Gar1p Binds to the Small Nucleolar RNAs snR10 and snR30 in Vitro through a Nontypical RNA Binding Element*

Claudia BagniDagger and Bruno Lapeyre§

From the Centre de Recherche de Biochimie Macromoléculaire and Institut de Génétique Moléculaire, 1919 Route de Mende, BP5051, 34293 Montpellier Cedex 01, France and Laboratoire de Biologie Moléculaire Eucaryote, 118 Rte de Narbonne, 31062 Toulouse, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The nucleolar proteins Gar1p and fibrillarin possess a typical nucleolar glycine/arginine-rich domain and belong to ribonucleoprotein particles. Both proteins are essential for yeast cell growth and are required for pre-rRNA processing. In addition, Gar1p is involved in pre-rRNA pseudouridylation, whereas fibrillarin is required for pre-rRNA methylation. Gar1p and fibrillarin are each associated with a different subset of the small nucleolar RNAs (snoRNAs). Gar1p is co-immunoprecipitated with the H/ACA family of snoRNAs, whereas fibrillarin is co-immunoprecipitated with the C/D family. However, attempts to demonstrate direct interactions between fibrillarin and snoRNAs have failed, and such interactions between Gar1p and the H/ACA snoRNAs had not yet been reported. Among the H/ACA snoRNAs associated with Gar1p, one can distinguish a large group of snoRNAs that are not essential in yeast and serve as guides for pseudouridine synthesis onto the pre-rRNA molecule. In contrast, the two snoRNAs snR10 and snR30 are required for normal cell growth and for pre-rRNA cleavage. We show here that Gar1p interacts in vitro directly and specifically with these two snoRNAs. Deletion analysis of Gar1p indicates that a major RNA binding element, which is extremely well conserved throughout evolution, lies in the middle of the protein. However, this domain alone binds poorly to the target RNAs and an accessory domain is required to restore efficient binding. The accessory domain can be either one of the glycine/arginine-rich domains or a second element of the core of the protein that is highly conserved between different species.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

It has recently been demonstrated that the numerous small nucleolar RNAs found in the nucleolus of eukaryotic cells can be sorted into two classes (1-3) if one sets apart the unique RNA, component of the mitochondrial RNA processing RNase that is involved in pre-rRNA processing (4). Members of the first class contain structural motifs called box C/D. These snoRNAs1 are associated with the nucleolar protein fibrillarin, and most of them are involved in targeting methylation onto the pre-rRNA through base pairing with this molecule (1, 5, 6). However, there is a subset of the C/D snoRNAs that do not appear to be involved in methylation, but are required for pre-rRNA processing. Members of the second class of snoRNAs contain a "hairpin-hinge-hairpin-tail" structure and are called the H/ACA snoRNAs (1, 7). They are associated with the nucleolar protein Gar1p and are required for targeting pseudouridylation onto the pre-rRNA molecule (8, 9). Among the H/ACA snoRNAs, snR10 and snR30 form a separate subset since they have been found to be required for normal pre-rRNA processing, and deletion of snR30 is lethal (10, 11), whereas the other H/ACA snoRNAs can be deleted without any significant phenotype. It is still controversial (snR10) or not yet determined (snR30) whether these two RNAs are also involved in targeting pseudouridylation onto the pre-rRNA (8, 9) or onto other RNA molecules.

Fibrillarin has long been known to be part of the snoRNP particles, since antisera from humans with autoimmune diseases contain antibodies that recognize fibrillarin and allow to precipitate the snoRNA U3 (12). It has now been demonstrated that fibrillarin is associated with all box C/D snoRNAs, even though attempts to show direct interactions have not been successful. These observations suggest that fibrillarin could be part of a larger structure, without directly contacting the snoRNAs.

