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• W2005042567 abstract "Article1 October 2001free access Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain Céline Verheggen Céline Verheggen IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author John Mouaikel John Mouaikel IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author Marc Thiry Marc Thiry Laboratoire de biologie cellulaire et tissulaire, Université de Liège, Liège, Belgium Search for more papers by this author Jean-Marie Blanchard Jean-Marie Blanchard IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author David Tollervey David Tollervey ICMB, The University of Edinburgh, Edinburgh, EH9 3JR UK Search for more papers by this author Remi Bordonné Remi Bordonné IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author Denis L.J. Lafontaine Denis L.J. Lafontaine ICMB, The University of Edinburgh, Edinburgh, EH9 3JR UK Present address: IRMW, FNRS-Université Libre de Bruxelles, B-1070 Brussels, Belgium Search for more papers by this author Edouard Bertrand Corresponding Author Edouard Bertrand IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Present address: IRMW, FNRS-Université Libre de Bruxelles, B-1070 Brussels, Belgium Search for more papers by this author Céline Verheggen Céline Verheggen IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author John Mouaikel John Mouaikel IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author Marc Thiry Marc Thiry Laboratoire de biologie cellulaire et tissulaire, Université de Liège, Liège, Belgium Search for more papers by this author Jean-Marie Blanchard Jean-Marie Blanchard IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author David Tollervey David Tollervey ICMB, The University of Edinburgh, Edinburgh, EH9 3JR UK Search for more papers by this author Remi Bordonné Remi Bordonné IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Search for more papers by this author Denis L.J. Lafontaine Denis L.J. Lafontaine ICMB, The University of Edinburgh, Edinburgh, EH9 3JR UK Present address: IRMW, FNRS-Université Libre de Bruxelles, B-1070 Brussels, Belgium Search for more papers by this author Edouard Bertrand Corresponding Author Edouard Bertrand IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France Present address: IRMW, FNRS-Université Libre de Bruxelles, B-1070 Brussels, Belgium Search for more papers by this author Author Information Céline Verheggen1, John Mouaikel1, Marc Thiry2, Jean-Marie Blanchard1, David Tollervey3, Remi Bordonné1, Denis L.J. Lafontaine3,4 and Edouard Bertrand 1,4 1IGMM, IFR 24, UMR 5535 du CNRS, 34293 Montpellier, Cedex 5, France 2Laboratoire de biologie cellulaire et tissulaire, Université de Liège, Liège, Belgium 3ICMB, The University of Edinburgh, Edinburgh, EH9 3JR UK 4Present address: IRMW, FNRS-Université Libre de Bruxelles, B-1070 Brussels, Belgium ‡D.L.J.Lafontaine and E.Bertrand contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:5480-5490https://doi.org/10.1093/emboj/20.19.5480 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis. Introduction The nucleus is a highly organized cellular compartment and >10 structures have been defined by electron or light microscopy in higher eukaryotes (reviewed in Matera, 1999). The nucleus of lower eukaryotes like yeasts appears less complex, and it is unclear whether the domains observed in higher eukaryotes are absent, perhaps being functionally replaced by other structures, or whether they have simply not been identified yet. While the role of the nuclear structures is not always known precisely, they are thought to perform specialized functions. The most conspicuous nuclear domain is the nucleolus, which is found in all eukaryotic cells and is the major site of ribosome synthesis. Another organelle that has been the focus of recent attention is the Cajal or coiled body (CB; Gall, 2000). CBs are found in vertebrates, invertebrates (Yannoni and White, 1997) and plants (Beven et al., 1995), but have not previously been identified in yeast. While their exact function