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. Author manuscript; available in PMC: 2016 Jun 2.
Published in final edited form as: Cell Metab. 2015 May 14;21(6):855–867. doi: 10.1016/j.cmet.2015.04.010

snoRNA U17 Regulates Cellular Cholesterol Trafficking

Sarah Jinn 1, Katrina A Brandis 1, Aileen Ren 1, Anita Chacko 1, Nicole Dudley-Rucker 1, Sarah Gale 1, Rohini Sidhu 1, Hideji Fujiwara 1, Hui Jiang 1, Brett N Olsen 1, Jean E Schaffer 1, Daniel S Ory 1,†
PMCID: PMC4456254  NIHMSID: NIHMS680180  PMID: 25980348

Summary

Cholesterol is required for the growth and viability of mammalian cells, and is an obligate precursor for steroid hormone synthesis. Using a loss-of-function screen for mutants with defects in intracellular cholesterol trafficking, a Chinese hamster ovary cell mutant with haploinsufficiency of the U17 snoRNA was isolated. U17 is a H/ACA orphan snoRNA, for which a function other than ribosomal processing has not previously been identified. Through expression profiling, we identified hypoxia upregulated mitochondrial movement regulator (HUMMR) mRNA as a target that is negatively regulated by U17 snoRNA. Upregulation of HUMMR in U17 snoRNA-deficient cells promoted formation of ER-mitochondrial contacts, decreasing esterification of cholesterol and facilitating cholesterol trafficking to mitochondria. U17 snoRNA and HUMMR regulate mitochondrial synthesis of steroids in vivo, 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.

Introduction

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 maintain sufficient levels of cholesterol, cellular synthesis and uptake of cholesterol is regulated by a negative feedback loop. When cholesterol levels are low, sterol regulatory element-binding proteins (SREBPs) upregulate genes required for de novo cholesterol synthesis and for internalization of low-density lipoprotein (LDL) cholesterol (Brown and Goldstein, 1999). Conversely, when cholesterol levels are elevated, the SREBP activity is suppressed and a small portion of the excess cholesterol is channeled to the mitochondria for synthesis of side-chain oxysterols. These oxysterols serve as ligands for Liver X Receptors (LXRs), nuclear hormone receptor transcription factors that promote expression of genes responsible for cholesterol efflux and elimination (Venkateswaran et al., 2000). As cellular cholesterol levels decline, oxysterol synthesis is reduced and this feed-forward pathway is silenced. Crosstalk between these transcriptional pathways provides for further fine-tuning of these homeostatic mechanisms (Zelcer et al., 2009).

Intracellular trafficking of cholesterol is dynamic and highly regulated. Receptor-mediated endocytosis of lipoprotein cholesterol or phagocytosis of apoptotic cells are accompanied by increased cholesterol flux through endolysosomes and mobilization to multiple organellar compartments, including the plasma membrane (PM) where ~60–80% of total cellular cholesterol resides. Expansion of the PM cholesterol pool increases cholesterol exposure and promotes rapid internalization of cholesterol that is independent of the endolysosomal routes. The arrival of PM cholesterol in the endoplasmic reticulum (ER) inhibits SREBP processing and stimulates re-esterification of cholesterol by the ER-resident acyl CoA:cholesterol acyltransferase (ACAT). PM cholesterol is also delivered to the mitochondria, where the mitochondrial enzyme sterol 27-hydroxylase converts cholesterol to 27-hydroxycholesterol (27-HC). In addition to activating LXR, 27-HC serves as an important modulator of ER-resident Insig proteins that are responsible for retention of SREBP transcription factors and that mediate rapid inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (Lange et al., 2008; Lange et al., 2009).

In steroidogenic tissues, trafficking of cholesterol to mitochondria involves multiple routes – from endolysosomes, PM and ER – which ensures an ample supply of cholesterol precursor for steroid hormone synthesis. Transfer of endolysosomal cholesterol to mitochondria is thought to occur in non-vesicular manner between closely apposed membranes and mediated by steroidogenic acute regulatory (StAR)-related lipid transfer (START) domain proteins (Alpy and Tomasetto, 2005). MLN46, which localizes in late endosomes, was found to facilitate cholesterol egress from late endosome to mitochondrial membranes (Charman et al., 2010). Similarly, StARD1, with its sterol-binding domain and mitochondrial-targeting sequences, has been shown to promote cholesterol uptake into the mitochondrial outer membrane (Bose et al., 2002b). On the other hand, the molecular basis for transfer of ER cholesterol to mitochondria, which may occur at contact sites in mitochondria-associated membranes, is not well understood (Rowland and Voeltz, 2012).

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 (Brandis et al., 2013). In the present study, we report the isolation and characterization of a mutant in which the locus encoding small nucleolar RNA hosting gene 3 (Snhg3) was disrupted. Unexpectedly, we found that the cholesterol trafficking phenotype was not due to reduced expression of the Snhg3 mRNA, but rather resulted from haploinsufficiency of the H/ACA small nucleolar RNA (snoRNA) U17, a small non-coding RNA produced from the Snhg3 introns. Here, we report a previously unrecognized, non-canonical function for U17 snoRNA in regulation of cellular cholesterol homeostasis and show that changes in expression of this small non-coding RNA are associated with postnatal maturation of steroidogenic tissues in vivo.

Results

Haploinsufficiency of Sngh3 disrupts intracellular cholesterol trafficking

To identify genes involved in regulation of cellular cholesterol homeostasis, we performed a genetic screen in CHO cells using an insertional mutagenesis strategy (Brandis et al., 2013). CHO cells were mutagenized by transduction at low multiplicity of infection with the ROSAβgeo promoter trap retrovirus, which encodes a splice acceptor, a neomycin-resistance cassette, and a poly(A) signal. Integration of the ROSAβgeo provirus downstream of an actively transcribed promoter and splice acceptor signal disrupts endogenous expression and confers resistance to neomycin (Friedrich and Soriano, 1991). Following neomycin selection, we employed a screen to identify cells deficient in trafficking of newly incorporated exogenous cholesterol. Mutagenized cells were cholesterol-starved for 24 hours, followed by incubation with LDL and treatment with amphotericin B. Amphotericin B is a polyene antibiotic that lyses cells through incorporation into cholesterol-rich membranes and formation of aqueous pores, and has been used previously to select for cholesterol trafficking mutants (Dahl et al., 1992; Frolov et al., 2001).