Gar1p has recently been shown to be associated with every H/ACA snoRNA (1), but no direct interaction has yet been reported. Gar1p, as well as Nop1p/fibrillarin, Nsr1p/nucleolin/gar2p, Ssb1p, and Nop3p all contain a similar domain rich in glycine and arginine residues referred to as the GAR domain (13). The function of this domain is still hypothetical. A related domain found in the hnRNP U protein was suggested to be directly involved in RNA binding (14), whereas the GAR domain of nucleolin was shown to play an accessory role in RNA binding, possibly by changing the conformation of the RNA (15, 16). Nsr1p (17), Ssb1p (18), and Nop3p (19) contain copies of the typical RNA recognition motif, and it is therefore conceivable that the GAR domain cooperates with these RNA recognition motifs to enhance their function. For fibrillarin, the role of the GAR domain is less obvious since this protein has not been shown to interact directly with any RNA. Curiously, Gar1p has two GAR domains, one at each end of the protein, that together represent almost 40% of the total protein. Deletion of the two GAR domains of Gar1p leaves a central domain of only 104 residues that has no similarity with known RNA binding motifs and is sufficient to support cell growth and to target a fusion protein to the nucleolus (20). This result prompted us to investigate whether this short peptide had retained the ability to bind the snoRNAs. Therefore, to decipher the structure of the snoRNA-nucleolar protein complexes and to better understand the relationship between their different components, we undertook the analysis of the interactions existing between Gar1p and other members of these complexes. The present study provides evidence that Gar1p is able to interact in vitro directly and specifically with the two snoRNAs, snR10 and snR30.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Strains and Plasmids-- The Saccharomyces cerevisiae haploid strain used in this study was described previously (13). S. cerevisiae cells were grown in minimal medium (SGal or SGlu: 0.67% yeast nitrogen base with 2% glucose or galactose) plus appropriate nutrients. Standard procedures for handling yeasts were essentially as described. The plasmids pT3/T7a18:10 and pT3/T7a18:30, which contain the genes coding for snR10 and snR30, were kindly provided by D. Tollervey (11). The different constructions used to produce the deleted Gar1p were derived from a GAR1 open reading frame inserted into pTZ18R.2 Digestion with various restriction enzymes and subcloning of the appropriate polymerase chain reaction fragments were used to produce several Gar1p deletions.

In Vitro Transcription and Translation-- Transcription using T7 RNA polymerase was performed according to the supplier's instructions (Promega). RNA was purified over a G-25 spin column, phenol extracted, and ethanol precipitated before translation. Biotinylated RNAs were obtained by performing the transcription in the presence of 100 µM biotin-21-UTP (CLONTECH). Conditions were optimized to obtain RNA molecules containing a similar amount of biotin, as determined by avidin-alkaline phosphatase assay (Bio-reaction). 35S-Labeled Gar1p derivatives were made in wheat germ extract according to the manufacturer (Promega) using 1 µg of mRNA in the presence of 15 µCi [35S]methionine (1200 Ci/mmol). The 35S proteins were stored at -20 °C and thawed and frozen several times without apparent loss of binding capacity.

RNA Protein Binding Assay-- 1 fmol of 35S-Gar1p derivative was incubated with 1 pmol of RNA in a total volume of 30 µl of binding buffer (20 mM Tris-Cl, pH 9.5, 20 mM MgCl2, 10 mM KCl, 0.25% Nonidet-P40) containing 4 µg of yeast tRNA. After 60 min at 30 °C, 350 µl of binding buffer and 7 µl of beads (Dynabeads M-280 streptavidin, Dynal) previously washed in the same buffer were added. After 90 min at room temperature, the beads were recovered using a magnet (Dynal MPC). The pellet was washed three times with 500 µl of binding buffer, and the bound proteins were eluted at 95 °C for 10 min in 15 µl of SDS-PAGE sample buffer. The supernatant was analyzed by 15% SDS-PAGE, and exposed on Kodak film for 1-3 days. For competition experiments, the competitor RNA was mixed with the biotinylated RNA prior to incubation with the labeled protein.

RNA Extraction-- Cultures were grown in liquid medium to an A600:nm of 1, then RNA was extracted as described previously (21) and analyzed by Northern blots.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Gar1p Binds Specifically to snR10 and snR30-- Gar1p had previously been shown to be co-immunoprecipitated with the two small nucleolar RNAs snR10 and snR30 (13). To assess whether Gar1p was able to interact directly with the snoRNAs, we used a previously described assay (22, 23). 35S-Labeled Gar1p protein made in wheat germ extract is incubated with synthetic biotinylated RNA and a large excess of tRNA as a competitor for nonspecific interactions. Biotin-containing complexes are purified by precipitation with streptavidin coupled to magnetic particles, then the bound proteins are released and analyzed by 15% SDS-PAGE. About 50% of the input Gar1p is reproducibly precipitated by either snR10 or snR30 when the RNA molecules are biotinylated (Fig. 1, lanes c and e). No Gar1p is precipitated when nonbiotinylated RNA molecules are used (lanes b and d), indicating that the binding of the RNA to the magnetic beads coupled to streptavidin is specific for biotinylated molecules. The specificity of the interaction between Gar1p and the two snoRNAs is demonstrated by comparison with the two nonspecific RNA substrates (5'ETS rRNA from S. cerevisiae or human U1 snRNA, lanes f and g).


