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Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP

2002; Springer Nature; Volume: 21; Issue: 14 Linguagem: Inglês

10.1093/emboj/cdf376

1460-2075

Niamh Cahill, Kyle Friend, Wayne Speckmann, Zhu‐Hong Li, Rebecca M. Terns, Michael P. Terns, Joan A. Steitz,

RNA Research and Splicing

Article15 July 2002free access Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP Niamh M. Cahill Niamh M. Cahill Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Kyle Friend Kyle Friend Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Wayne Speckmann Wayne Speckmann Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Zhu-Hong Li Zhu-Hong Li Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Rebecca M. Terns Rebecca M. Terns Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Michael P. Terns Michael P. Terns Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Joan A. Steitz Corresponding Author Joan A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Niamh M. Cahill Niamh M. Cahill Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Kyle Friend Kyle Friend Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Wayne Speckmann Wayne Speckmann Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Zhu-Hong Li Zhu-Hong Li Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Rebecca M. Terns Rebecca M. Terns Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Michael P. Terns Michael P. Terns Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA Search for more papers by this author Joan A. Steitz Corresponding Author Joan A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA Search for more papers by this author Author Information Niamh M. Cahill1, Kyle Friend1, Wayne Speckmann2, Zhu-Hong Li2, Rebecca M. Terns2, Michael P. Terns2 and Joan A. Steitz 1 1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, 06536 USA 2Department of Biochemistry and Molecular Biology, and Genetics, University of Georgia, Life Science Building, Athens, GA, 30602 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3816-3828https://doi.org/10.1093/emboj/cdf376 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Methylation of the ribose 2′-hydroxyl, the most widespread modification of ribosomal and splicesomal RNAs, is guided by the box C/D class of small nucleolar RNAs (snoRNAs). Box C/D small nucleolar ribonucleoproteins (snoRNPs) contain four core proteins: fibrillarin, Nop56, Nop58 and 15.5 kDa. We constructed U25 snoRNAs containing a single photoactivatable 4-thiouridine at each U position within the conserved box C/D and C′/D′ motifs. Proteins assembled on the snoRNA after injection into Xenopus oocyte nuclei were identified by cross-linking, and reconstituted particles characterized by functional rescue and mutational analyses. Our data argue that box C/D snoRNPs are asymmetric, with the C′ box contacting Nop56 and fibrillarin, the C box interacting with Nop58, and the D and D′ boxes contacting fibrillarin. No cross-link to 15.5 kDa was detected; its binding is disrupted by 4-thiouridine substitution in position 1 of the C box. Repositioning the guide sequence of U25 upstream of box D instead of D′ revealed that both C/D motifs have the potential to function as guide centers, but, surprisingly, there was no alteration in protein cross-linking. Introduction The 18S, 5.8S and 28S rRNAs are transcribed as one contiguous unit. Maturation of this transcript requires two critical events: the covalent modification of highly conserved regions of the rRNA, and a series of endo- and exonucleolytic cleavages liberating the mature rRNA products (Sollner-Webb et al., 1996; Venema and Tollervey, 1999). The two predominant covalent modifications are the addition of a methyl group to the 2′-hydroxyl of ribose sugars and the isomerization of uridines to pseudouridines (Maden, 1990). These modifications, as well as many essential nucleolytic cleavages of the pre-rRNA, are directed by small nucleolar ribonucleoproteins (snoRNPs) (reviewed