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INTRODUCTION |
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 3' 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 observed 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 (12-14). 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 3' 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 5' end structure, a feature which suggests that a
protein "cap" at the termini mediates the quality control function.
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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 5'
or 3' end, normal and mutant oligonucleotide primers were used to
prepare T7 promoter/5 S rRNA gene fusion constructs by PCR
amplification, and these were used as DNA templates for in
vitro runoff transcription reactions using T7 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 T7 RNA polymerase promoter
followed by a short portion of 5 S rRNA sequence beginning at the 5'
end and a reverse primer complementary to the 3' 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 µg) were
transcribed (32) in a 50-µl volume of reaction mixture containing 10 µl of 200 mM Tris-HCl, pH 7.5, 30 mM
MgCl2, 10 mM spermidine, 50 mM
NaCl, 1 µl of 500 mM
DTT,1 10 µl of 2.5 mM each of NTP and 50-70 units of T7 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 µl of 100 µM 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 × g for several hours to 5-6
A260 nm/ml using a Centricon-30
microconcentrator (Amicon, Beverly, MA). Small aliquots (40 µl) were
stored at
70 °C prior to use. In most experiments the RNA was
dephosphorylated using calf intestinal alkaline phosphatase and labeled
at the 5' end using T4 polynucleotide kinase and
[
-32P]ATP (34); for some experiments 3' end-labeled
RNA was prepared using T4 RNA ligase and cytidine[3',
5'-32P]bisphosphate (35). Approximately 100,000 cpm of
labeled 5 S rRNA or precursor in 10-20 µl of unlabelled yeast 5 S
rRNA-protein complex (5-6 A260 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% non-denaturing 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 5' or 3' 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 (~4 × 107
cells/ml), and cells were harvested by centrifugation for 5 min at
4000 × 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 µg/ml
histidine, 30 µg/µl 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 µl 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 µl
of 100 mM phenylmethylsulfonyl fluoride (PMSF), 10 µ l of
200 µ g/ml leupeptin, and 10 µl of 200 µg/ml pepstatin A), and
collected by centrifugation for 5 min at 6000 × 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 × 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 MgCl2)
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 × g,
and 25 ml were layered on a Ficoll step gradient (5-ml layers of 30, 40, and 50% Ficoll 400 in 1 mM MgCl2, 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 × 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
70 °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 MgCl2, 20 mM potassium
phosphate, pH 6.45, and PIC collected by centrifugation for 10 min at
10,000 × g and then lysed with 20 strokes of a Dounce
homogenizer in 3 ml of lysis buffer containing 400 mM NaCl,
1.5 mM MgCl2, 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 × 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 × 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 (~100 µl) using liquid nitrogen and could be stored at
70 °C for several weeks.
In Vitro RNA Processing--
For in vitro RNA
processing, the 5 S rRNA precursor was labeled at the 5' end using
T4 polynucleotide kinase and [
-32P]ATP
(34) or 3' end with cytidine[3',5'-32P]bisphosphate and
T4 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 µl of nuclear extract in a final volume of 20 µl containing 37.5 mM NaCl, 3 mM MgCl2, 100 mM KCl, 3 mM DTT, 10% glycerol, 0.25 mM PMSF, 50 µM 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 µl 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).
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RESULTS |
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
32P-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 T7 RNA polymerase (24). RNAs
labeled at either the 5' end with polynucleotide kinase after
pretreatment with alkaline phosphatase (34) or the 3' end using RNA
ligase (35) were equally efficient during RNA exchange (15, 24); the
one-step 3' 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 ~90-105% 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 (~10%). 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.