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Fig. 1.   Specific binding of Gar1p to snR10 and snR30. 35S-Labeled in vitro translated Gar1p was incubated with snoRNAs either containing or not containing biotin-substituted U residues. RNA bound proteins were released and analyzed by 15% SDS-PAGE followed by autoradiography. Lane a, input Gar1p corresponding to the amount used in each assay; lanes b-g, proteins purified using: b, unbiotinylated snR10; c, biotinylated snR10; d, unbiotinylated snR30; e, biotinylated snR30; f, biotinylated rRNA-ETS; g, biotinylated U1 RNA. The arrow indicates the position of Gar1p.

The effect of the ionic strength on the stability of the RNA-protein complex has been tested. In contrast with the hnRNP U protein, which retains binding at 500 mM NaCl (14) and hnRNP A1, which retains binding at 250 mM NaCl (24, 25), Gar1p exhibits decreased binding to the two snoRNAs at 100 mM KCl and no binding at 500 mM KCl (Fig. 2). This is in contrast with the finding that native Gar1p was still associated with the intact snR30 RNP at 600 mM KCl (26) (see "Discussion").


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Fig. 2.   Effect of the ionic strength on the binding of Gar1p to the RNAs. Gar1p was incubated with the biotinylated RNAs (either snR10 or snR30) in the presence of various concentrations of KCl, and analyzed as in Fig. 1. Lane a, input Gar1p used in the binding reactions; lanes b, c, and d, binding with snR10b; lanes e, f, and g, binding with snR30b using the indicated concentration of KCl.

Then, we carried out competition experiments using as competitor either the other snRNA or nonspecific RNA. 35SLabeled Gar1p protein was incubated with biotinylated snR10 (or snR30) in the presence of increasing amounts of nonbiotinylated snR30 (or snR10, respectively) (Fig. 3). The nonbiotinylated competitor has little effect when present in equimolar amounts, but a 20 times molar excess almost completely abolishes the binding of Gar1p to the biotinylated RNA. A nonspecific competitor (ETS rRNA) is unable to displace the interaction of Gar1p with the two snoRNAs, emphasizing the specificity of these interactions. It is unlikely that Gar1p possesses separate sites for each RNA since it is now known that Gar1p is associated with a whole set of snoRNAs that present a common structure (1, 7).


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Fig. 3.   Competition experiments. The same amount of Gar1p (lane a, input protein) was incubated with one biotinylated snoRNA with either no (lanes b and e), one time (lanes c, f, and h), or 20 times (lanes d, g, and i) excess of another unbiotinylated RNA. Lanes b-d, biotinylated snR10 is competed with increasing amounts of unbiotinylated snR30; lanes e-g, biotinylated snR30 competed with unbiotinylated snR10; or (lanes h and i) with ETS RNA as a nonspecific competitor.

The RNA Binding Determinants of Gar1p Lie in the Center of the Protein-- The two GAR domains located at the two ends of Gar1p (N-GAR and C-GAR) are the most recognizable features of this protein. These domains are present in several other nucleolar proteins involved in RNA metabolism. Moreover, a distantly related glycine-rich domain (hnRNP U) was proposed to interact directly with single-stranded DNA and homopolymeric RNA (14). We therefore investigated whether the GAR domains of Gar1p could interact with the two snoRNAs. A series of deletions of Gar1p were constructed, and the corresponding proteins were tested for the ability to bind the RNAs. Fig. 4A schematically presents the most informative of the constructions that have been tested and summarizes the results of the complementation experiments previously obtained (20) whenever they are available. The construction A (Fig. 4A), that lacks the two GAR domains, is retained by the biotinylated snoRNAs (Fig. 4B) although less efficiently than is the wild-type protein (wt), indicating that the GAR domains might contribute but are not absolutely required for the interaction. In contrast with the hnRNP U protein, the GAR domains are not sufficient under our experimental conditions to bind to the snoRNAs as exemplified by the construction E, which lacks part of the central domain but retains the GAR domains.