in Venema and Tollervey, 1999; Kiss, 2001; Terns and Terns, 2002). The snoRNPs involved in rRNA modification can be divided into two classes based on their conserved sequence elements and associated proteins. Both classes select the nucleotide to be modified through specific base pairing between the snoRNA and rRNA substrate. Box C/D snoRNPs guide 2′-O-methylation and are characterized by the conserved terminal sequence elements box C [RUGAUGA] and box D [CUGA], as well as a somewhat less conserved matching set of internal sequence elements referred to as C′ and D′ (Balakin et al., 1996; Tycowski et al., 1996; Kiss-Laszlo et al., 1998). In the extended duplex formed with the substrate, box D or D′ is located precisely five nucleotides from the residue that base-pairs to the rRNA nucleotide to be modified (Cavaille et al., 1996; Kiss-Laszlo et al., 1996; Tycowski et al., 1996). The box H/ACA snoRNPs guide pseudouridylation and are characterized by two conserved sequence elements, the H box [ANANNA] and the 3′-terminal ACA sequence, and form a short duplex with the rRNA on each side of the uracil to be modified (Balakin et al., 1996; Ganot et al., 1997; Ni et al., 1997; Bortolin et al., 1999). The 5′ and 3′ ends of box C/D snoRNAs form a terminal stem, bringing together the C and D boxes. This box C/D motif (Watkins et al., 2000) has been shown to be essential for snoRNA processing, stability, methylation activity, nucleolar localization and 5′ cap hypermethylation (Filipowicz and Pogacic, 2002; Terns and Terns, 2002). The C′ and D′ boxes are generally found within several nucleotides of one another (3–9 nucleotides) or are predicted to be brought together through the formation of an internal stem–loop (Kiss-Laszlo et al., 1998). The H/ACA snoRNAs likewise fold into a common hairpin–hinge–hairpin–tail structure (Balakin et al., 1996; Ganot et al., 1997), with the conserved sequence elements essential for snoRNA processing, stability, pseudouridylation activity and nucleolar localization (Filipowicz and Pogacic, 2002; Terns and Terns, 2002). Four core proteins are associated with each snoRNP class. The box C/D class contains fibrillarin (yeast Nop1p, the methyltransferase; Omer et al., 2002), Nop58, Nop56 and the 15.5 kDa protein (yeast Snu13p) (Schimmang et al., 1989; Tyc and Steitz, 1989; Gautier et al., 1997; Wu et al., 1998; Lafontaine and Tollervey, 1999; Lyman et al., 1999; Newman et al., 2000; Watkins et al., 2000). The proteins of the box H/ACA class are dyskerin (yeast Cbf5p, the pseudouridine synthase), Gar1, Nhp2 and Nop10 (Kiss, 2001; Filipowicz and Pogacic, 2002). A number of box C/D and box H/ACA snoRNAs guide two independent modification reactions making use of two separate antisense regions. This ability together with the symmetry observed for both the box C/D and box H/ACA snoRNAs at the RNA level has suggested that members of both classes of snoRNPs each possess two sets of core proteins (Watkins et al., 1998a; Venema and Tollervey, 1999). In support of this notion, electron micrographic studies of H/ACA snoRNPs revealed a rather symmetric bipartite particle, with an estimated mass consistent with two copies of each protein (Watkins et al., 1998a). We have undertaken site-specific cross-linking analyses to examine in greater detail the RNA–protein interactions of a modification guide box C/D snoRNP, Xenopus U25. In addition to providing a map of the interactions between core proteins and conserved box elements, the data surprisingly reveal that the core box C/D proteins assemble asymmetrically on the U25 snoRNA molecule. Results Fibrillarin, Nop56 and Nop58 assemble on and cross-link to the box C/D elements of U25 constructs injected into Xenopus oocytes To identify proteins interacting with the conserved box C and D sequences, the photoactivatable cross-linking agent 4-thiouridine (4SU) was inserted site-specifically into the U25 snoRNA by RNA ligation methods (Moore and Sharp, 1992). Constructs were designed such