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• W2045376768 abstract "Article1 June 1998free access Conserved Boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs Thilo Sascha Lang Thilo Sascha Lang Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author Anton Borovjagin Anton Borovjagin Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author E.Stuart Maxwell E.Stuart Maxwell Department of Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Susan A. Gerbi Corresponding Author Susan A. Gerbi Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author Thilo Sascha Lang Thilo Sascha Lang Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author Anton Borovjagin Anton Borovjagin Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author E.Stuart Maxwell E.Stuart Maxwell Department of Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA Search for more papers by this author Susan A. Gerbi Corresponding Author Susan A. Gerbi Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA Search for more papers by this author Author Information Thilo Sascha Lang1, Anton Borovjagin1, E.Stuart Maxwell2 and Susan A. Gerbi 1 1Division of Biology and Medicine, Brown University, Providence, RI, 02912 USA 2Department of Biochemistry, North Carolina State University, Raleigh, NC, 27695-7622 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3176-3187https://doi.org/10.1093/emboj/17.11.3176 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates. Introduction Normal cell structure and function requires that macromolecules travel to their correct subcellular destinations. A key organelle in the eukaryotic cell is the nucleolus, the site of ribosome biogenesis in eukaryotes (reviewed by Hadjiolov, 1985; Gerbi et al., 1990). However, little is known about transport of small RNAs to the nucleolus. Therefore, we studied the features in small nucleolar RNA (snoRNA) needed for nucleolar localization. The findings reported here suggest some general principles governing snoRNA localization. There are estimated to be 200 snoRNA species in nucleoli, which can be grouped into two major classes. The first class to be discovered was the Box C/D family of snoRNAs and is the focus of this report. snoRNA molecules in this family contain Boxes C and D sequence motifs that are conserved between different organisms and also between different snoRNAs within this family. All Box C/D class members are associated with fibrillarin, a protein found predominantly in the nucleolus (Tyc and Steitz, 1989; Baserga et al., 1991; Peculis and Steitz, 1994; Watkins et al., 1996). A few snoRNA molecules in the Box C/D class (including U3, U8, U14 snoRNAs) are essential for cell growth due to their critical roles in ribosomal RNA (rRNA) processing (reviewed by Gerbi, 1995; Maxwell and Fournier, 1995; Sollner-Webb et al., 1995; Venema and Tollervey, 1995). Recently, it has been discovered that a large number of other members of the Box C/D class are non-essential and are used as guide RNAs to direct individual sites of 2′-O-ribose methylation in rRNA (Cavaillé et al., 1996; Kiss-László et al., 1996, 1998; Maden, 1996; Nicoloso et al., 1996; Tollervey, 1996; Tycowski et al., 1996; Maden and Hughes, 1997; Smith and Steitz, 1997; J.Ni and M.J.Fournier, personal communication) and snRNA (K.T.Tycowski and J.A. Steitz, personal communication). The second major class of snoRNAs is the Box H/ACA family (Balakin et al., 1996). These snoRNAs contain the evolutionarily conserved Box H (ANANNA) and Box ACA (Balakin et al., 1996; Bousquet-Antonelli et al., 1997; Ganot et al., 1997b), and are associated with the nucleolar protein GAR1 (Balakin et al., 1996; Ganot et al., 1997b; Bousquet-Antonelli et al., 1997; F.Dragon, V.Pogacic and W.Filipowicz, personal communication). Some snoRNAs of the Box H/ACA family are required for rRNA processing (snR30: Morrissey and Tollervey, 1993; E1, E2 and E3: Mishra and Elicieri, 1997), but the vast majority are used as guide RNAs for pseudouridine modifications in rRNA (Ganot et al., 1997a; Ni et al., 1997; Smith and Steitz, 1997). The only snoRNA that does not fit into either class is 7-2/MRP, which is required for rRNA processing (Schmitt and Clayton, 1993; Chu et al., 1994; Lygerou et al., 1996). This paper reports results on nucleolar localization signals for snoRNAs (U8 and U14) of the Box C/D family. U8 snoRNA is transcribed from its own gene and is needed for 5.8S/28S rRNA processing (Peculis and Steitz, 1993, 1994; Peculis, 1997). U14 was the first snoRNA discovered to be encoded within the intron of another gene (Liu and Maxwell, 1990; Leverette et al., 1992; Xia et al., 1995). U14 snoRNA contains two domains (A and B) that are complementary to 18S