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Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe
Introduction
Materials And Methods
Preparation of ribosomal RNA precursor
Structure analysis by limited nuclease digestion
Electrophoretic mobility shift assay
Results
Discussion
Acknowledgement
References
Structural equivalence in the transcribed spacers of pre-rRNA transcripts in Schizosaccharomyces pombe
Received April 19, 1999; Revised and Accepted June 14, 1999
ABSTRACT The structure of the internal transcribed spacer 2 (ITS2) in Schizosaccharomyces pombe was re-evaluated with respect to phylogenetically conserved features in yeasts, features in other transcribed spacer regions as well as the binding of transacting factors which potentially play a role in ribosomal maturation. Computer analyses and probes for nuclease protection indicate a very simple core structure consisting of a single extended hairpin which includes the interacting termini of the mature 5.8S and 25S rRNAs. Comparisons with ITS2 sequences in greatly diverging organisms indicate that the same feature also can be recognized. This is especially clear in organisms that contain very short sequences in which the putative structures are much less ambiguous. Diversity between organisms is the result of changes in hairpin length as well as the addition of branched helices. Protein binding and gel retardation studies with the S.pombe ITS2 further indicate that, as observed in the 3[prime] external transcribed spacer (ETS) and ITS1 regions, the extended hairpin is not only the site of intermediate RNA cleavage during rRNA processing but also a site for specific interactions with one or more soluble factors. Taken together with other analyses on transcribed spacer regions, the present data suggest that the spacer regions all may act in a similar fashion, not only to organize the maturing terminal sequences, but also serve to organize specific soluble factors possibly acting with snoRNAs or in a manner which is analogous with that of the free snoRNPs.
INTRODUCTION
Eukaryotic rRNAs are cleaved from a large 35-45S pre rRNA nucleolar transcript which initially must be fully transcribed, modified through RNA methylation and other base conversions, and assembled into an 80-90S nucleolar ribonucleoprotein particle (1,2). Electron micrographs of chromatin spreads derived from actively transcribed nucleoli (3) and analyses of the nucleolar RNA precursors (4) have often demonstrated these nascent and intact rRNA transcripts. The processing of the transcripts, however, has usually been pictured as a cleavage pathway (5) of many rapid and independent steps beginning with the two external transcribed spacers (5[prime]ETS and 3[prime]ETS) and completed with cleavages in the internal transcribed spacers (ITS1 and ITS2). The ribosomal subunits also appeared to mature independent of each other and recent studies have shown (6) that some rRNA even can be produced in trans. The cleavage pathway itself is a conserved feature of eukaryotes as similar pathways have been demonstrated in many organisms (7).
Despite these observations, a number of more recent analyses now show that subunit maturation is much more interdependent then previously believed. For example, deletion of a conserved 3[prime]ETS structure has been found to dramatically affect the processing of the ITS2 and the 5.8S rRNA sequences which are over 3000 bases upstream (8,9) and deletion of the ITS2 spacer not only prevents the maturation of the large subunit, but severely affects maturation of the small subunit rRNA (10). Even changes to the position of termination appear to critically alter the maturation efficiency (11). All of these observations have been taken as evidence that interdependencies in rRNA maturation act as a quality control mechanism to help ensure that only functional rRNA is incorporated into ribosomes.
In order to identify features which underlie the observed interdependencies the transcribed spacer sequences are being re-examined for structures that interact with soluble constituents of the cell. In a recent study of the 3[prime]ETS, the hairpin structure which was found critical for 5.8S rRNA processing, also was demonstrated to form a complex when incubated with a cellular protein extract (9). More recently a similar hairpin structure was recognized in the ITS1 sequence (12). This hairpin was also shown to form a complex with cellular protein extract and is a site of intermediate cleavage (7). In the present study, the structure of the ITS2 region is re-evaluated and again a primary hairpin structure is demonstrated which also is shown to form a complex with cellular protein extract.