that every uridine position within each of the four conserved sequence elements was replaced individually with the zero-length cross-linker, 4SU (Sontheimer, 1994). The constructs also contained a single radioactive phosphate immediately 5′ of the photoactivatable nucleotide and were named with reference to the site of 4SU incorporation (Figure 1A). Figure 1.U25 snoRNA constructs and cross-linking analyses. (A) The Xenopus U25 snoRNA sequence is shown, highlighting in bold the conserved box elements. Three additional nucleotides were added to the 5′ end and two nucleotides to the 3′ end to extend the terminal stem (Tycowski et al., 1996). The antisense region (underlined) is shown base-paired to the Xenopus 18S rRNA sequence it targets. For site- specific cross-linking, a uridine within one of the conserved sequence elements was replaced with a 4SU (U) with a single radioactive label immediately 5′ of the 4SU, denoted by p. Constructs are named based on the site of 4SU incorporation. (B and C) SDS–polyacrylamide gel analyses of proteins cross-linked to the U25 constructs. After assembly of the injected RNAs in oocyte nuclei, photocross-linking and RNase digestion, proteins were resolved by 15% SDS–PAGE. The 40, 62 and 66 kDa cross-linked proteins indicated with an arrow are identified in Figure 2. RNA bands are indicated by an asterisk. The positions of molecular weight markers (kDa) are shown on the right. Download figure Download PowerPoint In vitro transcribed U25 snoRNA previously had been shown to reconstitute site-specific methylation activity within Xenopus oocytes (Tycowski et al., 1996). In a typical experiment, nuclei isolated from Xenopus oocytes and maintained under paraffin oil were microinjected with a 4SU-containing snoRNA transcript (Lund and Paine, 1990). After a 4 h incubation to allow snoRNP assembly, the nuclei were irradiated at 365 nm, the RNA was digested with RNase T1 and nuclease P1, and 32P-labeled proteins were resolved by SDS–PAGE. The amount of material injected, the assembly time and the extent of irradiation were all optimized to minimize non-specific cross-linking (Sontheimer, 1994; data not shown). Visualization of labeled protein bands was dependent upon irradiation at 365 nm and was sensitive to proteinase K digestion (data not shown). Figure 1B and C illustrates the cross-linking patterns generated with the six wild-type U25 constructs. Note that, technically, the C′-2 construct is not wild-type, since the C box nucleotide that has been substituted with 4SU is not a U, but rather a G in U25; we refer to it as 'wild-type' since the 4SU-containing sequence conforms to the consensus for box C/C′, UGAUGA (Kiss-Laszlo et al., 1998). Several cross-linked protein bands are seen with these six constructs, notably a protein migrating at ∼40 kDa with the D′ and D constructs (Figure 1B) as well as with the C′-1 and C′-2 constructs (Figure 1C, lanes 1 and 2). Three additional strongly cross-linked proteins were generated with the C′ and C constructs, migrating at ∼66, 62 and 31 kDa (Figure 1C). Surprisingly, the 66 kDa cross-link exhibits a striking specificity for only one of the two substituted positions in the C box (C-2 construct). The 40, 62 and 66 kDa cross-linked bands are of particular interest because their mobilities on SDS–PAGE correspond to those expected for the box C/D-associated proteins fibrillarin (40 kDa; Lapeyre et al., 1990), Nop56 (62–65 kDa; Newman et al., 2000; Watkins et al., 2000) and Nop58 (65–66 kDa; Newman et al., 2000; Watkins et al., 2000), respectively. The radioactivity migrating at ∼21 kDa (and a band occasionally running below this at ∼14 kDa; Figure 1B, lane 1) is RNA; undigested transcript migrates at 21 kDa (data not shown). Despite quite stringent RNase digestion conditions, such bands typically were seen, perhaps because a portion of the injected RNA forms aggregates resistant to nuclease action. The identities of the 40, 62 and 66 kDa proteins were verified by immunoprecipitation