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Molecular and Cellular Biology, February 2007, p. 1191-1206, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01287-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown} ,{dagger}

A. Cléry,1 V. Senty-Ségault,1 F. Leclerc,1 H. A. Raué,2 and C. Branlant1*

Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, Université Henri Poincaré, Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy, France,1 Section Biochemistry and Molecular Biology, Department of Chemistry, Faculty of Sciences and Institute Molecular Biology Science, BioCenter Amsterdam, Vrije Universiteit, Amsterdam, The Netherlands2

Received 14 July 2006/ Returned for modification 4 August 2006/ Accepted 20 November 2006


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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.


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INTRODUCTION
 
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.


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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 yU3{Delta}2,3,4 RNA variants were obtained by PCR-directed mutagenesis, using the oligonucleotide pairs given in Table S1 in the supplemental material and plasmid pUC18::T7-yU3A{Delta}2,3,4 as the matrix. PCR conditions were as previously described (38). For the in vivo test of the effect of mutations in RNA yU3A{Delta}2,3,4, pASZ11::yU3{Delta}2,3,4 plasmid derivatives (pASZ11::yU3A{Delta}2,3,4 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::yU3A{Delta}2,3,4 (38) with the SalI-EcoRI DNA fragment of the variant pUC18::T7-yU3A{Delta}2,3,4 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 [{alpha}-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 [{gamma}-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-yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 RNA associated with protein ProtA-Rrp9p was analyzed by Northern analysis as described above. The amounts of the immunoselected yU3A{Delta}2,3,4 variant (IV) RNAs compared to WT yU3A{Delta}2,3,4 (IWT) RNA were calculated as follows: (IV/IWT) x 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.


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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. 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 yU3A{Delta}2,3,4 RNA containing the yU3B/C sequence instead of the large cruciform structure of authentic yeast U3 snoRNAs (Fig. 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. 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. 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 x 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 x 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. 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. 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. 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).


Figure 1
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  		FIG. 1. The yU3A{Delta}2,3,4 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 in gray. Boxes and stems are numbered according to Marmier-Gourrier et al. (38). (C) The consensus motifs established for the C'/D and B/C motifs of all sequenced U3 snoRNAs and for the C/D motifs of the other yeast C/D snoRNAs are shown (38).


Figure 2
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  		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 to their GA, UGA, and PuUGA contents. Nucleotides in RNAs 1 to 31 are numbered according to the positions of the homolog nucleotides in WT yU3B/C RNA. The nucleotides corresponding to the nondegenerated sequences are shown in gray characters, and nucleotides in the degenerated sequence and nucleotides mutated during the RT-PCR cycles are shown in black. The GA pairs are underlined. (B) Enrichment in GA, UGA, and PuUGA motifs of RNAs from class I and class II compared to the N18 starting RNA mixture. Numbers given correspond to the total amount of GA, UGA, or PuUGA motifs in the sequences of the degenerated region of all the selected RNAs from class I or II, divided by the number of RNAs in each class. For the starting N18 RNA mixture, the statistical probability to get a GA, UGA, or PuUGA motif in a randomly degenerated 18-nucleotide sequence is given. (C) Test for Snu13p interaction with the selected RNAs by gel shift assay (EMSA). Complexes were formed by using 5 fmol of uniformly labeled RNA from class I or II and 60 pmol (2 µM) of protein Snu13p (+). The incubation conditions are described in Materials and Methods. RNP complexes were fractionated by electrophoresis on 6% polyacrylamide gels under nondenaturing conditions. The identity of the RNA used in each assay is indicated below each lane of the autoradiogram. Positions of the free RNA and the RNP complex are shown. (D) Estimation of the affinity of protein Snu13p for RNAs 1, 11, 16, and 19 by EMSA. RNA-protein complexes formed with 5 fmol of labeled RNA and increasing concentrations of protein Snu13p (as indicated below the lanes) were fractionated by gel electrophoresis as in panel C. The apparent Kd values (indicated above the autoradiograms) were calculated with SigmaPlot software (SPSS Science Software), by using the radioactivities measured in the bands of gel corresponding to the free and bound (RNP) RNA.

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. 3). This was not the case for the class II RNAs. Most of the structures proposed in Fig. 3A 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. 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. 1C) (38). A U·U pair is also found in the consensus established for the C/D motifs of yeast C/D snoRNAs (Fig. 1C) (69).


Figure 3
Figure 3
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  		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 RNAs is shown (RNA N18) as well as models of secondary structures for all the selected RNAs. Several of these models were verified experimentally as illustrated in panels B and C. Nucleotide numbering is as described in the legend of Fig. 2A. Nucleotides at degenerated positions and mutated nucleotides are in gray. The apparent Kd values given for each RNA were established by EMSA. The free energies of the proposed secondary structures, expressed in kcal/mol, were calculated by using M-fold software. (B) Footprinting analysis of the yU3B/C-Snu13p, RNA3-Snu13p, RNA5-Snu13p, and RNA10-Snu13p complexes. Complexes were formed as described in Materials and Methods, by using 5' end labeled RNAs. Free (–) and complexed (+) RNAs were digested with V1, T1, and T2 RNases under conditions described in Materials and Methods. The digestion products were fractionated by electrophoresis on a 10% polyacrylamide gel in the presence of 8 M urea. Lanes L correspond to the alkaline ladder. Positions of nucleotides and helices are indicated on the left of the autoradiograms. (C) Schematic representation of the data obtained in panel B. V1, T1, and T2 RNase cleavages are represented by arrows attached to squares, dots, and triangles, respectively. The number of symbols reflects the intensity of cleavages (one, two, and three for low, medium, and strong, respectively). Nucleotides protected in the presence of Snu13p are circled in gray. Nucleotides that are not cleaved are boxed.

