Terminal Structure Mediates 5 S rRNA Stability and Integration during Ribosome Biogenesis*

Formation of the eukaryotic ribosomal 5 S RNA-protein complex has been shown to be critical to ribosome biogenesis and has been speculated to contribute to a quality control mechanism that helps ensure that only normal precursors are processed and assembled into active ribosomes. To study the structural basis of these observations, the RNA-protein interface in the 5 S RNA-protein complex of the yeast (Saccharomyces cerevisiae) ribosome was examined based on a systematic introduction of targeted base substitutions in the RNA sequence. Most base substitutions had little or no effect on the efficiency of complex formation, but large effects were observed when changes disrupted helix I, the secondary structure formed between the interacting termini. Again, only modest effects were evident when the extended 3′ end of the mature RNA molecule was altered, but essentially no complex was formed when the 5′ end of the mature 5 S RNA sequence was artificially extended by one nucleotide.In vitro analyses demonstrated that this extension also dramatically altered the maturation of 5 S rRNA precursor molecules as well as the stability of the mature 5 S rRNA. Taken together, the results indicate that in the course of RNA maturation, the 5 S RNA-binding protein binds precisely over or “caps” the termini in a critical manner that protects the RNA from further degradation.

While first recognized as a discrete ribosomal component almost four decades ago, the function of the 5 S RNA remains unclear and often disputed (see Ref. 1). Nevertheless, numerous studies on 5 S RNAs from diverse origins have demonstrated intriguing evolutionary changes in the localization and expression of genes encoding this RNA, while molecular features have been shown to be highly conserved both with respect to the primary and secondary structures (see Refs. 2 and 3) and even aspects of the tertiary structure (4,5). Included in these features is a strong interaction between the RNA termini, which results in a 9 -10 base pair helix that is usually staggered at the 3Ј end (see Fig. 1). Previous studies on ribosomes, both in bacteria and eukaryotic organisms, also have shown that the 5 S RNA molecules can be dissociated from ribosomes as an RNA-protein complex (e.g. Refs. 6, 7). Three ribosomal proteins have been isolated from complex prepared using ribosomes of Escherichia coli (3); in eukaryotes a single ribosomal protein (YL3 or Rpl5p in yeast) of approximately equal total molecular weight has been ob-served to constitute the protein fraction (8). Mutations in either the yeast 5 S RNA (1) or the YL3 protein (9) result in strikingly unstable ribosomal subunits, consistent with a critical role in ribosome assembly (10).
Because the 5 S RNA-protein complex is relatively small and easily purified, it has been an attractive model for the study of RNA protein interactions in ribosomes and even ribonucleoprotein complexes, in general. Extensive studies on complexes from E. coli have characterized the binding sites for all three ribosomal proteins (see Ref. 11) and indicated interactions with three of the four helices (I, II, and IV) in the bacterial 5 S rRNA. Based on x-ray crystallography, recent analyses now show that the complex, together with a part of the 23 S rRNA, constitutes the central protuberance of the large ribosomal subunit, and at least in bacteria, the protein components mediate the integration of the 5 S RNA into the ribosome, permitting specific tertiary interactions with the 23 S rRNA (12)(13)(14). While crystal analyses are not available for eukaryotic ribosomes, past studies on the structure of the yeast 5 S RNA-protein complex, based on protection from ribonuclease digestion (8) or modification exclusion (15), have suggested that the helix formed by the termini of the 5 S rRNA molecule (helix I) represents the primary protein binding site with further influence by helix II and IV. Naturally arising differences in the nucleotide sequence that do not alter these helices appear not to affect the formation of this complex (16), an observation that suggests a recognition of secondary or tertiary structure (17) rather than the actual nucleotide sequence.
