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Mol Cell Biol. 2007 February; 27(4): 1191–1206.
Published online 2006 December 4. doi:  10.1128/MCB.01287-06
PMCID: PMC1800722

Analysis of Sequence and Structural Features That Identify the B/C Motif of U3 Small Nucleolar RNA as the Recognition Site for the Snu13p-Rrp9p Protein Pair[down-pointing small open triangle]

Abstract

The eukaryal Snu13p/15.5K protein binds K-turn motifs in U4 snRNA and snoRNAs. Two Snu13p/15.5K molecules bind the nucleolar U3 snoRNA required for the early steps of preribosomal processing. Binding of one molecule on the C′/D motif allows association of proteins Nop1p, Nop56p, and Nop58p, whereas binding of the second molecule on the B/C motif allows Rrp9p recruitment. To understand how the Snu13p-Rrp9p pair recognizes the B/C motif, we first improved the identification of RNA determinants required for Snu13p binding by experiments using the systematic evolution of ligands by exponential enrichment. This demonstrated the importance of a U·U pair stacked on the sheared pairs and revealed a direct link between Snu13p affinity and the stability of helices I and II. Sequence and structure requirements for efficient association of Rrp9p on the B/C motif were studied in yeast cells by expression of variant U3 snoRNAs and immunoselection assays. A G-C pair in stem II, a G residue at position 1 in the bulge, and a short stem I were found to be required. The data identify the in vivo function of most of the conserved residues of the U3 snoRNA B/C motif. They bring important information to understand how different K-turn motifs can recruit different sets of proteins after Snu13p association.

In eukaryotes, the 5.8S, 18S, and 25S (yeast)/28S (vertebrates) ribosomal RNAs (rRNAs) are transcribed by RNA polymerase I as a long precursor RNA (pre-rRNA) (for review, see reference 65). Mature rRNAs are then produced by an ordered series of cleavages, with simultaneous modifications of some of the bases and riboses (for reviews, see references 20 and 65). Several small nucleolar ribonucleoprotein particles (snoRNPs) containing a single small nucleolar RNA (snoRNA) and several proteins are involved in these maturation processes (for reviews, see references 2, 28, 31, 39, and 62). One of these RNPs (the U3 snoRNP) contains a highly conserved RNA and is essential for the early pre-rRNA cleavage steps (sites A′, A0, 1, 2, and 3 in Xenopus laevis [7, 8, 27] and sites A0, A1, and A2 in yeast [4, 23, 40, 42]). These early steps are needed for 18S rRNA production (23, 40, 59). Most of the other snoRNPs direct and catalyze nucleotide modifications. The C/D box snoRNPs are responsible for 2′ O methylations, whereas H/ACA snoRNPs catalyze pseudouridylations (for reviews, see references 2, 16, 28, 39, and 62). In spite of the presence of two C/D-like motifs (RUGAUGA/CUGA) in U3 snoRNA, no 2′ O-methylation guiding activity was attributed to this RNA. Its 5′ domain forms several base pair interactions with the 5′ external transcribed spacer and the 18S part of the pre-rRNA. These intermolecular interactions define the positions of early cleavages (sites A′, A0, 1, and 2 in X. laevis and sites A0, A1, and A2 in yeast) (5, 10, 9, 27, 40, 58, 59, 64). The 3′ domain of U3 snoRNA contains the two anchoring sites for the core proteins, namely, the phylogenetically conserved C′/D and B/C box pairs (26, 40, 51, 54). These two box pairs bind a common protein, denoted Snu13p in Saccharomyces cerevisiae and 15.5K in humans (38, 69). The Snu13p/15.5K proteins also interact with the spliceosomal U4 snRNA (47, 60). In the three-dimensional (3D) structure established for a 15.5K protein-U4 snRNA complex (68), the RNA was found to form a K-turn structure (68). The established 3D structures of the 30S and 50S ribosomal subunits (3, 55, 72) revealed that K-turn structures are frequent protein-binding motifs in RNAs (29). Conventional K-turn motifs contain a 3-nucleotide (nt) bulge bordered by two helices (I and II). Two noncanonical A·G and G·A pairs are located at the extremity of helix II (29, 68, 69). Several K-turn motifs in RNAs bind proteins of the L30 family (30, 47). Other members of this protein family are present in vertebrates (15.5K, NHP2 found in H/ACA snoRNPs [22], and SBP2 involved in selenocystein incorporation [1]). These proteins have a common homologue in archaea, the ribosomal protein L7Ae (13, 14, 32, 49, 53, 69). One important question is to establish how each of the L30 protein members of the eukaryotic cell recognizes its K-turn target(s). One discriminating parameter is the identity of the protruding residue (12, 21, 29, 37, 41, 68). However, one can expect additional RNA-specific determinants.

The most studied member of the L30 protein family is the Snu13p/15.5K protein and its archaeal homologue, protein L7Ae (32). Binding of these proteins to their target RNAs favors K-turn formation (15, 38, 63, 73). The current idea is that this structural transition allows the binding of other RNP proteins that recognize both protein Snu13p/15.5K and the remodeled RNA structure. By these combined RNA-protein and protein-protein interactions, the Snu13p/15.5K protein recruits the following: (i) the 61-kDa (hPrp31p) protein and the heterotrimeric 20/60/90K complex on U4 snRNA (44, 48); (ii) proteins Nop1p/fibrillarin, Nop56p, and Nop58p on C/D box snoRNAs (11, 70); and (iii) the Rrp9p/55K protein on the B/C motif of U3 snoRNA (19). A recent study identified the surface amino acids of the 15.5K protein that are required to recruit these various proteins (57).

In spite of numerous site-directed mutagenesis experiments that were performed on several Snu13p and 15.5K protein targets, the results of experiments using the systematic evolution of ligands by exponential enrichment (SELEX) to identify the most efficient RNA binding sequences of these proteins are still lacking. Such experiments successfully identified the crucial determinants for RNA recognition by the yeast ribosomal L30 protein (33). Here, we present SELEX experiments performed with a degenerated B/C motif of the yeast U3 snoRNA and the yeast Snu13p protein.

The information obtained was used to define the determinants required for Rrp9p binding on this B/C motif. Indeed, whereas protein Snu13p/15.5K binds both the C′/D and the B/C motifs of U3 snoRNA (38, 69), there is an asymmetric association of other snoRNP proteins. Binding of Snu13p/15.5K on the C′/D box motif likely allows the recruitment of the Nop1p, Nop56p, and Nop58p proteins, whereas its association with the B/C box motif allows the recruitment of protein Rrp9p/hU3-55K (19, 34, 57, 70). The U3-specific Rrp9p protein is essential in yeast (66). It is one of the few factors, already present in 90S preribosomes, that bridge 60S and 40S biogenesis (18, 45). Like U3 snoRNA, protein Rrp9p/hU3-55K is required for cleavages at sites A0, A1, and A2 (35, 66). The WD-40 repeats of protein Rrp9p/hU3-55K (35, 52, 66) are involved in its association with the Snu13p/15.5K-U3B/C complex (19), and a direct interaction between these two proteins was recently observed (57). An in vitro study on the binding of the human 55K protein to human U3 snoRNA revealed that the association depends upon several structural features of the B/C motif (19) (the size and stability of helices I and II, an internal loop, and helical structures located downstream from helix I). However, as some of these structural features are not found in yeast U3 snoRNA, the results obtained for the human 55K protein are not transposable to the yeast Rrp9p protein. Thus, by combining site-directed mutagenesis of U3 snoRNA with immunoselection experiments, we here identify the RNA determinants required for Rrp9p association with U3 snoRNA in vivo. The effects on U3 snoRNA activity of several mutations in the B/C motifs that alter Snu13p or Rrp9p binding were tested by a genetic approach. The role of most of the phylogenetically conserved residues of the U3 snoRNA B and C boxes in the formation of an active U3 snoRNP is described.

