Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 11

doi:10.1006/jmbi.2000.4015 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 302, 65±77

Ribosomal RNA Maturation in Schizosaccharomyces pombe is Dependent on a Large Ribonucleoprotein Complex of the Internal Transcribed Spacer 1 Atanas I. Lalev, Priyanka D. Abeyrathne and Ross N. Nazar* Department of Molecular Biology and Genetics University of Guelph, Guelph Ontario, Canada N1G 2W1

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a speci®c large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modi®cation exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses con®rmed an interaction with this sequence and indicated that this domain is critical to the ef®cient maturation of the precursor RNA. The protein constituents, puri®ed by af®nity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel puri®ed complex, prepared with af®nity-puri®ed proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identi®ed eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identi®ed as a pseudouridylate synthase. Additional RNA constituents were not detected. The signi®cance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. # 2000 Academic Press

*Corresponding author

Keywords: rRNA; RNA processing; internal transcribed spacer 1; nucleolus; ribosome

Introduction The ribosomal RNAs of eukaryote ribosomes are transcribed in the nucleolus as a large precursor molecule which interacts with both ribosomal and non-ribosomal proteins to form an 80 to 90 S ribonucleoprotein particle (Woolford & Warner, 1991; Raue & Planta, 1991). Processing of this precursor to form mature ribosomal subunits involves both the modi®cation of nucleotides through RNA methylation and base conversions as well as prerRNA cleavage to form the mature rRNAs. In the course of RNA cleavage at least four transcribed spacers are removed, two of which are external to the mature rRNA sequences (50 ETS and 30 ETS) and two which are internal (ITS1 and ITS2), separE-mail address of the corresponding author: [email protected] 0022-2836/00/010065±13 $35.00/0

ating the 18 S, 5.8 S and 25-28 S rRNAs. The role of these spacer regions and their removal in the course of rRNA processing has been the subject of considerable debate and experimentation. While clearly demonstrated to be important in rRNA maturation (e.g. Musters et al., 1990; Van der Sande et al., 1992; Van Nues et al., 1994, 1995), the wide divergence in both their sequences and size is surprising (see Nazar, 1982) and has led to a speculation that they may act as ``biological springs'' (Nazar et al., 1987) which bring the maturing termini together in an appropriate manner for the actual cleavage. Recent studies also have revealed unanticipated interdependencies between the spacers. For example, the 30 ETS structure was found to affect critically the processing of the ITS2 and the 5.8 S rRNA, sequences which are over 3000 bases upstream (Melekhovets et al., 1994). # 2000 Academic Press

66 Removal of the ITS2 region also affects not only the maturation of the 5.8 S and 28 S rRNAs but reduces the yield of the 18 S rRNA to about 10 % of normal levels. Deletions in the 50 ETS, which prevent 18 S rRNA maturation, also dramatically lower the production of the LSU RNAs (Good et al., 1997a; Intine et al., 1999). Taken together, all of these observations have led to a speculation that the formation of a 80-90 S nucleolar precursor particle and its subsequent processing acts as a quality control mechanism which helps to ensure that only functional rRNA is incorporated into ribosomes (Good et al., 1997a). To identify features which may underlie the interdependencies in rRNA processing, structural features in the spacer regions are being examined more closely. For example, in Schizosaccharomyces pombe a helical region in the conserved 30 ETS hairpin which correlates strongly with observed interdependencies in rRNA processing also appears to act as a binding site for soluble proteins or other factors (Hitchen et al., 1997). In a similar way a conserved core stem structure in the ITS1 sequence recently was observed to form a speci®c complex with protein or other soluble factors, a complex which could be critical to rRNA processing or interdependence in the processing steps (Lalev & Nazar, 1998). To examine these possibilities further, in this study the ITS1 ribonucleoprotein complex has been characterized in respect to both the protein components and the RNA sequences which interact with the proteins.

Results A speci®c ITS1-protein complex was demonstrated previously by gel retardation (Lalev & Nazar, 1998). The complexes could be detected using polyacrylamide or agarose gel electrophoresis; presumably, because of the large complex size, a better fractionation was observed with a 2 % (w/v) agarose gel. To prepare larger amounts of complex, in this study af®nity chromatography was examined as a means of puri®cation. Larger amounts (0.5-1.0 mg) of ITS1 RNA again were transcribed in vitro using T7 RNA polymerase. To use a poly C-agarose matrix, a poly C sequence was added to the DNA template sequence, which encoded a 12 nt poly G sequence at the 30 end of the internal transcribed spacer RNA. After puri®cation by gel electrophoresis the ITS1 RNA was annealed with poly C-agarose in binding buffer (0.15 M KCl, 10 mM Tris-HCl (pH 7.5)) with gentle agitation at 4  C for two hours. The RNA loaded matrix was washed and incubated in binding buffer with protein extract and carrier RNA at 4  C with gentle agitation for two hours before being loaded into a short column, washed and eluted with 0.3 M KCl followed with buffer containing 1 M KCl. Proteins which were eluted in each fraction were then characterized with respect to their ability to form ribonucleoprotein complex and the