is unknown, CBs are frequently located close to the nucleolus (Bohmann, 1995; see Matera, 1999 for review) and are likely to be involved in the biogenesis of small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs; Bohmann, 1995; Samarsky et al., 1998; Matera, 1999; Narayanan et al., 1999b). A major role of the nucleus is the synthesis of many types of RNA, all of which undergo nuclear processing, followed by transport to either the nuclear pore complex (NPC) for cytoplasmic export, or to specific sub-nuclear locations. Indeed, several post-transcriptional processing reactions require RNA factors, notably snRNAs and snoRNAs, which must be localized correctly. In addition to this initial post-transcriptional targeting event, recent experiments have suggested that the steady-state localization of these ribonucleoprotein particles (RNPs) is highly dynamic, and involves a constant and rapid flux of molecules in and out of the compartment in which they concentrate (Phair and Misteli, 2000). Despite their fundamental importance, the mechanisms responsible for targeting RNA to specific destinations within the nucleus are poorly understood. Most of our knowledge of the mechanisms in intra-nuclear RNA movement stems from analyses of nuclear mRNAs. Analyses of the movement of either bulk polyadenylated RNAs or of specific mRNAs suggest that they move through the nucleoplasm as single molecules by simple diffusion (Femino et al., 1998; Politz and Pederson, 2000). Moreover, pre-mRNAs are believed to assemble into RNP particles and undergo processing—capping, splicing and polyadenylation—at the transcription sites (Zhang et al., 1994; Custodio et al., 1999). During pre-mRNA splicing, export factors of the REF family bind to the matured molecule (Zhou et al., 2000). These recruit TAP, which allows docking of the complex onto the NPCs and subsequent transport to the cytoplasm (see Cullen, 2000 for a review). These and other results suggest a simple general model, in which RNAs assemble into RNP structures and are processed close to the site of transcription. During assembly and processing, the RNAs associate with localization factors that can anchor the RNP to its target compartment, which is reached by diffusion. A complication to this simple model is that RNAs do not always move directly from their transcription site to their final destinations. Newly synthesized snRNAs are proposed to traffic through the CBs prior to association with the NPCs and nuclear export (Matera, 1999; Smith and Lawrence, 2000). Following re-import, the snRNAs again co-localize with CBs and with nucleoli before being detected in the speckles, their final destination (Sleeman and Lamond, 1999). Other nuclear and cytoplasmic RNAs, including pre-tRNAs, the RNA component of RNase P, signal recognition particle (SRP) and telomerase, transiently localize to the nucleolus before reaching their final destinations (Bertrand et al., 1998; Pederson, 1998; Narayanan et al., 1999a). The transient association of these RNPs with nuclear substructures most likely reflects the localization of specific steps in their biogenesis (Pederson, 1998). In order to understand the mechanisms of intra-nuclear RNA transport better, we are studying the nucleolar targeting of snoRNAs. Most snoRNAs select sites of rRNA modification by base pairing to the pre-rRNAs. Based on conserved sequence elements, the snoRNAs are grouped into two families, termed box C/D and box H/ACA, which direct rRNA 2′-O-methylation or pseudouridine formation, respectively (Bachellerie and Cavaillé, 1997; Tollervey and Kiss, 1997). The box C/D motifs associate with four common snoRNP proteins: Nop1p/fibrillarin, Nop56p, Nop5p/Nop58p and Snu13p (Tyc and Steitz, 1989; Wu et al., 1998; Lafontaine and Tollervey, 1999, 2000; Watkins et al., 2000). Previous studies have shown that the box C/D motif is both necessary and sufficient for snoRNA processing, stability and nucleolar localization in vertebrates and yeast (Lange et al., 1998; Samarsky et al., 1998; Narayanan et al., 1999a and