MATERIALS AND METHODS

Strains and growth conditions.

The Escherichia coli TG1 strain was the host strain for plasmid construction and for protein production. The S. cerevisiae JH84 strain (MATa snr17a-Gald::URA3 snr17b::LEU2 his3 ade2 can1) (23), provided by J. Hughes, was used as previously described (38) to test for U3 snoRNA activity.

Recombinant protein preparation.

The S. cerevisiae glutathione S-transferase (GST)-Snu13p recombinant protein was prepared as previously described (14, 38). For Kd estimations, the GST part of the GST-Snu13p fusion protein was cleaved with the PreScission protease (Pharmacia) (14, 38).

Recombinant plasmid construction.

The DNA matrices used for in vitro production of the yU3Δ2,3,4 RNA variants were obtained by PCR-directed mutagenesis, using the oligonucleotide pairs given in Table S1 in the supplemental material and plasmid pUC18::T7-yU3AΔ2,3,4 as the matrix. PCR conditions were as previously described (38). For the in vivo test of the effect of mutations in RNA yU3AΔ2,3,4, pASZ11::yU3Δ2,3,4 plasmid derivatives (pASZ11::yU3AΔ2,3,4 U-A, G·G, del [deletion], Ins [insertion], C-G, G-C, A-U, G·U, U·C, G1A, G1C, G1U, del3, del4, del5, del6, RNA7, RNA16, and C/B) were produced by replacement of the SalI-EcoRI fragment of plasmid pASZ11::yU3AΔ2,3,4 (38) with the SalI-EcoRI DNA fragment of the variant pUC18::T7-yU3AΔ2,3,4 plasmids. To test for the effect of mutations in the full-length yU3A RNA, pASZ11::yU3A plasmid derivatives were generated by the same type of substitutions. For in vivo expression of the protein A (ProtA)-Rrp9-tagged protein, we used the pHIS3-ProtA::rrp9 plasmid kindly provided by H. A. Raué (66).

In vitro transcription.

Transcriptions were carried out as previously described (43) on 500 ng of PCR amplification product, followed by gel purification. For electrophoresis mobility shift assays (EMSA), transcripts were uniformly labeled by [α-32P]UTP (800 Ci/mmol; Amersham) incorporation during transcription (38). For secondary structure analysis, cold transcripts were prepared, and they were 5′ end labeled with [γ-32P]ATP and polynucleotide kinase (24).

Filter binding assays and EMSAs.

The wild-type (WT) or variant uniformly labeled yU3B/C RNAs used for EMSA were transcribed from PCR amplification products obtained by using oligonucleotides yU3B/C-5′ and yU3B/C-3′ (see Table S1 in the supplemental material) as the primers and a WT or variant pUC18::T7-yU3AΔ2,3,4 plasmid as the matrix. About 5 fmol of 32P-labeled transcript was incubated with protein Snu13p or GST-Snu13p in the presence of 2 μg of competitor tRNAs under conditions previously described (38). For filter-binding assays, filtration on a 0.45-μm-pore-size nitrocellulose filter (Bio-Rad) was at room temperature in a BioDot apparatus (Bio-Rad). For dissociation constant (Kd) determination, the amount of the free and complexed RNA was estimated using ImageQuant Software (Molecular Dynamics). The apparent Kd values were determined by using SigmaPlot software (SPSS Science Software).

SELEX experiment.

The starting DNA matrix containing a 18-nt long degenerated sequence was produced by PCR amplification using two partially complementary oligonucleotides: SELEX N18 with a 18-nt long degenerated sequence and SELEX-5′ that generates a T7 RNA polymerase promoter. PCR amplification was as previously described (38), except that MgCl2 was added at a 4 mM concentration in the incubation buffer. About 500 ng of amplified DNA was used for in vitro transcription with T7 RNA polymerase (38). Transcripts were purified by electrophoresis on a 6% denaturing polyacrylamide gel as described in Mougin et al. (43). Then, 0.2 nmol of transcript was used for the first round of selection. To eliminate RNA molecules having an affinity for the glutathione-Sepharose beads, the RNA mixture was first incubated with 30 μl of beads in the absence of the GST-Snu13 fusion protein. RNP complexes were then formed by a 30-min incubation at 4°C of 0.1 nmol of treated RNA with 0.01 nmol of purified GST-Snu13 fusion protein in 20 μl of buffer D (150 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM HEPES, pH 7.9). Two micrograms of a yeast tRNA mixture was used as competitor RNA (Boehringer). The mixture was then incubated with 15 μl of glutathione-Sepharose beads (Amersham) equilibrated in buffer D. After extensive washing with buffer D, the selected RNAs were released by a 30-min incubation at 37°C with 20 μg of proteinase K in buffer D. They were phenol-chloroform extracted, ethanol precipitated, and dissolved in sterile water. After hybridization with 50 pmol of SELEX-3′ primer, they were again ethanol precipitated and then reverse transcribed with 25 U of AMV reverse transcriptase (Q Biogene) for 30 min at 42°C. Then, 30 cycles of PCR amplification were performed in the presence of the SELEX-5′ primer and SELEX-3′ primer (50 pmol each). The amplified DNA fragments were gel purified and used as the matrix for in vitro transcription. At each cycle of the SELEX cycle, a filter-binding assay was performed by using uniformly labeled transcripts produced from the DNA pool and the GST-Snu13p protein. At the fourth cycle of the amplification-selection experiment, DNA fragments were cloned in plasmid pTAdv (Clontech), and 31 randomly selected clones were sequenced by the dideoxy sequencing method.

RNA secondary structure analysis by RNase footprinting.

About 5 fmol of 5′ end-labeled RNA and 1 μM Snu13p protein were used for formation of Snu13p-RNA complexes under the conditions described above for the EMSAs. RNase digestions of free and complexed RNAs were performed in buffer D for 6 min at 20°C with 0.8 U of T1 RNase (Roche), 2.4 U of T2 RNase (Gibco), or 0.001 U of V1 RNase (kemotex). The reactions were stopped as described in Jacquenet et al. (24). An alkaline hydrolysis of RNAs was performed for 5 min at 96°C using 10 fmol of RNA with 100 mM sodium bicarbonate. The cleavage products were fractionated by electrophoresis on a 10% polyacrylamide-8 M urea gel.