rRNA. Mutation of domain A alone or together with mutation in domain B, or genetic depletion of U14 snoRNA block rRNA processing at sites A1 and A2 in yeast, resulting in underaccumulation of 18S rRNA (Jarmolowski et al., 1990; Li et al., 1990; Li and Fournier, 1992; Liang and Fournier, 1995). Most snoRNAs are utilized either for rRNA processing or for rRNA modification, but U14 snoRNA is used for both events since domain B of U14 also guides the methylation of a nucleotide in the complementary region of 18S rRNA (Dunbar and Baserga, 1998), as predicted by Kiss-László et al. (1996). Another important region, domain Y, is found between domains A and B in yeast but not in metazoan U14 snoRNA, and it can be cross-linked to pre-rRNA (Morrissey and Tollervey, 1997). U14 snoRNA is transcribed in the nucleoplasm (E.Bertrand, D.A.Samarsky, M.J.Fournier and R.H.Singer, personal communication). How does U14 or any other Box C/D snoRNA finally reach the nucleolus, which is its functional compartment in the cell? The results reported here demonstrate that the sequences of Boxes C and D, as well as their position in the molecule, are critical for nucleolar localization of U14 snoRNA. Boxes C and D seem to be general nucleolar localization sequences for the Box C/D snoRNA family, as they are not only essential for U14, but also for U8 snoRNA. This is the first example of common nucleolar localization sequences that target several small RNA species to the nucleolus. Results Detection of snoRNA localization to nucleoli Nucleolar localization of U14 and U8 snoRNA was assayed by injection of fluorescein-labelled snoRNA into Xenopus oocyte nuclei with subsequent fluorescence microscopy. A similar procedure has been employed previously to assess the localization of various RNA molecules in living or fixed tissue culture cells (Wang et al., 1991; Jacobson and Pederson, 1997, 1998; Jacobson et al., 1995, 1997a,b). One advantage of the method described in our study is that RNA can be recovered from the injected oocytes for determination of its stability at defined times after injection. Two hours after injection, the nuclei were manually isolated from the oocytes, the nuclear envelope was manually removed and the contents were centrifuged onto a microscope slide. This procedure was originally developed to study lampbrush chromosomes (Gall et al., 1991). In addition to chromosomes, snurposomes that contain small nuclear RNAs (snRNAs; Wu et al., 1991) and multiple nucleoli containing amplified rDNA (Painter and Taylor, 1942; Brown and Dawid, 1968; Gall, 1968; Perkowska et al., 1968) are centrifuged onto the slide. Oocyte nucleoli vary in size (Wu and Gall, 1997), and may fuse into a multinucleolar cluster that can have a vacuolated appearance (Franke et al., 1981; Shah et al., 1996). The nucleoplasm is not centrifuged onto the slide, so the injected RNA which still remains in that compartment is not visualized. Fluorescence microscopy is valuable since it permits a direct qualitative assessment of nucleolar localization of the labelled RNA. Strong fluorescent signals depicting nucleolar localization of wild-type U14 snoRNA occurred within 2 h after injection of 1.4 ng transcript per oocyte nucleus (Figure 1). Similarly, fluorescein labelled wild-type U8 snoRNA (0.9 ng/oocyte) also localized to nucleoli within 2 h of injection into oocyte nuclei (Figure 2). In favorable preparations, signals from fluorescent U8 and U14 snoRNAs were found in ring-like structures within the nucleoli. Those structures appear to correspond to the dense fibrillar component, a compartment in nucleoli containing fibrillarin that surrounds rDNA (Shah et al., 1996). This supposition was supported by DAPI staining that shows DNA located in the center of the U8 or U14 labelled areas (e.g. Figures 1 and 2). Snurposomes do not contain DNA, and therefore the presence or absence of DNA can be assessed by microscopy to distinguish snurposomes from small nucleoli (Wu and Gall, 1997). Fluorescent U8 or U14 snoRNAs were never observed in structures lacking DNA. The nucleolar localization of fluorescent snoRNA was specific, as injection of the spliceosomal U2 snRNA in the same concentration as used for U8 or U14 snoRNA did not give nucleolar signals 2 h after injection (Figure 1). Figure 1.U14 snoRNA containing Boxes C and D localizes to nucleoli. Fluorescein-labelled U14 snoRNA or U2 snRNA was injected into the nuclei of Xenopus laevis oocytes. After 2 h, nucleoli were prepared. Nucleoli were analyzed by phase-contrast (PC) or fluorescence microscopy; the nucleolar rDNA is stained by DAPI. In some cases, there are multinucleolate clusters with two or more DAPI stained foci. Fluorescein-labelled wild-type U14 snoRNA