FIG. 8.

hU3-55k elements required for U3 snoRNA binding. (A) VSV-55k, VSV-55kΔC, 55kΔN-VSV, 55k-VSV, and 55k were transiently expressed in HeLa cells. Two days after transfection, cells were lysed and used for immunoprecipitations with anti-VSV (αVSV), antifibrillarin (αFibrillarin), and anti-U2B" (αU2B") antibodies. RNAs were extracted from immunoprecipitates (lanes 1 through 15) and total cell extracts (lanes 16 through 20), resolved by PAGE, and transferred to a Northern blot. Precipitated U3 snoRNA was detected by hybridization with an antisense U3 probe. Lanes 1 through 5, anti-VSV immunoprecipitations; lanes 6 through 10, antifibrillarin immunoprecipitations; lanes 11 through 15, anti-U2B" immunoprecipitations; lanes 16 through 20, RNA isolated from 5% of indicated total cell extracts used for immunoprecipitation. (B) Schematic representations of the VSV-tagged proteins used for transfection studies and a summary of their abilities to accumulate in the nucleolus and to associate with U3 snoRNA. Amino acid numbers are indicated, and different domains of hU3-55k (bipartite NLS, glutamic acid-rich region [E], and WD-40 repeats) are boxed. No, nucleolar accumulation; Nu, nuclear accumulation; Cy, cytoplasmic accumulation; +, binding; −, no binding.