The free energies of the 2D structures of the selected RNAs were obtained by an M-fold evaluation at 37°C in 1 M NaCl (25).

Test of variant U3 snoRNA functionality.

The S. cerevisiae strain JH84 transformed with recombinant plasmid pASZ11 coding the WT or mutated yU3AΔ2,3,4 RNA was grown for 48 h at 30°C in yeast extract-peptone-glycerol (YPG) liquid medium. After centrifugation and washing in yeast extract-peptone-dextrose (YPD) medium, the cells were transferred into liquid YPD medium and grown for 24 h at 30°C. Then, growth was either tested on YPD solid medium or in liquid YPD medium. For growth in liquid medium, 1.5 ml of cells grown as described above was transferred to 30 ml of YPD medium in order to get an A600 of about 0.1 U/ml. Growth was for 48 h at 37°C. Aliquots were collected at regular intervals, and their absorption at 600 nm was measured. Growth on plates was tested at three temperatures (20°C, 30°C, and 37°C). The sizes of colonies were examined after 48 h of incubation at 30°C and 37°C or after 72 h of incubation at 20°C.

The cellular stability of the variant yU3AΔ2,3,4 RNAs was studied by Northern blot analysis as previously described (40). The yU6 snRNA level was used for standardization. The 5′ end 32P-labeled oligonucleotides RT-yU3 and RT-yU6 (38) (see Table S1 in the supplemental material) were used as the probes for yU3AΔ2,3,4 and yU6 RNAs, respectively. The radioactivity in the bands of gel was quantified with a Molecular Dynamic PhosphorImager using ImageQuant software. The steady-state levels of variant yU3AΔ2,3,4 RNAs were expressed as a percentage of the steady-state level of the WT RNA (RNAvar relative concentration).

Immunoprecipitation and Western blot analysis.

The JH84 strain transformed with plasmid pHIS3-ProtA::Rrp9 and one of the pASZ11 derivatives was grown on YPG medium until stationary phase. Cells were then transferred on YPD medium and grown for 24 h. They were then washed with ice-cold water and lysed as previously described (66). A fraction of the extract was used to quantity the amount of variant yU3AΔ2,3,4 RNA by Northern blotting (see above). Another fraction was used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8%), followed by Western blot analysis of the ProtA-Rrp9 fusion protein using rabbit peroxidase antiperoxidase (Sigma) and an ECL Plus detection kit (Amersham, United Kingdom). A third fraction of the extract was used for immunoprecipitation assays that were carried out as described previously (6, 36), by using 400 ml of lysate and 30 ml of rabbit immunoglobulin G (IgG)-agarose beads (Sigma). The RNAs bound on the IgG-agarose beads were extracted by a 1-h incubation at 37°C with 200 μg of proteinase K treatment. After phenol extraction and ethanol precipitation, the amount of the variant yU3AΔ2,3,4 RNA associated with protein ProtA-Rrp9p was analyzed by Northern analysis as described above. The amounts of the immunoselected yU3AΔ2,3,4 variant (IV) RNAs compared to WT yU3AΔ2,3,4 (IWT) RNA were calculated as follows: (IV/IWT) × 100. The relative affinities of the ProtA-Rrp9p protein fusion for the variant RNAs were calculated by dividing the IV/IWT percent values by the values reflecting the RNA variant stability (RNAvar relative concentration). The binding capacity of the variant RNAs to ProtA-Rrp9p was expressed as a percentage of that found for the WT RNA.

RESULTS

First, we used the SELEX approach in order to define the characteristic features of K-turn motifs, which have the highest affinity for protein Snu13p.

Random in vitro selection of protein Snu13p RNA targets.

We previously showed that protein Snu13p binds the yU3B/C RNA (Kd of 230 nM) (14) (Fig. (Fig.1).1). This 55-nt RNA is derived from yeast U3 snoRNA. It forms a long stem-loop structure containing the B/C motif. Our previous studies also showed that the yU3AΔ2,3,4 RNA containing the yU3B/C sequence instead of the large cruciform structure of authentic yeast U3 snoRNAs (Fig. (Fig.1A)1A) is functional in vivo (38). By use of a DNA primer degenerated at 18 positions (SELEX N18 primer; see Materials and Methods), we produced a collection of degenerated yU3B/C RNAs by PCR amplification. The degenerated RNA region corresponded to the K-turn motif and the terminal loop in RNA yU3B/C (Fig. (Fig.1B).1B). Hence, the degenerated RNAs were all closed by a long helix formed by the invariant 5′ and 3′ sequences (stem 5 in yeast U3A snoRNA) (Fig. (Fig.1B).1B). The degree of degeneracy of the starting material was verified by sequence analysis (see Fig. S1 in the supplemental material). A GST-Snu13 protein fusion bound on glutathione-Sepharose beads was used for in vitro selection experiments (see Materials and Methods for experimental conditions). We used 5 μg of degenerated RNA mixture (1.6 × 1014 molecules) for the first round of selection. Assuming a perfectly statistical synthesis of the degenerated SELEX N18 DNA primer, the expected diversity of the starting RNA sequences was of 6.9 × 1010 molecules such that each possible RNA sequence was expected to be present 2,300 times. An [RNA]/[protein] ratio of 10 was found to be the most favorable ratio for the selection. Filtration assays revealed a strong increase of the affinity of Snu13p for the selected RNAs after four rounds of amplification-selection. The amplified cDNAs were cloned in plasmid pTAdv used to transform E. coli cells, and 31 clones were arbitrarily selected among the 100 clones obtained. Sequence analysis (Fig. (Fig.2A)2A) showed that two-thirds of the selected RNAs (class I RNAs) were significantly enriched in GA, UGA, and PuUGA motifs compared to random sequences (Fig. (Fig.2B).2B). The remaining third (class II RNAs) did not show this peculiar enrichment (Fig. 2A and B). Gel shift experiments demonstrated that only class I RNAs exhibited a significant affinity for protein Snu13p (Fig. (Fig.2C).2C). In class I RNA, in spite of the fact that position 38 was located outside of the degenerated sequence, a G residue was most frequently found at this position in place of the expected U residue (14 out of 19 selected RNAs). This G residue probably resulted from a miss-incorporation during one of the reverse PCR amplification steps. Two RNAs of class I also had mutations at position 18 (RNA3) and at positions 17 and 18 (RNA9), which were also located outside of the degenerated sequence. Finally, four RNAs were missing one residue in the degenerated region (RNAs 4, 5, 11, and 14), while two others gained an additional residue (RNAs 3 and 8).

FIG. 1.
The yU3AΔ2,3,4 RNA used for in vivo assays (38) (A) and the yU3B/C RNA used for in vitro assays (B). The secondary structures are according to Marmier-Gourrier et al. (38). In yU3B/C RNA, the sequence degenerated in the SELEX experiment is shown ...
FIG. 2.
Nucleotide sequences and affinities for Snu13p of the selected RNAs. (A) Alignment of the WT yU3B/C RNA sequence with the degenerated N18 RNA sequences and sequences of the 31 selected RNAs. Selected RNAs are divided into two classes, I and II, according ...

