FIG. 3.

Polyadenylated snoRNAs are unstable and do not associate with snoRNP proteins. (A) W303 cells were transformed with the C/D50 construct and grown in 2% raffinose-0.08% glucose (lane 1). Cultures were shifted to 2% galactose for 2 h (lane 2) and then transferred to 4% glucose-containing medium and incubated for 1 h more (lane 3). Northern blot analysis of 5 μg of total RNA was performed with a ³²P-labeled tag-specific oligonucleotide (αtag). The lower panel shows a control hybridization with a snR13 snoRNA-specific probe (αsnR13). (B) Strain YAF2, containing the NOP56-TAP fusion, was transformed with the C/D50 construct. After 2 h of growth in galactose, cells were lysed and immunoprecipitated with IgG beads. RNA was extracted from pellets (P) and supernatants (S) and run on a 6% polyacrylamide-urea gel. Lane T, RNA extracted from nonimmunoprecipitated extracts. Northern blot analysis was performed with αtag. The lower panel shows a control hybridization with a U5 snRNA-specific probe (αU5) which monitors the specificity of the immunoprecipitation. The migration of the size marker (MspI-digested pBR322) is indicated on the left; sizes are in nucleotides.