One of the CHO mutants that emerged after two rounds of LDL/amphotericin selection, mutant I5, was protected from amphotericin B-induced cell death and resistant to Perfringolysin O (PFO), a membrane cholesterol-binding toxin (Flanagan et al., 2009), but was not resistant to more general inducers of cell death, such as straurosporine and actinomycin D (Figure 1A). LDL uptake by mutant I5 was comparable to wild type (WT) CHO cells (Figure 1B), indicating that the phenotype was not simply a result of failure to internalize LDL cholesterol. The mutant also did not accumulate endolysosomal cholesterol in response to an LDL challenge (Figure S1A), indicating that cholesterol export from the endolysosomal compartment was not impaired, as is the case in Niemann-Pick C1 mutants (Millard et al., 2000). Further characterization of the I5 mutant revealed a defect in esterification of PM-derived cholesterol, which is comparable to the reduced rate of PM cholesterol esterification reported for the 3–6 cholesterol trafficking mutant isolated through a similar LDL/amphotericin selection (Dahl et al., 1994; Dahl et al., 1992; Jacobs et al., 1997) (Figure 1C). Since this cholesterol esterification assay measures the amount of 3H-cholesterol that is trafficked from the PM to the ER and converted to 3H-cholesteryl ester by the ER resident enzyme ACAT, it monitors the level of the ER cholesterol pool (Jacobs et al., 1997). A trivial explanation for the blunted cholesterol esterification might have been reduced ACAT expression or activity. However, ACAT activity was intact in the I5 mutant (Figure 1D). While increased hydrolysis of cholesteryl esters or increased cholesterol efflux might have contributed to the reduced rate of cholesterol esterification of PM-derived cholesterol, we did not find significant differences between mutant I5 and WT CHO cells either with respect to cholesterol content (total, free, or ester) or to the rate of cholesterol efflux (Figure S1B and C). We further found that LDL-stimulated cholesterol esterification (Figure S1D) – a measure of lysosome to ER trafficking – and LDL-stimulated pregnenolone production (Figure S1E) – a measure of lysosome to mitochondria cholesterol trafficking – are intact in the I5 mutant. This indicates that the esterification phenotype is specific to the pathway involved in the internalization of PM-derived cholesterol, which is distinct from post-lysosomal distribution of LDL-derived cholesterol. Together, these data suggest that the cholesterol esterification phenotype resulted from either reduced delivery of PM cholesterol to the ER or enhanced cholesterol trafficking from ER membranes (e.g., partitioning of cholesterol away from the ACAT-accessible cholesterol pool).

Figure 1. Mutant I5 is haploinsufficient for small nucleolar RNA (snoRNA) hosting gene 3 (Snhg3) and has altered cholesterol trafficking.

Figure 1

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(A) Cell death was quantified by propidium iodide (PI) staining of wild type (WT) CHO (black) and mutant I5 (white) cells in standard growth media (Ctr) or treated with cholesterol-binding toxins [10μg/ml AmphotericinB (AmB); 10μg/ml PerfringolysinO (PFO)] or treated with with apoptosis inducers [2μM ActinomycinD (ActD) and 80nM Staurosporine (Staur)]. (B) LDL uptake in WT, I5 and LDLA7 (negative control lacking LDLR) cells was determined by measuring fluorescence intensity following incubation with dil-LDL. RU, relative units. (C) Esterification of plasma membrane-derived cholesterol at the ER membrane was measured in I5 and 3–6 mutant cells relative to WT. (D) ACAT activity was measured in microsomes isolated from WT and I5 cells. (E) Genomic PCR of the I5 locus was performed using combinations of primers (below) specific for the locus (forward primer for 5′ exon of allele disrupted in I5, I5(F); reverse primer for 3′ exon of allele disrupted in I5, I5 (R), and ROSAβgeo reverse primer for proviral sequence, ROSA(R)). (F) Structure of Snhg3 locus in Cricetulus griseus, Mus musculus, and Homo sapiens. Exons are rectangles, snoRNAs are block arrows, and site of proviral integration is indicated by arrowhead. (G) Steady state levels of Snhg3 transcript in WT and I5 cells was assessed by qRT-PCR and normalized to β-actin. Data presented are mean+SE for 3 independent measurements. **, p < 0.01 and ***, p < 0.001 for mutant vs. WT

U17 snoRNA from the Snhg3 locus complements the altered cholesterol trafficking in I5 cells

5′ and 3′ RACE were used to clone the cDNA of the gene disrupted by proviral insertion in the I5 mutant. Southern blotting (Figure S1F) and PCR (Figure 1E) confirmed that a single provirus had integrated into one of two expressed alleles for an mRNA that lacked homology to murine and human sequences at the cDNA level. The lack of ORFs of substantial length (Figure S1G) and the absence of significant homology between the I5 cDNA sequence and other mammalian genes (Figure S1H) suggested that the I5 locus expresses a non-coding RNA. Using both genomic PCR and inverse PCR approaches, the 5.4 kb genomic I5 locus was cloned. Based on conserved promoter elements, exon/intron organization, and intronic orphan H/ACA snoRNAs U17a and U17b, we identified this locus as small RNA hosting gene 3 (Snhg3) (Pelczar and Filipowicz, 1998) (Figure 1F). Disruption of one allele resulted in a 50% reduction in expression of proviral-disrupted mRNA in mutant I5, consistent with a model of haploinsufficiency (Figure 1G). The U17 snoRNAs belong to the H/ACA snoRNA family characterized by the conserved H box (ANANNA) and ACA box (Kiss et al., 2010). In addition, U17 snoRNAs also contain two evolutionarily conserved elements m1 and m2 (Figure S1I). In the yeast U17 ortholog snR30, m1 and m2 elements base-pair with pre-rRNA sequences to mediate pre-rRNA processing for generation of 18S rRNA (Atzorn et al., 2004; Fayet-Lebaron et al., 2009). However, it is not known whether this function is conserved in higher eukaryotes.