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Fig. 4.   RNA binding activity of various deletions of Gar1p. A, schematic representation of wild-type Gar1p and of the most relevant deletion mutants. The two GAR domains are indicated as black boxes and the two motifs, Box-1 and Box-2, are indicated as thick gray lines. The RNA binding properties (this work) and the complementation abilities of the constructions (20) are summarized on the right. Numbers refer to the relevant residue positions according to the S. cerevisiae sequence. B, 35S-labeled Gar1p deletion mutants were incubated with biotinylated snR10 and snR30 RNAs and analyzed as described in Fig. 1. The peptides that contain a GAR domain migrate more slowly on SDS-PAGE than expected from their predicted molecular weight. The amount of input protein used in each binding assay is shown in the first lane of each panel (in). Lanes b, e, h, k, n, and q, proteins retained by snR10; lanes c, f, i, l, o, r, proteins retained by snR30. The apparent molecular weights of the peptides are indicated on the left.

The central region of Gar1p presents no remarkable features except for a stretch of basic amino acids rich in lysine and proline that resembles the nuclearization sequences found in many nuclear proteins. However, we have previously shown that this basic motif does not work as a nuclear targeting signal in S. cerevisiae and that its deletion does not impair the nucleolar accumulation of a reporter protein fused to Gar1p (20). Due to the advance of several genome sequencing projects, partial sequences for Gar1p from Drosophila (X71975), Arabidopsis thaliana (T21476) (27), and two species of Brassica (M64632 and M64633) are now available in addition to the sequences from S. cerevisiae and Schizosaccharomyces pombe that we published earlier (13, 28). An alignment of the sequences for the central 109 amino acids of Gar1p (wherever it is available) from the five different species is presented (Fig. 5). Two regions of very striking homology are denoted Box-1 and Box-2. For instance, the 25 amino acids of Box-1, exhibit an average of 90% identity (more than 98% similarity) between the different species. For Box-2, the alignment is also excellent between the yeasts and Drosophila, whereas there is a significant variation for half of the box in A. thaliana. Since there are some uncertainties in this portion of the sequence for this species, it is possible that the sequence identity is even greater than presented. In addition to Box-1 and -2, there are a number of residues throughout the whole core that are either well or absolutely conserved in all the species examined.


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Fig. 5.   Alignment of various Gar1p sequences. Sequences are from S. cerevisiae (Sc), S. pombe (Sp), Drosophila (Dr), A. thaliana (At), and Brassica napus or campestris (Br). The two parts of the figure correspond to the two domains, I and II (see "Discussion") and are contiguous. Residues that are identical to the S. cerevisiae sequence are represented by a dot. The two best conserved motifs, Box-1 and Box-2, are underlined with a thick line. The other well conserved positions are also boxed with the almost invariant residues boxed and shaded. When relevant, the consensus amino acid is represented below the sequence and boxed when it is a highly conserved position. The well conserved valine residue, which is mutated in a gar1ts allele (29), is indicated by a vertical arrowhead. Numbers refer to the S. cerevisiae sequence and correspond to the major deletions (see the text and also Fig. 4A). A few residues from the A. thaliana sequence are either unknown (x) or uncertain (lowercase letters).

A deletion that removes a portion of Box-2 (Fig. 4A, construction B; see also Fig. 5, position 124) almost completely abolishes the binding to either snR10 or snR30 (Fig. 4B, lanes h and i). However, even when Box-2 is entirely removed, the presence of a GAR domain (construction C) can rescue in part the RNA binding activity. In contrast, any deletion affecting Box-1 abolishes the binding activity (construction D) even when the two GAR domains are present (construction E).

Interestingly, the S. pombe Gar1p, which can substitute for its homologue in S. cerevisiae, is also able to interact in vitro with the two snoRNAs snR10 and snR30 from the budding yeast.3 This result supports the idea that identical segments between the two proteins might bear the RNA binding activity. Recently, a thermosensitive allele of Gar1p has been isolated and described (29). This mutant has multiple mutations that lead to six residue changes, from which the one(s) responsible for the phenotype has not yet been mapped. However, a valine at position 52 (Fig. 5) is the only residue affected that is invariant in the 5 species examined, making likely that this position is essential for the function of the protein.

