Aileen
authors.initials: A
authors.lastname: Ren
authors.forename: Anna
authors.initials: A
authors.lastname: Goldberg
authors.forename: Jean E
authors.initials: JE
authors.lastname: Schaffer
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Ory
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D056426:Genetic Loci / Q000235:genetics; D057895:Haploinsufficiency / Q000187:drug effects / Q000235:genetics; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010713:Phosphatidylcholines / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism
chemical_list: D010713:Phosphatidylcholines; D020537:RNA, Small Nucleolar; D020718:Ribonucleoproteins, Small Nucleolar; D000666:Amphotericin B; D002784:Cholesterol
keywords: Cholesterol; Cholesterol Regulation; Intracellular Trafficking; Lipid Transport; Noncoding RNA; Small Nucleolar RNA (snoRNA)