We generated and expressed in the I5 genetic background a series of constructs designed to determine the element within the Sngh3 locus – mRNA or embedded snoRNAs – whose deficiency is responsible for the altered cholesterol trafficking in this mutant. GEN, is a construct containing the entire genomic locus of murine Snhg3, including 2kb of the endogenous promoter followed and 1kb of downstream sequences; A or B are GEN constructs lacking U17a or U17b, respectively; AB is a GEN construct lacking both U17a and U17b; cDNA is a full length Snhg3 cDNA sequence driven by a CMV promoter; SNO+ is a construct for expression of the U17b snoRNA from the second intron of a modified human beta-globin gene; and MUT+ is a construct for expression of the mutated U17b snoRNA from the second intron of a modified human beta-globin gene (Figure 2A and B). Clonal cell lines stably expressing these constructs were generated, analyzed for the expression of exogenous murine U17 snoRNA and Snhg3 mRNA by qRT-PCR (Figure 2C and D), and phenotyped by esterification of PM-derived cholesterol. Expression of the entire genomic Snhg3 locus complemented the cholesterol trafficking phenotype in the I5 mutant (Figure 2E, GEN). Likewise, expression of ΔA or ΔB constructs also complemented the I5 mutant esterification defect, suggesting that U17a and U17b are functionally equivalent (Figure S2A and B). On the other hand, mRNA expression alone failed to complement the mutant phenotype (Figure 2E, ΔAB and cDNA). Consistent with these findings, expression of only U17 snoRNA in a heterologous background (SNO+) was sufficient to rescue the cholesterol trafficking phenotype. Taken together, the findings from the complementation experiments indicated that deficiency of U17 snoRNAs, but not the Snhg3 mRNA, was responsible for the altered cholesterol trafficking in mutant I5.

Figure 2. U17 snoRNA of Snhg3 locus complements the altered cholesterol trafficking in the I5 mutant.

Figure 2

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(A) Structures of plasmid constructs for complementation are shown with exons as rectangles and snoRNAs as block arrows. (B) Sequences that are within U17 snoRNA m1/m2 elements are shown in gray boxes. Nucleotides mutated in the MUT+ construct shown in black. (C, D) Clonal lines expressing the various constructs were analyzed by qRT-PCR for expression of the murine U17 snoRNA (C) and Snhg3 mRNA (D), each normalized to β-actin. MUT+1 and MUT+2 are independent clonal lines expressing the MUT+ construct, in which both m1 and m2 elements are mutated. (E) Clones were assessed for esterification of PM-derived cholesterol. Data presented are mean + SE for 4 independent measurements. ***, ρ <0.001 for comparisons indicated

The m1/m2 elements are necessary for U17 snoRNA function

Based on the known functions of H/ACA snoRNAs, we hypothesized that U17 forms a snoRNP by associating with known H/ACA core proteins and recognizes targets via RNA:RNA interaction with its single-stranded antisense element. To test whether murine U17 snoRNA is present in H/ACA small nucleolar ribonucleoprotein (snoRNP) complexes, epitope-tagged H/ACA core proteins were transiently expressed, immunoprecipitated, and analyzed for associated snoRNAs by qPCR. N-terminal FLAG-tagged Dyskerin or C-terminal MYC-tagged Gar1 were enriched ~10–20 fold with U17b, ACA28, and E3, which are H/ACA class snoRNAs, but not with box C/D snoRNAs such as U33 and U60 (Figure S2C); (Brandis et al., 2013; Michel et al., 2011; Wang et al., 2002; Xiao et al., 2009). Canonical H/ACA snoRNAs use the distal loop of a pseudouridylation pocket to designate a target uridine for modification to pseudouridine (Xiao et al., 2009). While target pseudouridyation sites have not been identified for U17 snoRNA, the U17 m1/m2 boxes located in the proximal loop of 3′ hairpin are highly conserved elements that are reported to be essential in Saccharomyces cerevisiae for mediating 18S rRNA processing via directly binding to pre-18S rRNA (Atzorn et al., 2004). To determine if the m1/m2 elements in U17 snoRNA are necessary for correction of the cholesterol trafficking phenotype in the I5 mutant, 7 of 9 bases from m1 and 5 of 7 bases from m2 were mutated in the SNO+ construct. Clonal cell lines stably expressing mutated U17 snoRNA from the second intron of a modified human beta-globin cassette were isolated (Figure 2A and B, MUT+). MUT+ clones expressing m1/m2-double mutated U17 snoRNA at levels comparable to WT U17 snoRNA in SNO+ clones were phenotyped with respect to esterification of PM-derived cholesterol (Figure 2E). While mutation in m1/m2 did not interfere with the formation of U17 snoRNP (Figure S2D and F), it did abrogate ability of U17 snoRNA to rescue cholesterol trafficking phenotype in the I5 mutant. Taken together, these findings suggest that the m1/m2 boxes are essential for U17 snoRNA function in regulation of cholesterol trafficking, potentially by guiding the U17 snoRNP complex to RNA targets.

Haploinsufficiency of U17 snoRNA does not affect 18S rRNA maturation

In Saccharomyces cerevisiae, absence of the yeast ortholog of U17 snoRNA, snR30, led to defects in processing of pre-rRNA and decrease in the amount of mature 18S rRNA (Fayet-Lebaron et al., 2009). To determine if haploinsufficiency of U17 snoRNA affects 18S rRNA processing in CHO cells, cleavage of 47S pre-rRNA at the A′ site that is mediated by U17 snoRNA was studied by S1 nuclease protection assay (Figure 3A). Cleavage at this site was quantified by the relative amounts of long uncleaved species and short cleaved species of biotin-labeled probes after S1 nuclease digestion following hybridization with total RNA. No differences were found in the ratio of uncleaved to cleaved species between WT and I5 (Figure 3B and C). RNA from cells with transient knockdown of U3 snoRNA-associated protein 18 (UTP18) was used as a positive control (Holzel et al., 2010), and led to significant accumulation of uncleaved species and reduction in cleaved species, as evidence of defects in 18S rRNA production. A similar result was obtained from an S1 nuclease protection assay comparing rRNA processing in WT and U17 knockdown in NIH3T3 fibroblasts (Figure S3). Consistent with the results from S1 nuclease protection assay, there was no difference in the amount of mature 18S rRNA between WT and mutant I5 when normalized to 28S rRNA, whose maturation process is independent of that for 18S rRNA (Figure 3D and E). Thus, 18S rRNA formation is unaffected by U17 snoRNA haploinsufficiency. While it is possible that U17 snoRNA may mediate 18S rRNA processing in the mammalian system, one intact allele is sufficient to support this function, and U17 snoRNA’s role in cholesterol trafficking is genetically separable from a possible role in rRNA processing.

Figure 3. Haploinsufficiency of U17 snoRNA does not affect 18S rRNA maturation.