ITS1 Ribonucleoprotein Complex

macromolecular constituents which were present. Control columns without annealed RNA or with RNA of unrelated sequence also were prepared. As shown in Figure 1 (left panel), this chromatographic procedure did effectively purify protein constituents which speci®cally interacted with the ITS1 sequence. The last (1.0 M KCl) fraction provided constituents which formed an ITS1-protein complex (lane d) as observed with the original protein extract (lane b). In contrast, very little complex was formed when the 0.3 M KCl fraction was used (lane c). Equivalent fractions from columns with no RNA or an unrelated RNA sequence were unable to form the complex (Figure 1, right panel). For example, when an unrelated plant (lane e) or plasmid (lane g) sequence-derived RNA was used as the RNA ligand during af®nity chromatography, the protein complex was not retained and no complex could be formed (lanes f and h, respectively). The protein constituents in the eluted fractions also were characterized using SDS-gel electrophoresis. As shown in Figure 2, when compared with the original protein extract the protein pro®les for the eluted fractions remained relatively complex with the high salt fraction (1.0 M) being the least heterogeneous. Proteins ranged in size from 20-200 kDa MW, with the relative molar concentrations varying greatly. To better de®ne the actual protein components which speci®cally associate with the ITS1 RNA sequence, ITS1-protein complex was prepared and puri®ed by gel electrophoresis. The complexes were detected by autoradiography (Figure 3, left); the band was excised, soaked in loading buffer and placed in one of the individual slots positions of an SDS-polyacrylamide gel for protein analysis. As shown in Figure 3 (right), the protein pro®le remained complex, consistent with a large ribonucleoprotein complex, but most of the individual protein components now were present in similar concentrations as evident from the more uniformly stained bands. At least 20 protein components were observed again ranging in size from 20200,000 Da. In contrast, when RNA was extracted from the same puri®ed RNP band, the ITS1 RNA was clearly evident but only traces of other RNA components were observed (results not shown). To characterize further or identify the protein components, peptide mapping was undertaken using Maldi-Tof mass spectroscopy (see Kuster & Mann, 1998). The protein bands were excised, reduced, 5-alkylated and digested with trypsin ``in gel'' (Shevchenko et al., 1996) and the peptide masses were determined using a Voyage-DE STR Maldi-Tof mass spectrometer (PE Biosystems, Foster City, CA, USA). The ProFound server (Rockefeller University) was used to search protein databases in order to identify the proteins and the Pfam server (Washington University, St. Louis, USA) was used to identify putative RNA binding motifs. As indicated in Table 1, eight proteins were tentatively identi®ed as RNA binding proteins. At least four different RNA-binding motifs were pre-

67

ITS1 Ribonucleoprotein Complex

Figure 1. Ribonucleoprotein formation after protein fractionation by af®nity chromatography. (a) ITS1 RNA or (e) unrelated plant or (g) plasmid-encoded RNAs were transcribed in vitro and used as ligands for the fractionation of a S. pombe cellular protein extract as described in Materials and Methods. Fractions from an ITS1 RNA-loaded column (left panel) eluted with 0.3 M (c) and 1.0 M (d) KCl were analyzed for ability to form ribonucleoprotein by incubation with labelled ITS1 RNA as previously described (Lalev & Nazar, 1998). The free and protein bound fractions were separated by agarose gel electrophoresis. Free RNA (a) and RNA incubated with the initial cell protein extract (b) were included for comparison. Fractions (right panel) eluted with 1.0 M KCl from columns loaded with (f) plant or (h) plasmid gene encoded RNA were analyzed for ability to form ribonucleoprotein complex as described above. (a) ITS1 RNA and (b) ribonucleoprotein are included for comparison.

sent and two of the proteins (gi3123239 and gi4049510) contained more than one RNA binding site. All of these observations were consistent with a protein complex containing multiple binding sites for spacer and perhaps maturing RNAs. In addition to putative RNA binding proteins one of the bands (10) was found to correspond with an putative pseudouridylate synthase (gi3850107). The presence of this type of protein activity also was entirely consistent with rRNA maturation. While

all the identi®ed proteins were only characterized through motif searches, six of the eight proteins with RNA binding motifs were also predicted to be nuclear in localization. Of ®ve additional proteins, which could be identi®ed with equal certainty but did not contain recognizable RNA binding motifs, three (gi2661614, gi3650404 and gi3024016) also were predicted to be localized in the nucleus. All of these observations again were fully consistent with a role in ribosome biogenesis.