All the selected RNAs with significant Snu13p affinities form a conventional K-turn.

All the class I RNAs could form a canonical K-turn structure (Fig. (Fig.3).3). This was not the case for the class II RNAs. Most of the structures proposed in Fig. Fig.3A3A were verified experimentally by enzymatic probing, using 5′end labeled transcripts. T1 and T2 RNases were used to identify single-stranded regions. Double-stranded and stacked RNA regions were defined by V1 RNase digestion. Examples of cleavage patterns are given for RNAs 3, 5, and 10 and for the WT yU3B/C RNA (Fig. (Fig.3B).3B). Only three of the selected RNAs, denoted RNAs 1 to 3, formed a K-turn structure with an orientation similar to that in the B/C motif (bulge in the 3′-strand) (Fig. 3A, B, and C). Their 5-nt-long terminal loops were closed by one or two base pairs, and as in the B/C motif, the closing pair was a G-C pair. The 16 other RNAs had the bulge in the 5′ strand, and except for RNAs 9 and 11, the GA dinucleotide at positions 17 and 18 in the invariable sequence of the degenerated RNAs was always used as residues 1 and 2 in the bulge, respectively. U, G, and A residues were always selected at positions 3, 4, and 5 in the K-turn motif, respectively, except, in RNA5, which had a G residue at position 3 instead of a U residue. In these RNAs with a bulge in the 5′ strand, the two residues of the GA dinucleotide at positions 6 to 7 in the K-turn motif were most frequently the conserved A39 residue and the unexpected G38 residue in the nondegenerated sequence, respectively. Hence, the requirement of a GA dinucleotide at positions 6 to 7 in the K-turn motifs likely explains the selection of RNAs with an unexpected mutation at position 38. Interestingly, in all the selected RNAs, except for RNA17, a U·U pair follows the noncanonical A·G and G·A pairs in helix II, and this is independent of the orientation of the K-turn motif (a C·U pair is found in RNA17). The presence of this U·U pair is the only sequence conservation found in helix II. In agreement with this finding, a U·U pair is found in both the C′/D and the B/C motifs of yeast U3 snoRNAs, and it is highly conserved in all yeast species (Fig. (Fig.1C)1C) (38). A U·U pair is also found in the consensus established for the C/D motifs of yeast C/D snoRNAs (Fig. (Fig.1C)1C) (69).

FIG. 3.FIG. 3.
All the selected RNAs having a significant affinity for Snu13p can form a K-turn structure. (A) Secondary structures proposed for the selected RNAs. The yU3B/C RNA structure is shown for comparison; the conserved part of this structure in the N18 degenerated ...

The Snu13p affinity for the selected RNAs parallels the stability of their 2D structures.

An M-fold evaluation of the free energies of the 2D structures of the selected RNAs (at 37°C in 1 M NaCl) shows that they range from −11.1 to −16.4 kcal/mol (Fig. (Fig.3A).3A). By using gel mobility shift assays, we measured the apparent dissociation constants of the complexes that were formed by the class I RNAs and a recombinant Snu13p protein, which was not fused to GST (Fig. (Fig.2D2D and and3A).3A). Interestingly, among the 16 RNAs with the bulge in the 5′ strand, Snu13p showed a higher affinity for the five RNAs exhibiting the lowest free energies (RNAs 15 to 19, Kd values of 30 nM) (Fig. (Fig.3A).3A). Interestingly, although the free energy of the structure formed by RNA3 with a bulge on the opposite strand is −15.3 kcal/mol, its estimated apparent Kd for Snu13p was high (250 nM) (Fig. (Fig.3A).3A). This lower affinity compared to RNAs with a bulge in the 5′ strand may be explained by the short length of helix I. Altogether, the data revealed a strong influence of the stability of helices I and II on Snu13p affinity. This observation has to be viewed in the light of our footprinting data on the RNP complexes formed with the selected RNAs (Fig. (Fig.3C).3C). Indeed, Snu13p generates a strong protection against RNase action of both stems I and II. Only the terminal loop remained accessible in the complexes. As expected, all the selected RNAs, except RNA5, had a U residue at position 3 in the bulge. In agreement with previous findings (14, 38), a G residue can be accommodated at this position since RNA5 has a G residue at position 3. However, in spite of its high thermodynamic stability (ΔG of −14.2 kcal/mol), RNA5 has a lower affinity for protein Snu13p (Kd of 250 nM) compared to RNAs containing a U residue at position 3.

The orientation of the B/C motif is crucial for cell growth.

As most of the selected RNAs had the 3-nt bulge sequence in their 5′ strand instead of its location in the 3′ strand in the authentic B/C motif, it was tempting to test for the functionality of U3 snoRNAs carrying a B/C motif in an opposite orientation. To this end, we used the in vivo test for U3 snoRNA activity that we developed previously (38). In this test a truncated version of U3 snoRNA (yU3AΔ2,3,4 RNA) (Fig. (Fig.1A)1A) is subjected to site-directed mutagenesis and is expressed in yeast cells under conditions such that no genomic U3 snoRNA is expressed. For this purpose, the S. cerevisiae JH84 strain with a single active U3 gene (U3A gene) under the control of the Gal10 element (23) was transformed with recombinant pASZ11 plasmids encoding the WT or variant yU3AΔ2,3,4 RNAs. The effects on the growth at 20, 30, and 37°C of cells expressing three distinct yU3AΔ2,3,4 RNAs with B/C motifs in an opposite orientation to that in the WT RNA were tested by this approach (Fig. (Fig.4B).4B). In RNA yU3AΔ2,3,4:RNA16, the authentic B/C motif was replaced by the K-turn motif of the SELEX RNA16 (Fig. (Fig.4A).4A). RNA16 has a long stem II and was one of the four SELEX RNAs showing the highest affinity for Snu13p (Fig. (Fig.3A).3A). With RNA yU3AΔ2,3,4:RNA7 (Fig. (Fig.4A),4A), we tested the effect of the substitution of the B/C motif with a SELEX motif showing an affinity for Snu13p in the same range as that of the B/C motif (RNA7, Kd of 200 nM) (Fig. (Fig.3A).3A). Finally, we built a yU3AΔ2,3,4:C/B RNA with an inversion of the authentic B/C K-turn and terminal loop sequence (Fig. (Fig.4A).4A). None of these three modified yU3AΔ2,3,4 RNAs was able to ensure growth at any of the tested temperatures (Fig. (Fig.4B).4B). The cellular stability of these variant yU3AΔ2,3,4 RNAs was tested by Northern blot analysis of total cellular RNA, using oligonucleotide RT-yU3 as the probe for yU3AΔ2,3,4 RNA and oligonucleotide RT-yU6 as a probe for the invariant yU6 snRNA. As evidenced in Fig. Fig.4C,4C, the in vivo stabilities of the mutated RNAs were strongly diminished compared to that of the WT RNA, especially for the two yU3AΔ2,3,4 RNAs with the SELEX motifs 16 and 17 (11 and 17% of the WT stability for the yU3AΔ2,3,4:RNA16 and yU3AΔ2,3,4:RNA7, respectively) (Fig. (Fig.4C).4C). We concluded that the orientation of the B/C motif plays a crucial role in U3 snoRNA stability and activity.