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. 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. 2D and 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. 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. 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. 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 ({Delta}G 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 (yU3A{Delta}2,3,4 RNA) (Fig. 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 yU3A{Delta}2,3,4 RNAs. The effects on the growth at 20, 30, and 37°C of cells expressing three distinct yU3A{Delta}2,3,4 RNAs with B/C motifs in an opposite orientation to that in the WT RNA were tested by this approach (Fig. 4B). In RNA yU3A{Delta}2,3,4:RNA16, the authentic B/C motif was replaced by the K-turn motif of the SELEX RNA16 (Fig. 4A). RNA16 has a long stem II and was one of the four SELEX RNAs showing the highest affinity for Snu13p (Fig. 3A). With RNA yU3A{Delta}2,3,4:RNA7 (Fig. 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. 3A). Finally, we built a yU3A{Delta}2,3,4:C/B RNA with an inversion of the authentic B/C K-turn and terminal loop sequence (Fig. 4A). None of these three modified yU3A{Delta}2,3,4 RNAs was able to ensure growth at any of the tested temperatures (Fig. 4B). The cellular stability of these variant yU3A{Delta}2,3,4 RNAs was tested by Northern blot analysis of total cellular RNA, using oligonucleotide RT-yU3 as the probe for yU3A{Delta}2,3,4 RNA and oligonucleotide RT-yU6 as a probe for the invariant yU6 snRNA. As evidenced in Fig. 4C, the in vivo stabilities of the mutated RNAs were strongly diminished compared to that of the WT RNA, especially for the two yU3A{Delta}2,3,4 RNAs with the SELEX motifs 16 and 17 (11 and 17% of the WT stability for the yU3A{Delta}2,3,4:RNA16 and yU3A{Delta}2,3,4:RNA7, respectively) (Fig. 4C). We concluded that the orientation of the B/C motif plays a crucial role in U3 snoRNA stability and activity.


Figure 4
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  		FIG. 4. The correct orientation of the B/C motif is essential for growth. (A) The yU3A{Delta}2,3,4 RNA variants (yU3A{Delta}2,3,4:RNA16, yU3A{Delta}2,3,4:RNA7, and yU3A{Delta}2,3,4:C/B) are shown. Helices are numbered as in Fig. 3. (B) Test of the effects of an inversion of the orientation of the B/C motif in RNA yU3A{Delta}2,3,4 on cell growth. JH84 S. cerevisiae cells were transformed with plasmids encoding the WT yU3A{Delta}2,3,4 RNA (WT) or variant yU3A{Delta}2,3,4 RNAs with an RNA16 or RNA7 motif instead of the B/C motif or carrying an inversion of the B/C motif. After 24 h of growth in liquid YPD medium, cells were spread on YPD plates and allowed to grow for 48 h at 30°C and 37°C and for 72 h at 20°C. (C) Test of the stability of the variant yU3A{Delta}2,3,4 RNAs in transformed cells grown as described in panel B. Total cellular RNA was extracted as described in Materials and Method. The cellular stability of the RNA variants was assessed by Northern blot analysis using the 5' end 32P-labeled oligonucleotide RT-yU3. The RT-yU6 oligonucleotide complementary to yU6 snRNA was used for standardization of the results. The identities of the variant yU3A{Delta}2,3,4 RNAs expressed in the cells are indicated above the lanes of the autoradiogram. The positions of the yU3A{Delta}2,3,4 RNA and yU6 snRNA are shown. The radioactivity in the bands of gel was measured on a PhosphorImager by using ImageQuant software. The concentration of the yU3A{Delta}2,3,4 RNA variants in cellular extracts is expressed as a percentage of that found for the WT yU3A{Delta}2,3,4 RNA. The values obtained ([variant yU3A{Delta}2,3,4 RNA]/[WT yU3A{Delta}2,3,4 RNA] x 100) that reflect the RNA variant stabilities (RNAvar relative concentration) are given below each lane.

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. 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 yU3A{Delta}2,3,4 RNAs carrying these mutations were stable in vivo (Fig. 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 yU3A{Delta}2,3,4 RNA variants and the yU3A{Delta}2,3,4 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.


