FIGURE 3.

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).