and the A/V/B deletion of U14 localize to nucleoli (FL, green). U2 snRNA, a splicing RNA that is not endogenous to the nucleolus, was used as a negative control and did not localize to nucleoli. U14 snoRNA with Box C or Box D substituted by unrelated sequences did not localize to nucleoli. Bar is 10 μm. Download figure Download PowerPoint Figure 2.Boxes C and D are needed for U8 snoRNA nucleolar localization. Fluorescein-labelled wild-type U8 snoRNA localizes to nucleoli, but U8 with Box C or Box D substituted by unrelated sequences does not. Other details as in Figure 1. Bar is 10 μm. Download figure Download PowerPoint Various controls demonstrated that the fluorescent signals we observed in nucleoli are not due to degradation of fluorescent snoRNA and subsequent reutilization of the label by other nuclear components. For example, injection of fluorescein-UTP alone did not label the nucleoli (data not shown). Moreover, direct assays demonstrated the stability of injected 32P-labelled wild-type U8 or U14 snoRNA, as will be described below. The conclusions that U8 and U14 snoRNAs are transported to nucleoli after injection into the nucleoplasm, unlike U2 snRNA that remains in the nucleoplasm, are supported by fractionation of manually isolated oocyte nuclei. Two hours after injection into oocyte nuclei, almost 40% of the U8 and U14 snoRNAs in the nuclei are found in nucleoli (U8, 37.2%; U14, 36.8%), unlike U2 snRNA where only background levels of 3% are in nucleoli. The amount of RNA injected may have saturated the nucleolar localization machinery, thus explaining why some of the injected snoRNA remains in the nucleoplasm. Competition experiments to be described below indicate that the nucleolar localization machinery can in fact be saturated. The cytological approach used in our experiments reveals that the injected snoRNA is in nucleoli rather than associated with chromosomes and snurposomes that also pellet with the nucleolar fraction; these components of the nucleolar fraction cannot be distinguished from one another by the nuclear fractionation described. Thus, fluorescence microscopy is an excellent way to assay the nucleolar localization of U14 and U8 snoRNAs. Moreover, the subcompartments within the nucleoli can be cytologically resolved. Fluorescein-labelled snoRNA can function in rRNA processing The data just presented indicate that fluorescein labelled U8 or U14 snoRNAs can specifically localize to nucleoli, but did not reveal whether these snoRNAs were in a functional compartment of the nucleolus and active there. Therefore, we have tested whether fluorescein-labelled snoRNA can function in rRNA processing after injection into oocyte nuclei. After Xenopus oocytes are depleted of endogenous U8 snoRNA by injection of an antisense oligonucleotide followed by endogenous RNase H digestion, the 28S rRNA processing pathway is hindered as previously reported (Peculis and Steitz, 1993, 1994; Peculis, 1997). 28S and 32S disappear and novel species of 32*S and 36*S pre-rRNAs appear; the amount of 20S also decreases. These changes can be rescued by subsequent injection of intact U8 snoRNA. Controls showed that fluorescein-labelled U8 snoRNA rescued 28S rRNA processing as effectively as unlabelled U8 snoRNA (Figure 3). Figure 3.Fluorescein-labelled U8 and U14 snoRNAs are functional. Gel electrophoresis was carried out for precursor and mature rRNAs from Xenopus oocytes after no depletion or depletion of U8 or U14 snoRNA by injection of antisense oligonucleotides. After depletion of U14 snoRNA, newly synthesized 18S rRNA fails to appear and the amount of its 20S precursor increases relative to 28S and 32S. After depletion of U8 snoRNA, 28S and 32S disappear and novel species of 32*S and 36*S appear; and 20S pre-rRNA decreased in amount. These effects of U8 snoRNA depletion have been reported by Peculis and Steitz (1993, 1994). Fluorescein-labelled or -unlabelled U8, or U14 snoRNAs at 1.15 ng/oocyte could restore rRNA processing when injected after the antisense oligonucleotide. Download figure Download PowerPoint Similar experiments with U14 snoRNA depletion demonstrated that U14 is needed for 18S rRNA production; in the absence of U14 snoRNA in the Xenopus oocyte, newly synthesized 18S rRNA fails to appear and instead its 20S precursor accumulates relative to the 28S and 32S levels (Figure 3). Production of 18S rRNA was restored by subsequent injection of U14 snoRNA that was synthesized in vitro with or without fluorescein UTP (Figure 3). Therefore, not only are fluorescein labelled U14 or U8 snoRNAs able to localize in nucleoli (Figures 1 and 2), but they can also carry out their functions there in rRNA processing. A terminal core structure