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. 2002 Dec;22(23):8342–8352. doi: 10.1128/MCB.22.23.8342-8352.2002

FIG. 5.

FIG. 5.

The conserved sequence of stem II of the box C/D motif is required for nucleolar localization. (A) Time-dependent localization of snoRNPs in HeLa nuclei. Fluorescent U14 snoRNA transcripts were microinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C, and images were taken at 2, 30, 60, and 90 min after injection. Three examples of each time point are shown. The time point is indicated above each set of pictures. (B) Injected fluorescent U14 transcripts and 70-kDa dextran differentially localize upon injection into HeLa nuclei. Fluorescent U14 snoRNA transcripts and FITC-labeled 70-kDa dextran were coinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C, and fluorescent images of the labeled RNA and dextran were taken at 2 and 90 min after injection. Three examples at each time point are shown. The time point is indicated above the panels. The Alexa 546-labeled snoRNA is shown in red, and the FITC-labeled dextran image is shown in green. The overlay of the snoRNA and dextran images is shown at the bottom of the figure. (C) The internal loop and conserved stem II of the box C/D motif are both essential for nucleolar localization. Fluorescent snoRNA and snRNA transcripts were microinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C for 90 min, and fluorescent images were taken. Three examples for each injected RNA are shown. The injected RNA is indicated to the right of the images. (D) Mutant U14 snoRNAs injected into HeLa nuclei are stable. Unlabeled U14 mutC and FITC-labeled 70-kDa dextran were coinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C for 90 min. The U14 snoRNA was subsequently detected by fluorescence in situ hybridization. The Cy3 in situ-hybridized U14 snoRNA (U14 FISH) is shown in red, and the FITC-labeled dextran image is shown in green. The injected cell is shown on the right, and the control, uninjected cell is on the left-hand side of the panel. The overlay of the snoRNA and dextran image is shown at the bottom of the figure.