MATERIALS AND METHODS
Preparation of ribosomal RNA precursor
A DNA template for the ITS2 region in Schizosaccharomyces pombe rDNA was prepared by PCR amplification using primers specific for the 3[prime] end of the 5.8 rRNA (ATGCCTGTTTGAGTGTC), begining 20 nt from the 3[prime] end, and the 5[prime] end of the 25S rRNA (GCGAATAACTATACGAA), ending 55 nt downstream of the 5[prime] end. This 374 bp fragment was cloned in the pTZR19 plasmid for expression by bacteriophage T7 RNA polymerase after cleavage with EcoRI restriction endonuclease, as previously described (13,14). The precursor RNA transcript was purified on a 6% denaturing polyacrylamide gel and labeled at the 5[prime] end using bacteriophage T4 polynucleotide kinase and [[gamma]-32P]ATP (15), after dephosphorylation with calf intestinal alkaline phosphatase (16). The phosphatase was inactivated by heating to 75°C for 10 min in 5 mM EDTA (pH 8.0) before the RNA was labeled and the labeled RNA again was purified on a 6% denaturing polyacrylamide gel. To prepare just the central extended stem RNA in ITS1, the same methodology was applied using primers specific for the 5[prime] (GATGAGGTGTTG) and 3[prime] (GTTAAGGTTCAA) ends of the 222 nt sequence.
Structure analysis by limited nuclease digestion
The RNA structure was probed by partial nuclease digestion essentially as described by Van Ryk and Nazar (17). For pancreatic or T1 ribonuclease digests, the in vitro transcribed and labeled ITS2 precursor RNA was dissolved renatured in physiological-like buffer (0.2 M KCl, 50 mM MgCl2, 10 mM Tris, pH 7.5) or dissolved in denaturing buffer (7 M urea, 1 mM EDTA, 20 mM Na citrate, pH 5.0). Aliquots (10 µl) containing 10 µg of carrier RNA (Torula) were incubated for 5 min in the presence of different amount of enzyme at room temperature in physiological-like buffer or 50°C in denaturing buffer. For S1 nuclease digests, the RNA was dissolved and renatured in 0.3 M KCl, 1 mM ZnSO4, 30 mM NaOAc, pH 5.1 containing 5% glycerol. Aliquots (10 µl) containing 10 µg of carrier RNA were digested for 5 min at room temperature. Digestion was terminated by the addition of 200 µl of 0.1% SDS, 0.3 M NaOAc, pH 5.1 followed immediately with 200 µl of phenol:chloroform (50:50 v/v). Each sample was mixed by vortex for 2 min, the phases were separated by brief centrifugation in a microfuge and the RNA in the aqueous layer was precipitated with 2.5 vol of ethanol. The digested RNA pellets were dissolved in 10 µl of distilled water, 10 µl of loading buffer (formamide containing 0.5% xylene cyanol and 0.05% bromphenol blue) were added, the solution was heated for 3 min at 90°C and applied to an 8% polyacrylamide sequencing gel. After fractionation at 1200 V for 6-12 h, the fragments were detected by autoradiography.
Electrophoretic mobility shift assay
A whole cell protein extract was prepared from S.pombe, strain h- leu1-32 ura 4-D18 cells and used for gel retardation studies based on the methods described by Henninghausen and Luban (18) and Jazwinski (19). Logarithmically growing cells (A550nm = 0.6, 1 1) were harvested by centrifugation, washed with water, suspended in 10 ml ice-cold breaking buffer (0.4 M KC1, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 10 mM Tris-HC1, pH 7.9) and broken by vortex for 30 min with an equal volume of glass beads (30 s cycles alternating with 30 s on ice). The lysate was cleared by centrifugation at 100 000 g in a Beckman (Fullerton, CA) Ti 70 rotor for 1 h at 4°C, diluted with glycerol (15%, final concentration) and divided into 100 µl aliquots for storage at -85°C. The protein concentration was ~10 µg/µl when standardized against bovine serum albumin.