Figure 5
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  		FIG. 5. Helix II contains determinants for Snu13p and Rrp9p binding. (A) The mutations generated in RNA yU3B/C and yU3A{Delta}2,3,4 are indicated. (B) Test of the Snu13p affinity for the yU3B/C variant RNAs by EMSA. RNA-protein complexes were formed and purified as described in the legend to Fig. 2C and D. The identities of the RNA variants used are indicated above the autoradiograms. The concentration of protein Snu13p used in each assay is given below each lane. The apparent Kd values calculated with the SigmaPlot software are given above the autoradiograms. (C) Test for the effects of mutations in stem II of RNA yU3A{Delta}2,3,4 on yeast growth. JH84 S. cerevisiae cells were transformed with plasmids encoding the full-length yU3A snoRNA (yU3A), the WT yU3A{Delta}2,3,4 RNA (WT), or variant yU3A{Delta}2,3,4 RNAs carrying substitutions in the U·U pair of stem II (A-U, G·U, U·C, and G-C) or the conversion of the G-C pair in stem II into a C-G pair. After 24 h of growth in liquid YPD medium, cells were spread on YPD plates and allowed to grow for 48 h at 30°C and 37°C and for 72 h at 20°C. (D) Test of the stability of the variant yU3A{Delta}2,3,4 RNAs. JH84 cells transformed with plasmid pHIS3-ProtA::Rrp9 expressing the ProtA-Rrp9 fusion protein and a pASZ11 derivative expressing the WT or a variant yU3A{Delta}2,3,4 RNA were grown in liquid YPG medium and then transferred into YPD liquid medium. After 24 h of growth, clear lysates were prepared. The stability of the RNA variants in the extracts was assessed by Northern blot analysis using the 5' end 32P-labeled oligonucleotide RT-yU3. The RT-yU6 oligonucleotide complementary to yU6 snRNA was used for standardization of the results. The identities of the variant yU3A{Delta}2,3,4 RNAs expressed in the cells are indicated above the lanes of the autoradiograms. The positions of yU3A{Delta}2,3,4 and yU6 RNAs are shown as described in the legend of Fig. 4. The radioactivity in the bands of gel was measured on a PhosphorImager by using the ImageQuant software, and the values obtained were used to determine the relative concentrations of the variant and WT RNAs ([variant yU3A{Delta}2,3,4 RNA]/[WT yU3A{Delta}2,3,4 RNA] x 100) (RNAvar relative concentration), which are given below each lane. (E) Test of the effect of mutations in stem II on Rrp9p protein binding. Parts of the cellular extracts described in panel D were subjected to immunoselection with IgG-agarose beads. The coimmunoselected WT or variant yU3A{Delta}2,3,4 RNAs were fractionated by gel electrophoresis and their amounts (IWT and IV, respectively) were estimated by Northern blot analysis using oligonucleotide yU3 as the probe. The amounts of immunoselected yU3A{Delta}2,3,4 variant RNAs compared to WT yU3A{Delta}2,3,4 RNA [(IV/IWT) x 100] are indicated below each lane. Western blot analysis of the total amount of protein A-Rrp9p protein bound to the beads in each assay show no significant variation from one assay to the other. Thus, the relative affinities of the protein A-Rrp9p fusion for the variant RNAs (ProtA-Rrp9p relative affinities) were calculated as described in Materials and Methods and are given below each lane.

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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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, yU3A{Delta}2,3,4 WT, or the variant yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 RNA and yU6 snRNA, respectively (Fig. 5D). The concentrations of yU3A{Delta}2,3,4 RNA variants in total RNAs from the transformed cells were expressed as a percentage of the concentration of WT yU3A{Delta}2,3,4 RNA in total RNA of the control cells (RNAvar relative concentration) (Fig. 5D). Association of the ProtA-Rrp9p protein with the various yU3A{Delta}2,3,4 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. 5E). After phenol extraction of the remaining part of the beads, the bound RNAs were analyzed by Northern blot analysis (Fig. 5E, RT-yU3 probe). As a control, an immunoselection assay was performed on cells expressing the yU3A{Delta}2,3,4 RNA but not the ProtA-Rrp9p fusion protein (Fig. 5E, first lane). The specificity of the immunoselection was demonstrated by use of the yU6 snRNA probe (RT-yU6) (Fig. 5E). The amounts of immunoselected yU3A{Delta}2,3,4 variant RNAs compared to the amount found for the WT yU3A{Delta}2,3,4 RNA [(IV/IWT) x 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. 5E). Based on the values obtained for growth at 30°C (Fig. 5E), none of the U·U substitutions tested had a significant effect on protein Rrp9p association with RNA yU3A{Delta}2,3,4. 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. 6, the phenotype observed was even stronger, since the growth defect was observed at all the tested temperatures.