In all the eukaryotes that have been examined, the 5 S rRNA molecule is transcribed as a slightly longer precursor molecule with a short sequence extension at the 3Ј end (l2 nucleotides in Saccharomyces cerevisiae. Although this feature seems unnecessary and wasteful, the extra sequence is removed by nuclease cleavage during RNA maturation and integration into the ribosomal structure. Studies in the toad (18), fly (19), yeast (20), and mammalian cells (21) have documented this maturation process and provided some detail regarding the underlying mechanism. For example, in Drosophila the extra nucleotides appear to be removed by a single rapid endonucleolytic step (22,23), while in yeast it is primarily or entirely removed by an exonuclease (24). Studies of the 5 S rRNA maturation process in yeast also indicate that, although the process is essentially independent of the nucleotide composition, it is surprisingly dependent on the length and higher order structure of the extended sequence. Transcripts that are not normally terminated or terminated much later are highly unstable and not incorporated into ribosomes (25). Furthermore, this is equally true when helical structure is introduced into this extended sequence (24). Taken together, these observations have led to the suggestion that the precursor sequence and its removal during RNA maturation serve, at least in part, as a quality control mechanism that helps ensure that mutated transcripts are not integrated into active ribosomes.
Since the 5 S rRNA-binding protein appeared to interact primarily with the helix formed by the termini of the 5 S RNA molecule, in this study specific changes were introduced into this region to further examine the influence on protein binding and the integration of the molecule into stable ribosomes. The results indicate an unusual and critical dependence on the 5Ј end structure, a feature which suggests that a protein "cap" at the termini mediates the quality control function.

EXPERIMENTAL PROCEDURES
Construction and Expression of mutant 5 S rRNA Genes-Mutations were introduced into a yeast 5 S rRNA gene (26) containing a structural marker mutation (27) by using mutant oligonucleotide primers during PCR amplification; a modified two-step PCR strategy (28) was used to introduce mutations into coding sequence. Mutant oligonucleotides and normal primers were synthesized using a Cyclone Plus automated DNA synthesizer (Millipore Corp., Milford, MA). Mutant genes were cloned into pYF404, a high-copy (30 -40 copies per cell) yeast shuttle vector (29), and the mutant sequence was confirmed by DNA sequencing (30). To express the mutant 5 S rRNA genes, the recombinant plasmids were purified and used to transform a LEU2-deficient yeast strain (AH22) as described by Hinnen et al. (31).
Preparation of Mutant 5 S rRNAs and Precursors-To prepare mutant 5 S rRNAs with internal nucleotide changes, the transformed cells were grown with shaking under selective conditions at 30°C. Whole cell RNA was prepared by SDS-phenol extraction, and the 5 S rRNAs were fractionated at room temperature by electrophoresis on 12% non-denaturing polyacrylamide gels (27).
To synthesize precursor molecules or 5 S rRNAs with changes at the 5Ј or 3Ј end, normal and mutant oligonucleotide primers were used to prepare T 7 promoter/5 S rRNA gene fusion constructs by PCR amplification, and these were used as DNA templates for in vitro runoff transcription reactions using T 7 RNA polymerase. PCR amplification was carried out using 30 cycles of 1 min at 94°C, 1 min at 45-55°C, and 1 min at 72°C with a forward primer consisting of a T 7 RNA polymerase promoter followed by a short portion of 5 S rRNA sequence beginning at the 5Ј end and a reverse primer complementary to the 3Ј end of the precursor molecule. Recombinant plasmids containing normal or mutant 5 S rRNA genes were used as templates.
Agarose gel-purified PCR products (typically 0.2-1.0 g) were transcribed (32) in a 50-l volume of reaction mixture containing 10 l of 200 mM Tris-HCl, pH 7.5, 30 mM MgCl 2 , 10 mM spermidine, 50 mM NaCl, 1 l of 500 mM DTT, 1 10 l of 2.5 mM each of NTP and 50 -70 units of T 7 RNA polymerase (kindly provided by Dr. T. O. Sitz, Virginia Polytechnical Institute and State University). After incubation at 37°C for 2 h, the reaction was stopped with the addition of 5 l of 100 M EDTA and the mixture was immediately extracted twice with an equal volume of phenol/chloroform/isoamylalcohol (25:24:1) before being precipitated with ethanol. To remove contaminating nucleoside triphosphates, incompletely transcribed products, and the DNA template, the transcript was gel-fractionated on an 8% denaturing polyacrylamide gel, stained with methylene blue (33), and eluted by homogenization in diluted SDS buffer (27).