Figure 3

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RNA was prepared from WT and I5 cells and from cells transfected with scrambled siRNA (Ctr) or siRNA against U3 small nucleolar RNA associated protein 18 (UTP18 siRNA), which is an 18S rRNA processing factor essential for the cleavage of the 5′-external transcribed spacer (5′ ETS) leader sequence. (A) Scheme shows 47S pre-rRNA with A′ site where U17 is known to direct cleavage. Alignment of 5′ biotin-labeled S1 probe to uncleaved and cleaved 5′ ETS results in protection of probes of different lengths shown. (B) Protected probes were resolved on denaturing PAGE gel and resulting blot was probed with streptavidin-HRP. (C) Graph shows intensity ratio of uncleaved to cleaved probe in (B) quantified by Image J. (D) Amounts of 18S rRNA in WT CHO and mutant I5 were assessed by formaldehyde agarose gel electrophoresis and ethidium bromide (EtBr) staining. (E) Graph shows quantification of levels of 18S rRNA in (D). Data presented are mean + SE for three independent measurements **, ρ<0.01

Regulation of HUMMR by U17 snoRNA

To investigate downstream U17 snoRNA targets and to confirm, using an independent approach, that U17 snoRNA deficiency perturbs cholesterol trafficking, we knocked down U17 snoRNA in murine NIH3T3 cells by stable expression of a shRNA targeting Snhg3 or overexpression of U17 snoRNA using the SNO+ construct (Figure 4A). Knockdown of U17 snoRNA reduced PM cholesterol esterification by 40% compared to WT cells (Figure 4A). Curiously, more robust U17 snoRNA knockdown could not be achieved in the NIH3T3 cells, suggesting that reduction in U17 snoRNA expression beyond 50% may be deleterious to cell survival (Fayet-Lebaron et al., 2009). Conversely, overexpression of U17 snoRNA resulted in ~1.5-fold increase in PM cholesterol esterification relative to WT.

Figure 4. Regulation of HUMMR by U17 snoRNA.

Figure 4

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(A) Esterification of plasma membrane-derived cholesterol was measured in NIH3T3 cells (WT) and in NIH3T3-derived cell lines with stable knockdown (KD1, KD2) or overexpression of U17 (SNO+1, SNO+2) (left graph). Levels of U17 snoRNA were assessed by qRT-PCR (right graph). (B) Diagram depicts genes identified as those up-regulated in KD (green) and down-regulated in SNO+ (red) relative to WT. (C) Alignment of conserved human, hamster and murine sequences on HUMMR mRNA that are complementary to m1/m2 elements on U17 snoRNA. (D) HUMMR mRNA abundance in WT, KD and SNO+ was quantified by qRT-PCR. (E) HUMMR protein level in WT, KD and SNO+ was measured by western blotting (above) and quantified (below). (F) qRT-PCR quantification of co-precipitated of U17 snoRNA (black), mutated U17 snoRNA (white) and U33 snoRNA (gray) in pulldown of MS2-tagged HUMMR mRNA in the presence or the absence of GST-tagged MS2 binding protein (MS2BP-GST). (G) HUMMR mRNA newly synthesized during a 30 min pulse labeling with ethenyl uridine in WT CHO and mutant I5 were quantified by qRT-PCR (left). HUMMR mRNA was then quantified during chase in media lacking label to determine half-life in WT CHO and mutant I5 (right). Data presented are mean + SE for 3 independent measurements. ns, non-significant. *, ρ<0.05; **, ρ<0.01; and ***, ρ<0.001 for KD or SNO vs. WT or for comparisons indicated

These experiments provided murine cell lines that were well suited for analysis of potential downstream targets using commercially available microarrays. We hypothesized that if U17 snoRNA regulates cholesterol homeostasis by modulating the expression of target mRNAs, genes that are differentially expressed by the loss-of-function or gain-of-function of U17 snoRNA would be candidate U17 targets. One possible model is negative regulation of targets by U17 snoRNA, in which increasing gene dosage of U17 snoRNA from KD to WT to SNO+ would lead to decreases in target levels. Comparison of the transcriptomes of KD and SNO+ cell lines with WT identified 145 genes differentially upregulated in KD and 22 genes differentially downregulated in SNO+, relative to WT (Figure 4B). Of these, only 3 genes that were upregulated in KD were also downregulated in SNO+. Of these, we chose to focus on hypoxia up-regulated mitochondrial movement regulator (HUMMR), which contained sequences complementary to the U17 snoRNA m1/m2 motifs (Figure 4C) and whose expression inversely correlated with U17 snoRNA expression both at the mRNA and protein level (Figure 4D and E). This relationship was also validated in the I5 mutant, which revealed a 3-fold increase in HUMMR mRNA expression relative to WT CHO (Figure S4). The increased HUMMR expression normalized when mutant I5 was rescued by stable expression of U17 snoRNA in SNO+ cell line and when U17 snoRNA was stably re-introduced to U17 KD cells (KD+).

To determine if U17 regulates HUMMR mRNA through a physical interaction, we transiently expressed U17 snoRNA and HUMMR mRNA tagged with the MS2 aptamer at the 3′ UTR and used GST-tagged MS2 binding protein (MS2BP-GST) in pull-down experiments (Yoon et al., 2012). Pulldown of HUMMR by MS2-binding protein affinity purification resulted in significant enrichment of U17 snoRNA compared to the box C/D snoRNA U33, which was used as an unrelated snoRNA control (Figure 4F) Enrichment was significantly greater with WT U17 snoRNA compared to m1/m2-mutated U17 snoRNA, suggesting the m1/m2 element is required for interaction between U17 snoRNA and HUMMR mRNA. To determine how steady-state level of HUMMR mRNA is increased by reduction in U17 snoRNA expression, we measured the de novo transcription and half-life of HUMMR mRNA in WT CHO and mutant I5 by pulse/chase labeling with ethenyl uridine, a clickable uracil analog. Although there were no difference in HUMMR transcription between WT CHO and mutant I5 (Figure 4G, left), the half-life of HUMMR mRNA in mutant I5 (t1/2 167 min) was ~5-fold greater than in WT CHO (t1/2 34 min) (Figure 4G, right), suggesting the HUMMR mRNA was stabilized by the down-regulation of U17 snoRNA.