of the RNase-digested, cross-linked samples with antibodies targeting the predicted box C/D snoRNP components (Speckmann et al., 2002). The specificity of these antibodies for Xenopus fibrillarin, Nop56 and Nop58 is confirmed by immunoprecipitation and western blot in Figure 2A and B. The 40 kDa band cross-linked to the D′ element was precipitated selectively by anti-fibrillarin antibodies (Figure 2C, lanes 1 and 2), but not by the control anti-Sm antibodies (Figure 2C, lanes 3 and 4). Although the 62 and 66 kDa cross-linked bands migrate close to one another, precipitation with anti-Nop56 and anti-Nop58 antibodies clearly demonstrated that the 62 kDa protein, cross-linked by the C′-1 construct, is Nop56, while the 66 kDa protein, cross-linked to C-2, is Nop58 (Figure 2D). Figure 2.Identification of the 40, 62 and 66 kDa cross-linked proteins. (A) Antibodies generated against Xenopus Nop56, Nop58 and fibrillarin were used for immunoprecipitation of in vitro translated, 35S-labeled Nop56, Nop58 and fibrillarin proteins. Immunoprecipitations with pre-immune and immune sera are shown. Total lanes (T) show 20% of the 35S-labeled protein included in the assays. (B) Immunoblot analysis using oocyte crude nuclear extracts was performed with the Nop56, Nop58 or fibrillarin antisera as indicated. (C and D) After injection of the D′, C′-1 or C-2 construct, the irradiated and RNase-digested nuclear extracts were subjected to immunoprecipitation prior to SDS–PAGE using anti-Sm (Y12) or the antisera characterized in (A) and (B). The resulting pellets (P) and 1/10 of the supernatants (S) were resolved on 12.5% SDS–polyacrylamide gels. The bands identified as fibrillarin (Fib), Nop56 and Nop58 are indicated with arrows. RNA bands are indicated with an asterisk. The positions of protein molecular weight markers are shown on the right. Download figure Download PowerPoint Mutation of the conserved boxes alters cross-linking to Nop56, but not to fibrillarin Site-specific U25 constructs with mutations in the four conserved sequence elements were generated to examine their effects on protein association (Figure 3A). It is well known that mutations in the C or D box destabilize box C/D snoRNAs (Venema and Tollervey, 1999; Weinstein and Steitz, 1999), as we confirmed in oocyte injection experiments (see below). In contrast, mutation of the internal C′ or D′ element does not alter the stability of the snoRNA (Kiss-Laszlo et al., 1998), allowing analysis of alterations in protein binding as revealed by cross-linking. Figure 3.Proteins cross-linked to mutant U25 constructs. (A) Mutant constructs are named with reference to the site of 4SU incorporation, as in Figure 1A, followed by (in parentheses) the box element that has been mutated. Mutated sequences are underlined. (B–D) The labeling of gels resolving proteins cross-linked to the mutant constructs is as in Figure 2B and C. Download figure Download PowerPoint Mutation of the D′ element from C4SUGA to C4SUCU [D′(D′mt)] did not alter cross-linking to fibrillarin (Figure 3B), whereas mutation of the D element from C4SUGA to C4SUCC [D(Dmt)] resulted in the loss of fibrillarin cross-linking (Figure 3C, lane 2). This is most probably a consequence of snoRNA destabilization; during the 4 h incubation, >80% of the box D mutant relative to wild-type constructs was degraded (data not shown). Likewise, the D′(D′mt, Dmt) construct, with 4SU within the internal D′ element and both the D′ and D boxes mutated (Figure 3C, lane 3), generated no cross-linked fibrillarin, but rather greatly enhanced the appearance of a 45 kDa cross-linked protein (a cross-link also occasionally seen with wild-type snoRNAs, particularly at incubation times <4 h; Figures 2C and D, and 3C, and data not shown). Mutation of the C′ element from 4SUGAGGA to 4SUCCGGA resulted in the loss of cross-linked Nop56, but fibrillarin cross-linking was still observed (Figure 3D, lane 2). Thus, Nop56's interaction with the C′ box appears to be very sequence