Figure 6
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  		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 II of RNA yU3A on cell growth. JH84 S. cerevisiae cells were transformed with plasmid pASZ11::yU3A derivatives encoding the full-length U3 snoRNA (yU3A) or variant yU3A RNAs carrying a U·C pair instead of the U· U pair in stem II or the conversion of the G-C pair in stem II into a C-G pair (C-G). After 24 h of growth in liquid YPD medium, cells were spread on YPD plates and allowed to grow for 48 h at 30°C and 37°C and for 72 h at 20°C. (C) Test of the stability of the variant yU3A RNAs: the transformed cells were grown as described in panel B and total RNAs were extracted as described in Materials and Methods. The stability of the RNA variants in the extracts was assessed by Northern blot analysis using the 5' end 32P-labeled oligonucleotide RT-yU3. The RT-yU6 oligonucleotide complementary to yU6 snRNA was used for standardization of the results. The positions of the WT and variant yU3A RNAs and yU6 snRNA are indicated on the left of the autoradiograms. By measuring the radioactivity in the bands of gel, the relative cellular concentrations of the WT and variant yU3A RNAs ([variant yU3A RNA]/[WT yU3A RNA] x 100) were established (RNAvar relative concentration). They are given below each lane.

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. 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. 5B). In contrast, when G-C was replaced with C-G in helix II of RNA yU3A{Delta}2,3,4 a strong thermosensitive phenotype was observed on glucose. Growth was reduced at 30°C and abolished at 37°C (Fig. 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. 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. 5E, for growth at 30°C, only trace amounts of the ProtA-Rrp9p protein fusion were found to be associated with the yU3A{Delta}2,3,4 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. 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. 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. 3A). We confirmed the small influence of the identity of the residue at position 1 for Snu13p binding by gel shift experiment (Fig. 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 yU3A{Delta}2,3,4 was replaced by an A, C, or U residue (Fig. 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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.


Figure 7
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  		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 Snu13p binding. RNA-protein complexes were formed and purified as described in the legends of Fig. 2C and D. Protein concentrations are given below each lane. The identities of the mutants used and the established apparent Kd values are given above the autoradiograms. (C) Test of the effect on cell growth of substitutions at position 1 in the B/C motif of RNA yU3{Delta}2,3,4. The S. cerevisiae JH84 cells transformed with plasmid pASZ11 derivatives were grown as described in the legend of Fig. 5C. (D) Test of the in vivo stability of yU3A{Delta}2,3,4 variant RNAs with a base substitution at position 1 in the B/C motif. The test was performed as described in the legend of Fig. 5D. (E) Test of the effect on Rrp9p binding of base substitutions at position 1 in the B/C motif of RNA yU3A{Delta}2,3,4. The test was performed as described in the legend of Fig. 5E.

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. 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. 8C, when these mutations were introduced in the yU3A{Delta}2,3,4 RNA, only the yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4 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. 8D showed that the absence of functionality of the yU3A{Delta}2,3,4 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. 8E) revealed a low level of association of the ProtA-Rrp9p fusion protein with the yU3A{Delta}2,3,4 RNA carrying the substitution of A-U for G-C. Only trace amounts of the fusion protein were associated with the yU3A{Delta}2,3,4 RNAs carrying the substitution of G·G for G-C or the G-C pair deletion (Fig. 8E). Hence, the G-C pair closing the internal loop is important for Snu13p binding and for Rrp9p association.


Figure 8
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  		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 ({Delta}G-C) and insertion (Ins) made in helix I of the B/C motif and deletions made in the terminal loop (del3, del4, del5, and del6) are shown. In all the variant RNAs, nucleotide numbering in the internal and terminal loop is according to that in the WT B/C motif. (B) EMSAs of the affinity of Snu13p for the yU3{Delta}2,3,4:Ins RNA. Assays were performed as described in the legends of Fig. 2C and D. (C) Effects of mutation in the terminal loop of the B/C motif of yU3A{Delta}2,3,4 RNA on cell growth. JH84 S. cerevisiae cells were transformed with pASZ11 plasmid derivatives expressing the various yU3A{Delta}2,3,4 RNA mutants described in panel A. The test was performed as described in the legend of Fig. 5C. (D) Test of the stability of the variant yU3A{Delta}2,3,4 RNAs carrying the mutations described in panel A. The test was performed as described in the legend of Fig. 5D. (E) Test of the effect of the mutations described in panel A on Rrp9p protein binding. The test was performed as described in the legend of Fig. 5E.

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 yU3A{Delta}2,3,4 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. 8A, RNA yU3A{Delta}2,3,4: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. 8B). However, a yU3A{Delta}2,3,4:Ins RNA carrying this insertion (Fig. 8C, Ins) did not ensure growth at any of the tested growth temperatures. The mutated yU3A{Delta}2,3,4:Ins RNA was significantly unstable, since its cellular amount was half of that found for the WT yU3A{Delta}2,3,4 RNA (Fig. 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 yU3A{Delta}2,3,4: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 (yU3A{Delta}2,3,4-del6 RNA variant) completely abolished growth at any of the tested temperatures (Fig. 8C). The yU3A{Delta}2,3,4 variant RNAs with 3- or 4-nt-long deletions (yU3A{Delta}2,3,4-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 yU3A{Delta}2,3,4 RNA carrying the 5-nt deletion (yU3A{Delta}2,3,4-del5 RNA variant) was expressed. The del3, del4, and del5 RNA variants were only slightly less stable than the WT yU3A{Delta}2,3,4 RNA (Fig. 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. 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 yU3A{Delta}2,3,4-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 yU3A{Delta}2,3,4-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.