Table 1. RNA-binding sites in ITS1 RNP proteins identi®ed by Maldi-Tof mass spectrometry Banda 1 3 4 5 11 13 14 15

Proteinb gi6014422 gi6523773 gi1723536 gi3123239 gi4049510 gi3287868 gi6066728 g93024013

a

Peptidesc 9 5 6 19 5 4 5 5

MMd 249,000 92,000 84,000 72,000 38,000 35,000 35,000 35,000

Predictede MW 220,000 99,000 88,000 79,000 49,000 40,000 38,000 34,500

RNAf binding sites DEAD box G patch rrm site 4 rrm sites 2 rrm sites rrm site Hypotheticalg S1 domain

Bands of protein fractionated by SDS/gel electrophoresis as shown in Figure 3. Protein identi®ed by peptide mapping using the ProFound server (http://prowl.rockefeller.edu/cgi-bin/ProFound) based on calculated posterior probability (Zhang & Chait, 1995). c Number of peptides matched in the identi®cation. d Molecular mass (kDa) of the identi®ed protein. e Molecular mass (kDa) predicted from the SDS/gel electrophoresis. f RNA-binding sites as identi®ed by sequence alignments using the Pfam server (http://pfam.wustl.edu) based on the Pfam protein families database (Bateman et al., 2000). g A hypothetical RNA binding protein in the S. pombe protein database PomPep. b

68

Figure 2. Fractionation of af®nity-puri®ed proteins. Aliquots of the original cellular protein extract (Total), or af®nity-puri®ed protein fraction, eluted with 0.3 (0.3 M) or 1.0 (1.0 M) M KCl, were diluted with an equal volume of loading buffer, heated to 65  C and fractionated on a SDS/10% polyacrylamide gel as described by Laemmli (1970). The positions of ``Bench Mark'' protein ladder (Bio Rad Laboratories, Richmond, CA, USA) molecular mass standards, as separately fractionated with the initial cellular protein extract, are indicated at the left.

Previous structural analyses of the ITS1 in S. pombe pre-rRNA (Lalev & Nazar, 1998) identi®ed a central extended hairpin structure which contained a helical domain of limited sequence homology with a similar structure in the 30 ETS and an snRNA protein-binding site. A sequence homology also has been noted with respect to the central extended hairpin in the ITS2 of S. pombe (see Figure 4). This hairpin alone retains the ability to form a ribonucleoprotein complex with cellular protein (Lalev & Nazar, 1998). To identify critical nucleotides in the ITS1-protein interaction, modi®cation exclusion analyses (Nazar & Wildeman, 1983) were undertaken with both the intact ITS1 RNA and only the central extended hairpin structure. The RNA was labeled at the 50 end using

ITS1 Ribonucleoprotein Complex

g[32P] ATP and bases were modi®ed using RNA chemical sequencing reagents (Peattie, 1979). As shown in Figure 5, when modi®ed RNA was incubated with af®nity-puri®ed protein and the resulting ribonucleoprotein complex was fractionated by gel electrophoresis, a reduced amount of ribonucleoprotein was evident which correlated with the degree of RNA modi®cation. At a higher concentration of diethylpyrocarbonate less then 50 % of the RNA was able to form RNP (lane d) when compared with unmodi®ed RNA (lane b). The RNA in both the RNP and free RNA fractions was eluted, puri®ed by SDS/phenol extraction and treated with aniline to cleave the chemically modi®ed residues (Peattie, 1979; Nazar & Wildeman, 1983). The fragments were fractionated on polyacrylamide sequencing gels as shown in Figure 6. As originally reported by Peattie (1979), diethylpyrocarbonate-induced cleavage was primarily directed at the purine residues but some cleavage was observed at pyrimidine sites. A careful comparison of the protein-associated and RNPexcluded RNA fragments indicates partially excluded residues in two regions of the sequence. As shown in the two expanded ®elds (b and c), these reductions were similar when the complex was examined using either the original protein extract or the af®nity-puri®ed protein. When images of replicate experiments were analyzed and the average relative reactivity at each base determined, the two areas of modi®cation exclusion are clear, between U224-U237 and C253A258. More important, when these sites are examined with respect to the secondary structure (Figure 4), the sites appear on opposite sides of the apical helix in the extended central stem. This is the same region which contains conserved nucleotides and previously was speculated to constitute a protein binding site (Lalev & Nazar, 1998). The present observations and their relationship with past speculation were intriguing but it remained unclear if these observations bore any biological signi®cance. To clarify this question, a speci®c mutation was introduced into the conserved helical domain and the mutated rDNA was expressed in vivo to detect any consequences for rRNA biosynthesis. Previous studies have developed and veri®ed an expression system with which about 50 % of the cellular pre-rRNA transcripts are mutant and about half of the cellular rRNA is derived from the mutant genes (Abou Elela et al., 1994, 1995). As illustrated in Figure 7 (lane a, right), using a neutral structural tag in the 5.8 S rRNA, plasmid-derived RNA is easily detected by gel electrophoresis. As also shown in Figure 7, when RNA from S. pombe cells expressing the ITS1 mutant sequence was fractionated, the analyses indicate the sequence was critical and no mature plasmid-derived 5.8 S rRNA was observed. The three independently isolated transformants which were analyzed (lanes b-d) all contained little or no mutant RNA. In addition, when the high molecular weight RNA components were analyzed