specific, whereas fibrillarin interacts with the C′ box in a sequence-independent fashion. More extensive mutation of the D′ element [D′(D′fullmt): C4SUGA to G4SUCC] (Figure 3D, lane 3) or mutation of both the D′ and C′ elements, with the 4SU in either element [C′-1(D′mt, C′mt) and D′(D′mt, C′mt)], likewise revealed no alteration in the cross-linking of fibrillarin (Figure 3D, lanes 4 and 5). The sequence requirements for Nop58 binding were not assessed since mutation of the C box element destabilizes the snoRNA in the oocyte (Caffarelli et al., 1998). Nop56 and Nop58 cross-linking occurs in fibrillarin-containing snoRNPs We next asked whether any of the additional cross-linked bands visualized in Figures 1, 2 and 3 might be previously unrecognized, but genuine snoRNP proteins. For instance, there is a major cross-linked protein at 31 kDa, a doublet migrating at ∼97 kDa, and the 45 kDa band that is enhanced with the mutant constructs. To determine whether these proteins are a part of fibrillarin-containing snoRNPs, nuclei were injected with a 4SU-containing U25 snoRNA, incubated, and irradiated as above. Only after immunoprecipitation with anti-fibrillarin antibodies were snoRNPs disassembled by RNase treatment and the cross-linked proteins analyzed. As expected, fibrillarin itself was strongly labeled in the immunoprecipitate, whether cross-linked to the wild-type D′ construct (Figure 4A, lane 1), either D′ mutant [D′(D′mt) and D′(D′fullmt)] (Figure 4A, lanes 3 and 5) or the C′ wild-type and mutant construct (Figure 4B, lanes 1 and 3). Importantly, both labeled Nop56, generated with the C′-1 construct (Figure 4B, lane 1), and labeled Nop58, generated with the C-2 construct (Figure 4C, lane 3), were precipitated by anti-fibrillarin antibodies even though fibrillarin does not cross-link to the C-1 or C-2 constructs (Figure 4C, lanes 2 and 4, and Figure 1C). In contrast, the strong doublet at 97 kDa generated by either the C′-1 wild-type or mutant construct (Figure 4B, lanes 2 and 4) was not precipitated, indicating that these two proteins interact non-specifically with the U25 snoRNA or perhaps represent assembly factors that are not part of a mature snoRNP. Likewise, the cross-linked 45 kDa protein generated with mutant constructs (Figure 3) was not precipitated with anti-fibrillarin (Figure 4B, lanes 3 and 4, and data not shown) nor was the 31 kDa protein generated by the C-1 or C-2 construct (Figure 4C, lanes 1 and 3). We conclude that the 97, 45 and 31 kDa cross-linked proteins are not components of mature fibrillarin-containing box C/D particles. Figure 4.Anti-fibrillarin antibodies co-immunoprecipitate cross-linked fibrillarin, Nop56 and Nop58. After injection of nuclei with the D′, D′(D′mt), D′(D′fullmt), C′-1, C′-1(C′mt), C-1 or C-2 construct, cross-linked samples were immunoprecipitated with the anti-fibrillarin monoclonal antibody (72B9). The pellets (P) and supernatants (S) were then RNase digested and equal amounts were resolved on 12.5% SDS–polyacrylamide gels. Labeling is as in Figures 2 and 3. Download figure Download PowerPoint The 15.5 kDa protein is not detected by cross-linking, but is present in oocyte assembled snoRNPs The fourth known box C/D-associated protein, the 15.5 kDa protein, is essential for the stability of a box C/D snoRNP (Watkins et al., 2000). However, it did not appear in any of our cross-linking profiles (Figures 1, 2, 3 and 4; data not shown). This was surprising since structural information predicts that the first nucleotide of the C box, which is replaced with 4SU in the C-1 construct, should closely contact the 15.5 kDa protein, participating in several hydrogen bonds and forming extensive van der Waals interactions (Vidovic et al., 2000). Instead, no cross-links to any bona fide box C/D-associated protein were detected with the C-1 construct; only the 31 kDa non-specifically cross-linked band was seen (Figure 4C, lanes 1 and 2). There are two potential explanations. First, the 4SU of the C-1 construct, when