Assay for the Formation of 5 S rRNA-Protein Complexes-The ability to form a 5 S rRNA-protein complex was assayed using an exchange reaction with normal unlabeled complex and labeled mutant RNA as previously described (24). The 5 S rRNA-protein complex was prepared from purified ribosomes (4,15) and concentrated by centrifugation at 5000 ϫ g for several hours to 5-6 A 260 nm /ml using a Centricon-30 microconcentrator (Amicon, Beverly, MA). Small aliquots (40 l) were stored at Ϫ70°C prior to use. In most experiments the RNA was dephosphorylated using calf intestinal alkaline phosphatase and labeled at the 5Ј end using T 4 polynucleotide kinase and [␥-32 P]ATP (34); for some experiments 3Ј end-labeled RNA was prepared using T 4 RNA ligase and cytidine[3Ј, 5Ј-32 P]bisphosphate (35). Approximately 100,000 cpm of labeled 5 S rRNA or precursor in 10 -20 l of unlabelled yeast 5 S rRNA-protein complex (5-6 A 260 nm /ml) was incubated for 1-2 h on ice. For subsequent analyses, a half-volume of loading buffer (45 mM Tris-HCl, pH 8.3, 45 mM boric acid, 1 mM EDTA, 10% glycerol, 0.05% xylene cyanol, and 0.05% bromphenol blue) was added, and the labeled 5 S rRNA precursor protein complex was fractionated on an 8% nondenaturing polyacrylamide slab gel (4°C) and visualized by autoradiography. For quantitative analyses, the radioactivity in the free and protein-bound fractions was determined by scintillation counting. As previously observed, both unlabeled and 5Ј or 3Ј end-labeled normal 5 S RNAs were incorporated into ribonucleoprotein with equal efficiency.
Isolation of Yeast Nuclei and Preparation of Nuclear Extracts-Yeast nuclei were isolated as described by Aris and Blobel (36) with minor modifications (27). S. cerevisiae, strain AH22, was grown at 30°C in YEPD medium (1% (w/v) Bacto-yeast extract, 2% (w/v) Bacto-peptone, 2% (w/v) glucose) medium to an absorbency of 1 at 600 nm (ϳ4 ϫ 10 7 cells/ml), and cells were harvested by centrifugation for 5 min at 4000 ϫ g. The pellets were resuspended in 30 ml of cold SPH medium (1 M sorbitol, 1% (w/v) glucose, 0.2% (w/v) Bacto-yeast nitrogen base without amino acids, 20 g/ml histidine, 30 g/l leucine, 25 mM HEPES (free acid), and 50 mM Tris-HCl) and again collected by centrifugation. To prepare spheroplasts, 3 g of cells were resuspended in 27 ml of SPH medium containing 10,000 units of lyticase (Sigma) and 60 l of 1 M DTT and incubated at 30°C for about 1 h. Spheroplast formation and cell lysis were closely monitored microscopically for optimum yields. The spheroplasts were chilled rapidly on ice for about 5 min, layered on a 5-ml cushion (1.5 M sorbitol, 2% (w/v) Ficoll 400, 25 mM MES-Tris, pH 6.5) containing protease inhibitor mixture (PIC; 20 l of 100 mM phenylmethylsulfonyl fluoride (PMSF), 10 l of 200 g/ml leupeptin, and 10 l of 200 g/ml pepstatin A), and collected by centrifugation for 5 min at 6000 ϫ g. The pellet was resuspended in 15 ml of wash buffer (1.2 M sorbitol, 2% (w/v) Ficoll 400, 25 mM MES-Tris, pH 6.5), containing PIC, and collected by centrifugation for 5 min at 3000 ϫ g. To lyse the spheroplasts, the pellet was dispersed in 27 ml of lysis buffer (20% (w/v) Ficoll 400, 20 mM potassium phosphate, pH 6.45, 1 mM MgCl 2 ) containing PIC using 20 strokes of a Dounce homogenizer with a loose-fitting pestle. The lysate was chilled on ice for 10 min and cleared by centrifugation for 15 min at 13,000 ϫ g, and 25 ml were layered on a Ficoll step gradient (5-ml layers of 30, 40, and 50% Ficoll 400 in 1 mM MgCl 2 , 20 mM potassium phosphate, pH 6.45, and PIC) formed in a SW28 rotor ultraclear tube (Beckman Instruments Inc., Fullerton, CA). The gradients were fractionated at 18,000 rpm (58,400 ϫ g) and 2°C for 60 min. Nuclei, visible in the 40% layer and at the 30 -40% and 40 -50% interfaces, were collected with a Pasteur pipette, frozen in liquid nitrogen, and stored at Ϫ70°C.