HUMMR acts downstream of U17 snoRNA to regulate PM cholesterol esterification

HUMMR was previously identified as an outer mitochondrial membrane (OMM) protein that serves as an adaptor for mitochondrial movement (Li et al., 2009). Based on the cholesterol trafficking phenotype in the I5 mutant and knockdown cell lines, we hypothesized that HUMMR acts downstream of U17 snoRNA to regulate PM-derived cholesterol esterification. To test this hypothesis, we generated stable NIH3T3 cell lines in which HUMMR expression was either knocked down (shHUM) or overexpressed (HUM). HUM clones phenocopied HUMMR up-regulation by U17 snoRNA knockdown and led to decrease in cholesterol esterification (Figure 5A). Conversely, shHUM clones phenocopied HUMMR down-regulation by U17 snoRNA overexpression by increasing cholesterol esterification (Figure 5B). These results indicated that HUMMR modulates delivery of cholesterol to the ACAT-accessible pool in a pathway that is either parallel to or downstream of U17 snoRNA-mediated cholesterol trafficking. To differentiate between these possibilities, cell lines with double knockdown of both U17 snoRNA and HUMMR were generated. Knockdown of HUMMR expression either in the mutant I5 (Figure 5C) or the NIH3T3 fibroblasts with U17 snoRNA KD (Figure 5D) resulted in increased esterification relative to scrambled controls. In both cases, knockdown of up-regulated HUMMR was sufficient to restore cholesterol esterification to that of WT. This is consistent with the cholesterol trafficking phenotype exhibited by knockdown of HUMMR alone, and suggests that HUMMR functions downstream of U17 snoRNA in this pathway.

Figure 5. HUMMR acts downstream of U17 snoRNA to regulate PM cholesterol esterification.

Figure 5

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(A and B) Graphs in left side of panels show esterification of PM-derived cholesterol in clonal NIH3T3 lines with stable overexpression (HUM) or knockdown (shHUM) of HUMMR. Cell lines from empty vector (EV) and scrambled shRNA plasmid (shSCR) were controls. The levels of HUMMR protein quantified from western blotting are shown on the right. (C and D) Stable knockdown of HUMMR in mutant I5 cell line (C) or in NIH3T3 U17 KD cell line (D). Clones were assessed by esterification phenotype relative to clones that received shSCR (left). Levels of HUMMR mRNA are shown on right. Data presented are mean + SE for 3 independent measurements. (E) Levels of 27-HC and pregnenolone in WT and U17-KD NIH3T3 lines and in WT CHO and I5 mutant were quantified. (F) Cholesterol from isolated mitochondria were measured in WT, U17-KD and SNO+ in NIH3T3-derived cell lines. (G–I) Following a pulse with 3H-cholesterol, ER cholesteryl ester (G) and mitochondrial cholesterol (H) were measured in WT CHO and mutant I5 cells. Correlation between ER cholesteryl ester decrease and mitochondrial cholesterol increase is graphed (I). SE on plots in G and H are contained within the symbols. *, ρ<0.05; **, ρ<0.01; and ***, ρ<0.001 for comparisons indicated

HUMMR attenuates esterification of PM-derived cholesterol by diverting cholesterol to mitochondria

The blunted esterification of PM-derived cholesterol in the setting of upregulation of HUMMR, an outer mitochondrial membrane adaptor protein, raised the possibility that the altered cholesterol esterification in the U17 snoRNA-deficient I5 mutant reflects diversion of cholesterol from the ACAT-accessible pool in the ER to the mitochondria, rather than a defect in delivery of PM cholesterol to the ER. We reasoned that HUMMR might facilitate cholesterol flux to mitochondria by promoting close apposition between ER and mitochondrial membranes. Indeed, it is known that lipid trafficking between ER and mitochondria occurs at sites where ER membrane and the OMM are in close contact due to formation of specialized complexes, referred to as mitochondrial-associated ER membranes (MAM) (Hayashi et al., 2009; Prinz, 2014; Rowland and Voeltz, 2012). We found that cell lines with upregulated HUMMR expression – CHO I5 mutant and NIH3T3 U17 snoRNA KD cells – exhibited increased mitochondrial uptake of cholesterol, assessed by increased conversion of cholesterol by mitochondria-specific enzymes to 27-hydroxycholesterol in NIH3T3 (Figure 5E, left) or pregnenolone in the CHO-derived I5 mutant (Figure 5E, right) (Charman et al., 2010; Kennedy et al., 2012), consistent with the different mitochondrial enzymes expressed in each cell type. Similarly, HUMMR overexpression resulted in increased formation of 27-HC in the NIH3T3 cells (Figure S5A). Cholesterol levels in isolated mitochondria were also elevated in U17 snoRNA KD relative to WT NIH3T3 (Figure 5F). Increased level of 27-HC suggests that synthetic capacity for oxysterols in mitochondria is unperturbed in the setting of MAM induction by HUMMR up-regulation. Furthermore, both the U17 snoRNA knockdown and overexpressor cell lines had normal MitoTracker staining (Figure S5B), providing further evidence for actively respiring mitochondria. While Mitosox staining was elevated ~2-fold in the cells with HUMMR upregulation (Figure S5C), there was no evidence that the increased HUMMR expression causes cell death (Figure 1A).

To address whether PM-derived cholesterol that fluxes through the ER is being diverted to mitochondria, we measured rates of cholesterol esterification and delivery of cholesterol to mitochondria following a pulse of 3H-cholesterol to the PM. Cholesterol esterification was decreased and mitochondrial cholesterol increased in the U17 snoRNA-haploinsufficient cells (I5 mutant) compared to WT cells (Figure 5G and H). We further found a significant linear relationship (R2=0.89, Figure 5I) between the reduction in cholesteryl ester and the increase in mitochondrial cholesterol. This relationship is not stoichiometric, in that the absolute reduction in CE mass was only ~1% of the increase in mitochondrial cholesterol mass (note axes in Figure 5I), a finding consistent with prior studies showing that only a small fraction of the total cholesterol that transits through the ER exceeds threshold for esterification by ACAT (Chang et al., 1997). The total amount of cholesterol in the ER and mitochondria compartments is roughly equivalent, and the rate of ester formation is used here as a proxy for the change in total ER cholesterol, for which accurate quantification is not feasible. In agreement with our finding that the difference in intracellular cholesteryl ester is specific to the pathway involved in the internalization of PM-derived cholesterol, LDL-stimulated production of pregnenolone (Figure S1E) – a measure of lysosome to mitochondria trafficking – is also intact in the I5 mutant.