references therein). Additionally, the box C/D motif localizes the snoRNAs to CBs (Samarsky et al., 1998), and this is probably an intermediate step on their way to the nucleolus (Narayanan et al., 1999b). In the present study, we took advantage of the yeast model system to carry out a genetic analysis of the roles of the snoRNP proteins in localizing the box C/D snoRNAs to the nucleolus. We report that correct localization requires all four core box C/D proteins, as well as additional nucleolar factors. We further show that the nucleolar transport pathway involves a novel sub-nuclear structure, the nucleolar body (NB), which shares similarities with CBs. Results Overexpression of artificial box C/D snoRNAs induces a specific block in the nucleolar localization pathway The characterization of the various pathways for nuclear RNA export has greatly benefited from competition experiments, in which large amounts of a particular RNA are introduced into cells in order to specifically saturate and block a given pathway. To determine whether box C/D snoRNA localization is also a saturable process dependent on trans-acting factors, and to analyse at which step localization would be blocked, we overexpressed an artificial snoRNA in yeast. U14/MS2x2 contained two binding sites for the phage MS2 coat protein, flanked by the box C/D motif of yeast U14 snoRNA. Because the box C/D motif is both necessary and sufficient for RNA processing and nucleolar targeting, all other sequences of U14 were removed (see Supplementary data, available at The EMBO Journal Online). To stabilize this snoRNA further, the terminal stem of the box C/D motif was extended from 9 to 15 or 22 bp (Huang et al., 1992). U14/MS2x2 was cloned within the actin intron on a multicopy plasmid, and transcription was driven by a strong constitutive promoter (Figure 1A). As controls, we used the same RNA expressed from a centromeric vector, and a similar intronic RNA lacking boxes C and D. Northern blot analysis revealed that the artificial snoRNA was processed and accumulated as expected (see Figure 3C). A minor band of slower mobility, corresponding to unspliced precursors, was also detected. The RNA 5′ end was determined by primer extension (see Supplementary data). The major 5′ end corresponded to the authentic U14 site, but minor forms extended by few nucleotides were also detected, most likely because the long terminal stem slowed final trimming. We have also tested the functionality of U14/MS2x2 as a methylation guide, and found that this snoRNA was indeed capable of efficiently methylating a target RNA at the expected site (see Supplementary data). Figure 1.Overexpressed artificial box C/D snoRNA accumulates in the NB. (A) Schematic of the expression vectors used. The snoRNA is represented by a stem–loop in red. Pm, GPD promoter; BP, branch point of the actin intron; RPR1 leader and trailer, promoter and terminator of the RNase P gene. (B) Localization of the snoRNA reporters by light microscopy. Each field is 5 × 5 μm. The snoRNA (red) was detected by fluorescent in situ hybridization, the nucleolus was labelled with a Gar1–GFP fusion protein (green) and the DNA was stained with DAPI (blue). The artificial snoRNA was expressed from either the RNase P gene (RPR1) on a 2 μ plasmid (CD-2m/e), or the actin intron on a 2 μ plasmid (CD-2m/i), or on a centromeric vector (CD-Cen). A similar RNA lacking boxes C and D was expressed from the actin intron on a 2 μ plasmid, and used as a control (ΔCD). (C) Localization of the snoRNA reporter by electron microscopy. The nucleolus (NU) of different yeast strains (upper left panel: CD-Cen; upper right panel: CD-2m/i) was composed of a fibrillar and a granular component. Upon snoRNA overexpression (CD-2m/i), an additional structure of spherical shape was obvious in the nucleolus (the NB). This fibrillar body exhibited strong immunogold labelling after in situ hybridization using a BrdU-labelled MS2 oligonucleotide probe (lower panel). NP, nucleoplasm. Download figure Download PowerPoint Localization of the