FIG. 4.
The correct orientation of the B/C motif is essential for growth. (A) The yU3AΔ2,3,4 RNA variants (yU3AΔ2,3,4:RNA16, yU3AΔ2,3,4:RNA7, and yU3AΔ2,3,4:C/B) are shown. Helices are numbered as in Fig. Fig.3.3. (B) Test ...

Growth of yeast cells does not parallel the Snu13p affinity for the B/C motif.

Based on our finding of a U·U pair in most of the K-turn motifs obtained in the SELEX experiment, we considered the possible functional importance of this U·U pair in the B/C motif of U3 snoRNA. First, its importance for in vitro binding of protein Snu13p was tested using uniformly labeled yU3B/C RNAs (5 fmol) carrying base substitutions in the U·U pair (G·U, A-U, U·C, and G-C). These RNAs were incubated with increasing amounts of protein Snu13p and subjected to gel shift assays (Fig. (Fig.5B).5B). Conversion of the U·U pair into a G·U pair was found to have a very limited effect on the measured apparent Kd (250 nM versus 230 nM), but its conversion into a U·C, an A-U, and, especially, a G-C pair had a marked negative effect on the Snu13p affinity (Kd values of 600, 850, and >1,500 nM, respectively). Thus, experiments were designed in order to test whether base substitutions in the U·U pair affect U3 snoRNA activity. The yU3AΔ2,3,4 RNAs carrying these mutations were stable in vivo (Fig. (Fig.5D).5D). Surprisingly, the A-U and G-C mutations, which had a strong negative effect on the in vitro binding of Snu13p, only decreased growth at 37°C and had, respectively, a limited and strong negative effect. In contrast, in spite of its limited effect on the in vitro Snu13p affinity, replacing U·U with U·C led to a strong thermosensitive phenotype. We confirmed these data by comparing the growth kinetics in liquid medium at 37°C of JH84 cells expressing the three yU3AΔ2,3,4 RNA variants and the yU3AΔ2,3,4 WT RNA. The A600 value was followed during 48 h of growth (see Fig. S2 in the supplemental material). On the basis of these results, we concluded that the in vivo activity of U3 snoRNA does not parallel the affinity of the B/C motif for protein Snu13p.

FIG. 5.
Helix II contains determinants for Snu13p and Rrp9p binding. (A) The mutations generated in RNA yU3B/C and yU3AΔ2,3,4 are indicated. (B) Test of the Snu13p affinity for the yU3B/C variant RNAs by EMSA. RNA-protein complexes were formed and purified ...

The substitution of U·C for the U·U pair in the B/C motif does not alter Rrp9p binding.

One possible explanation for the discrepancy between in vitro and in vivo data concerning the U·U pair substitution could be the involvement of a cooperative binding of Snu13p and Rrp9p on the B/C motif in vivo. Therefore, we tested the in vivo binding of protein Rrp9p on the four yU3AΔ2,3,4 RNA variants with U·U substitutions. To this end, in addition to the endogenous WT Rrp9p protein, we expressed a protein A-Rrp9p fusion (ProtA-Rrp9p) in JH84 cells. Under these conditions, the level of yU3AΔ2,3,4 RNA variants associated with the ProtA-Rrp9p protein could be measured by immunoselection of the RNA-ProtA-Rrp9p complexes using IgG-agarose beads. JH84 cells were transformed with one of the pASZ11 derivatives expressing RNAs yU3A, yU3AΔ2,3,4 WT, or the variant yU3AΔ2,3,4 RNAs and with the pHIS3-ProtA::rrp9 plasmid (66). They were grown on glucose for 24 h. By using Western blot analysis, we verified that the ProtA-Rrp9p expression was similar in each cell culture (data not shown). The stability of the yU3AΔ2,3,4 RNA variants was estimated by Northern blot analysis of the RNAs extracted from 20% of each cell extract. As above, we used the RT-yU3 and RT-yU6 oligonucleotides as the probes for the yU3AΔ2,3,4 RNA and yU6 snRNA, respectively (Fig. (Fig.5D).5D). The concentrations of yU3AΔ2,3,4 RNA variants in total RNAs from the transformed cells were expressed as a percentage of the concentration of WT yU3AΔ2,3,4 RNA in total RNA of the control cells (RNAvar relative concentration) (Fig. (Fig.5D).5D). Association of the ProtA-Rrp9p protein with the various yU3AΔ2,3,4 RNAs was tested by incubation of equal amounts of each cell extract (30 U at A600) with IgG-agarose. The binding of similar amounts of the ProtA-Rrp9p fusion protein on the agarose beads in the different assays was verified by Western blot analysis performed on aliquot fractions of the beads (Fig. (Fig.5E).5E). After phenol extraction of the remaining part of the beads, the bound RNAs were analyzed by Northern blot analysis (Fig. (Fig.5E,5E, RT-yU3 probe). As a control, an immunoselection assay was performed on cells expressing the yU3AΔ2,3,4 RNA but not the ProtA-Rrp9p fusion protein (Fig. (Fig.5E,5E, first lane). The specificity of the immunoselection was demonstrated by use of the yU6 snRNA probe (RT-yU6) (Fig. (Fig.5E).5E). The amounts of immunoselected yU3AΔ2,3,4 variant RNAs compared to the amount found for the WT yU3AΔ2,3,4 RNA [(IV/IWT) × 100] were calculated. The relative affinities of the ProtA-Rrp9p fusion for the various RNA variants were calculated by dividing the IV/IWT percent values by the values established as the RNAvar relative concentrations. Finally, the capacities of the RNA variants to bind ProtA-Rrp9p were expressed as a percentage of the capacity of the WT RNA (Fig. (Fig.5E).5E). Based on the values obtained for growth at 30°C (Fig. (Fig.5E),5E), none of the U·U substitutions tested had a significant effect on protein Rrp9p association with RNA yU3AΔ2,3,4. Therefore, in spite of the low affinity of protein Snu13p for B/C motifs with a substitution of A-U for U·U or of G-C for U·U, protein Rrp9p is bound to these motifs in vivo.

To confirm that the phenotype observed for the substitution of U·C for U·U was not due to our utilization of a truncated version of the yU3A snoRNA, this substitution of U·C for U·U was generated in plasmid pASZ11::yU3A encoding the full-length yeast U3A snoRNA. As shown in Fig. Fig.6,6, the phenotype observed was even stronger, since the growth defect was observed at all the tested temperatures.

FIG. 6.
Effect on cell growth of substitutions of U·C for U·U and of C-G for G-C in helix II of the B/C motif of the full-length yU3A snoRNA. (A) The mutations generated in RNA yU3A are indicated. (B) Test for the effects of mutations in stem ...