Detailed Characterization of the Strain Carrying the Deletion B-- Since the deletion B was sufficient to support the growth of a yeast cell (20) while its ability to bind RNA in vitro was strongly impaired, we undertook a more careful examination of the strain harboring the construction B as the sole source of Gar1p. Microscopic examination reveals that the morphology of these cells is strongly altered (Fig. 6). Some are very large and rounded, whereas others are abnormally elongated with a shape resembling the cells starved for nitrogen (30). Moreover, the budding site selection of these cells is deeply affected, and newly formed cells are heterogeneous in shape. We isolated the abnormal cells by micro manipulation to ascertain that the observed phenotype was not due to a loss of the plasmid carrying the modified GAR1 gene. In addition, we found that at 37 °C the division of these cells is strongly impaired; abnormally shaped cells and even dead cells are more prominent in the culture.


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Fig. 6.   Cell morphology of wild-type and mutant B strains. Microscopic analysis of S. cerevisiae cells carrying the Gar1p wild-type and mutated construct B. Cells were grown on minimal medium at 30 °C, and living cells were examined with a microscope using interferential contrast (Nomarski). Top panel, wild-type cells (Wt) as a control; bottom panel, mutant cells corresponding to the deletion B (MutB) of GAR1.

To assess the effect of the various deletions of Gar1p on rRNA accumulation, the steady state levels of 18 S and 25 S rRNA were examined. No difference was observed for the deletions A and B as compared with the wild-type strain. However, for deletion E, synthesis of 18 S rRNA was completely abolished (data not shown) as previously reported for a strain depleted of Gar1p (13) or for a strain expressing a conditional allele of GAR1 (29). Finally, we addressed the question of the fate of the snoRNAs in vivo in the different GAR1 mutants. RNAs prepared from different mutants were analyzed by Northern blots, using clones corresponding to snR10 or snR30 as probes (Fig. 7). Neither snR10 nor snR30 appear to be destabilized in the mutant strains. This confirms previous report that in strains deprived of Gar1p by the use of a GAL inducible version of Gar1p (13), or carrying a thermosensitive allele of Gar1p, snoRNA steady state is not changed (29).


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Fig. 7.   Steady state levels of snR10 and snR30 in gar1 mutants. Total RNA was extracted from wild-type and mutant strains (harboring either the deletion A or B of GAR1, as indicated), then either 5 µg (wild-type) or 10 µg (mutant A and B) were analyzed by Northern blot hybridization using probes for snR10 and snR30. The position of these RNA species are given by the arrows on the left.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The nucleolus contains a very large number of species of snoRNAs that can be present in the cell in both small (10-15 S) and large (70-90 S) particles, indicating that snoRNPs could exist in the cell as individual particles or that they could associate with each other and/or with the pre-rRNA following a complex pattern of affinities and interactions (31). To understand how the processing machinery operates, it is necessary to provide a better description of the composition of the snoRNPs and of the interactions between their constituents.

We report here that Gar1p binds directly and specifically to snR10 and snR30 in vitro. It is noteworthy that this binding is salt-sensitive in contrast to the previous report that the association between Gar1p and the snoRNP snR30 was resistant to high salt washes (26). One possibility is that the optimal interaction between Gar1p and its RNA target requires another protein with which Gar1p could associate. Another possibility is that Gar1p alone binds its target, but to be fully active, it requires some protein modifications that the in vitro translated protein is lacking. For instance, the arginine residues of the GAR domain have been shown to be dimethylated in mammalian nucleolin (32). This modification could stabilize the interaction of the protein with its target RNA.

The most striking feature of the Gar1p sequence is the presence of two GAR domains, which are present in several nucleolar proteins involved in pre-rRNA processing. However, we previously showed that the two GAR domains of Gar1p are not necessary for the cell growth and are not required to target a reporter protein to the nucleolus (20). In this report, deletion analysis has revealed that the two GAR domains are not sufficient to bind specifically to the snoRNAs and are not essential for the binding but that they contribute to the binding when they are associated with another RNA binding domain. Together, these observations support the hypothesis that the GAR domains have an accessory role in binding the RNA and are dispensable in vivo.