For gel retardation studies, aliquots of labeled precursor RNA (0.5 ng; 20 000 c.p.m.) were incubated (20 µl, total volume) for 10 min with 5 µl of protein extract, 4 µl of 5× binding buffer [0.5 M KCl, 25 mM MgCl2, 2.5 mM DTT, 60 mM Tris-HC1 (pH 8.0), containing 40% glycerol] and 6 µg mouse liver whole cell RNA, to eliminate non-specific interactions. After incubation, the solutions were cleared by microfuge centrifugation for 1 min and applied to a non-denaturing 5.5% polyacrylamide or 2% agarose gel (30 mM boric acid, 30 mM Tris-HCl, pH 8.0) for fractionation using 100 V at 4°C. Following electrophoresis the gels were placed on X-ray film to detect the bands by autoradiography.
RESULTS
As a result of their extensive study on the higher order structure of the ITS2 region in the yeast (Saccharomyces cerevisiae) 35S precursor RNA, Yeh and Lee (20) proposed a model for the ITS2 region with a high degree of secondary structure consisting of several stable hairpins. In attempting to further evaluate this yeast model through a phylogenetic comparison with S.pombe, we found that computer analyses using the mfold web server (http://www.ibc.wustl.edu/~zuker/rna/form1.cgi ) based on the algorithms of Zuker and co-workers (21,22), predicted a somewhat different, less complex but also highly base-paired structure (Fig. 1). Equally important, these analyses basically predicted a simple extended hairpin structure similar to the conserved extended hairpin in the 3[prime]ETS (8) and the central hairpin found in the ITS1 sequence (12). As indicated in Figure 1, the central ITS2 hairpin essentially represents an extension to a previously well documented interaction between the 3[prime] end of the 5.8S rRNA and the 5[prime] end of the 25-28S rRNA (23-25), with the remaining sequence simply forming small branches on each side.
Figure 1. Estimate of the secondary structure of the 1TS2 sequence in the 35S pre-rRNA from S.pombe. The structure was based on a prediction using the mfold web server (http://www.ibc.wustl.edu/~zuker/rna/form1.cgi ) and the results of limited digestion with pancreatic (white arrows) S1 (gray arrows) and T1 (black arrows) ribonuclease as illustrated in Figure 2. The larger arrows indicate strong cleavage and the smaller arrows indicate moderate cleavage. The large black arrowheads represent previously mapped termini in precursor intermediates (7) and the shaded interaction between the 5.8S and 2.5S rRNAs is based on the original studies of Pace and co-workers (25) and a compilation of large subunit RNA sequences (35). The broken lines indicate the extent of the central hairpin structure used in gel shift analyses.
To confirm the estimate for the S.pombe ITS2, the actual structure was probed using partial digestion by pancreatic, T1 and S1 nucleases. RNA for this study was prepared using a cloned ITS2 region template and bacteriophage T7 RNA polymerase (13); the ITS2 RNA was labeled at the 5[prime] end with polynucleotide kinase (15) or at the 3[prime] end with RNA ligase (26) as required. Since the 3[prime] end terminal of the T7 RNA polymerase transcribed RNA is heterogenous, the results were primarily based on 5[prime] end-labeled substrate and confirmed where required, with 3[prime] end-labeled RNA. A range of enzyme concentrations was used in order to distinguish primary from secondary cleavage sites. As illustrated by the example gels shown in Figure 2, and all the experiments summarized in Figure 1, the results were entirely consistent with the computer generated prediction. Primary cleavages were clearly evident at the end of each hairpin and many bulges but were not observed in the base-paired regions. For example, as shown in Figure 2, with pancreatic ribonuclease (left), the strongest cleavages occurred at U168 U225, U247 and U275 in one of the hairpin loops and two major bulges. With S1 nuclease (center), the strongest cleavages at U71, U176 and A226 occurred in a second hairpin loop and the same major bulges and with T1 ribonuclease (right), the major cleavage (G167) also occurred at the large bulge near the end of the extended hairpin. The only unusual feature was the large polypyrimidine-rich bulge near the mature termini (U5-C11/A290-U294) which was not readily susceptible to either S1 or pancreatic RNase digestion, an observation which suggests a higher order structure. Indeed, such a loop in the yeast 5S rRNA also has been shown to resist nuclease attack due to higher order interactions (e.g. 17).