The nuclear extracts were prepared from frozen nuclei using a modification of the procedure described by Hennighausen and Lubon (37). The nuclei were diluted 10-fold in cold buffer containing 1 mM MgCl 2 , 20 mM potassium phosphate, pH 6.45, and PIC collected by centrifugation for 10 min at 10,000 ϫ g and then lysed with 20 strokes of a Dounce homogenizer in 3 ml of lysis buffer containing 400 mM NaCl, 1.5 mM MgCl 2 , 0.1 mM EGTA, 0.5 mM DTT, 5% glycerol, 0.5 mM PMSF, and 10 mM HEPES-KOH, pH 7.9. The lysed nuclei were kept on ice for 30 min before centrifugation for 60 min at 100,000 ϫ g (31,000 rpm) using a Beckman Type 70Ti rotor. After dialysis at 4°C for 2-4 h against 50 volumes of buffer containing 75 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 20% glycerol, 0.5 mM PMSF, and 20 mM HEPES-KOH, pH 7.9, the supernatant was cleared further by centrifugation at 25,000 ϫ g for 15 min. The protein concentration in the supernatant was typically 0.2-0.5 mg/ml. The supernatant was frozen in small aliquots (ϳ100 l) using liquid nitrogen and could be stored at Ϫ70°C for several weeks.
In Vitro RNA Processing-For in vitro RNA processing, the 5 S rRNA precursor was labeled at the 5Ј end using T 4 polynucleotide kinase and [␥-32 P]ATP (34) or 3Ј end with cytidine[3Ј,5Ј-32 P]bisphosphate and T 4 RNA ligase (35). The labeled RNA was purified on an 8% denaturing polyacrylamide gel, detected by autoradiography, and recovered by homogenization as described above. For the RNA processing reactions, labeled RNA (10,000 cpm) was incubated at 30°C with 10 l of nuclear extract in a final volume of 20 l containing 37.5 mM NaCl, 3 mM MgCl 2 , 100 mM KCl, 3 mM DTT, 10% glycerol, 0.25 mM PMSF, 50 M EDTA, and 30 mM HEPES-KOH, pH 7.9. Reactions were stopped by the addition of 0.4 ml of stop buffer (0.3 M sodium acetate, 0.1% SDS, 10 mM EDTA, and 40 mg/ml of yeast tRNA as a carrier). The RNA was extracted once with phenol/chloroform/isoamylalcohol (25:24:1), ethanol-precipitated, ethanol-washed, and vacuum-dried. For product analyses, the samples were resuspended in 5 l of loading buffer (formamide containing 0.03% xylene cyanol and 0.03% bromphenol blue dyes), heated for 2 min at 90°C, and fractionated on an 8% polyacrylamide gel containing 8 M urea before being exposed to x-ray film for autoradiography.
Immunoblot Analyses-For immunoblotting, whole cell protein extracts (38) or purified YL3/5 S rRNA complex protein was fractionated on a 12% SDS-polyacrylamide resolving gel with a 5% stacking gel and transferred to nitrocellulose. The membrane was rinsed in TBST (20 mM Tris-HCl, 137 mM sodium chloride, 0.3% Tween 20, pH 7.6), stained (39) with India ink (1:250 dilution in TBST), destained, and blocked for 1 h with 5% dried milk powder in TBST (blocking buffer). The filter was then incubated with antibody (anti-Lhp1p diluted 1:1000 in blocking buffer) for 1 h, washed three times with TBST, and incubated for 1 h with a horseradish peroxidase-linked secondary antibody (Amersham Biosciences) diluted 1:2500 in blocking buffer. After four washes the membrane was incubated in ECL (Amersham Biosciences) detection reagents as described by the manufacturer and exposed on X-OMAT AR film (Kodak).