69

ITS1 Ribonucleoprotein Complex

Figure 3. Protein constituents of gel puri®ed ribonucleoprotein. ITS1 RNA (ÿ) transcribed in vitro and labelled at the 50 end, was incubated with af®nity-puri®ed protein (1.0 M fraction) and unrelated carrier RNA (‡) and fractionated on a non-denaturing 6 % polyacrylamide gel (left). The ribonucleoprotein band (indicated on the left) was excised and applied (RNP) to a SDS 10 % polyacrylamide gel (right) together with the initial af®nity-puri®ed protein extract (Protein), for electrophoretic fractionation. The proteins were visualized by silver stain, the image was captured using a Gel Doc 1000 image analysis system (Bio-Rad Laboratories, Richmond, and the molecular weight of each band was estimated (indicated in parentheses) as shown in Figure 2.

using a previously well characterized 18 S rRNA speci®c probe for plasmid-derived RNA (Intine et al., 1999), little or no mature 18 S rRNA was present and even precursor molecules were severely reduced. The presence of mutant DNA was con®rmed by PCR ampli®cation and DNA sequence analysis (results not shown). Finally, to more directly implicate the critical sequence in RNP formation, mutant DNA also was expressed in vitro to prepare mutant ITS1 RNA. As shown in Figure 8, when the mutant sequence was compared with normal RNA, the amount of RNP which could be formed also was very signi®cantly reduced and the ribonucleoprotein which formed appeared as a satellite band to the free RNA, possibly because it was less stable and gradually dissociated in the course of electrophoresis. It is clear that under these conditions in which an excess of protein was present, some RNP formed, but the binding obviously was compromised severely, again indicating that the mutated sequence not only affects rRNA processing but also has an important role in protein binding.

Discussion The processing of pre-rRNA transcripts in eukaryotic cells has been linked with a variety of nucleolar snoRNAs and proteins (Lafontaine & Tollervey, 1995). At least three small nucleolar ribonucleoprotein particles (snoRNps) containing different snoRNA (U3, U14 and snR30) have been shown to be essential for the synthesis of the 18 S rRNA (Kass et al., 1990 Li et al., 1990; Hughes & Ares, 1991; Morrissey & Tollervey, 1993) and two large families of snoRNAs also have been shown to participate in the modi®cation of all the three mature rRNAs (see Bachellerie & Cavaille, 1997; Ni et al., 1997). Protein components that are associated with the snoRNAs molecules, including Noplp, So¯p and Garlp also have been shown to be required for 18 S rRNA synthesis (Tollervey et al., 1991; Girard et al., 1992; Jansen et al., 1993) and a number of nuclease activities have been linked with speci®c steps in transcript cleavage during rRNA maturation. For example, protein constituents of two related endoribonucleases, RNAse P

70

ITS1 Ribonucleoprotein Complex

Figure 4. Sequence and estimate of the secondary structure for the ITS1 sequence in the 35 S pre-RNA from S. pombe. The darkly shaded residues are conserved in the central extended stems of both the ITS1 and 1TS2 sequence: the lightly shaded area indicates base modi®cations which were partially excluded in the ribonucleoprotein (see Figure 6). Changes in the nucleotide sequence as characterized in Figures 7 and 8 are indicated in bold letters. The estimate of the secondary structure is taken from Lalev & Nazar (1998); the large arrowheads indicate known cleavages in rRNA processing (Good et al., 1997b).

and RNAse MRP have been linked by genetic analyses (Schmitt & Clayton, 1992; Stolc & Altman, 1997) and RNAse III-like endonucleases have been shown directly to cleave the 30 ETS at known intermediate processing sites in both Schizosaccaromyces

cerevisiae and S. pombe (Abou Elela et al., 1996; Rotondo et al., 1997). A 30 to 50 exoribonuclease, Rrp4p, also has been linked with the 30 end maturation of the 5.8 S rRNA (Mitchell et al., 1996) and, more recently, Rrp4p protein has been shown to be

ITS1 Ribonucleoprotein Complex

Figure 5. Effect of diethylpyrocarbonate modi®cation on ribonucleoprotein formation. The central stem in the S. pombe ITS1 RNA sequence was transcribed in vitro, labelled at the 50 end and chemically modi®ed with diethlypyrocarbonate as described in Materials and Methods. (b) Unmodi®ed RNA or (c) RNA modi®ed with 1 ml or (d) 2 ml diethlypyrocarbonate was incubated with af®nity puri®ed protein and unrelated carrier RNA to form ribonucleoprotein and fractionated an a 6 % polyacrylamide gel. The positions of the free and protein-associated RNA are indicated on the right; unmodi®ed RNA is included on the left (a).