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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 {alpha}2 in the RNA recognition motif of the L7Ae protein subfamily is involved in K-turn recognition, and this {alpha}-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. 3A). It is also supported by the negative effect on Snu13p association of substitutions in the U·U pair of the yeast (Fig. 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. 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 yU3A{Delta}2,3,4-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 yU3A{Delta}2,3,4 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. 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. 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 yU3A{Delta}2,3,4 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 yU3A{Delta}2,3,4: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. 1C). However, as a G residue is also conserved at position 1 in the C'/D motif of U3 snoRNA (38) (Fig. 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. 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.


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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.


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Maturation des ARN et Enzymologie Moléculaire, UMR 7567, Université Henri Poincaré, Nancy I, BP 239, 54506 Vandoeuvre-lès-Nancy, France. Phone: 33 383684303. Fax: 33 383684307. E-mail: christiane.branlant{at}maem.uhp-nancy.fr. Back

{triangledown} Published ahead of print on 4 December 2006. Back

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


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REFERENCES
 
    1
  1. Allmang, C., P. Carbon, and A. Krol. 2002. The SBP2 and 15.5 kD/Snu13p proteins share the same RNA binding domain: identification of SBP2 amino acids important to SECIS RNA binding. RNA 8:1308-1318.[Abstract]
    2. 2
  2. Bachellerie, J. P., J. Cavaille, and A. Huttenhofer. 2002. The expanding snoRNA world. Biochimie 84:775-790.[Medline]
    3. 3
  3. Ban, N., P. Nissen, J. Hansen, P. B. Moore, and T. A. Steitz. 2000. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science 289:905-920.[Abstract/Free Full Text]
    4. 4
  4. Beltrame, M., Y. Henry, and D. Tollervey. 1994. Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA. Nucleic Acids Res. 22:5139-5147.[Abstract/Free Full Text]
    5. 5
  5. Beltrame, M., and D. Tollervey. 1995. Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis. EMBO J. 14:4350-4356.[Medline]
    6. 6
  6. Berges, T., E. Petfalski, D. Tollervey, and E. C. Hurt. 1994. Synthetic lethality with fibrillarin identifies NOP77p, a nucleolar protein required for pre-rRNA processing and modification. EMBO J. 13:3136-3148.[Medline]
    7. 7
  7. Borovjagin, A. V., and S. A. Gerbi. 1999. U3 small nucleolar RNA is essential for cleavage at sites 1, 2 and 3 in pre-rRNA and determines which rRNA processing pathway is taken in Xenopus oocytes. J. Mol. Biol. 286:1347-1363.[CrossRef][Medline]
    8. 8
  8. Borovjagin, A. V., and S. A. Gerbi. 2001. Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing. Mol. Cell. Biol. 21:6210-6221.[Abstract/Free Full Text]
    9. 9
  9. Borovjagin, A. V., and S. A. Gerbi. 2004. Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction. RNA 10:942-953.[Abstract/Free Full Text]
    10. 10
  10. Borovjagin, A. V., and S. A. Gerbi. 2005. An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA. Nucleic Acids Res. 33:4995-5005.[Abstract/Free Full Text]
    11. 11
  11. Cahill, N. M., K. Friend, W. Speckmann, Z. H. Li, R. M. Terns, M. P. Terns, and J. A. Steitz. 2002. Site-specific cross-linking analyses reveal an asymmetric protein distribution for a box C/D snoRNP. EMBO J. 21:3816-3828.[CrossRef][Medline]
    12. 12
  12. Chao, J. A., and J. R. Williamson. 2004. Joint X-ray and NMR refinement of the yeast L30e-mRNA complex. Structure 12:1165-1176.[Medline]
    13. 13
  13. Charpentier, B., S. Muller, and C. Branlant. 2005. Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation. Nucleic Acids Res. 33:3133-3144.[Abstract/Free Full Text]
    14. 14
  14. Charron, C., X. Manival, A. Clery, V. Senty-Segault, B. Charpentier, N. Marmier-Gourrier, C. Branlant, and A. Aubry. 2004. The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity. J. Mol. Biol. 342:757-773.[CrossRef][Medline]
    15. 15