Figure 2. Nuclease-sensitive sites in the ITS2 pre-ribosomal RNA sequence of S.pombe. The RNA was transcribed, labeled and partially digested under physiological-like (a-c) as described in Materials and Methods. The digests were fractionated on denaturing 8% (w/v) polyacrylamide gels with the fragments detected by autoradiography. The RNA was digested with 10-5 (a), 10-4 (b) or 10-3 (c) U/mg of pancreatic ribonuclease (left panel), 5 (a), 2.5 (b) or 125 (c) U/mg of S1 nuclease (center panel), or 0.02 (a), 0.2 (b) or 1 (c) U/mg of T1 ribonuclease (right panel). Digests under denatured conditions (P and T) or by partial base hydrolysis (B) were used to identify the cleavage positions; a sample incubated in the absence of enzyme is included on each gel (C).
While common features previously have been described for closely related organisms (e.g. 26-30), to date the known wide diversity in ITS2 nucleotide composition and length has concealed any conserved core features. In the present study, comparisons with many other ITS2 sequences of smaller, similar or large size were undertaken to see if a central hairpin could be identified. Again the updated mfold web server was used to derive estimates of the secondary structure and both computer databank and actual sequence analyses were undertaken to provide widely divergent examples shown in Figure 3.
Figure 3. A comparison of estimates for the secondary structure of the ITS2 sequence in pre-rRNA transcripts from diverse organisms. The structures were based on predictions using the mfold web server (http://www.ibc.wustl.edu/~zuker/rna/form1.cgi ) and the S.cerevisiae and Homo sapiens sequences were taken from Veldman et al. (24) and Gonzales et al. (48), respectively. The sequences for M.hapla and V.dahliae were determined in our laboratory. The shaded interaction between the 5.8S and 25S rRNAs are based on the original studies of Pace and co-workers (25) and a compilation of large subunit RNA sequences (35).
As illustrated with the human ITS2 sequence, a similar but more extended hairpin can be recognized in many larger ITS2 sequences but alternate putative structures, which also can be suggested, leave these comparisons somewhat ambiguous. To minimize this problem a search was undertaken for shorter ITS2 regions in which the structure could be predicted more accurately. As also shown in Figure 3, studies in our laboratory (31,32) have identified significantly shorter ITS2 sequence (167 nt), in a plant wilt pathogen, Verticillium dahliae, and a very short ITS2 (109 nt) a plant nematode pathogen, Maloedigyne hapla. In each case computer-aided analyses predict a simple central hairpin structure with one or more branch hairpins. This notion is also consistent with bacterial ribosomal RNA in which the 5.8S rRNA sequence is not cleaved from the 23S rRNA (33,34) and forms a still shorter hairpin in the core structure of the RNA from the bacterial large subunit (35).
As noted earlier, the critical conserved stem structure in the 3[prime]ETS is the site of one or more intermediate cleavage steps (8) and forms a ribonucleoprotein complex with cellular protein, the interacting site resembling a protein binding site in U1 snRNA (9). As noted in Figure 1, a more recent study now indicates that during RNA processing, a cleavage site in the S.pombe ITS2 sequence (large arrowhead) also is located in the extended hairpin structure (A). Furthermore, another recent study (7) indicates that, as observed with the 3[prime]ETS region, deletions of the ITS2 sequence can dramatically affect rRNA processing at distinct sites.
