Figure 4. Regulation of HUMMR by U17 snoRNA.
(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