containing Boxes C and D is sufficient for nucleolar localization of U14 snoRNA To describe intrinsic elements of U14 snoRNA necessary for its nucleolar localization, substitutions, deletions or insertions in the U14 sequence were made by PCR mutagenesis. Table I summarizes the sequence of mature wild-type U14 snoRNA (Leverette et al., 1992) as well as the various mutated U14 constructs used here. Table 1. Mutations in U14 and U8 snoRNAs The sequence of the murine processed U14 snoRNA is from Watkins et al. (1996). Boxes C and D are highly conserved in many snoRNAs and a consensus sequence has been derived for each of them (Xia et al. 1997). Regions A and B are complementary to 18S rRNA, and V is a variable region (Xia et al. 1997). One extra U (bold font) was added to the 5′ end to fully close the terminal stem of U14. Nucleotides that are the same as in wild-type U14 snoRNA are shown in the sequence alignment by dots, and deletions are indicated by dashes. Substitutions or insertions are indicated by lower case letters and several are the same as used by Watkins et al. (1996) and Xia et al. (1997). In partial mutants of either Box C or Box D, the two most highly conserved nucleotides (GA) of each Box were replaced by cytosine. The double mutants Box C-2, 3 and Box D-5, 6 are not shown in this table, as they are simply the sum of the individual single mutations Box C-2 plus Box C-3 or Box D-5 plus Box D-6. Similarly, just the double mutant +6(5′, 3′) is shown. The A/V/B deletion is the same one used by Watkins et al. (1996). The Xenopus U8 snoRNA sequence is from Peculis and Steitz (1993) and consensus sequences for conserved Boxes C and D (Xia et al., 1997) are indicated. The Box C mutation is the same one used by Peculis and Steitz (1994). After half of the sequence of U14 snoRNA was deleted [A/V/B deletion construct, spanning domains A and B and the variable (V) region], the truncated U14 snoRNA containing only 39 nucleotides still localized to nucleoli as well as the full-length wild-type U14 snoRNA (Figure 1). Nucleolar localization was seen when the A/V/B deletion transcript was injected at the same concentration as used for wild-type U14. This was true even when the concentration was adjusted so that equimolar amounts were injected, compensating for its decreased molecular weight. The 49 nucleotides that had been deleted in the A/V/B construct are unimportant for nucleolar localization even though they included regions A and B that are complementary to 18S rRNA and necessary for rRNA processing and modification. The residual core structure containing the terminal stem and adjacent Boxes C and D had sufficient information for nucleolar localization of U14 snoRNA. Since the phylogenetically conserved Boxes C and D were present in the residual core structure of U14 snoRNA, mutations were made in Box C or D to assay their effect on nucleolar localization. Replacement of the entire Box C or Box D in full-length U14 snoRNA by unrelated sequences prevented its nucleolar localization (Figure 1). Hence, Boxes C and D are critical for nucleolar localization of U14 snoRNA. Boxes C and D also are nucleolar localization elements for U8 snoRNA In order to examine whether Boxes C and D are general signals for nucleolar localization of other snoRNAs of the Box C/D family, the localization of U8 snoRNA was investigated. Fluorescein-labelled wild-type U8 snoRNA that is injected into Xenopus oocyte nuclei can localize to nucleoli (Figure 2). In contrast, when the sequences of Box C or Box D in U8 snoRNA (Peculis and Steitz, 1994) were replaced by unrelated sequences, nucleolar localization was abolished (Figure 2). Therefore, Boxes C and D are necessary for nucleolar localization of both U8 snoRNA and U14 snoRNAs. It was important to show that the failure to observe fluorescent signals after injection of Box C and D mutants was not due to their lack of stability. We ruled out this trivial explanation for Box C and D mutants of U8 and U14 and all other U14 mutants studied here as follows. A dilution series shows that just 25% of the amount of wild-type U14 snoRNA usually injected (1.4 ng/oocyte) can still be detected (Figure 4). The stability was determined for 32P-snoRNA constructs 2 h after their injection into oocyte nuclei (Table II). Although the mutated U14 snoRNAs are generally somewhat less stable than wild-type U14 snoRNA, they are still sufficiently stable (>25%) to have been detected, had they so localized in nucleoli 2 h after injection into oocyte nuclei. Clearly, an advantage of the method used here is that the amounts of labelled mutant U14 snoRNAs can be determined in the oocyte following injection. Figure 4.Dilution of injected snoRNA. Wild-type