Deletion analysis has revealed a complex organization of the core of the protein in two major domains. Domain I is 60 residues long, extends from residues 37 to 96, and encompasses 9 of the highly invariant positions as well as Box-1. Domain II extends from residues 97 to 146 and contains 5 highly conserved residues and Box-2. It is noticeable that although Domain I and II do not contain any canonical motifs, Box-1 exhibits some similarities with Snm1p, a protein component of the RNase mitochondrial RNA processing (33) with a putative yeast DNA-polymerase and with several ATP-synthases. It is thus possible that, in addition to its RNA binding activity, this domain bears also some catalytic activity. The well conserved residues located upstream from Box-1 must also play a major role since a deletion removing these residues up to position 54 (Fig. 5) abolishes the complementation activity (20).

Domain I is able to complement the growth of a cell lacking the wild-type Gar1p to accumulate a reporter protein to the nucleolus, and to specifically bind the snoRNAs in vitro but only when it is linked to either a GAR domain or to Domain II. Domain II, alone or associated with the two GAR domains, does not have any of these properties either in vitro or in vivo. It is interesting to note that construction B, which contains Domain I and II but lacks three residues of Box-2, has very little RNA binding activity in vitro. However, it is sufficient to support cell growth and nucleolar targeting in vivo. We conclude from these experiments that Box-2 is required for the RNA binding in vitro, but this requirement can be overridden by the addition of a GAR domain. It seems that in vivo the lack of a complete Box-2 can also be partially compensated, even in the absence of a GAR domain, probably due to other proteins. For instance, other nucleolar GAR proteins may compensate for a defect in unwinding activity. Alternatively, this function could be achieved by RNA helicases since it has recently been shown that an allele for the putative RNA helicase Rok1p was synthetically lethal with a thermosensitive allele of GAR1 (34). It is likely that Box-2 performs other functions in vivo, since its deletion is lethal for the cell (20). At least two different hypothesis could explain the abnormal morphology observed for the cells harboring the construction B. It is conceivable that an abnormal Gar1p leads to a defect in ribosome synthesis that alters the translation of certain factors involved in cell proliferation and bud site selection. A more direct consequence of the Gar1p mutation could result from a change in nucleolar organization and/or function. It has been observed that the location of the nucleolus depends on cell polarity since it is always located on the opposite side of the spindle pole body, that in itself points toward the future bud site. It is an interesting possibility that a modification of the nucleolus could result in an abnormal cell polarity.

The finding that the major RNA binding element coincides with Domain I, which is also required for nucleolar targeting as well as for the essential functions or Gar1p, supports the view that the snoRNAs and the protein could be co-transported to the nucleolus and that the association between Gar1p and the snoRNAs is likely to be essential for the complex to perform its function. Since the interaction between Gar1p and the snoRNAs in vitro is salt-sensitive, it is conceivable that Gar1p interacts in vivo with some of the other proteins that have been reported to be associated with the snR30 RNP to provide a tighter interaction. Interestingly, depletion of Gar1p (13, 29), does not lead to snoRNA instability. This result suggests that Gar1p does not belong to the core of the particles and that other proteins may bind the RNAs and protect them from being degraded in the gar1 mutant cells. The characterization of these proteins should allow the description of the structure of the snoRNPs to build up.

    ACKNOWLEDGEMENTS

We thank I. Mattaj for the gift of U1 snRNA and U1A plasmids and D. Tollervey for providing SNR10 and SNR30 genes. We are grateful to F. Amalric for his continuous support and to colleagues in Toulouse for their help and fruitful discussions.

    FOOTNOTES

* This research was supported by the Science Program of the Commission of the European Community (contract SCI*-0259-C) and by the CNRS.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of an EMBO long term fellowship. Present address: Dipartimento di Biologia, Universita' di Roma Tor Vergata, Via della Ricerca Scientifica 00133 Roma, Italia. E-mail: Bagni{at}utovrm.it.

§ To whom correspondence should be addressed. Tel.: 33-467-61-36-80; Fax: 33-467-04-02-31; E-mail: lapeyre{at}jones.igm.cnrs-mop.fr.

1 The abbreviations used are: snoRNAs, small nucleolar RNAs; snRNA, small nuclear RNA; RNP, ribonucleoprotein; snoRNP, small nucleolar RNP; hnRNP, heterogeneous nuclear RNP; PAGE, polyacrylamide gel electrophoresis; GAR domain, glycine/arginine-rich domain.

2 J. P. Girard and B. Lapeyre, unpublished observations.

3 C. Bagni and B. Lapeyre, unpublished observations.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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