snoRNA was determined by fluorescent in situ hybridization with an oligo probe specific for the MS2 sequences, while the nucleolus was visualized with a functional Gar1p–GFP (green fluorescent protein) fusion (Trumtel et al., 2000; Figure 1B). The artificial snoRNA expressed from the centromeric vector localized correctly to the nucleolus (Figure 1B, CD-Cen). In contrast, the intronic RNA lacking boxes C and D was seen in dispersed foci (3–10) in the nucleoplasm (Figure 1B, ΔCD). These are likely to be sites of transcription and processing, as the use of a chromosomal reporter reduced the number of foci to one (data not shown). The artificial snoRNA expressed from the multicopy vector was also localized to the nucleolus, but surprisingly was strongly concentrated in a single sub-nucleolar region (Figure 1B, CD-2m/i). A similar concentration was seen with an artificial snoRNA expressed from a highly active RNA polymerase III promoter (Figure 1A and B, CD-2m/e; Good and Engelke, 1994). Exonucleases can gain direct access to the termini of this transcript, eliminating the requirement for pre-mRNA splicing to produce mature snoRNA molecules. Thus, sub-nucleolar concentration did not depend on a particular snoRNA maturation pathway, but was dependent on snoRNA expression levels. The sub-nucleolar localization of the overexpressed box C/D snoRNAs was further analysed by electron microscopy (Figure 1C, upper right panel). The snoRNA-overexpressing cells showed normal overall morphology, with dense fibrillar and granular components, but contained an additional structure, which we called the NB (Figure 1C). This body was spherical, and in most cells was present at a single copy per nucleus. Notably, the NB was contiguous with the dense fibrillar compartment, in which the snoRNAs are normally located. To demonstrate that the NB corresponded to the site of snoRNA concentration observed in fluorescence microscopy, in situ hybridization was performed at the ultrastructural level (Figure 1C, lower panel). Strong enrichment of the immunogold label was seen in the NB, showing it to be the site of snoRNA accumulation. We conclude that overexpression of the artificial snoRNA blocked a late step in the localization pathway. Rather than becoming dispersed in the dense fibrillar region of the nucleolus, the snoRNAs were restricted to the NB. The NB is involved in the trafficking of endogenous box C/D snoRNA To determine whether transport of endogenous snoRNAs also involves transit through the NB, we utilized in situ hybridization to examine the localization of the box C/D snoRNAs U14 and U3 in the presence of the artificial snoRNA (Figure 2A). U14 and U3 localized throughout the nucleolus in wild-type cells, but became concentrated in the NB in cells overexpressing U14/MS2x2 (Figure 2A, U3 and U14). U14 largely co-localized with the artificial snoRNA reporter whereas a substantial pool of the U3 snoRNA was visible outside the NB, reflecting differential sensitivity to retention in the NB. The common C/D snoRNP proteins Nop1p and Nop5/Nop58p also became highly enriched in the NB (shown for Nop1p in Figure 2A), indicating that many or all box C/D snoRNAs are co-localized. Retention in the NB appeared to be specific for box C/D snoRNAs, as localization of a box H/ACA snoRNA, snR10, and an associated protein, Gar1p, was unaffected (data not shown and Figure 1B, right panels). Figure 2.The NB is a structure hosting several box C/D snoRNAs, and is related to CB. (A) Overexpression of an artificial snoRNA leads to accumulation of endogenous snoRNA in the NB. Yeast cells overexpressing the multicopy, artificial snoRNA were simultaneously labelled for the artificial snoRNA (red), and for endogeneous U14 (green, U14), U3 (green, U3) or Nop1–GFP (green, Nop1). (B) Human SMN localizes in the NB. Human SMN (green) was fused to GFP, and expressed in either wild-type cells (U3), or in cells overexpressing the artificial snoRNA (CD-2m/i). The NB was detected with a probe against the artificial snoRNA (red, CD-2m/i), and the nucleolus