To directly assess the effect of HUMMR expression on the extent of ER-mitochondrial contacts, we assessed the relative distribution of mitochondria and ER in WT, U17 snoRNA KD, and U17 snoRNA SNO+ lines using electron microscopy (Figure 6A). Closely apposed interfaces (<40nm) formed by ER wrapping around mitochondria were increased in U17 snoRNA KD as compared to WT cells (Figure 6B). Moreover, the combined length of the interfaces formed per mitochondria was increased in U17 snoRNA KD relative to WT (Figure 6C). Although not statistically significant, a trend towards fewer and shorter interfaces was observed in U17 snoRNA SNO+ relative to WT. Enrichment of GRP75, a MAM protein in the crude mitochondrial fraction of U17 snoRNA KD relative to WT NIH3T3 (Figure 6D), suggested that the enhanced mitochondrial contacts with the ER were associated with formation of MAM structures (Szabadkai et al., 2006).

Figure 6. HUMMR attenuates esterification of PM-derived cholesterol by diverting cholesterol to mitochondria.

Figure 6

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(A) Representative electron micrographs from WT, U17 KD and U17 SNO+ cells show distribution of ER in relation to mitochondria. Arrowheads indicate mitochondrial membranes that are in close proximity (<40nm) to ER membranes. Scale bar, 500nm (B) The number of ER-outer mitochondrial membrane interfaces per μm2 is shown for WT, KD and SNO+. (C) The length of interface normalized by mitochondrial (mito) perimeter is shown for WT, KD and SNO+. (D) Western blotting of crude mitochondrial and cytosolic fractions from NIH3T3 WT and KD cells was probed for the MAM glucose-regulated protein 75 (GRP75); calnexin (CNX); HUMMR; and heat shock protein 90 (HSP90). For quantification of the mitochondrial fraction (right panel), GRP75 and CNX densitometry was normalized to mitochondrial DNA content. Data presented are mean +SE. *, ρ<0.05; and **, ρ<0.01

To determine whether cholesterol transfer to the mitochondria can be similarly augmented by other mechanisms that enhance ER-mitochondrial contacts, we measured mitochondrial cholesterol following a cholesterol pulse in the presence and the absence of tunicamycin – an inhibitor of glycosylation and ER stress inducer that also promotes ER-mitochondrial contacts. When WT NIH3T3 cells were treated with 0.5μg/ml tunicamycin for 4hrs – a concentration at which ER-mitochondrial contacts and ER stress are induced (Bravo et al., 2011), esterification of PM-derived cholesterol was decreased and mitochondrial cholesterol content was increased (Figure S5D–F). While it is possible that tunicamycin-induced ER stress could have affected ACAT activity and stimulated cholesterol transfer to mitochondria, we found that inhibition of ACAT alone was unlikely to explain the enhanced trafficking of cholesterol to mitochondria (Figure S5G). These findings indicate that induction of ER-mitochondrial contacts – whether by HUMMR-dependent or -independent mechanisms – is sufficient to promote channeling of ER cholesterol to mitochondria.

Reciprocal regulation of U17 snoRNA and HUMMR in steroidogenic tissue

HUMMR is highly expressed in steroidogenic tissues and is regulated by the hypothalamic-pituitary-gonadal axis in mice (Matsumoto et al., 2009; Zhou et al., 2011). Since cholesterol is an obligate precursor for mitochondrial steroid synthesis, we examined the U17 snoRNA-HUMMR pathway in steroidogenic tissues. Tissues with higher HUMMR expression (e.g., testes, ovaries, and adrenal glands) demonstrated relatively lower expression of U17 snoRNA (Figure 7A). We hypothesized that if U17 snoRNA negatively regulates HUMMR expression, then during gonadal maturation U17 snoRNA expression would be expected to decrease as HUMMR expression increases with age, promoting synthesis of steroid hormones increases in gonadal tissues. To test this reciprocal relationship, we profiled expression of these genes in ovarian tissue of mice during postnatal development. As HUMMR mRNA abundance increased from postnatal day 5 to week 8, U17 snoRNA expression steadily declined (Figure 7B). This reciprocal relationship was consistent with a role for U17 snoRNA in gonadal tissue maturation through regulation of HUMMR and cholesterol channeling to mitochondria. To establish a causal relationship between decrease in U17 snoRNA and increase in HUMMR mRNA and steroid hormone production in vivo, we utilized antisense locked nucleic acid (LNA) oligonucleotides to transiently knockdown U17 snoRNA (Michel et al., 2011). Anti-U17 or anti-GFP (control) LNA were injected intraperitoneally in 3-week-old mice every other day for a week (three injections), and ovarian tissue was harvested 2 days following the last injection. Mice injected with anti-U17 LNA showed ~30% knockdown of U17 snoRNA relative to anti-GFP control (Figure 7C), which resulted in a ~2-fold increase in HUMMR mRNA and protein level (Figure 7D and E). Upregulation of HUMMR led to ~1.5–1.6-fold increase in pregnenolone and progesterone in ovarian tissue in the U17 knockdown group relative to control (Figure 7F and G). Together, these findings provide support for a physiological role for U17 snoRNA/HUMMR in regulation of steroid hormone production in ovarian tissue in vivo.

Figure 7. Regulation of steroidogenesis in ovarian tissue by U17 snoRNA and HUMMR.

Figure 7

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(A) HUMMR mRNA and U17 snoRNA were quantified in tissues of eight-week-old mice by qRT-PCR (normalized to Gapdh). 3β-hydroxysteroid dehydrogenase (3βHSD) is shown as a marker of steroidogenic ceIls. (B) U17 snoRNA and HUMMR mRNA expression in postnatal developing ovaries was assessed by qRT-PCR and normalized to 18S rRNA. 1 wk corresponds to postnatal day five. (C–G) Effect of treatment with GFP LNA (control, n=7) or U17 LNA (U17 snoRNA, n=10) in 3 week-old mice. U17 snoRNA (C) and HUMMR mRNA (D) expression were assessed by qPCR (normalized to Gapdh). HUMMR protein expression (E) was determined by western blotting. (F and G) Levels of pregnenolone (F) and progesterone (G) in ovarian tissue were quantified by mass spectrometry-based assays. (H) Model of U17 snoRNA-HUMMR cholesterol trafficking pathway. In the presence of U17 snoRNA (left panel), HUMMR expression is suppressed. Cholesterol cycling between the PM and ER is directed to an ACAT-accessible pool and is esterified, with only basal level of cholesterol flux to mitochondria. Deficiency of U17 snoRNA (right panel) alleviates suppression of HUMMR expression. Because HUMMR promotes ER-mitochondrial contacts, trafficking of PM-derived cholesterol to mitochondria is increased. *, ρ<0.05; **, ρ<0.01 and ***, ρ<0.001 for comparisons indicated