present in cell extracts predominantly as part of a nuclease complex which Tollervey and coworkers (1991) have called the exosome (Mitchell et al., 1997), an apparently conserved eukaryotic RNA processing complex containing multiple 30 to 50 exonucleases. This complex, which has been characterized in S. cerevisiae, consists of ®ve essential proteins, Rrp4p, Rrp41p, Rrp42p, Rrp43p and Rrp44p, all with distinct exonuclease activities. The complex which was isolated and characterized in this study is different from the previously reported RNPs in several important respects. Most notable is the new complex is much bigger, and at least as isolated, does not contain additional RNAs as an integral part of its structure. Although this complex includes a known cleavage site in the protein/RNA interface (see Figure 4), all attempts to demonstrate associated nuclease activities, at least under previously described buffer conditions, have been essentially negative with only traces which

71 could not be linked precisely with the known processing steps (results not shown). The protein size distribution is very broad with many of the constituents having molecular masses in excess of those associated with ribosomal proteins. The gelpuri®ed complex contains many proteins in essentially equal amounts, an observation which suggests a stable and speci®c complex rather than simply an aggregation of less soluble proteins. This is further demonstrated by its stability to electrophoretic fractionation. The presence of at least eight proteins with RNA-binding motifs suggests a protein complex with multiple binding sites for spacer or maturing RNA and the presence of a putative pseudouridylate synthase provides additional evidence that this complex is important in pre-rRNA maturation. Although the role of the individual protein components or the complex, itself, is unclear, at least one site of interaction in the ITS1 sequence is obviously critical to rRNA maturation and/or rRNA stability (see Figure 6). Since this same site also is very important for complex formation these observations clearly underline the potential importance of the complex with respect to rRNA processing. Taken together, all the results are consistent with at least three roles for this complex. The complex could simply act to protect the nascent transcript from random nuclease digestion, it could act to help or chaperone the correct assembly of the rRNAs into ribosomal subunits or it could serve as a ``work bench'' with a robotic-like or guide function to orient other protein or RNA components which actively participate in the maturation of ribosomes. The size of the complex and the large number of proteins are reminiscent of the splicesomal complex found in mRNA processing but snRNA components could not be detected. If they actually are equivalent the RNAs were lost in the course of protein puri®cation or, as previously speculated (Lalev & Nazar, 1998), the transcribed spacers themselves may play this role. When the protein-binding site in the ITS1 sequence is altered, the absence of mature RNA or of large amounts of precursor, as demonstrated by the hybridization analyses of the ITS1 mutant (Figure 7) indicates that, at least indirectly, the complex acts to stabilize the nascent RNA and is essential for ef®cient maturation. Therefore, it is clear that the complex does act to prevent the rapid degradation of the precursor RNA molecules. The rapid turnover of mutant RNA again suggests that at least one important role is ``quality control'' which helps ensure that only functional RNA is incorporated into mature ribosomes (see Good & Nazar, 1997a). The presence of a pseudouridylate synthase-like protein further indicates the complex contains or, at least, does interact with proteins that function in rRNA maturation. This is consistent with the more expanded ``work bench'' model but must be con®rmed with direct evidence. Further studies of interactions with other regions of RNA or soluble factors will be essential to explore the alternate

72

ITS1 Ribonucleoprotein Complex

Figure 6. Probing of the protein-binding site by modi®cation exclusion. Labelled ITS1 RNA was prepared, modi®ed with diethypylrocarbronate and used to form ribonucleoprotein as shown in Figure 4. The gel puri®ed protein-associated (RNP) or free RNA (RNA) bands were eluted, extracted with SDS/plenol and the modi®ed bases were cleaved with aniline. (a) The resulting fragments were fractionated on 8 % polyacrylamide sequencing gels for comparison and image analyses. Both ribonucleoprotein formed with the (b) original cellular protein extract and (c) af®nity-puri®ed protein were examined. The gel images were captured and used to determine the level of modi®cation in protein-associated RNA as compared with the free RNA fraction. The average for three replicate experiments with af®nity-puri®ed protein is presented as a histogram.

possibilities for additional function. In the interim, the identi®cation of this large spacer RNA/protein complex is likely an essential step in our understanding of the assembly and role of the 80-90 S nucleolar preribosomal RNP.

