Components of U3 snoRNA-containing complexes shuttle between nuclei and the cytoplasm and differentially localize in nucleoli: implications for assembly and function

Abstract

U3 small nucleolar RNA (snoRNA) and associated proteins are required for the processing of preribosomal RNA (pre-rRNA) and assembly of preribosomes. There are two major U3 snoRNA-containing complexes. The monoparticle contains U3 snoRNA and the core Box C/D snoRNA-associated proteins and an early preribosome-associated complex contains the monoparticle and additional factors that we refer to as preribosome-associated proteins. To address how and where the U3 snoRNA-containing preribosome assembles and how these processes are temporally and spatially regulated, we have examined the dynamics and distribution of human U3 complex-associated components in cells with active or inactive transcription of rDNA. We found that U3 complex-associated proteins shuttle between the nucleus and the cytoplasm independent of the synthesis and export of preribosomal particles, suggesting that the shuttling of these proteins may either provide opportunities for their regulation, or contribute to or modulate ribosome export. In addition, monoparticle and preribosome associated components predominantly localize to different nucleolar substructures, fibrillar components, and granular components, respectively, in active nucleoli, and partition separately into the two components during nucleolar segregation induced by inhibition of pol I transcription. Although the predominant localizations of these two sets of factors differ, there are significant areas of overlap that may represent the sites where they reside as a single complex. These results are consistent with a model in which U3 monoparticles associate with the fibrillar components of nucleoli and bind pre-rRNA during transcription, triggering recruitment of preribosome-associated proteins to assemble the complex necessary for pre-rRNA processing.

Figure 1.

U3 snoRNP proteins shuttle between nuclei and the cytoplasm at different rates. (A) Untransfected HeLa were fused in heterokaryon assays with HeLa transfected with constructs encoding either negative (GFP-UBF) or positive (GFP-PTB) controls, U3 snoRNP core proteins GFP-fibrillarin or -U3-55K (left) or the preribosome-associated proteins GFP-Imp3, -Imp4, -Mpp10, -Rcl1, or -Sof1 (right). Two cell heterokaryons were analyzed 2 h after fusion. DNA was labeled with DAPI to delineate nuclei and nucleoli. Thick arrows indicate nucleoli of transfected cells, thin arrows the nucleoli of untransfected cells. Bar, 10 μm. (B) Quantitative analyses of the heterokaryon assays represented in A were done to compare the relative shuttling rates of the proteins analyzed in A. For all constructs besides GFP-PTB, the F_R (y-axis) was measured as fluorescence intensity in the untransfected cell nucleolus (F_U)/fluorescence intensity in the transfected cell nucleolus (F_T) within a same-sized area. For GFP-PTB, nucleoplasmic rather than nucleolar fluorescence intensity was used.

Figure 2.

Nucleocytoplasmic shuttling of U3 snoRNP proteins does not require rDNA transcription or Crm1-mediated export. (A) Untransfected HeLa were fused in heterokaryon assays with HeLa transfected with constructs encoding either core proteins GFP-fibrillarin or -U3-55K or the preribosome-associated proteins GFPImp3, -Imp4, -Mpp10, -Rcl1, or -Sof1. The cells were pretreated for 2 h and grown postfusion for 2 h in the presence or absence (untreated) of 0.04 μg/ml actinomycin D. Quantitative analysis was done to compare the relative shuttling rates of the proteins. The F_R (y-axis) was measured as fluorescence intensity in the untransfected cell nucleolus (F_U)/fluorescence intensity in the transfected cell nucleolus (F_T) within a same sized area. Error bars indicate standard deviations. (B) Heterokaryon assays were done as described above except that the cells were pretreated for 1 h prefusion and grown for 2 h postfusion in the presence of 0.2 ng/ml leptomycin B. Quantitation was done as described above.

Figure 3.

In active nucleoli, U3 snoRNA and the core proteins localize to different subnucleolar structures than preribosome-associated proteins. (A) The subcellular localization of U3 snoRNA HHeHe (top) in HeLa cells was determined by in situ hybridization against U3 snoRNA and immunolabeling with antibodies against fibrillarin (top). The subcellular localization of core U3 snoRNP proteins (bottom four panels) was determined by double immunolabeling HeLa cells with antibodies against fibrillarin (middle) and antibodies against either Nop56, Nop58, or U3-55K (left). Overlay images are shown in the right panels. Arrows indicate Cajal bodies. Bar, 10 μm. (B) Subcellular localization of preribosome-associated U3 snoRNA-associated proteins was determined by transfecting HeLa cells constructs encoding either GFP-Imp3, -Imp4, -Mpp10, or -Rcl1 (left column) and immunolabeling the cells with antibodies against fibrillarin (middle column). Overlay images are shown in the right column. Arrows indicate Cajal bodies. Bar, 10 μm.

Figure 4.

In transcriptionally inactive nucleoli, U3 snoRNA and the core proteins localize to different subnucleolar structures than preribosome-associated proteins. (A) To inhibit transcription by RNA polymerase I, HeLa cells were grown for 3 h in the presence of 0.04 μg/ml actinomycin D before fixation. The subcellular localization of U3 snoRNA (top) was determined by in situ hybridization against U3 snoRNA and immunolabeling with antibodies against fibrillarin (top). The subcellular localization of core U3 snoRNA-associated proteins (bottom four panels) under these conditions was determined by double immunolabeling HeLa cells with antibodies against fibrillarin (middle) and antibodies against either Nop56, Nop58, or U3-55K (left). Overlay images are shown in the right panels. Arrows indicate fibrillar caps of segregated nucleoli. Bar, 10 μm. (B) The subcellular localization of preribosome-associated U3 snoRNA-associated proteins was determined by transfecting HeLa cells with GFP-Imp3, -Imp4, -Mpp10, or -Rcl1, treating them as described above, and then immunolabeling the cells with antibodies against fibrillarin. Overlay images are shown in the right panels. Arrows indicate fibrillar caps of segregated nucleoli. Bar, 10 μm.

Figure 5.

Model for the assembly of U3 monoparticles and the formation and function of the U3 snoRNA-containing preribosome in the context of subnucleolar and subnuclear structures.