  15. Cojocaru, V., S. Nottrott, R. Klement, and T. M. Jovin. 2005. The snRNP 15.5K protein folds its cognate K-turn RNA: a combined theoretical and biochemical study. RNA 11:197-209.[Abstract/Free Full Text]
    16. 16
  16. Decatur, W. A., and M. J. Fournier. 2002. rRNA modifications and ribosome function. Trends Biochem. Sci. 27:344-351.[CrossRef][Medline]
    17. 17
  17. Dobbyn, H. C., and R. T. O'Keefe. 2004. Analysis of Snu13p mutations reveals differential interactions with the U4 snRNA and U3 snoRNA. RNA 10:308-320.[Abstract/Free Full Text]
    18. 18
  18. Grandi, P., V. Rybin, J. Bassler, E. Petfalski, D. Strauss, M. Marzioch, T. Schafer, B. Kuster, H. Tschochner, D. Tollervey, A. C. Gavin, and E. Hurt. 2002. 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10:105-115.[CrossRef][Medline]
    19. 19
  19. Granneman, S., G. J. Pruijn, W. Horstman, W. J. van Venrooij, R. Luhrmann, and N. J. Watkins. 2002. The hU3-55K protein requires 15.5K binding to the box B/C motif as well as flanking RNA elements for its association with the U3 small nucleolar RNA in vitro. J. Biol. Chem. 277:48490-48500.[Abstract/Free Full Text]
    20. 20
  20. Granneman, S., and S. J. Baserga. 2004. Ribosome biogenesis: of knobs and RNA processing. Exp. Cell Res. 296:43-50.[CrossRef][Medline]
    21. 21
  21. Hamma, T., and A. R. Ferre-D'Amare. 2004. Structure of protein L7Ae bound to a K-turn derived from an archaeal box H/ACA sRNA at 1.8 Å resolution. Structure 12:893-903.[Medline]
    22. 22
  22. Henras, A., Y. Henry, C. Bousquet-Antonelli, J. Noaillac-Depeyre, J. P. Gelugne, and M. Caizergues-Ferrer. 1998. Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs. EMBO J. 17:7078-7090.[CrossRef][Medline]
    23. 23
  23. Hughes, J. M., and M. Ares, Jr. 1991. Depletion of U3 small nucleolar RNA inhibits cleavage in the 5' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA. EMBO J.10:4231-4239.[Medline]
    24. 24
  24. Jacquenet, S., A. Mereau, P. S. Bilodeau, L. Damier, C. M. Stoltzfus, and C. Branlant. 2001. A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H. J. Biol. Chem. 276:40464-40475.[Abstract/Free Full Text]
    25. 25
  25. Jaeger, J. A., D. H. Turner, and M. Zuker. 1989. Improved predictions of secondary structures for RNA. Proc. Natl. Acad. Sci. USA 86:7706-7710.[Abstract/Free Full Text]
    26. 26
  26. Jeppesen, C., B. Stebbins-Boaz, and S. A. Gerbi. 1988. Nucleotide sequence determination and secondary structure of Xenopus U3 snRNA. Nucleic Acids Res. 16:2127-2148.[Abstract/Free Full Text]
    27. 27
  27. Kass, S., K. Tyc, J. A. Steitz, and B. Sollner-Webb. 1990. The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing. Cell 60:897-908.[CrossRef][Medline]
    28. 28
  28. Kiss, T. 2001. Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs. EMBO J. 20:3617-3622.[CrossRef][Medline]
    29. 29
  29. Klein, D. J., T. M. Schmeing, P. B. Moore, and T. A. Steitz. 2001. The kink-turn: a new RNA secondary structure motif. EMBO J. 20:4214-4221.[CrossRef][Medline]
    30. 30
  30. Koonin, E. V., P. Bork, and C. Sander. 1994. A novel RNA-binding motif in omnipotent suppressors of translation termination, ribosomal proteins and a ribosome modification enzyme? Nucleic Acids Res. 22:2166-2167.[Abstract/Free Full Text]
    31. 31
  31. Kressler, D., P. Linder, and J. de La Cruz. 1999. Protein trans-acting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 19:7897-7912.[Free Full Text]
    32. 32
  32. Kuhn, J. F., E. J. Tran, and E. S. Maxwell. 2002. Archaeal ribosomal protein L7 is a functional homolog of the eukaryotic 15.5kD/Snu13p snoRNP core protein. Nucleic Acids Res. 30:931-941.[Abstract/Free Full Text]
    33. 33
  33. Li, H., and S. A. White. 1997. RNA apatamers for yeast ribosomal protein L32 have a conserved purine-rich internal loop. RNA 3:245-254.[Abstract]
    34. 34
  34. Lubben, B., C. Marshallsay, N. Rottmann, and R. Luhrmann. 1993. Isolation of U3 snoRNP from CHO cells: a novel 55-kDa protein binds to the central part of U3 snoRNA. Nucleic Acids Res. 21:5377-5385.[Abstract/Free Full Text]
    35. 35
  35. Lukowiak, A. A., S. Granneman, S. A. Mattox, W. A. Speckmann, K. Jones, H. Pluk, W. J. Venrooij, R. M. Terns, and M. P. Terns. 2000. Interaction of the U3-55k protein with U3 snoRNA is mediated by the box B/C motif of U3 and the WD repeats of U3-55k. Nucleic Acids Res. 28:3462-3471.[Abstract/Free Full Text]
    36. 36