To evaluate the possibility that the extended ITS2 hairpin also interacts with one or more soluble cellular components, ITS2 RNA binding was examined using gel retardation. Again a synthetic ITS2 region was made using bacteriophage T7 RNA polymerase labeled at the 5[prime] end with polynucleotide kinase, and incubated with a soluble protein extract of S.pombe cells. To eliminate non-specific interactions, whole cell RNA extracted from mouse liver also was added to each sample. As shown in Figure 4, under the physiological-like gel conditions that were used to fractionate any resulting complexes, a retarded band was clearly evident (lane b) even in the presence of large amounts of unrelated whole cell mouse liver RNA. When a relatively small amount of unlabeled ITS RNA (lane c) was incubated with labeled RNA and protein, competition was clearly evident and the complex was largely dissociated. In contrast, when an equal amount of unrelated synthetic RNA with a similar molecular weight was incubated with labeled RNA and protein, no competition was evident (lane d).
Figure 4. RNA-protein interactions with the ITS2 pre-ribosomal RNA sequence of S.pombe. The RNA was transcribed with T7 RNA polymerase and labeled at the 5[prime] end with [[gamma]-32P]ATP and polynucleotide kinase as described in Materials and Methods. The purified RNA was incubated in the absence (a) or presence (b-d) of 25 µg of S.pombe soluble protein extract and 6 µg of mouse liver RNA, together with 100 ng of unlabeled ITS2 RNA (c) or 100 ng of an unrelated T7 RNA polymerase transcript (d) of similar size (212 nt). The resulting complexes were fractionated by gel electrophoresis and both free (RNA) and complexed (RNP) RNA were detected by autoradiography.
To further define the degree of sequence dependence, two additional types of competition binding were examined. As shown in Figure 5, when other spacer regions from the S.pombe rDNA were used in competition studies the results indicated significant or no binding equivalence, depending on the spacer that was used. In the left panel, with either the 5[prime]ETS (lane c) or 3[prime]ETS (lane d), no competition was evident. In contrast, when ITS1 was substituted (lane d in the right panel), substantial competition was evident and at high concentrations the labeled RNA was fully competed as observed with the unlabeled ITS2, itself (lane c). A similar study with ITS2 regions of more distant organisms, also revealed a limited degree of binding equivalence. As shown in Figure 6, when unlabeled ITS1 from another yeast, S.cerevisiae (lane d), a distantly related fungus, V.dahliae (lane e), or a soil nematode, M.hapla (lane f), was used, all were able to compete with the S.pombe ITS1 sequence to some degree. Where unlabeled S.pombe RNA was able to fully displace the labeled RNA (lane c), the more closely related S.cerevisiae RNA displaced ~75% of the labeled S.pombe ITS1 molecules and the more distantly related examples displaced <50% of the labeled molecules even with a 100-fold excess of unlabeled RNA (Fig. 6).
Figure 5. Effect of other transcribed spacer regions on RNA-protein interactions with the ITS2 pre-ribosomal RNA sequence of S.pombe. The transcribed and labeled ITS2 pre-ribosomal RNA was incubated in the absence (a) or presence (b-d) of 25 µg of S.pombe soluble protein extract and 6 µg of mouse liver RNA, together with 100 ng of unlabeled 5[prime] (c) and 3[prime] (d) ETS (left panel) or unlabeled ITS1 (d) and ITS2 (c) ITS (right panel). The resulting complexes were fractionated by gel electrophoresis and both free (RNA) or complexed (RNP) RNA were detected by autoradiography.
Figure 6. Effect of diverse ITS2 spacer regions on RNA-protein interactions with the ITS2 pre-ribosomal RNA sequence of S.pombe. The transcribed and labeled ITS2 pre-ribosomal RNA was incubated in the absence (a) or presence (b-d) of 25 µg of S.pombe soluble protein extract and 6 µg of mouse liver RNA together with 100 ng of unlabeled ITS1 RNA from S.pombe (c), S.cerevisiae (d), V.dahliae (e) or M.hapla (f). The resulting complexes were fractionated by gel electrophoresis and both free (RNA) or complexed (RNP) RNA were detected by autoradiography.