The conserved G-C pair in helix II is important for Rrp9 association.

The G-C pair next to the U·U pair in the B/C motif of yeast U3A snoRNA is almost universally conserved in U3 snoRNA (Fig. (Fig.1C)1C) (38). As it was not conserved in the RNAs obtained in the SELEX experiment, a role in Snu13p binding was not expected. As a confirmation, conversion of this G-C pair into a C-G pair in RNA yU3B/C had no significant effect on Snu13p affinity (Kd of 220 nM) (Fig. (Fig.5B).5B). In contrast, when G-C was replaced with C-G in helix II of RNA yU3AΔ2,3,4 a strong thermosensitive phenotype was observed on glucose. Growth was reduced at 30°C and abolished at 37°C (Fig. (Fig.5C).5C). Here also, a stronger phenotype was observed when this substitution of C-G for G-C was introduced in the full-length yU3A snoRNA (Fig. (Fig.6B).6B). Growth was affected at all the tested temperatures and especially at 37°C. The immunoselection approach described above was then used to test whether the conserved G-C pair in helix II of the B/C motif is required for protein Rrp9p association. As illustrated in Fig. Fig.5E,5E, for growth at 30°C, only trace amounts of the ProtA-Rrp9p protein fusion were found to be associated with the yU3AΔ2,3,4 RNA carrying the substitution of C-G for G-C in helix II. Thus, we concluded that the G-C pair that follows the U·U pair in helix II of the B/C motif is strongly implicated in Rrp9p association.

The G residue at position 1 in the bulge is essential for protein Rrp9p association.

A G residue is highly conserved at position 1 in the K-turn structure formed by the B/C motif of U3 snoRNA (38). In the RNA obtained in the SELEX experiment, 15 out of 16 RNAs with the bulge in the 5′ strand had a G residue at position 1 in the bulge (Fig. (Fig.3A).3A). However, we hypothesized that the data obtained were biased by the preferential usage of an existing GA dinucleotide in the constant region of the degenerated RNAs (Fig. (Fig.3A).3A). Indeed, in agreement with previous data suggesting that the identity of the residue at position 1 has a limited influence on the Snu13p affinity (47, 61, 69), the three selected RNAs which had a bulge in the 3′ strand had an A or an C residue at position 1 (Fig. (Fig.3A).3A). We confirmed the small influence of the identity of the residue at position 1 for Snu13p binding by gel shift experiment (Fig. (Fig.7B).7B). Protein Snu13p had a slightly better affinity for B/C motifs with a purine at position 1 compared to a pyrimidine. However, the difference of affinity was limited (Kd value of 230 nM versus 400 nM for pyrimidines). In contrast, no growth was observed at any of the tested temperatures, when the G residue at position 1 in the B/C motif of RNA yU3AΔ2,3,4 was replaced by an A, C, or U residue (Fig. (Fig.7C).7C). We thus used the same strategy as above to establish whether this growth defect was due to the absence of protein Rrp9p association. As illustrated in Fig. 5D and E, whereas the mutated yU3AΔ2,3,4 RNAs were stable in vivo, no binding of the ProtA-Rrp9p protein was detected for the G-to-C and G-to-U substitutions, and only trace amounts of the fusion protein were found for the G-to-A substitution. Hence, we concluded that the presence of a G residue at position 1 in the B/C motif is essential for protein Rrp9p association and, as a consequence, for growth. Here again, the growth defect obtained upon replacement of this G residue in RNA yU3AΔ2,3,4 was not linked to the truncations present in this RNA, since Venema et al. (66) also found a growth defect upon replacement of this G residue in the full-length yeast U3A snoRNA.

FIG. 7.
Presence of a G residue at position 1 in the internal loop of the B/C motif is needed for Rrp9p binding. (A) The substitutions generated at position 1 in the B/C motif (G1A, G1C, and G1U). (B) EMSAs of the effect of the substitutions at position 1 on ...

The G-C pair closing the B/C motif is highly important for both Snu13p and Rrp9p association.

As mentioned above, the internal loops of the three selected RNAs with a bulge in the 3′ strand (Fig. (Fig.3A)3A) were all closed by a G-C pair. Accordingly, our previous data (14) showed a lower affinity of protein Snu13p for a B/C motif closed by an A-U pair instead of the G-C pair (Kd of 350 nM versus 230 nM). The decrease in affinity was found to be even stronger after substitution of a G·G pair for the G-C pair or after deletion of the G-C pair (Kd value of 1,000 nM and 1,200 nM, respectively). As illustrated in Fig. Fig.8C,8C, when these mutations were introduced in the yU3AΔ2,3,4 RNA, only the yU3AΔ2,3,4 RNA with the substitution of U-A for G-C could ensure growth on glucose. However, a strong thermosensitive phenotype was observed for this RNA variant. In addition, the yU3AΔ2,3,4 RNAs with the replacement of G-C by G·G and with the G-C pair deletion were not functional. The Northern blot analysis illustrated in Fig. Fig.8D8D showed that the absence of functionality of the yU3AΔ2,3,4 RNA variants with a mutated G-C pair was not due to their instability. Expression of the ProtA-Rrp9p protein in the cells and immunoselection assays on IgG beads (Fig. (Fig.8E)8E) revealed a low level of association of the ProtA-Rrp9p fusion protein with the yU3AΔ2,3,4 RNA carrying the substitution of A-U for G-C. Only trace amounts of the fusion protein were associated with the yU3AΔ2,3,4 RNAs carrying the substitution of G·G for G-C or the G-C pair deletion (Fig. (Fig.8E).8E). Hence, the G-C pair closing the internal loop is important for Snu13p binding and for Rrp9p association.

FIG. 8.
Complex effects on Snu13p and Rrp9p protein binding and cell growth of the suppression or the extension of helix I in the B/C motif. (A) Nucleotide substitutions (A-U or G·G) deletions (ΔG-C) and insertion (Ins) made in helix I of the ...

An extended helix I prevents Rrp9p association.

As RNAs with a highly stable helix I were predominantly obtained in the SELEX experiment, we tested the functionality of a yU3AΔ2,3,4 RNA with a B/C motif in the correct orientation containing an extended helix I. To this end, three additional base pairs were inserted between the G-C pair closing the internal loop and the terminal loop, so that stem I was extended from one to four base pairs (Fig. (Fig.8A,8A, RNA yU3AΔ2,3,4:Ins). As expected, the gel shift assays performed with the yU3B/C:Ins RNA demonstrated that this insertion strongly increase the affinity for protein Snu13p (Kd of 20 nM instead of 230 nM) (Fig. (Fig.8B).8B). However, a yU3AΔ2,3,4:Ins RNA carrying this insertion (Fig. (Fig.8C,8C, Ins) did not ensure growth at any of the tested growth temperatures. The mutated yU3AΔ2,3,4:Ins RNA was significantly unstable, since its cellular amount was half of that found for the WT yU3AΔ2,3,4 RNA (Fig. (Fig.8D).8D). In addition, when the ProtA-Rrp9p protein was expressed in the cells, only trace amounts of this protein were found to be associated with the yU3AΔ2,3,4:Ins RNA. Hence, increasing the size of helix I in the B/C motif prevents Rrp9p association.