  36. Lygerou, Z., P. Mitchell, E. Petfalski, B. Seraphin, and D. Tollervey. 1994. The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins. Genes Dev. 8:1423-1433.[Abstract/Free Full Text]
    37. 37
  37. Mao, H., S. A. White, and J. R. Williamson. 1999. A novel loop-loop recognition motif in the yeast ribosomal protein L30 autoregulatory RNA complex. Nat. Struct. Biol 6:1139-1147.[CrossRef][Medline]
    38. 38
  38. Marmier-Gourrier, N., A. Clery, V. Senty-Segault, B. Charpentier, F. Schlotter, F. Leclerc, R. Fournier, and C. Branlant. 2003. A structural, phylogenetic, and functional study of 15.5-kD/Snu13 protein binding on U3 small nucleolar RNA. RNA 9:821-838.[Abstract/Free Full Text]
    39. 39
  39. Maxwell, E. S., and M. J. Fournier. 1995. The small nucleolar RNAs. Annu. Rev. Biochem. 64:897-934.[CrossRef][Medline]
    40. 40
  40. Mereau, A., R. Fournier, A. Gregoire, A. Mougin, P. Fabrizio, R. Luhrmann, and C. Branlant. 1997. An in vivo and in vitro structure-function analysis of the Saccharomyces cerevisiae U3A snoRNP: protein-RNA contacts and base-pair interaction with the pre-ribosomal RNA. J. Mol. Biol. 273:552-571.[CrossRef][Medline]
    41. 41
  41. Moore, T., Y. Zhang, M. O. Fenley, and H. Li. 2004. Molecular basis of box C/D RNA-protein interactions: cocrystal structure of archaeal L7Ae and a box C/D RNA. Structure 12:807-818.[Medline]
    42. 42
  42. Morrissey, J. P., and D. Tollervey. 1993. Yeast snR30 is a small nucleolar RNA required for 18S rRNA synthesis. Mol. Cell. Biol. 13:2469-2477.[Abstract/Free Full Text]
    43. 43
  43. Mougin, A., A. Gregoire, J. Banroques, V. Segault, R. Fournier, F. Brule, M. Chevrier-Miller, and C. Branlant. 1996. Secondary structure of the yeast Saccharomyces cerevisiae pre-U3A snoRNA and its implication for splicing efficiency. RNA 2:1079-1093.[Abstract]
    44. 44
  44. Mougin, A., A. Gottschalk, P. Fabrizio, R. Luhrmann, and C. Branlant. 2002. Direct probing of RNA structure and RNA-protein interactions in purified HeLa cell's and yeast spliceosomal U4/U6·U5 tri-snRNP particles. J. Mol. Biol. 317:631-649.[CrossRef][Medline]
    45. 45
  45. Nissan, T. A., J. Bassler, E. Petfalski, D. Tollervey, and E. Hurt. 2002. 60S pre-ribosome formation viewed from assembly in the nucleolus until export to the cytoplasm. EMBO J. 21:5539-5547.[CrossRef][Medline]
    46. 46
  46. Nolivos, S., A. J. Carpousis, and B. Clouet-d'Orval. 2005. The K-loop, a general feature of the Pyrococcus C/D guide RNAs, is an RNA structural motif related to the K-turn. Nucleic Acids Res. 33:6507-6514.[Abstract/Free Full Text]
    47. 47
  47. Nottrott, S., K. Hartmuth, P. Fabrizio, H. Urlaub, I. Vidovic, R. Ficner, and R. Luhrmann. 1999. Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA. EMBO J. 18:6119-6133.[CrossRef][Medline]
    48. 48
  48. Nottrott, S., H. Urlaub, and R. Luhrmann. 2002. Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins. EMBO J. 21:5527-5538.[CrossRef][Medline]
    49. 49
  49. Omer, A. D., S. Ziesche, H. Ebhardt, and P. P. Dennis. 2002. In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex. Proc. Natl. Acad. Sci. USA 99:5289-5294.[Abstract/Free Full Text]
    50. 50
  50. Oruganti, S., Y. Zhang, and H. Li. 2005. Structural comparison of yeast snoRNP and spliceosomal protein Snu13p with its homologs. Biochem. Biophys. Res. Commun. 333:550-554.[CrossRef][Medline]
    51. 51
  51. Parker, K. A., and J. A. Steitz. 1987. Structural analysis of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol. Cell. Biol. 7:2899-2913.[Abstract/Free Full Text]
    52. 52
  52. Pluk, H., J. Soffner, R. Luhrmann, and W. J. van Venrooij. 1998. cDNA cloning and characterization of the human U3 small nucleolar ribonucleoprotein complex-associated 55-kilodalton protein. Mol. Cell. Biol. 18:488-498.[Abstract/Free Full Text]
    53. 53
  53. Rozhdestvensky, T. S., T. H. Tang, I. V. Tchirkova, J. Brosius, J. P. Bachellerie, and A. Huttenhofer. 2003. Binding of L7Ae protein to the K-turn of archaeal snoRNAs: a shared RNA binding motif for C/D and H/ACA box snoRNAs in Archaea. Nucleic Acids Res. 31:869-877.[Abstract/Free Full Text]
    54. 54
  54. Samarsky, D. A., and M. J. Fournier. 1998. Functional mapping of the U3 small nucleolar RNA from the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 18:3431-3444.[Abstract/Free Full Text]
    55. 55