with a probe against U3 (red, U3). Download figure Download PowerPoint Figure 3.Antagonistic effects of Srp40p and Nsr1p on snoRNA trafficking. (A) Srp40p is required to accumulate box C/D snoRNA in the NB. The multicopy artificial snoRNA (red) was detected in either wild-type cells (Wild-type), or in isogenic cells deficient in SRP40p (srp40-Δ). SRP40p was fused to YFP (green) and detected in the NB (SRP40::YFP). (B) Nsr1p is required to distribute box C/D snoRNA in the entire nucleolus. The centromeric artificial snoRNA (red) was detected in either wild-type cells (Wild-type), in isogenic cells expressing the N-terminal domain of NSR1p (Nsr1-Nter), or following inactivation of RNA polymerase I (Pol I). The nucleolus was visualized with a Gar1–GFP fusion protein (Gar1, green), and the localization of the N-terminal domain of Nsr1p was visualized through a fusion with YFP (Nsr1-Nter::YFP, green). (C) SnoRNA expression levels in Srp40p and Nsr1p-deficient strains. Northern blots were probed with sequences specific for the artificial snoRNA (upper panels), or for SCR1 RNA for normalization (lower panels). The band corresponding to the mature snoRNA is indicated (91 bases, U14/MS2x2) and an arrow points to the precursor (∼250 bases). The molecular weight markers are shown on the left. Nsr1 (left panel) and Srp40 (right panel) strains were expressing U14/MS2x2 from a centromeric or 2 μ plasmid, respectively. Lane 1, wild-type cells; lane 2, Nsr1-Nter; lane 3, Nsr1-Δ; lane 4, Srp40-Δ; lane 5, wild type. Download figure Download PowerPoint We conclude that an excess of the artificial snoRNA saturates the localization machinery, and that the NB represents an intermediate site in the localization of many, and possibly all, box C/D snoRNAs. The NB shows similarities to the CB In vertebrates, U3 snoRNA transits through the CB en route to the nucleolus (Narayanan et al., 1999b), suggesting possible similarities to the NB. To test this hypothesis, we expressed a CB marker in yeast. The archetypal human CB marker is p80 coilin, but database searches have failed to identify homologues in yeast, Drosophila or Caenorhabditis elegans. In contrast, another CB marker, the survival of motor neuron protein (SMN), has homologues in many eukaryotes, including the fission yeast Schizosaccharomyces pombe (Hannus et al., 2000), although there is no obvious homologue in Saccharomyces cerevisiae. SMN was originally reported to localize in nucleoplasmic structures called gems (gemini of CB; Liu and Dreyfuss, 1996), but gems co-localize with CB in the vast majority of cells (Carvalho et al., 1999). As the distinction, if any, is unclear, we will use the term ‘CB’ to refer to the gems/CB compartment. Human SMN was fused to GFP and the fusion protein was shown to co-localize with the endogeneous protein in mammalian cells (data not shown). To avoid artifacts due to overexpression in yeast cells, the fusion was expressed at low levels, from a centromeric vector, with transcription driven at the basal level of the inducible MET25 promoter. When co-expressed in yeast with the artificial snoRNA, SMN–GFP was seen as a small dot in the nucleus of living cells. Fixation and hybridization with the MS2 probe showed that SMN–GFP co-localized with the NB (Figure 2B, CD-2m). In cells that did not express the artificial snoRNA, SMN–GFP was still concentrated in a small nucleolar region, as shown by double labelling with a probe specific for U3 (Figure 2B, U3). This suggests that the NB is a CB-like domain, which exists even in the absence of snoRNA overexpression. Srp40p and Nsr1p have antagonistic activities on the localization of box C/D snoRNAs to the NB In order to identify the trans-acting factors required for box C/D snoRNA localization, we analysed the localization of U14/MS2x2 in several mutant strains. The mammalian Nopp140 protein associates with both box H/ACA and box C/D snoRNAs, and localizes in CBs and nucleoli (Yang et al., 2000). Strains lacking the yeast homologue of Nopp140, Srp40p, are viable but mildly impaired in