While it was attractive to speculate that the distal end of the extended hairpin was forming the ribonucleoprotein complex as previously observed with the 3[prime]ETS (9), other regions of the ITS2 sequence could serve as a binding site or contribute significantly to it. To provide direct evidence for an interaction with the distal end, a DNA template for the upper half of the extended hairpin structure was prepared by PCR amplification, inserted into a vector containing a bacteriophage T7 RNA poly-merase promoter and again expressed using the T7 RNA polymerase. As shown in Figure 7, when labeled hairpin RNA was incubated with the soluble protein extract of S.pombe cells and mouse liver whole cell RNA, a ribonucleoprotein complex was again clearly evident in the presence of a large amount of unrelated RNA (right panel, lane b). Also, as in the case of intact ITS2 RNA, the labeled complex was competitively dissociated with unlabeled hairpin fragment (right panel, lane c) but was not affected by a specific unrelated RNA sequence of similar size (right panel, lane d). Furthermore, as shown in Figure 7 (left panel), the hairpin sequence alone was able to effectively compete with the intact ITS2 RNA and could fully dissociate the intact ribonucleoptrotein complex (left panel, lane d). Therefore, as previously observed with the 3[prime]ETS structure, the extended hairpin in ITS2 not only represents a site for RNA processing, but also a specific site for protein binding, presumably one or more trans-acting factors for ribosome maturation.
Figure 7. RNA-protein interactions with the central extended hairpin structure in the ITS2 pre-ribosomal RNA sequence of S.pombe. The central and extended hairpin (right panel) or intact ITS2 RNA (left panel) was transcribed, labeled and incubated in the absence (a) or presence (b-d) of 25 µg of S.pombe soluble protein extract and 6 µg of mouse liver RNA, together with 100 ng of unlabeled hairpin RNA (c), unlabeled hairpin [left (d)] or an unrelated T7 RNA polymerase transcript [right (d)]. The resulting complexes were fractionated by gel electrophoresis and both free (RNA) and complexed (RNP) RNA were detected by autoradiography.
DISCUSSION
While in recent years very significant progress has occurred with respect to rRNA processing (36) and even rRNA modification (37,38), the role of the transcribed spacer regions remains an intriguing puzzle. Although extensive phylogenetic comparisons have indicated a very wide diversity in the sequence and size of these spacers (39), mutational analysis in many laboratories (e.g. 7,8,40-42) provide strong evidence for important functions. Based on structural analyses, we have speculated previously (43) that the secondary structure formed by these sequences may act as a `biological spring' to organize and bring cleavage domains together for specific cuts. This allows for a great diversity in spacer size and composition to accomplish essentially the same task in each individual organism. Our more recent studies of spacer function (7,8), however, have identified more general consequences in rRNA processing and potentially an important role in the `quality control' of ribosome biogenesis. In both the 3[prime]ETS and ITS1 sequence, central extended hairpin structures also have been identified which interact with soluble protein factors that presumably mediate in both localized and general rRNA processing. The present study provides a third example of the same group of observations. The ITS2 sequence dramatically affects both local and distant rRNA processing events (7,44). As illustrated in Figure 3, ITS2 appears to be organized around an extended hairpin structure; it is the site of one or more intermediate cleavages and, as illustrated in Figure 4, it also is a site for soluble factor interaction.