Discussion

In this study we performed a forward genetic screen that identified a non-canonical role for the H/ACA snoRNA U17 in the regulation of cellular cholesterol homeostasis. We established that haploinsufficiency of the intron-embedded U17 snoRNA decreased esterification of PM-derived cholesterol by channeling cholesterol away from the ACAT-accessible pool in the ER and instead to the mitochondria. Through expression profiling, we found that the U17 snoRNA negatively regulates the expression of HUMMR, a mitochondrial adaptor protein that participates in mitochondrial motility. In U17 snoRNA-deficient cells, upregulation of HUMMR promotes ER-mitochondrial contacts that facilitate oxysterol and steroid synthesis, revealing a previously unanticipated mechanism for regulation for cholesterol flux to mitochondria. Our in vivo studies showing that expression of U17 snoRNA and HUMMR is developmentally regulated in postnatal steroidogenic tissues and that antisense-mediated down-regulation of U17 snoRNA in ovarian tissue results in up-regulation of HUMMR and accelerated production steroid hormones suggest that the U17 snoRNA-HUMMR pathway serves a physiological role in regulation of steroidogenesis and gonadal tissue maturation.

Recently, non-coding RNAs have emerged as potent regulators of cholesterol homeostasis. Several microRNA species have been characterized that specifically target cholesterol efflux, a key step in the reverse cholesterol transport pathway. MicroRNA-33 has been shown to regulate reverse cholesterol transport through modulation of expression of ABCA1, the principal cell surface sterol transporter responsible for cholesterol efflux and HDL biogenesis (Marquart et al., 2010; Rayner et al., 2010). This microRNA is embedded within an intron of SREBP2, and inhibits ABCA1, a canonical LXR target, providing yet another mechanism for crosstalk between the SREBP and LXR transcriptional pathways. MiR-26, an LXR-suppressed miRNA, targets cholesterol efflux through inhibition of expression of ABCA1 and ADP-ribosylation factor-like 7 (ARL7) (Sun et al., 2012), and activation of the farnesoid X receptor (FXR) has been shown to upregulate miR144, a microRNA that lowers ABCA1 expression and HDL levels in vivo (de Aguiar Vallim et al., 2013). More recently, we identified a requirement for the U60 box C/D snoRNA in intracellular cholesterol trafficking, which appears to modulate cholesterol trafficking through remodeling of membrane phospholipid composition (Brandis et al., 2013).

U17 snoRNA is the first H/ACA snoRNA to be implicated in regulation of cholesterol homeostasis. H/ACA snoRNAs are known to target rRNAs, tRNAs, and snRNAs for pseudouridylation through binding target sequences via their distal loop. However, emerging evidence suggests that orphan H/ACA snoRNAs that lack complementarity to conventional targets may serve to modify messenger RNAs (Karijolich and Yu, 2011; Schwartz et al., 2014). There is also evidence that snoRNAs may be processed like microRNAs and suppress the expression of target mRNAs (Ender et al., 2008). These findings raise the possibility that U17 snoRNA, which also lacks complementarity to canonical pseudouridylation targets, may function through modulation of unconventional targets. Indeed, we found mRNAs that are differentially regulated by loss-of-function or gain-of-function of U17 snoRNA, one of which was HUMMR. HUMMR does not have conserved sites that are complementary to the distal loop of U17 hairpin pocket, which would be anticipated for a role in pseudouridylation, and we have been unable to detect pseudouridylated residues on the endogenous HUMMR mRNA (not shown). Rather, we identified sites in HUMMR that are complementary to m1/m2 elements located in the proximal loop of 3′ hairpin of U17 snoRNA. Our observation that exogenous expression of the snoRNA with mutated m1/m2 elements fails to suppress HUMMR expression and rescue the cholesterol trafficking phenotype in mutant I5 indicates that m1/m2 are functionally relevant motifs of U17 snoRNA. Moreover, the m1/m2 motifs are required for physical interaction between U17snoRNA and HUMMR mRNA. Future studies will be needed to determine the precise domains within the HUMMR RNA required to support regulation by U17 snoRNA. Delineation of the proteome that associates with U17 snoRNA, beyond the H/ACA snoRNA core proteins, may also shed light on U17-directed modifications of target RNAs.

Trafficking of cholesterol to mitochondria occurs through multiple channels from endolysosomes, PM, and ER compartments. In contrast to the transfer of cholesterol from endolysosomal membranes to outer mitochondria membranes (Alpy and Tomasetto, 2005; Charman et al., 2010), the mechanisms of cholesterol trafficking from the ER to mitochondria are less well characterized. Contact sites in the MAM, which are sites of lipid exchange between ER and mitochondria, might likewise mediate cholesterol transfer (Vance, 1990). Our findings support a model in which the coordinated actions of U17 snoRNA and HUMMR regulate the formation of such ER-mitochondria contact sites (Figure 7H). Under basal conditions U17 snoRNA destabilizes HUMMR mRNA and lowers HUMMR protein expression, thereby restricting cholesterol movement to mitochondria. By contrast, in the setting of decreased U17 snoRNA expression or U17 snoRNA-deficiency – as is the case for the CHO I5 mutant isolated in our genetic screen – HUMMR mRNA and protein expression are increased, facilitating MAM formation and ER to mitochondria cholesterol flux. Although HUMMR is induced by HIF-1 under hypoxic conditions, all phenotypic assessments in our study were performed under normoxic conditions. Thus, regulation of HUMMR expression by U17 snoRNA appears not to be restricted to hypoxic conditions. The reduced cholesterol esterification phenotype, therefore, is likely a consequence of diversion of the rapidly cycling PM-derived cholesterol from an ACAT-accessible pool to the MAM contact sites. Mitofusin 2 is an OMM protein that has been reported to have a role in mitochondrial fusion, tethering of ER to mitochondria, and most recently axonal mitochondria trafficking (Chen et al., 2003; de Brito and Scorrano, 2008; Misko et al., 2010). In these settings, HUMMR was shown to interact with miro1, miro2 and Milton, adaptor proteins that also link mitochondria to the motor protein kinesin. Whether Mfn2 and HUMMR participate in a common tethering complex remains to be determined. On the other hand, there is no evidence that HUMMR interacts either physically or functionally with StAR D1, another OMM protein critical for acute steroidogenesis through its role in promoting cholesterol flux from OMM to IMM (Bose et al., 2002a, b).