growth (Meier, 1996). Deletion of the SRP40 gene did not affect the nucleolar localization of U14/MS2x2 expressed from a centromeric vector (data not shown). However, overexpression of U14/MS2x2 in the -Δ strain did not lead to accumulation in the NB (Figure 3A, srp40-Δ). Northern blot analysis showed similar levels of U14/MS2x2 in the srp40-Δ strain and the isogenic control (Figure 3C). Srp40p was fused to yellow fluorescent protein (YFP), and the fusion protein was shown to be present in the NB in strains overexpressing U14/MS2x2 (Figure 3A, SRP40::YFP). This suggested that Srp40p is required in the NB to retain box C/D snoRNAs. Nopp140 is a member of a family of nucleolar proteins containing a serine-rich domain interspersed with basic clusters of amino acids, which also includes vertebrate nucleolin (Sicard et al., 1998). Strains lacking the putative yeast homologue of nucleolin, Nsr1p, are viable but impaired in growth and 40S ribosomal subunit synthesis (Lee et al., 1991; Lin et al., 1994). Two Nsr1p-deficient strains were tested for their effects on snoRNA localization. The nsr1-Δ strain lacks the gene entirely (Lin et al., 1994), while the Nsr1-Nter strain lacks the C-terminal region but retains the Srp40p-like serine-rich domain (Lee et al., 1991). In the nrs1-Δ strain expressing U14/MS2x2 snoRNA from a centromeric vector, ∼25% of the cells showed snoRNA accumulation in the NB, as compared with 10% in the control (data not shown). This phenotype was exacerbated by expression of the N-terminal fragment of Nsr1p, with 60% of cells accumulating U14/MS2x2 in the NB (Figure 3B, Nsr1-Nter). As judged by northern blot analysis, this effect was not due to an alteration of snoRNA levels (Figure 3C), and it appeared specific for box C/D snoRNAs as the localization of Gar1p–GFP was unaffected (Figure 3B, Nsr1-Nter). We conclude that Nsr1p promotes the dispersal of box C/D snoRNAs from the NB to the nucleolus and that its N-terminal domain shows some dominant activity in snoRNA retention. This domain was fused to YFP, and we observed that it was slightly enriched in the NB, although it could also be detected throughout the nucleus (Figure 3B, Nsr1-Nter::YFP). The core box C/D snoRNP proteins, Snu13p, Nop1p, Nop56p and Nop5/58p, are all required for the nucleolar localization of box C/D snoRNA Box C/D snoRNAs have been shown to associate tightly with four core snoRNP proteins: Nop1p/fibrillarin, Nop56p, Nop5/58p and Snu13p (Wu et al., 1998; Lafontaine and Tollervey, 1999, 2000; Watkins et al., 2000). Furthermore, the box C/D motif is necessary and sufficient to promote assembly with all four proteins in vitro (Newman et al., 2000; Watkins et al., 2000). It was, therefore, likely that one or more of these proteins would play a role in box C/D snoRNA localization. To investigate this possibility, we constructed Gal-dependent alleles for each of the corresponding genes. Endogenous U14 was concentrated in the nucleolus of wild-type cells, but was delocalized to the nucleoplasm following depletion of either Nop1p or Nop56p (Figure 4A). Depletion of Nop1p was more effective in delocalizing U14; following Nop1p depletion, U14 was evenly distributed between the nucleolus and the nucleoplasm, whereas it remained largely nucleolar following Nop56p depletion. To rule out the possibility that these effects could be an indirect consequence of a block in ribosome biogenesis, we analysed the localization of U14/MS2x2 in an RNA polymerase I mutant that stopped rRNA synthesis. In such mutants, inactivation of RNA polymerase I affects the nucleolar ultrastructure globally, and this leads to nucleolar segregation (Oakes et al., 1993; Trumtel et al., 2000). In our case, this resulted in the concentration of both Gar1p and the snoRNA reporter in two small areas of the nucleolus, but importantly without mislocalization of snoRNA in the nucleoplasm (Figure 3C). Figure 4.Nop1p, Nop56p, Nop5/58p and Snu13p are all required for efficient localization of box C/D snoRNAs in the nucleolus. (A) Mislocalization of endogenous U14 