Complete deletion of the terminal loop in the B/C motif does not abolish growth and Rrp9p binding.

As we previously showed that deletions of residues in the terminal loop of the yU3B/C RNA have strong deleterious effects on the binding of protein Snu13p in vitro (14), we tested whether this dramatic decrease in Snu13p affinity can abolish growth. Surprisingly, among the various mutants tested, only the deletion of six residues including the G residue involved in formation of the G-C base pair (yU3AΔ2,3,4-del6 RNA variant) completely abolished growth at any of the tested temperatures (Fig. (Fig.8C).8C). The yU3AΔ2,3,4 variant RNAs with 3- or 4-nt-long deletions (yU3AΔ2,3,4-del3 or -del4 RNAs, respectively) supported some growth at 20°C. However, they led to a strong thermosensitive phenotype. Surprisingly, growth was almost normal at any of the tested temperatures when the yU3AΔ2,3,4 RNA carrying the 5-nt deletion (yU3AΔ2,3,4-del5 RNA variant) was expressed. The del3, del4, and del5 RNA variants were only slightly less stable than the WT yU3AΔ2,3,4 RNA (Fig. (Fig.8D).8D). The data obtained upon testing the association of the ProtA-Rrp9p protein with the del3, del4, del5, and del6 variant RNAs were in perfect correlation with the observed growth phenotypes (Fig. (Fig.8E).8E). The fusion protein did not bind to the del6 variant RNA, and the amount of ProtA-Rrp9p protein bound to the del3 and del4 variant RNAs was small. In contrast, the amount of fusion protein bound to the del5 variant RNA was almost half of that found for the WT RNA. Interestingly, in the yU3AΔ2,3,4-del5 RNA variant, the G-C base pair is replaced by a G and a C residue linked by a phosphodiester bond. This replacement might be compatible either with direct binding of protein Rrp9p or with a cooperative binding of the Snu13p-Rrp9p protein pair. Thus, we tested the possible direct binding of protein Rrp9p on the yU3AΔ2,3,4-del5 RNA variant by using an in vitro transcribed yU3B/C-del5 RNA and an in vitro translated Rrp9p protein. No direct binding could be detected. Hence, here again data are in favor of a cooperative binding of proteins Snu13p and Rrp9p on the B/C motif in vivo.

DISCUSSION

Formation of Snu13p-RNA complexes depends upon stem stability.

On the basis of the strong sequence similarity of their RNA binding domains, the archaeal L7Ae and eukaryal Snu13p/15.5K, NHP2p, and SBP2 proteins form a subfamily within the L30 protein family. We present here the first SELEX experiment performed on one member of this subfamily. As expected, the 19 selected RNAs, which have a significant affinity for protein Snu13p, have the ability to form a K-turn structure. Only one of them had a G residue at position 3 in the internal loop. Accordingly, based on the 15.5K and Snu13p 3D structures (14, 50, 68), both the O6 carbonyl of a G residue at position 3 and the O4 carbonyl of a U residue at position 3 can form a hydrogen bond with Lys88 in the nucleotide-binding pocket of protein Snu13p. However, formation of hydrophobic interactions with amino acids of the binding pocket is probably limited by the large size of the nucleobase in the case of a G residue (14).

All the selected RNAs showing an affinity for Snu13p could form a helix I and a helix II. Accordingly, we previously showed that one base pair should close the K-turn structure of the B/C motif, for efficient binding of Snu13p (14). Also in agreement with our observation, the impossibility of forming helix I was recently shown to be the reason for the absence of an interaction of the 15.5K protein with the C′/D′ motif of C/D box snoRNAs (61). In contrast to Snu13p, the archaeal L7Ae protein was found to be able to bind K-loop motifs, in particular, the K-loop motifs formed by the C′/D′ motif of archaeal C/D box sRNAs (14, 46). Two possible explanations for the more restricted RNA specificity of protein Snu13p compared to its archaeal counterpart have been proposed (21, 50). First, helix α2 in the RNA recognition motif of the L7Ae protein subfamily is involved in K-turn recognition, and this α-helix is rigidified in protein Snu13p compared to protein L7Ae (50). Secondly, the replacement of the VSR sequence of the eukaryal Snu13p/15.5K proteins by an (I/L)EV sequence in archaeal proteins may explain why archaeal protein can associate with K-loop motifs (21).

One important piece of information brought by our SELEX experiment is the direct correlation between the stability of stems I and II and the apparent Kd values of the Snu13p-RNA complexes. By enzymatic probing we previously showed that K-turn formation is induced by the RNA-protein interaction (38). This assumption is supported by recent chemical probing experiments combined with computer simulations (15) and also by fluorescent resonance energy transfer experiments (73). Altogether, these experiments demonstrated that the sharp angle of the phosphodiester bond in the K-turn structure is imposed by the RNA-protein interaction. As this chaperone activity of protein Snu13p/15.5K depends upon stacking of the two A·G and G·A pairs and residues at positions 1 and 2 in the bulge on stems I and II, it is reasonable to find that the efficiency of the RNA-protein interaction depends upon the stability of these stems. This proposal is also in agreement with previous data showing that mutations which disrupt helix I or II in the C/D motif of U14 snoRNA abolishes 15.5K protein binding (70). The strong dependence of Snu13p affinity upon the stability of helices I and II explains why the authentic B/C motif was not selected among the winner RNAs in the SELEX experiment. Indeed, only 3 RNAs with the bulge in the same orientation as that of the B/C motif were obtained, against 16 RNAs with the bulge in the opposite orientation.

A U·U base pair is preferentially selected in stem II.

Eighteen of the 19 selected RNAs contained a U·U pair stacked on the two sheared pairs of the K-turn motif. Even though in some of the RNAs one of the two U residues was a constant residue, the functional importance of a U·U pair is supported by its selection in the three RNAs with a bulge in the 3′ strand (Fig. (Fig.3A).3A). It is also supported by the negative effect on Snu13p association of substitutions in the U·U pair of the yeast (Fig. (Fig.5B)5B) and human B/C motifs (19). This finding is in agreement with the presence of a U·U pair in the consensus sequences established for both the B/C and the C′/D motifs of all sequenced U3 snoRNAs and also for the C/D motifs of C/D box snoRNAs (38, 69) (Fig. (Fig.1C).1C). Conversely, a low level of conservation of the U·U pair is observed in the C′/D′ motifs of C/D box snoRNAs that do not bind the Snu13p/15.5K protein. Among the identified Snu13p/15.5K RNA targets, only U4 snRNA has a G-C pair instead of a U·U pair. This difference may explain why mutations in the nucleotide-binding pocket of Snu13p have a stronger negative effect on U4 snRNA binding than on U3 snoRNA association (17). Indeed, one can imagine that due to the presence of an unfavorable G-C pair in stem II, the U4 snRNA-Snu13p interaction is more strongly dependent on nucleotide-amino acid interactions in the binding pocket.