buried in the 15.5 kDa protein, may be constrained such that it is unable to adopt a position optimal for cross-linking. Secondly, substitution of the first U within the C box with 4SU may adversely affect the binding of the 15.5 kDa protein to U25. A gel shift assay was therefore employed to compare the binding of recombinant 15.5 kDa protein to the C′-1, C-1 and C-2 constructs (Figure 5A). Whereas all three RNAs formed a shifted complex with the 15.5 kDa protein in the presence of an ∼10-fold excess of protein, both the C′-1 and C-2 constructs were fully shifted compared with only 50% of the C-1 construct. We conclude that the substitution of 4SU in the first position of the C box significantly lowers the RNA's affinity for the 15.5 kDa protein and could explain the absence of a cross-link. This explanation is supported by the inability of the C-1 construct to rescue methylation activity, as described below. Figure 5.The 15.5 kDa protein assembles on U25 constructs in the oocyte. (A) Substitution of 4SU at the C-1 position adversely affects the binding of the 15.5 kDa protein. The C′-1, C-1 or C-2 RNA was incubated either alone (lane 1) or in the presence of recombinant 15.5 kDa protein at the concentrations indicated (see Materials and methods). Complexes were resolved on an 8% non-denaturing polyacrylamide gel, with the RNA alone (RNA) and the shifted RNA complex (RNP) indicated. (B) Approximately 20 nuclei isolated under paraffin oil were co-injected with the D construct (21 fmol/nucleus) and a T7 epitope-tagged recombinant 15.5 kDa protein (12.5 μM; gift of T.Hirose). The nuclei were incubated, irradiated and immunoprecipitated with anti-T7 tag antibody or mouse IgG. Samples were RNase digested, and equal fractions of the pellets (P) and supernatants (S) were resolved on a 12.5% SDS–polyacrylamide gel as in Figures 2,3,4. Download figure Download PowerPoint To demonstrate that the 15.5 kDa protein nonetheless was assembling onto the other U25 constructs, recombinant human 15.5 kDa protein containing a T7 epitope tag was co-injected with the D box construct. Cross-linked nuclear extracts, prepared as above, were incubated with a monoclonal antibody that targets the T7 tag followed by RNase digestion. Figure 5B shows that labeled fibrillarin was co-immunoprecipitated by this antibody, demonstrating that the 15.5 kDa protein had indeed assembled onto the injected RNA construct. The large amount of undigested RNA (*) precipitated was most probably due to mixing the protein and RNA prior to injection, leading to aggregates resistant to digestion. The cross-linking constructs reconstitute methylation activity The oocyte system provides the opportunity to assess whether snoRNPs assembled with the 4SU-containing U25 RNAs are functional. Endogenous U25 snoRNA was depleted from a pool of oocytes by injecting an antisense DNA oligonucleotide that elicits selective degradation of U25 by endogenous RNase H and is then itself hydrolyzed (Prives and Foukal, 1991). These oocytes next received a U25 cross-linking construct co-injected with a 32P- labeled artificial methylation substrate (Tycowski et al., 1998; Figure 6A), containing a 12 nucleotide sequence identical to that which is methylated in 18S rRNA (see Figure 1A). Site-specific methylation of the substrate within the sequence complementary to the U25 snoRNA was assayed by exploiting the fact that RNase H does not cleave a site that is 2′-O-methylated when targeted by an RNA–DNA chimera (Yu et al., 1997). Figure 6.U25 constructs reconstitute methylation activity in vivo. (A) The substrate used to assay the methylation activity of substituted U25 snoRNPs is shown, with underlining indicating sequences complementary to the U25 snoRNA and the arrow indicating the nucleotide targeted for methylation. (B) The methylation activity of reconstituted U25 snoRNPs was assessed by co-injecting a 4SU-containing U25 snoRNA construct with the methylation substrate into nuclei 18 h after oocytes were