  55. Schluenzen, F., A. Tocilj, R. Zarivach, J. Harms, M. Gluehmann, D. Janell, A. Bashan, H. Bartels, I. Agmon, F. Franceschi, and A. Yonath. 2000. Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102:615-623.[CrossRef][Medline]
    56. 56
  56. Schultz, A., S. Nottrott, K. Hartmuth, and R. Luhrmann. 2006. RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac small nuclear ribonucleoproteins. J. Biol. Chem. 281:28278-28286.[Abstract/Free Full Text]
    57. 57
  57. Schultz, A., S. Nottrott, N. J. Watkins, and R. Luhrmann. 2006. Protein-protein and protein-RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs. Mol. Cell. Biol. 26:5146-5154.[Abstract/Free Full Text]
    58. 58
  58. Segault, V., A. Mougin, A. Gregoire, J. Banroques, and C. Branlant. 1992. An experimental study of Saccharomyces cerevisiae U3 snRNA conformation in solution. Nucleic Acids Res. 20:3443-3451.[Abstract/Free Full Text]
    59. 59
  59. Sharma, K., and D. Tollervey. 1999. Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing. Mol. Cell. Biol. 19:6012-6019.[Abstract/Free Full Text]
    60. 60
  60. Stevens, S. W., and J. Abelson. 1999. Purification of the yeast U4/U6.U5 small nuclear ribonucleoprotein particle and identification of its proteins. Proc. Natl. Acad. Sci. USA 96:7226-7231.[Abstract/Free Full Text]
    61. 61
  61. Szewczak, L. B., J. S. Gabrielsen, S. J. Degregorio, S. A. Strobel, and J. A. Steitz. 2005. Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein. RNA 11:1407-1419.[Abstract/Free Full Text]
    62. 62
  62. Terns, M. P., and R. M. Terns. 2002. Small nucleolar RNAs: versatile trans-acting molecules of ancient evolutionary origin. Gene Expr. 10:17-39.[Medline]
    63. 63
  63. Turner, B., S. E. Melcher, T. J. Wilson, D. G. Norman, and D. M. Lilley. 2005. Induced fit of RNA on binding the L7Ae protein to the kink-turn motif. RNA 11:1192-1200.[Abstract/Free Full Text]
    64. 64
  64. Tyc, K., and J. A. Steitz. 1992. A new interaction between the mouse 5' external transcribed spacer of pre-rRNA and U3 snRNA detected by psoralen crosslinking. Nucleic Acids Res. 20:5375-5382.[Abstract/Free Full Text]
    65. 65
  65. Venema, J., and D. Tollervey. 1999. Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet. 33:261-311.[CrossRef][Medline]
    66. 66
  66. Venema, J., H. R. Vos, A. W. Faber, W. J. van Venrooij, and H. A. Raue. 2000. Yeast Rrp9p is an evolutionarily conserved U3 snoRNP protein essential for early pre-rRNA processing cleavages and requires box C for its association. RNA 6:1660-1671.[Abstract]
    67. 67
  67. Verheggen, C., D. L. Lafontaine, D. Samarsky, J. Mouaikel, J. M. Blanchard, R. Bordonne, and E. Bertrand. 2002. Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments. EMBO J. 21:2736-2745.[CrossRef][Medline]
    68. 68
  68. Vidovic, I., S. Nottrott, K. Hartmuth, R. Luhrmann, and R. Ficner. 2000. Crystal structure of the spliceosomal 15.5kD protein bound to a U4 snRNA fragment. Mol. Cell 6:1331-1342.[CrossRef][Medline]
    69. 69
  69. Watkins, N. J., V. Segault, B. Charpentier, S. Nottrott, P. Fabrizio, A. Bachi, M. Wilm, M. Rosbash, C. Branlant, and R. Luhrmann. 2000. A common core RNP structure shared between the small nucleolar box C/D RNPs and the spliceosomal U4 snRNP. Cell 103:457-466.[CrossRef][Medline]
    70. 70
  70. Watkins, N. J., A. Dickmanns, and R. Luhrmann. 2002. Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP. Mol. Cell. Biol. 22:8342-8352.[Abstract/Free Full Text]
    71. 71
  71. Watkins, N. J., I. Lemm, D. Ingelfinger, C. Schneider, M. Hossbach, H. Urlaub, and R. Luhrmann. 2004. Assembly and maturation of the U3 snoRNP in the nucleoplasm in a large dynamic multiprotein complex. Mol. Cell 16:789-798.[CrossRef][Medline]
    72. 72
  72. Wimberly, B. T., D. E. Brodersen, W. M. Clemons, Jr., R. J. Morgan-Warren, A. P. Carter, C. Vonrhein, T. Hartsch, and V. Ramakrishnan. 2000. Structure of the 30S ribosomal subunit. Nature 407:327-339.[CrossRef][Medline]
    73. 73
  73. Wozniak, A. K., S. Nottrott, E. Kuhn-Holsken, G. F. Schroder, H. Grubmuller, R. Luhrmann, C. A. Seidel, and F. Oesterhelt. 2005. Detecting protein-induced folding of the U4 snRNA kink-turn by single-molecule multiparameter fluorescent resonance energy transfer measurements. RNA 11:1545-1554.[Abstract/Free Full Text]

Molecular and Cellular Biology, February 2007, p. 1191-1206, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01287-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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