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Fig. 1.
Formation of 5 S rRNA-protein complexes by
RNA exchange. Normal or mutant 5 S rRNAs were prepared, labeled,
and incubated with purified 5 S RNA-protein complex as described under
"Materials and Methods." Protein-bound and free RNA fractions were
separated on an 8% nondenaturing polyacrylamide gel, visualized by
autoradiography and quantified by scintillation counting. The
nucleotide changes in each RNA are indicated in bold letters
together with the amount of ribonucleoprotein complex expressed as a
percentage of that which was observed with normal 5 S rRNA. Values
represent averages for three to six replicate experiments. The helical
regions also are identified (I-V) together with the RNA-protein
interface as initially determined by ribonuclease protection studies
(dark shading) and extended (light shading) using
modification exclusion (4, 15, 16).
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Fig. 2.
Effect of a 5' end nucleotide extension on
the formation of the 5 S rRNA-protein complex by RNA exchange.
Purified cellular 5 S rRNA (A), in vitro
transcribed 5 S rRNA with an extra guanylic acid residue at the 5' end
(B), and in vitro transcribed normal 5 S rRNA
(Ctl) was labeled and incubated with purified 5 S RNA
protein-complex as described under "Experimental Procedures."
Protein-bound (RNP) and free (RNA) fractions were
separated on an 8% polyacrylamide gel and visualized by
autoradiography.
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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 3' end, in
the course of the additional analyses the 5' 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 5'
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 3' 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 5' end (lanes c and d), again only
trace amounts of ribonucleoprotein were evident, with complex formation
being almost totally inhibited.

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Fig. 3.
Comparative effects of terminal alterations
on the formation of 5 S RNA-protein complexes by RNA exchange.
Normal (Ctl) and mutant 5 S rRNAs (a-d) were
expressed in vitro, labeled, incubated with purified 5 S
RNA-protein complex, and fractionated as described in Fig. 2. The
terminal structure for each RNA is shown below and the positions of the
5 S RNA-protein complexes (RNP) and free RNA
(RNA) are indicated on the right.
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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 5' 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 terminal-modified 5 S RNA molecules also
were subjected to partial cleavage with T1 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.

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Fig. 4.
Partial T1
ribonuclease digests of 5 S rRNAs with altered termini.
Cellular 5 S rRNA (Ctl) and mutant RNA with a 5' end
nucleotide extension and a U121 deletion at the 3' end
(A) or only a U121 deletion at the 3' end
(B) were labeled with cytidine
[3',5'-32P]bisphosphate and partially digested under
physiological-like conditions with 0 (a), 0.4 (b), or 2 (c) units of T1
ribonuclease per mg of RNA. Normal RNA, digested under denaturing
conditions (M) also was included as a nucleotide marker.
Major cleavage sites are identified at the right.
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Processing and Stability of the 5' 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 5' 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 ~15 min, but a
5'-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 5' end. About 50% of this population was degraded after 105 min of
incubation. Abbreviated gel fractionations that would include short
fragments of the 5' end did not reveal any specific processing at the
5' terminal (results not shown).

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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 5' end
were prepared by runoff transcription using T7 RNA
polymerase, labeled at the 5' 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.
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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 3' end (A) was partially degraded, and RNA
with a one nucleotide extension at the 5' 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).

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Fig. 6.
Stability of 5 S rRNAs with altered
termini. Normal 5 S rRNA (Ctl) and RNA with a
nucleotide deletion (U121) at the 3' end (A) or
a deletion at the 3' end and a single guanylic acid residue extension
at the 5' end (B) were prepared by runoff transcription
using T7 RNA polymerase, labeled at the 5' 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.
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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).

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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.
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DISCUSSION |
Past studies on the maturation of the ribosomal 5 S RNA in
S. cerevisiae (25) have indicated that proper termination
coupled 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 evidence
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 5' 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 5' 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 (45-47). The La protein binds principally
via its conserved N-terminal domain (NTP) to the UUUOH
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 3'
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 3' 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 3' end
was much less critical than at the 5' end. Although less strongly
bound, a natural slightly shorter processing variant 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 5' end, also fully caps the 3' 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 5' end extension remains an
intriguing structural feature of this essential ribosomal RNA-protein complex.