Influence of the 5 S RNA Sequence on the Formation of
Ribonucleoprotein Complex-Past studies on the eukaryotic 5 S RNA-protein complex from yeast ribosomes indicated a single protein constituent, which based on peptide mapping and partial sequence analyses (8,40) was identified as the YL3 protein.
Subsequent studies showed that this complex could be efficiently labeled through RNA exchange by incubating unlabelled complex in the presence of 32 P-labeled 5 S RNA (see Fig.  2, Ctl). This efficient exchange also has provided for a simple assay of essential structural features in the 5 S RNA and previously was combined with nuclease digestion (4,8) or modification exclusion (15) to determine general features in the protein interface. As indicated in Fig. 1, both approaches identified the universally conserved terminal helix (I) as a primary binding site but also suggested interactions or at least protein contact with helix II and IV. To more directly access the contributions of individual nucleotides, in the present study mutations were introduced systematically in a yeast 5 S rRNA gene containing a neutral structural marker (Y5A99) mutation (27), either by the methods of Kunkel (41) or by a modified two-step PCR procedure (28). Normal or mutant RNAs were purified from cells transformed with mutant 5 S RNA genes or prepared in vitro using T 7 RNA polymerase (24). RNAs labeled at either the 5Ј end with polynucleotide kinase after pretreatment with alkaline phosphatase (34) or the 3Ј end using RNA ligase (35) were equally efficient during RNA exchange (15,24); the one-step 3Ј end label was used in most experiments. As shown Fig. 2B, when compared with natural 5 S RNA (Ctl), the in vitro prepared 5 S RNA also was equally efficient in RNA exchanges. As summarized in Fig. 1, the efficiency of RNA exchange varied dramatically depending on the specific mutant RNA. Most changes had little or no effect on the exchange efficiency with levels of exchange ϳ90 -105% of that observed with normal yeast 5 S rRNA. Some changes, however, (e.g. Y5A33 and Y5U90i5) did have modest but reproducible effects with 75-85% levels of exchange, while still others (e.g. Y5G116 and Y5C118) resulted in very little RNA exchange (ϳ10%). Consistent with the past studies based on nuclease digestion (4,11) or modification exclusion (12), the mutational analyses indicated that the strongest effects were all linked to structural disruptions in helix I, providing clear and direct evidence that helix I constitutes the primary protein binding site.
Influence of the Terminal Structure on the Formation of Ribonucleoprotein Complex-To further explore essential features in helix I, additional changes were introduced in this region. Since 5 S RNA molecules normally have a sequence extension at the 3Ј end, in the course of the additional analyses the 5Ј end also was extended by one nucleotide. Surprisingly, this change had the most striking effect. As shown in Fig. 2, with one additional guanylic acid residue at the 5Ј end (A), only a slight trace of complex was evident. This was in strong contrast with normal, in vivo (Ctl) or in vitro (B) transcribed 5 S RNA. The special nature of this change was further underlined when compared with other changes at the termini. As shown in Fig. 3, when the 3Ј end extension was removed (lane a) or when the residue was changed (lane b) only moderate effects were evident. In contrast, when these changes were introduced in a 5 S RNA molecule containing the nucleotide extension at 5Ј end (lanes c and d), again only trace amounts of ribonucleoprotein were evident, with complex formation being almost totally inhibited.
Internal changes in helix would be expected to disrupt the secondary structure of this helix, an effect that is likely the cause of the observed strong reduction in complex formation. Indeed past structural analyses (1, 42) have confirmed such changes in the secondary structure. The changes at the termini, however, would not be expected to disrupt the helix. In view of the dramatic effects, with a 5Ј end extension, the RNA sequences were further confirmed by sequence determination, and the possibility of structural rearrangements were eliminated by computer-aided modeling and actual secondary structure determination. The only interaction that could be envisaged would be a base pair extension in helix I but this G-U base pair at the end of an open helix clearly is unstable. Nevertheless, to fully exclude a structural rearrangement, the terminalmodified 5 S RNA molecules also were subjected to partial cleavage with T 1 ribonuclease (Fig. 4). Since in vitro synthesized RNA contains the same terminal heterogeneity, doublet bands were evident with the two synthetic molecules (A and B) but the primary cleavage sites remained identical to those observed in the natural 5 S rRNA molecule (Ctl). Such results again indicated that the effect on complex formation was not the result of a structural rearrangement but was due to a direct hindrance of protein binding.