U14 snoRNA was diluted to 50% or 25% of the amount generally used (1.4 ng/oocyte) prior to injection into oocyte nuclei. Nucleolar labelling is seen 2 h after injection even with the 25% dilution. Other details as in Figure 1. Bar is 10 μm. Download figure Download PowerPoint Table 2. Stability of U14 and U8 wild-type and mutant snoRNAs RNA Stability (% of wild-type 2 h after injection) U14 snoRNA A/V/B 81 Box C full 60 Box C-2,3 59 Box C-2 100 Box C-3 86 Box D full 64 Box D-5,6 92 Box D-5 68 Box D-6 84 +6(5′) 103 +6(3′) 97 +6(5′,3′) 82 open stem 1 74 open stem 2 45 open stem 3 43 mini C/D 61 U8 snoRNA Box C 99 Box D 75 32P-labelled capped U14 or U8 snoRNA were isolated 2 h after injection into Xenopus oocyte nuclei and quantified after gel electrophoresis (Materials and Methods). The amount of mutant snoRNA relative to wild-type snoRNA remaining in the nucleus 2 h after injection of equivalent amounts of each is expressed as a percentage. Competition reveals that U14 snoRNA nucleolar localization can be saturated Competition experiments were carried out using U14 snoRNA as the fluorescent probe and other unlabelled transcripts as the competitor. Since a 100-fold molar excess of competitor was used, the amount of fluorescent probe was decreased to 0.5 ng/oocyte (about one third of the usual amount) to reduce the total amount of RNA being injected to a net excess of 35 times the usual amount. Unlabelled wild-type U14 snoRNA competes effectively with fluorescein-labelled U14 snoRNA for nucleolar localization, indicating that limiting components needed for its nucleolar localization become saturated (Table III). U2 snRNA is unable to compete with fluorescent U14 snoRNA for its nucleolar localization. However, U8 snoRNA is a competitor for U14 nucleolar localization, suggesting that the sequences they share in common, namely Boxes C and D, may be responsible for the competition. This is supported by the observation that U14 snoRNA carrying a double mutation (U14 Box C/D) that substitutes sequences in both Boxes C and D does not appreciably hinder the nucleolar localization of fluorescent U14 wild-type snoRNA. Finally, a truncated and mutated construct of U14 (mini C/D to be discussed later), which has just Boxes C and D and no other wild-type sequences, can compete the nucleolar localization of fluorescent U14 snoRNA. Therefore, to sum up, the components needed for U14 snoRNA localization can be saturated by an excess of any molecule tested that has Boxes C and D. Table 3. Competition of U14 snoRNA nucleolar localization Probe Competitor Competition U14 U14 strong U14 U2 none U14 U8 moderate U14 U14 Box C/D weak U14 U14 mini C/D strong 0.5 ng/oocyte of fluorescein-labelled wild-type U14 snoRNA was co-injected into Xenopus oocyte nuclei with a 100-fold molar excess of unlabelled transcripts (competitor), and slides with nucleoli were prepared 2 h later. The qualitative degree of competition is indicated. Importance of the spatial position of Boxes C and D for nucleolar localization of U14 Is the spatial relationship or position of Boxes C and D with respect to the 5′- and 3′-termini of U14 snoRNA important for nucleolar localization? Increasing the distance of Box C from the 5′ end [+6(5′)] or Box D from the 3′ end [+6(3′)] by six nucleotides prevents processing of U14 snoRNA (Xia et al., 1997). These same insertions abolished localization of U14 snoRNA to nucleoli 2 h after oocyte injection (Figure 5). This effect could be due to the staggered alignment of Box C sequences relative to Box D sequences within the secondary structure of U14 snoRNA after inserting six nucleotides between the terminal stem and either Box C or Box D. To attempt to realign Box C with Box D, a double mutant [+6(5′/3′)] was made from the single mutations just described. However, even for this double mutant, no nucleolar localization of U14 snoRNA was seen (Figure 5). This suggests the importance of the distance of Box C and Box D to the terminal stem or to the ends of the molecule for nucleolar localization. The latter seems more probable, as it will be shown below that the terminal stem is not required for nucleolar localization. Figure 5.The spatial position of Boxes C and D but not the terminal stem is important for nucleolar localization of U14 snoRNA. Spacers of 6 cytosine residues between Box C and the 5′ end [+ 6(5′)] or between Box D and the 3′ end [+6(3′)] alone, or together [+6 (5′/3′)], impaired U14 localization to nucleoli. Substitution of sequences to open the terminal stem had no deleterious effect on nucleolar localization [open stem 1 and open stem 2 mutations shown here; the results for open stem 3 (data not shown) look the same]. The mini C/D construct