Materials and Methods Preparation of ITS1 RNAs Normal and mutant ITS1 RNA for RNP formation and af®nity chromatography were prepared by in vitro

ITS1 Ribonucleoprotein Complex

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Figure 7. Effect of ITS1 sequence mutations on the processing and stability of the S. pombe pre-RNA. Whole cell RNA was extracted from S. pombe cells (WT) or cells expressing normal ``tagged'' rDNA (a) and rDNA containing a mutant ITS1 sequence (b-d) as described in Figure 4. Aliquots of RNA were fractionated on a 2 % (w/v) agarose gel and transferred to nylon for hybridization analysis (left) or an 8 % polyacrylamide sequencing gel for 5.8 S rRNA analysis (right). The 32P-labelled oligomer probe, speci®c for the plasmid-derived tagged 18 S rRNA, was that described previously by Good et al. (1997a); the position of the 18 S rRNA, as initially detected by methylene blue stain, is indicated at the right. The 5.8 S rRNA were detected directly by methylene blue stain; the position of the normal (5.8 S rRNA) and plasmid-derived RNA (Mutant) are indicated on the right. (b)-(d) RNA from three separate transformants is shown together with RNA from untransformed cells (WT).

transcription using T7 RNA polymerase (Melton et al., 1984; Lee & Nazar, 1997). The templates for transcription assays were subclones of the pTZ19R plasmid containing the normal ITS1 sequence (Lalev & Nazar, 1998) or mutant sequences derived from it. Transcription reactions were performed with 0.4-2.0 mg of template DNA, 2 mM ATP, CTP, GTP and UTP, and 100 units of T7RNA polymerase in 100 ml of reaction buffer (20 mM MgCl2, 20 mM DTT, 1 mM spermidine, 40 mM Tris-HCl (pH 8.1) and 0.01% (w/v) Triton X-100). Affinity chromatography RNA-binding proteins were puri®ed by af®nity chromatography using ITS1 RNA bound to a poly C agarose support (Sigma-Aldrich Co., St. Louis, MI, USA). The RNA ligand was prepared by in vitro transcription as described above using T7RNA polymerase and a pTZ19R plasmid template containing a T7RNA polymerase promoter and the ITS1 intragenic sequence with a 12 nucleotide poly G sequence at its 30 end. The original plasmid ®rst was modi®ed with the insertion of a poly C/G cluster in the multiple cloning site sequence. Two oligonucleotides, AGCTTCCCGGGGGGGGGG and CCCCCCCCCGGGA were synthesized using a Cyclone Plus DNA synthesizer (Milligen/Biosearch, San Rafael, CA, USA), annealed to each other and cloned into a HindIII and SmaI endonuclease cleaved vector. A PCR ampli®ed S. pombe ITS1 sequence (Lalev & Nazar, 1998) was cloned into this modi®ed vector after cleavage with SmaI endonuclease and ®nally the resulting clone was

cleaved with EcoRI restriction enzyme to provide the linearized template which encoded ITS1 RNA with 12 guanylic acid residues at the 30 end. To prepare the column, 0.1 g of matrix was suspended in 1.5 ml of H2O, allowed to swell for 20 minutes with occasional agitation and washed three times with 1 ml of H2O, followed with 2  RNA binding buffer (0.3 M KCl, 20 mM Tris-HC; (pH 7.5)). Labelled RNA (0.5-1 mg, 100,000 cpm) was dissolved in 0.5 ml of binding buffer, heated brie¯y at 65  C and gently mixed with the matrix for two hours at 4  C. The suspension was applied to a small column (0.5 cm  5 cm) and washed with 10 ml of binding buffer. The radioactivity in the eluents was used to calculate the annealing ef®ciency (normally about 50 mg RNA per 100 ml swollen matrix). Proteins for af®nity chromatography or gel mobility shift assays were prepared as described by Jazwinski, 1990 and Lalev & Nazar, 1998. The proteins were extracted from logarithmically growing S. pombe, strain hÿ leu 1-32 ura 4-D18. Cells were harvested (A550 ˆ 0.6, 1 l) by centrifugation, washed with water, suspended in 10 ml of ice-cold breaking buffer (0.4 M KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 10 mM Tris-HCl (pH 7.9)) and broken by vortex for 30 minutes with an equal volume of glass beads (30 second cycles alternating with 30 seconds on ice). The lysate was cleared by centrifugation at 100,000 g in a Beckman (Fullerton, CA, USA) Ti70 rotor for one hour at 4  C, and glycerol was added (15 %, ®nal concentration) for storage at ÿ85  C. The protein concentration was approximately 10 mg/ml when standardized against bovine serum albumin. For

74

ITS1 Ribonucleoprotein Complex buffer (0.5 M KCl, 25 mM MgCl2, 25 mM DTT, 60 mM Tris-HCl, (pH 8.0), containing 40 % glycerol) and 6 mg of calf liver rRNA, to eliminate non-speci®c interactions. After incubation, the solutions were cleared by microfuge centrifugation for one minute and applied to a 2 % (w/v) agarose gel for fractionation at 4  C. Following electrophoresis the gels were exposed to X-ray ®lm to detect the bands. Electrophoretic analyses of protein constituents

Figure 8. Effect of ITS1 sequence mutations on the formation of ribonucleoprotein. ITS1 was transcribed in vitro from a (a) normal or (c) mutant DNA template, labelled at the 50 end and incubated with af®nity-puri®ed protein and unrelated carrier RNA. (b) and (d) The ribonucleoprotein was fractionated on a 2 % agarose gel and detected by autoradiography. The position of the free (RNA) and normal ribonucleoprotein complex (RNP) are shown at the right.