Defects due to U·U substitutions are partially compensated in vivo.

Our observation that the substitutions of A-U for U·U and G-C for U·U in the B/C motif have a limited effect on cell growth but a strong deleterious effect on Snu13p affinity reveals that the affinity of protein Snu13p for the B/C motif is not the unique parameter involved in the in vivo assembly of proteins on the B/C motif. One possible explanation is a cooperative binding of the Snu13p and Rrp9p proteins on the B/C motif, as found for binding of the human 15.5K and hPrp31p proteins on U4 snRNA (56). We also assume that cooperative binding explains the in vivo association of proteins Snu13p and Rrp9p on RNA yU3AΔ2,3,4-del5. Indeed, growth of cells transformed with this RNA was nearly normal, in spite of the inability of protein Snu13p to bind the K-loop formed by the truncated B/C motif of this RNA in vitro. In addition, recent data revealed that the in vivo assembly of proteins on both the B/C and C′/D motifs of U3 snoRNA and the C/D motif of the C/D box snoRNAs is a complex process. It involves proteins which are not found in the mature particles (67, 71). Therefore, the yU3AΔ2,3,4 RNAs with substitutions in the U·U pair that we produced may be useful to complete the study of the in vivo mechanism of assembly of proteins on the B/C motif.

The G-C pair in stem II and residue G1 are key determinants for Rrp9p association.

The second base pair in stem II of the B/C motif is a universally conserved G-C pair (38, 69) (Fig. (Fig.1C).1C). Conversely, a C-G pair is universally conserved at the corresponding position of the C′/D motif of the U3 snoRNA and of the C/D motif of C/D box snoRNAs (38, 69) (Fig. (Fig.1C).1C). This C-G pair was shown to be required for recruitment of proteins Nop56p, Nop58p, and Nop1p on the U14 snoRNA (70). This asymmetry of G-C versus C-G in stem II of the C′/D and B/C motifs of U3 snoRNA was proposed to be involved in asymmetric protein recruitment on these two motifs (70). Here, we bring an experimental demonstration of the requirement of the G-C pair in stem II for efficient recruitment of protein Rrp9p. Interestingly, whereas the negative effect of the absence of a G-C pair is temperature dependent for the truncated yU3AΔ2,3,4 RNA, this effect is seen at any of the tested temperatures for the full-length yeast U3 snoRNA. Granneman et al. (19) found only a limited effect of the corresponding substitution during in vitro assembly of protein hU3-55k on the B/C motif of human U3 snoRNA, which has a smaller 3′ domain compared to yeast U3 snoRNA. Therefore, the importance of the G-C pair in stem II of the B/C motif for Rrp9p association seems to be dependent upon the overall context of the U3 snoRNA 3′ domain. The absence of functionality of RNA yU3AΔ2,3,4:C/B with an inverted B/C motif may be explained by the wrong orientation of the G-C pair in helix II. Indeed, in this RNA the G residue of this base pair is on the same side as the bulge present in the K-turn motif.

In agreement with previous data (66), our results also reveal a strong dependence of Rrp9p association on residue G1 in the bulge. This may explain the strong conservation of the identity of this residue throughout U3 snoRNA evolution (38, 69) (Fig. (Fig.1C).1C). However, as a G residue is also conserved at position 1 in the C′/D motif of U3 snoRNA (38) (Fig. (Fig.1C),1C), residue G1 in the B/C motif cannot be a determinant for the asymmetric association of proteins on the B/C and C′/D motifs. Interestingly, the 2′-OH of the ribose moiety of residue at position 1 in the internal loop was shown to play an essential role in the association of protein 15.5K with the X. laevis U25 C/D box snoRNA (61). However, the identity of the base was not found to be important. Hence, this residue may bridge the two Snu13p/15.5K and Rrp9p proteins by interaction of its ribose with protein Snu13p/15.5K and of its base with protein Rrp9p. This intricate interaction of proteins Snu13p and Rrp9p with the B/C motif may be favored by direct an interaction of the two proteins (57).

The reduced size of helix I and the orientation of the K-turn motif may be determinants for Rrp9p association.

Whereas the replacement of the unique G-C pair corresponding to helix I in the B/C motif by a 4-bp stem increases Snu13p affinity by a factor of 10, this mutation abolishes Rrp9p association and cell growth. Hence, protein association on the B/C motif does not require the presence of an optimal Snu13p binding site. Accordingly, mutations in the B/C motif of human U3 snoRNA, which converted an asymmetric loop in stem I into a base-paired region and thus increased the size of stem I, abolished binding of protein hU3-55k (19). Our footprinting analysis of RNAs 5 and 10 (Fig. (Fig.3C)3C) shows that, in the presence of a stable helix I, protein Snu13p wraps helices I and II and the internal loop. Under these conditions, residue G1 is likely buried inside the RNA-protein complex. Hence, the requirement of a highly reduced stem I in the B/C motif may reflect the needed interaction of protein Rrp9p with residue G1. Consequently, the presence of two stable helices I and II in the C′/D motif may prevent protein Rrp9p association.

Our results showing that only one of the deletions that we generated in the terminal loop of the B/C motif does not alter cell growth, whereas it completely impairs Snu13p binding in vitro, is intriguing. We assume that the defined conformation of the B/C motif which is required for Rrp9p binding is achieved in this variant RNA. Binding of protein Snu13p to this motif should be facilitated by its interaction with Rrp9p (57) and may be, by the implication of other proteins, involved in U3 snoRNP biogenesis (71).

Conclusions.

We discovered three very important parameters for binding of protein Rrp9p on the B/C motif: the G-C pair in stem II, residue G1 in the internal loop, and the presence of a highly reduced helix I. As the implicated G-C pair and G1 residue are located on opposite sides of the K-turn motif, one role of protein Snu13p may be to diminish the distance between these two determinants by forcing the RNA to adopt the K-turn structure. All the mutations in U3 snoRNA that abolished Rrp9p association with the B/C motif also blocked cell growth. Hence, binding of protein Rrp9p on the B/C motif has a crucial role for pre-rRNA processing. The present data also reveal a complex interplay of interactions between proteins Snu13p and Rrp9p and the B/C motif and show that optimization of the Snu13p binding site in the B/C motif is deleterious for Rrp9p binding and, consequently, for U3 snoRNA activity. Finally, we demonstrated the crucial role of the orientation of the B/C motif in U3 snoRNA for cell growth.

Supplementary Material

[Supplemental material]

Acknowledgments

V. Igel-Bourguignon is acknowledged for her excellent technical assistance. B. Charpentier is acknowledged for helpful discussion. We thank S. Sonkaria for careful reading of the manuscript.

A.C. was a fellow from the French Ministère de la Recherche et des Nouvelles Technologies. This work was supported by the Centre National de la Recherche Scientifique; the French Ministère de la Recherche et des Nouvelles Technologies; the ACI Biologie Cellulaire, Moléculaire et Structurale, grant BCMS226; and the PRST Bioingénierie of the Conseil Régional Lorrain.

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 December 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

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