Processing and Stability of the 5Ј End-extended 5 S rRNA-Previous studies (24) on the maturation of the yeast 5 S rRNA have indicated that the 5 S RNA-binding protein (YL3) does not directly influence the processing of 5 S rRNA precursors, but it  does play a significant role in protecting this rRNA from further degradation by "housekeeping" nucleases. To examine the influence of the 5Ј end extension on RNA maturation and stability, both 5 S rRNA precursor and mature RNAs were incubated with a cell extract, previously demonstrated to effectively process 5 S rRNA precursors in vitro (24). As shown in Fig. 5, the normal precursor (A) was fully processed to stable mature 5 S rRNA in ϳ15 min, but a 5Ј-extended molecule (B) revealed differences in both the maturation profile and product stability. Basically, two populations of product were evident, a more stable normal population and a rapidly degraded second population. Even the normal population, however, appeared less stable than product without the additional nucleotide at the 5Ј end. About 50% of this population was degraded after 105 min of incubation. Abbreviated gel fractionations that would include short fragments of the 5Ј end did not reveal any specific processing at the 5Ј terminal (results not shown).
Differences in stability were further observed when matured RNAs were reincubated with the same extract. As shown in Fig. 6, when RNAs with differences at the termini were compared in vitro, the results correlated strongly with observations in vivo, and the efficiency of ribonucleoprotein complex formation. After 60 min of incubation, normal RNA (Ctl) was essentially undergraded. In contrast, RNA with a change at the 3Ј end (A) was partially degraded, and RNA with a one nucleotide extension at the 5Ј end (B) was almost completely degraded. As reported in a previous study for other mutations, the yield or stability of the 5 S RNA again correlated closely with its ability to form a complex with the 5 S rRNA-binding protein (YL3).
In eukaryotes virtually all nascent polymerase III transcripts initially appear to associate with the La protein or Lhp1p in S. cerevisiae (see Ref. 43). Apparently, the 5 S rRNA interacts with the ribosomal 5 S rRNA-binding protein after a transient association with the La protein (10,45), which then directs the 5 S rRNA to the nucleolus (10,46). Since the YL3 protein used in this study was purified as a 5 S rRNA protein complex, there was some possibility that La protein might be present. To eliminate this possibility an immunoblot analysis was undertaken using anti-Lhp1p protein (47). A shown in Fig.  7, no La protein was observed even when the gel was heavily loaded and the resulting membrane was somewhat overexposed. In a whole cell extract the La protein is clearly present, but even traces were not present in the protein preparation (RNP). DISCUSSION Past studies on the maturation of the ribosomal 5 S RNA in S. cerevisiae (25) have indicated that proper termination cou-pled with efficient processing is critical to the integration of 5 S rRNA transcripts into stable ribosomes. Such studies also suggested that rRNA processing, at least in part, acts as a "quality control" mechanism that helps to ensure that only normal rRNA precursors are effectively processed and assembled into active ribosomes. Subsequent studies (24) have further suggested that the 5 S RNA-binding protein (YL3) may not influence the maturation process directly but rather that it plays an important role in protecting the nascent 5 S RNA molecules from further degradation by housekeeping nucleases. The mutational analyses described in this report provide direct evi-FIG. 5. Effect of a 5 end nucleotide extension on the processing of the 5 S rRNA precursor in vitro. Normal (A) and mutant (B) precursor RNA containing an extra guanylic acid residue at the 5Ј end were prepared by runoff transcription using T 7 RNA polymerase, labeled at the 5Ј end and purified by gel electrophoresis as described under "Experimental Procedures." The labeled precursor RNAs or the mature RNA molecule (Ctl) were incubated for 0 -105 min with nuclear extract; the processed RNA was further extracted with SDS/phenol, and the fragments were fractionated by gel electrophoresis before visualization by autoradiography.
