HsQ96HR8 (hNaf1) is the human ortholog of yeast Naf1p. (A) Expression of HsQ96HR8 improves growth of yeast following Naf1p depletion. Yeast strain GAL::naf1, in which the NAF1 gene is transcribed from the GAL1-10 promoter, was transformed with an empty expression vector (line empty vector) or derivatives thereof containing wild-type yeast NAF1 (line scNaf1p) or the human HsQ96HR8 gene (line hNaf1) transcribed from the GAR1 promoter and tagged with the ZZ sequence encoding two IgG-binding domains. The resulting transformed strains were grown in galactose-containing medium, washed, and grown for a further 24 h in glucose-containing medium. Serial dilutions of samples from galactose- or glucose-grown cultures were plated on galactose- or glucose-containing plates, respectively. (B) Expression of HsQ96HR8 increases steady-state levels of yeast H/ACA proteins Cbf5p, Gar1p, and Nop10p or (C) steady-state levels of yeast H/ACA snoRNAs, following depletion of endogenous yeast Naf1p. The transformed GAL::naf1 strains described in A were grown in galactose-containing medium, washed, and transferred to glucose-containing medium to repress transcription of chromosomal NAF1. Samples from galactose-grown cultures (GAL, lanes 1,5,9) or from cultures grown for 24 h (lanes 2,6,10), 48 h (lanes 3,7,11), or 72 h (lanes 4,8,12) were collected, from which total proteins (B) or RNAs (C) were extracted. In panel B, total proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a cellulose membrane. Specific proteins were detected by enhanced chemiluminescence using antibodies listed in the “Materials and Methods” section. In panel C, total RNAs were separated by denaturing acrylamide gel electrophoresis and transferred to nylon membranes. Specific RNAs were detected by hybridization with antisense oligonucleotide probes. Signal intensities were measured by PhosphorImager scanning. Values (indicated below each lane) are expressed as percentages of the intensities obtained in the case of galactose-grown samples. (D) HsQ96HR8 interacts with yeast box H/ACA snoRNP proteins. Transformed GAL::naf1 strains expressing the ZZ tag alone (lanes 1,2), tagged yeast Naf1p (lanes 3,4), or tagged human HsQ96HR8 expressed from the TDH3 promoter (lanes 5,6) were grown in galactose-containing medium, washed, and transferred to glucose-containing medium for 24 h. Total extracts were prepared and immunoprecipitation experiments were carried out using IgG-Sepharose beads. After precipitation and washing, beads were resuspended in protein denaturing buffer. Aliquots of the resulting supernatants (IP, lanes 2,4,6) and 1/20th of the corresponding input extract (Input, lanes 1,3,5) were submitted to SDS-PAGE and Western analysis as described in B. The upper band detected in lane 4, anti-Nhp2p Western, probably corresponds to a breakdown product of ZZ-tagged yeast Naf1p. (E) H/ACA snoRNA levels in naf1Δ cells expressing wild-type yeast Naf1p (lane 1) or human HsQ96HR8 (lane 2) from a plasmid. RNAs were analyzed as described for panel C.

hNaf1 interacts with components of box H/ACA snoRNPs, box H/ACA scaRNPs, and telomerase. Immunoprecipitations were carried out using purified anti-hNaf1 antibodies (αhNaf1) or non-immune serum (NIS) and HeLa cell extracts. (A) Western analysis of hNaf1-associated proteins. After precipitation and washing, protein A-agarose beads were resuspended in protein denaturing buffer. Aliquots of the resulting supernatants (lanes 2,3) or 3% of the corresponding input extract (Input, lane 1) were submitted to SDS-PAGE and Western analysis as described in Figure 1B. The strong bands labeled “IgG” correspond to immunoglobulins detached from the beads during pellet resuspension after the precipitation. (B) Detection of small RNAs associated with hNaf1. RNAs extracted from the pellets obtained following precipitation (lanes 2,3) or from an amount of input extract (Input) corresponding to 3% of that used for precipitations were analyzed by Northern blot (to detect snRNAs, snoRNAs, and scaRNAs) as described in the legend of Figure 1C, or RNase protection to detect hTR.

hNaf1 is required for normal accumulation of box H/ACA RNP and telomerase components. (A) Western analysis of the consequences of the reduction of fibrillarin, hNaf1, or dyskerin levels obtained by RNA interference. HeLa cells were mock-transfected (No siRNA) or transfected with double-stranded siRNAs targeting fibrillarin, hNaf1, or dyskerin mRNAs and grown for 60 h. Total proteins were then extracted, separated, and analyzed by the Western blot procedure as described in the legend of Figure 1. (B) Fluorescence in situ immunodetection of hNaf1 (red) and fibrillarin (green) in HeLa cells mock-transfected (No siRNA) or transfected with double-stranded siRNAs targeting hNaf1 or dyskerin mRNAs. Bar, 10 μm. (C) Analysis of small RNA levels following siRNA treatment. HeLa cells were transfected with siRNAs as described in A. After 60 h of incubation, total RNAs were extracted. U17, U19, U92, U3, and U6 (acting as an internal control) were then separated and analyzed by Northern blot, while U13 and hTR were analyzed by RNase protection. U17, U19, and U3, on the one hand, and U92, on the other, were analyzed on different blots. For both procedures, equal amounts of total RNA were used for all samples. Signal intensities were measured by PhosphorImager scanning. Values (indicated below each lane) are expressed as percentages of the intensities obtained in the case of mock-transfected samples (No siRNA).

hNaf1 is located in numerous discrete nucleoplasmic foci. Subcellular localization of hNaf1 in HeLa cells. Bar, 10 μm.