RNA binding, 10 ml of protein extract was diluted with 20 ml of chromatography buffer (5 mM MgCl2, 0.3 mM PMSF, 2 mM DTT, 10 mM Tris-HCI (pH 7.5), containing 5 % (w/v) glycerol and 0.01 % Triton X-100) and incubated on ice for ten minutes with 5-10 mg of unrelated carrier RNA (calf liver rRNA). The solution was cleared by centrifugation at 12,000 RPM using a Beckman JA10 rotor and mixed gently with RNA-loaded column matrix for two hours at 4  C. For af®nity chromatography the RNA-loaded matrix suspension again was applied to a small column (0.5 cm  5 cm) and washed with 10-20 ml of chromatography buffer containing 0.133 M KCl. The bound protein was eluted in two steps; an intermediate fraction was eluted with ®ve, 200 ml applications of chromatography buffer containing 0.3 M KC1 and a high salt fraction with ®ve, 200 ml applications of chromatography buffer containing 1 M KC1. Electrophoretic mobility shift assay Ribonucleoprotein complexes were assayed by gel retardation as previously described (Henninghausen & Luban, 1997; Lalev & Nazar, 1998). Aliquots of in vitro transcribed labelled ITS1 precursor RNA (1-2 ng/20,000 CPM) were incubated (20 ml, total volume) for ten minutes with 5 ml of protein extract, 4 ml of 5  binding

For analyses of column eluates, the protein constituents were fractionated directly on 10 % SDS/polyacrylamide gels using a 5 % stacking gel as described by Laemmli (1970). Aliquots (10 ml) were mixed directly with equal volumes of loading buffer, heated in boiling water for three minutes and fractionated for 2.5 hours at 70 volts using a Mini-PROTEAN Cell (Bio-Rad Laboratories, Hercules, CA USA). The protein bands were visualized using silver stain essentially as described by Merril et al. (1981). Gels were ®xed for one hour in 12 % (w/v) trichloroacetic at 55  C, washed overnight at room temperature in 10 % (v/v) methanol/10 % (v/v) acetic acid with gentle agitation and then deionized water for one hour. The washed gels were soaked in 12 mM AgNO3 for 20 minutes followed by brief rinsing with water. Finally, the image was developed with 0.28 M NaCO3/0.018 % (v/v) formaldehyde with changes at ten seconds and one minute, the reaction was stopped with 3 % acetic acid and the gels were washed with water for one hour. Images were captured using a Gel Doc 1000 (Bio-Rad Laboratories, Richmond, CA, USA). For ribonucleoprotein constituents, the complex was puri®ed on a 5 % polyacrylamide gel using the labelled RNA and autoradiography to identify the position. The band was excised, soaked for two hours at room temperature in loading buffer and placed vertically in a position corresponding to a normal slot in the 5 % stacking layer of a SDS/10 % polyacrylamide protein analysis gel as described above. The stacking layer was polymerized with the excised band in place, the protein constituents again were fractionated for 2.5 hours at 70 volts and the bands were visualized using silver stain. In some analyses markers were applied in an adjacent lane to estimate the molecular masses. Protein identification by Maldi-Tof mass spectrometry To identify protein constituents in the ribonucleo protein complex, the complex was puri®ed on a 5 % polyacrylamide gel and the excised band was fractionated further by SDS/polyacrylamide gel electrophoresis as described above. The bands were localized using silver stain and, after development, washed with 1 % acetic acid and rinsed with water. For peptide digests the bands were excised and chopped with a clean blade, washed with 50 % (w/v) acetonitrile/ammonium bicarbonate, in gel reduced, S-alkylated with iodoacetamide in ammonium bicarbonate and ®nally dehydrated with acetonitrile essentially as described by Shevchenko and co-workers (1996). The gel particles were rehydrated in 50 mM ammonium bicarbonate, 0.05 % CaCl2 containing 10 ng/ml of modi®ed sequencing grade trypsin (Promega, Madison, WI, USA) and incubated overnight at 37  C. Following digestion, the peptides were extracted with 100 mM ammonium bicarbonate, acidi®ed with 1 % (v/v) (glacial acetic acid and adsorbed on C18 reverse