FIG. 6. Stability of 5 S rRNAs with altered termini. Normal 5 S rRNA (Ctl) and RNA with a nucleotide deletion (U 121 ) at the 3Ј end (A) or a deletion at the 3Ј end and a single guanylic acid residue extension at the 5Ј end (B) were prepared by runoff transcription using T 7 RNA polymerase, labeled at the 5Ј end, and purified by gel electrophoresis as described under "Experimental Procedures." The RNAs were incubated for 0 -60 min with nuclear extract; the RNAs and any fragments were then extracted with SDS/phenol, fractionated by gel electrophoresis, and visualized by autoradiography. dence for such a relationship. Mutant RNAs that retain an affinity for the 5 S rRNA-binding protein (see Fig. 1) are readily observed in the mature ribosomes of cells transformed with the corresponding mutant 5 S rRNA genes (24), while mutant 5 S RNA molecules that inefficiently bind the cognate ribosomal protein in vitro are degraded in vivo and are not readily incorporated into mature stable ribosomes.
The observations in this study, while fully consistent with the previous reports, also provide new detail about the nature of the eukaryotic 5 S rRNA-protein complex and the way in which the 5 S rRNA-binding protein probably acts to stabilize and protect the RNA molecule from further degradation. A number of the changes indicate that structure associated with the 5 S rRNA termini is important to the RNA-protein interaction, but a single nucleotide addition at the 5Ј end is sufficient to sterically hinder the protein interaction to a degree that almost prevents the interaction entirely. We suggest that in the course of rRNA maturation the YL3 protein (8, 48) binds tightly over or "caps" the termini, thereby protecting them from further degradation. As a result, this precise fit is critical to 5 S rRNA stability and perhaps even ribosome integration. As illustrated in Figs. 3, 5, and 6, when the tight fit is disrupted by a single nucleotide extension at the 5Ј end, the protein interaction is largely disrupted and the nascent RNA remains easily susceptible to nuclease degradation.
Although the La protein clearly was not a factor in the present experiments (Fig. 7), as already noted, it apparently does transiently associate with the nascent 5 S rRNA prior to ribosomal protein binding and nucleolar integration (45)(46)(47). The La protein binds principally via its conserved N-terminal domain (NTP) to the UUU OH motif that results from transcription termination (49), a sequence that would be removed during 5 S rRNA processing. As La protein also has been speculated to act as a molecular chaperone that protects the 3Ј end from degradation and facilitates assembly processes, this notion could be combined with the present findings to suggest a model in which the La protein protects the 3Ј end until it is displaced by the ribosomal protein to induce rRNA processing. In the case of pre-tRNAs, some members of the Lsm protein family have been shown to be essential for the efficient association of the La-homologous protein (50). At present there is no evidence that these proteins affect the 5 S RNA complexes but some Lsm proteins do affect rRNA processing (51) further raising the possibility of other transient interactions.
A question that remains is why the effect of an extension at the 3Ј end was much less critical than at the 5Ј end. Although less strongly bound, a natural slightly shorter processing var-iant also has been reported to form a complex with the YL3 protein (44), and our past study on the yeast 5 S RNA-protein complex has shown that the longer precursor RNAs form stable complexes with the YL3 protein (24) as well. The same study further indicated that in S. cerevisiae, the 5 S RNA is processed by an exonuclease activity that is limited primarily or entirely by the helix I structure. As a result it seems attractive to speculate that the YL3 protein, fixed critically relative to the 5Ј end, also fully caps the 3Ј end when the extended terminal has been removed, and as suggested above for the La protein, perhaps may even act to displace the exonuclease enzyme in the course of RNA processing. Whatever the case, the critical 5Ј end extension remains an intriguing structural feature of this essential ribosomal RNA-protein complex. FIG. 7. Immunoblot analysis of the 5 S rRNA-binding protein. Whole cell and YL3/5 S rRNA complex (RNP) proteins were fractionated on an SDS-polyacrylamide gel and transferred to nitrocellulose for immunoblot analysis with anti-Lhp1p. The membrane, after staining with India ink, and the exposed film, after immunoblotting, are shown as left and right panels, respectively. Positions of size markers (M) and the La protein are indicated on the right.