of 35 nucleotides can localize to nucleoli almost as well as wild-type U14 snoRNA; it is derived from the A/V/B deletion but has substituted sequences to open the terminal stem, so that Boxes C and D are the only wild-type sequences that remain. Other details as in Figure 1. Bar is 10 μm. Download figure Download PowerPoint The stem of the terminal core structure is not critical for nucleolar localization As described above (Figure 1), a residual core structure lacking regions A/V/B is still able to localize to nucleoli. Which sequences and structures of this core structure are essential for nucleolar localization? The A/V/B deletion mutant has a terminal stem bounded by Box C on one side and Box D on the other side, and connected by a small loop. We created three different mutations to open the terminal stem of the full-length U14 molecule (Table I). In open stem 1, the sequences of the left side of the stem are replaced by their complement to hinder base-pairing; in open stem 2, the sequences of both sides of the stem are replaced by cytosine residues; and in open stem 3, they are replaced by guanosine residues. For all three mutations, nucleolar localization occurred, though somewhat less than for wild-type U14 snoRNA (Figure 5; data not shown for open stem 3). Therefore, the terminal stem does not appear to be essential for nucleolar localization. The role of the terminal stem was also examined in the residual core structure of the A/V/B deletion. As for open stem 3 above, the terminal stem was opened by substituting both sides of the stem with guanosine residues. The small internal loop was also replaced by unrelated nucleotides. Therefore, the only wild-type sequences that remain in this mini C/D construct of 35 nucleotides are Boxes C and D (Table I). Amazingly, the mini C/D construct could still localize to nucleoli, though not as well as wild-type U14 snoRNA (Figure 5). Specific nucleotides within Boxes C and D are essential localization elements in U14 snoRNA Recently it was shown that consensus sequences of both Box C and Box D also are required for U14 processing (Watkins et al., 1996) and that certain single highly conserved nucleotides within both Boxes are critical for its processing (Xia et al., 1997). We have tested point substitutions of the most highly conserved and important nucleotides in Boxes C and D for their effect on nucleolar localization of U14 snoRNA. Among snoRNAs of the Box C/D class of numerous eukaryotic organisms, the nucleotides in position 2 (G) and 3 (A) of Box C are 100% conserved (Xia et al., 1997). We replaced these nucleotides, individually (Table I) or both, by cytosine. Both the double and single substitutions in Box C of U14 snoRNA greatly reduced localization to nucleoli (Figure 6). Similarly, positions 6 (A) and 5 (G) of Box D are conserved 100% and 99.3%, respectively (Xia et al., 1997). These nucleotides were also replaced by cytosine (Table I). Again, the double and single substitutions within Box D greatly diminished nucleolar localization (Figure 6). Thus, the GA nucleotides in Box C (2,3) and Box D (5,6) are necessary for nucleolar localization, and their substitution compromises this localization. Overall, the data from this study show that the sequences of Boxes C and D are crucial elements for nucleolar localization of Box C/D snoRNAs. Figure 6.Highly conserved nucleotides within Box C and Box D are needed for nucleolar localization of U14 snoRNA. Nucleolar localization of fluorescein-labelled U14 mutated in the two most highly conserved nucleotides of Box C or D was analyzed. Substitution of both positions (Box C-2/3 or Box D-5/6) as well as single substitutions (Box C-2, Box C-3, Box D-5, Box D-6) hindered U14 snoRNA from localizing to nucleoli. Other details as in Figure 1. Bar is 10 μm. Download figure Download PowerPoint Discussion Injected snoRNA is localized to the nucleolus and is functional in rRNA processing We have injected fluorescein-labelled snoRNA into Xenopus oocyte nuclei, and have determined certain principles for nucleolar localization of Box C/D snoRNAs by fluorescence microscopy. In favorable preparations, fluorescein-labelled U8 or U14 snoRNA localized to ring-like structures within the oocyte nucleoli. These rings surround rDNA that was detected by DAPI staining; the rings probably correspond to the dense fibrillar comp" @default.
• W2045376768 created "2016-06-24" @default.
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• W2045376768 date "1998-06-01" @default.
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• W2045376768 title "Conserved Boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs" @default.
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