injected with an antisense U25 DNA oligonucleotide. Nuclear RNA was isolated 4 h later and incubated with the chimeric oligonucleotide chim-U25 and RNase H as indicated. Lanes 1 and 2 show the uninjected substrate incubated in the absence or presence of RNase H. In lanes 3 and 4, the endogenous U25 snoRNA was not depleted. The amount of residual full-length substrate (full) and the cleaved substrate (cleaved) provides a qualitative assessment of the extent of site-specific methylation. In lanes 19–30, the full-length product was more degraded than in lanes 1–18. The activity of each construct was reproduced in three separate experiments. Download figure Download PowerPoint Figure 6B shows that uninjected substrate can be fully cleaved in the presence of RNase H and the chimeric oligonucleotide (Figure 6B, lanes 1 and 2). Substrate recovered from undepleted oocytes was methylated by the endogenous U25 snoRNA, as revealed by its partial protection from cleavage by RNase H (Figure 6B, lanes 3 and 4). In oocytes depleted of the U25 snoRNA, the substrate was not methylated (Figure 6B, lanes 5 and 6). When 4SU-containing constructs having the wild-type U25 sequence were injected, methylation activity (appearance of uncleaved full-length substrate) was observed (Figure 6B, lanes 8, 18, 20 and 26). Only the C-1 construct showed no rescue (lane 16), presumably because its binding of the 15.5 kDa protein is perturbed, as shown in Figure 5A. The C-2 construct was active in methylation, although its activity appeared weak in comparison with the other wild-type constructs (lane 18) in this non-quantitative assay. SnoRNA constructs with mutations in the C, C′, D′ or D boxes exhibited either partial (lane 10) or complete loss (lanes 12, 14, 24, 28 and 30) of methylation activity, with all double mutants showing complete loss. One surprise was the D′(D′mt) construct, whose methylation activity was comparable reproducibly with that of the wild-type D′ construct (lanes 20 and 22). Although the G and A nucleotides are the most conserved nucleotides within the D′ box (Kiss-Laszlo et al., 1998), the activity of this mutant suggests that they may not be required for methylation. We conclude that the incorporation of 4SU within the individual box elements (with the exception of the C-1 position) does not disrupt the proper assembly of a box C/D snoRNP or adversely affect the ability of the synthetic snoRNA to reconstitute site-specific methylation. It therefore seems likely that cross-linking takes place within functional reconstituted snoRNPs. The position of the guide sequence does not alter the asymmetric cross-linking of Nop56 and Nop58 Finally, we asked whether the exclusive cross-linking of Nop56 and Nop58 to boxes C′ and C, respectively, might reflect the position of the guide sequence, which in U25 is located upstream of box D′ (Figure 1A). The antisense element was thus relocated 5′ to the D box (Figure 7A) by switching the 19 nucleotide sequence that lies between the C′ box and D box of U25 with the 15 nucleotide sequence between the C box and D′ box. The C′-1 position and the C-2 position were again substituted with 4SU and the constructs analyzed by cross-linking (Figure 7B). Nop56 cross-linked exclusively to box C′ and Nop58 to box C in the switched (SW) constructs, the same pattern as observed for wild-type U25. Moreover, the construct with the D box guiding methylation was functional in rescuing methylation activity in the oocyte (Figure 7C), showing that U25 possesses two potentially functional guide centers—at the D and D′ boxes. Figure 7.The C′ box cross-links to Nop56 and the C box to Nop58, regardless of the location of the guide sequence. (A) U25 constructs were designed with the 15 nucleotide sequence between the C box and D′ box switched for the 19 nucleotide sequence between the C′ box and the D box. The constructs are schematized and named as in Figure 1A, with SW indicating switched. (B) Cross-linking and analyses of labeled proteins wer