In the previous comparison of factor binding in ITS1 and 3[prime]ETS, similarities were noted in the distal helical region which also shared some features with known protein binding sites in the U1snRNA (9,12). In the present study some structural similarity between the ITS1 and ITS2 regions in S.pombe also was detected by gel retardation analysis (Fig. 5). To explore these potential relationships further, the sequences at the distal end of all three regions were examined more closely. As shown in Figure 8, two interesting similarities can be noted. In the upper comparison all three structures are observed to share a limited conservation in respect to residues previously shown to be similar in the U1snRNA protein-binding site and also to affect factor binding in the 3[prime]ETS (9). In the lower comparison, a more extensive similarity is observed between the ITS1 and ITS2 sequences in the S.pombe rDNA. Indeed either or both of these similarities may be the explanation for competition in the gel retardation analyses (Fig. 5). If this is the case, the inverted orientation of the conserved regions (lower comparison) would predict alternate orientations in the factor binding as well. In any event, both comparisons provide specific targets for future analyses by site-directed mutagenesis.
Figure 8. Comparison of sequence similarity in the central extended hairpin structure from the ITS1, ITS2 and 3[prime]ETS regions in the 35S pre-rRNA from S.pombe. The upper comparison indicates nucleotides that are conserved in all three spacers; the lower comparison indicates additional identical features in the ITSs. Equivalent nucleotide residues are indicated by shading.
The competition studies with divergent ITS1 sequences (Fig. 6) indicate that some features that are important for complex formation were present in all the examples even though the sizes of these spacers varied greatly (Fig. 3). Sequence comparisons have not identified obvious similarities so further speculations regarding putative binding sites are not likely to be useful until the binding site is specifically identified in the S.pombe ITS2 sequence.
In past studies of ITS2 function (44), Raue and co-workers have systematically introduced mutations into the ITS2 spacer of the rDNA in S.cerevisiae, demonstrating a number of critical cis-acting processing elements. Although their working structure model was somewhat different with more branches to the central stem, a number of regions which constitute the extended central stem proposed in Figure 3, were shown to be critical to rRNA processing. As observed in our own studies of the 3[prime]ETS (9) changes in the upper end of the central helix corresponding with or close to the similarities illustrated in Figure 8, had dramatic effects on rRNA processing (26,44), possibly because they disrupt the complex which was demonstrated in Figure 4.
The competitive studies with divergent ITS sequences (Fig. 6) that at least some features which are important for complex formation are present in all the examples even though these spacers vary widely in size and sequence (Fig. 3). Even though attempted, sequence comparison did not identify obvious similarities other then the extended stem. Further speculation as to the putative binding sites is unlikely to be useful until the binding site is identified specifically in the S.pombe ITS2 sequence.
If the three spacer structures (ITS1, ITS2 and 3[prime]ETS) are indeed functionally equivalent as suggested in this study, an obvious question which remains is any relationship with the 5[prime]ETS spacer. While a conserved hairpin has been reported immediately adjacent to the mature 18S rRNA sequence site-directed mutations have not demonstrated a significant role in rRNA processing (45) and our repeated attempts to detect a complex by gel retardation have been negative (results not shown). A second striking difference, however, is the fact that unlike the other spacers the 5[prime]ETS is known to interact with the U3 snoRNA which also appears to initate RNA processing. Because of sequence similarity, we previously suggested (9) that the 3[prime]ETS extended hairpin structure may act in a fashion analogous to a snoRNP, during 3[prime] end maturation. While the three more distal transcribed spacers could all act in this fashion, the requirement of an external snoRNA for efficient 5[prime]ETS processing may be specially related to its initiation function, which also may provide a means of regulating rRNA processing. Indeed past studies (46,47) have shown that a U3-depleted extract processes an rRNA substrate much less efficiently. Whatever the case, the present study strongly suggests that despite the greater diversity in composition and size, at least the other three spacers may be much more equivalent in their structure and function than previously believed.
ACKNOWLEDGEMENT
This study was supported by the Natural Sciences and Engineering Research Council of Canada.
REFERENCES
*To whom correspondence should be addressed. Tel: +1 519 824 4120; Fax: +1 519 837 2075; Email: rnnazar{at}uoguelph.ca
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