75

ITS1 Ribonucleoprotein Complex phase support, using ZipTips (Millipore Corp. Bedford, MA, USA) or batch adsorption. The resin was washed with 2 % acetonitrile/1 % acetic acid and the peptides were eluted for mass analysis with 65 % acetonitrile/1 % acetic acid. The samples were subjected to Maldi-Tof mass spectrometry at the Mass Spectrometry laboratory of the Molecular Medicine Research Centre at the University of Toronto using a Voyage-DE STR Maldi-Tof mass spectrometer (PE Biosystems, Foster City, CA, USA). Proteins in each sample were identi®ed from the resulting peptide masses by peptide mapping using the ProFound server (Rockefeller University) to search protein data bases. Both peptide matches and the observed molecular masses were used to identify the most probable proteins. For RNA-binding sites, the Pfam server (Washington University, St. Louis, USA) was used to search the Pfam protein families database. Modification exclusion analyses Critical residues in protein binding sites within the RNA spacer were determined by modi®cation exclusion as described (Peattie & Herr, 1981; Nazar & Wildeman, 1983). After dephosphorylation with calf intestinal alkaline phosphotase in vitro transcribed ITS1 precursor RNA or just the central extended stem regions were labelled at the 50 end using bacteriophage T4 polynucleotide kinase and g[32P] ATP and puri®ed in a denaturing 6 % (w/v) polyacrylamide gel (Lalev & Nazar, 1998). The labelled RNA (5  105 cpm) together with 10 mg of carrier RNA (tRNA) was dissolved in 200 ml of reaction buffer (1 mM EDTA, 50 mM NaAc, (pH 4.5)), 1 ml of diethylpyrocarbonate was added and the solution was heated at 90  C for three minutes. The mixture was cooled, 20 ml of 3 M NaAc were added to stop the reaction and the RNA was precipitated with 2.5 volumes of ethanol. The modi®ed RNA was dissolved in 20 ml of water and incubated with 4 ml of 5  binding buffer, 6 mg of calf liver rRNA and 5 ml of protein extract to form complex. The solution was then cleared by microfuge centrifugation and the RNP was puri®ed by electrophoresis on a non-denaturing 6 % polyacrylamide gel at 4  C. The free and complexed labelled RNA bands were detected by autoradiography and eluted by gel homogenization. After SDS/phenol extraction, each RNA fraction again was puri®ed by electrophoresis on a denaturing 6 % polyacrylamide gel, dissolved in 20 ml of freshly prepared aniline solution (1 M analine in 2.5 % acetic acid) and incubated at 60  C for 20 minutes in the dark. The mixture was cooled, the reaction was stopped with 200 ml of 0.3 M NaAc and the RNA was precipitated with 0.5 ml of ethanol. The RNA fragments were dissolved in loading buffer (50 % formamide, 0.001 % xylene cyanol, 0.001 % bromophenol blue) and fractionated on a denaturing 8 % polyacrylamide sequencing gel. Construction and expression of mutant rRNA genes Site speci®c mutations were introduced into the ITS1 sequence of a S. pombe rRNA transcriptional unit that had previously been subcloned (Abou Elela et al., 1994) into the pFL20 shuttle vector and ``tagged'' with a PstI restriction site in the 18 S rRNA sequence (Good et al., 1997a) and a four-base insert in the 5.8 S rRNA (Abou Elela et al., 1995). The base substitutions were introduced by two-step PCR ampli®cation (Good & Nazar, 1992)

using a pTZ19R plasmid template containing the ITS1 region with adjacent 30 end 18 S and 50 end 5.8 S rRNA sequences. The resulting mutated and ampli®ed DNA was used to replace the normal sequence in the shuttle vector containing the tagged S. pombe rDNA transcriptional unit. The recombinant subsequently were ampli®ed in Escherichia coli, strain C490 and used to transform S. pombe strain hÿ leu 1-32 ura 4-D18 using the method described by Prentice (1992). Each mutation initially was con®rmed by DNA sequencing (Sanger et al., 1977) and subsequently the presence of mutant rDNA in transformed cells was con®rmed again by PCR ampli®cation of the ITS1 rDNA region followed once more by DNA sequencing. Characterization of the expressed mutant ribosomal RNAs Transformed S. pombe cells expressing normal or mutant rRNAs were grown with constant aeration at 30  C in minimal medium broth (Leupold, 1970). For all analyses, the cells were rapidly cooled with ice, harvested by centrifugation and extracted with SDS/phenol after disruption by vortexing with an equal volume of glass beads (Van Ryk et al., 1992). For 5.8 S rRNA analyses, the RNA was fractionated on an 8 % (v/w) (polyacrylamide gel and stained with methylene blue to detect the separated RNA components (Abou Elela et al., 1995). For 18 S rRNA or precursor analyses, the RNA was fractionated on a 1.5 % agarose/0.2 M formaldehyde gel and transferred to nylon by capillary blotting (Good et al., 1997a). The membrane was stained with methylene blue to con®rm that equivalent amounts of RNA were transferred and then hybridized with a 32P-labelled oligomer probe speci®c for the tagged 18 S rRNA transcribed from the plasmid-associated rDNA (Good et al., 1997a).

Acknowledgments This study was supported by the Natural Sciences and Engineering Research Council of Canada.

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Edited by D. Draper (Received 17 May 2000; received in revised form 29 June 2000; accepted 1 July 2000)