In steroidogenic cells, trafficking of cholesterol to mitochondria is critical for generation of pregnenolone, the first committed step in steroid synthesis through the actions of the mitochondrial cytochrome P450scc (Miller, 2011). The reciprocal relationship between U17 snoRNA and HUMMR mRNA abundance in the developing mouse ovary suggests that the U17 snoRNA-HUMMR pathway may play a role in regulating steroid hormone production and postnatal gonadal maturation. Enrichment of HUMMR in steroidogenic tissues and its regulation under hypothalamic-pituitary-gonadal axis is consistent with such a role (Matsumoto et al., 2009; Zhou et al., 2011). Our findings that the developmental decline in U17 snoRNA expression is concomitant with the de-repression of HUMMR expression and the steroidogenic demand, and that in vivo knockdown of U17 snoRNA increases HUMMR expression and steroid synthesis, implicates U17 snoRNA as an upstream regulator that modulates mitochondrial cholesterol import. Together, these findings strongly supports a physiological role for U17 snoRNA in steroid hormone production and gonadal maturation via regulation of HUMMR.

Experimental procedures

Additional details of methods are available online in the Supplemental Experimental Procedures.

Cell culture

CHO-K1 cells and NIH3T3 cells (American Type Culture Collection) were cultured as previously described (Millard et al., 2000; Ory et al., 1996). For esterification assay and measurement of RNA expression, cells were maintained in 5% lipoprotein deficient media (LPDS) (CoCalico Biologicals).

Generation of Amphotericin-resistant CHO cell mutants

Mutant CHO-K1 clones (Friedrich and Soriano, 1991; Ory et al., 1996) were isolated following the amphotericin B/LDL selection as described (Brandis et al., 2013). The number of retroviral insertions within the mutant cell genome was assessed by Southern blotting. Briefly, genomic DNA was digested with restriction enzymes and separated by 0.8% agarose gel electrophoresis, transferred to nylon membranes, and probed with a 32P-labeled probe (5′-CTCAGGTCAAATTCAGACGG-3′) corresponding to the ROSAgeo proviral sequence.

Cell death assays

Cell death was assessed by membrane permeability to PI staining as described previously (Listenberger et al., 2001). The percentage of PI-positive cells was determined by flow cytometry or by Tecan Infinite M200 at excitation/emission wavelengths of 535nm/620nm.

Measurement of PM-derived cholesterol esterification

Measurement of esterification of PM-derived cholesterol was performed as described (Brandis et al., 2013). Cells were split from confluency by one-third and grown for 24 hours. These cells were then seeded at 2.5 × 104 per well in a 6 well dish for 24 hours. Media was changed to 5% LPDS for 24 hours. Cells were then pulsed for a given time with 1 μCi/ml [3H]-cholesterol in 1 ml 5% LPDS media. Cells were washed with cold Tris-buffered saline for 10 min at 4°C. Lipids were extracted from each well in 3 ml 3:2 hexane:isopropanol and dried under nitrogen. 14C-cholesteryl oleate was added to lipid extracts as a recovery standard. Cholesteryl esters (CE) were separated from cholesterol by thin layer chromatography (TLC) in a 130:30:2 heptane/ethyl ether/acetic acid solvent using 100 μg and 30 μg cholesterol and CE as carriers, respectively, and visualized by iodine. Percent recovery of [3H]-CE was assessed by recovery of [14C]-cholesteryl oleate. [3H]-cholesterol esterification was measured as a ratio of recovery-normalized [3H]-CE to total [3H]-cholesterol.

Measurement of trafficking of PM-derived mitochondrial cholesterol to mitochondria

Cells were plated and pulsed with [3H]-cholesterol as described for the cholesterol esterification assay. Mitochondria were isolated using the Mitochondria Kit for Cultured Cells (Thermo), solubilized in 0.1N NaOH, and incorporation of [3H]-cholesterol into mitochondria determined by scintillation counting.

Microarray sample preparation and data analysis

Total RNA were isolated by TRIzol reagent (Invitrogen) from WT, U17 snoRNA KD (ShSnhg3 stable clone), and U17 snoRNA OE (pMDβglobinU17b stable clone) cells grown in LPDS media for 24 hours, and integrity of RNA was assessed by BioAnalyzer 2100 (Agilent Technolgies). cDNA was prepared with oligodeoxyribose thymidine (oligo-dT) to select for poly-A RNA. Microarray data was generated with using Illumina mouse-6 beadschip microarray at Genome Technology Access Center (Washington University School of Medicine). The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE67569 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67569). Significance analysis of microarrays tool was used to find differentially expressed genes in comparison between WT and KD (comparison 1) and WT and OE (comparison 2), at false discovery rate of 0%. Genes overlapping between two comparisons and whose direction of expressional change is opposite were selected.

Measurement of nascent mRNA and mRNA half-life

Amount of newly synthesized mRNA was assessed by click-it nascent RNA capture kit (Invitrogen). Briefly, cells were incubated with 0.5mM of ethylene uridine in DMEM for 30 min and harvested for total RNAs. For half-life measurement, cells were chased in normal growth media for indicated time points followed by RNA harvest. EU-labeled RNAs were then chemically clicked to biotin, pulled down by streptavidin bead and purified. Isolated RNAs were reversed transcribed and quantified by qRT-PCR.

Statistics

Results are expressed as mean ± SE. For group comparisons, the statistical significance of differences in mean values was determined by a two-tailed Student’s t test using Graphpad Prism 6 for Mac OS X (version 6.0b). A p value of 0.05 or less was considered significant.

Supplementary Material

1
2

Highlights.

  • Haploinsufficiency of U17 snoRNA alters intracellular cholesterol trafficking

  • HUMMR acts downstream of U17 snoRNA to regulate cholesterol delivery to mitochondria

  • U17 snoRNA and HUMMR developmentally regulate steroidogenesis in vivo.

Acknowledgments

This work was supported by grants to D.S.O. (HL1033001) and J.E.S. (DK064989), and by the Washington University Diabetes Research Center (P30 DK020579). S.J. was supported by NIH training grant T32 HL727534. A.R. was supported by a Science Education Grant from the Howard Hughes Medical Institute.

Footnotes

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Supplementary Materials

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NIHMS680180-supplement-1.docx (177.4KB, docx)
2
NIHMS680180-supplement-2.docx (4.1MB, docx)

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