in Nop1p- and Nop56p-depleted strains. U14 snoRNA (red) was detected in wild-type cells (U14/wt), or following depletion of Nop56p (U14/Gal::Nop56) or Nop1p (U14/Gal::Nop1). In the right image of each panel, the DNA has been stained with DAPI. The nucleolus is visible as the nuclear region that stains poorly with DAPI. (B) Mislocalization of the artificial snoRNA reporter in strains depleted for Nop1p, Nop56p or Nop58p. The multicopy, intronic, artificial snoRNA (red) was detected in wild-type cells (CD-2m), or following depletion of Nop5p/Nop58p (CD-2m/Gal::Nop58), Nop1p (CD-2m/Gal::Nop1) or Nop56p (CD-2m/Gal::Nop56). For comparison, a similar intronic RNA lacking boxes C and D was stabilized by inactivating the gene for debranching enzyme (ΔCD-2m/dbr). (C) Mislocalization of the artificial snoRNA in strains depleted for Snu13p. The artificial snoRNA (red, CD-2m/e) was expressed from the RPR1 gene on a 2 μ plasmid, and detected in wild-type strains (Wild-type), or following depletion of Snu13p (Gal::Snu13). A similar RNA lacking boxes C and D was used as a control (red, ΔCD-2m/e; Wild-type). The nucleolus was visualized with a Gar1–GFP fusion protein (Gar1, green). Download figure Download PowerPoint The steady-state levels of endogenous box C/D snoRNAs are strongly affected on depletion of either Nop58p or Snu13p (Lafontaine and Tollervey, 1999; Watkins et al., 2000), precluding similar analysis for these proteins. Long terminal stems were, however, shown to bypass some requirements for snoRNA stability (Huang et al., 1992), and the terminal stem of U14/MS2x2 (15 or 22 bases) stabilized this RNA in the absence of Nop5p/Nop58p (Figure 4B and data not shown). In addition, expression of U14/MS2x2 from a multicopy plasmid allowed us to test whether the core box C/D proteins were required to localize the snoRNA to the NB before its dispersal throughout the nucleolus. Depletion of Nop1p, Nop5/Nop58p or Nop56p delocalized the snoRNA reporter to the nucleoplasm, and the effects were again more pronounced for Nop1p (Figure 4B). The NB was not visible after depletion of any of the proteins, indicating that each is required for snoRNA accumulation in this compartment. Snu13p binds directly the box C/D motif, but also binds to the first stem–loop of U4 snRNA and is, therefore, essential for pre-mRNA splicing (Watkins et al., 2000). The intronic construct was, therefore, not suited for analysing Snu13p, and the snoRNA reporter was expressed from the RNA polymerase III promoter. The analysis of Snu13p is further complicated by the fact that depletion of the protein affects not only box C/D snoRNA accumulation, but also destabilizes box H/ACA snoRNAs and snRNAs at late time points (Watkins et al., 2000). However, when GAL::snu13 cells are shifted to glucose for only 6–8 h, the effects are specific for box C/D snoRNAs (Watkins et al., 2000). In wild-type cells, the overexpressed artificial snoRNA localized in the NB (Figure 4C, wild type). In contrast, 6 h following the glucose shift, ∼20% of the GAL::snu13 cells showed altered distribution of the snoRNA reporter: accumulation in the NB was lost and a large fraction of the snoRNA became delocalized in the nucleoplasm (Figure 4C, GAL::snu13). In the same cells, the localization of Gar1p–GFP was unaffected, indicating that the mislocalization was specific for the box C/D class of snoRNAs. The resulting snoRNA localization resembled that of the ΔCD RNA expressed from the RNase P promoter (Figure 4C, ΔCD), suggesting that the localization activity of the box C/D motif was inactivated. A complete set of core snoRNP proteins was thus required for nucleolar accumulation of the box C/D snoRNAs, indicating that full assembly of the sno" @default.
• W2005042567 created "2016-06-24" @default.
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• W2005042567 date "2001-10-01" @default.
• W2005042567 modified "2023-10-18" @default.
• W2005042567 title "Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain" @default.
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