A Chaperone for Ribosome Maturation*
Atanas I.
Lalev and
Ross N.
Nazar
From the Department of Molecular Biology and Genetics, University
of Guelph, Guelph, Ontario N1G 2W1, Canada
Received for publication, February 6, 2001
 |
ABSTRACT |
The nascent pre-rRNA of eukaryotic ribosomes is
fully transcribed and assembled into an 80-90 S nucleolar particle
before being cleaved into mature ribosomal RNA. The interdependence of steps in the processing of this precursor RNA indicates that RNA processing, at least in part, acts as a quality control mechanism that
helps ensure that only functional RNA is incorporated into mature ribosomes. In search of structural components that underlie this
interdependence using the Schizosaccharomyces pombe
internal transcribed spacer 1 (ITS) as a ligand for affinity
chromatography of ITS1-specific proteins, we have isolated a large
spliceosome-like protein complex, a ribosome assembly chaperone (RAC)
of 20 or more polypeptides (Lalev, A. I., Abeyrathne, P. D.,
and Nazar, R. N. (2000) J. Mol. Biol. 302, 65-77).
When the ITS2 spacer was used in the present study to isolate
ITS2-specific proteins, the same proteins were identified
consistent with a complex containing multiple specific binding
sites. Subsequent competition binding studies indicated that the
protein complex actually contains independent binding sites for all
four of the transcribed spacers in the pre-rRNA. Because disruption of
protein-binding sites in these spacer RNAs is known to severely affect
rRNA processing, taken together these results suggest that the RAC
complex is a chaperone for ribosome maturation acting as a "rack"
on which critical structure is organized.
 |
INTRODUCTION |
The introns of eukaryotic mRNA precursors interact with
snRNAs1 and many proteins to
form a large "spliceosome" complex (1) that catalyzes both
the excision of the intron and the splicing of the remaining RNA to
produce the mature coding sequence (2). Ribosomal RNA transcripts also
represent large precursor molecules from which transcribed spacers are
excised to form the RNA components of the mature ribosomal subunits.
Although RNA splicing is not necessary in this process, several snoRNAs
also have been implicated in the cleavage of the pre-rRNA (3) and
a U3 snoRNA·5'-ETS complex has been postulated to
be essential for the initiation of rRNA processing (4).
Initially, studies of rRNA processing reported a split processing
scheme for the independent maturation of the large and small subunit
RNAs (5). Indeed, some mature rRNA is observed when the sequences are
not linked genetically (6). More recent studies, however, have begun to
demonstrate interdependences that dramatically affect the
efficiency of maturation and/or the stability of the products. For
example, an extended hairpin structure in the 3'-ETS has been shown to
be critical not only to the processing of the 3'-end in the large
subunit rRNA but also to the maturation of the 5.8 S RNA some 3000 nucleotides upstream of it (7). In addition, the deletion of the ITS2
sequence has been shown to be critical to the maturation of the large
subunit but also to have severe effects on the level of 18 S rRNA
production (8), and as already indicated, a U3
snoRNA·5'-ETS complex appears to be essential for the initiation of
pre-rRNA processing overall. Because the pre-rRNA is fully transcribed
and assembled into a large 80-90 S nucleolar precursor particle before
rRNA processing is initiated (9), these observations pose intriguing
questions about the nature and role of the nucleolar precursor
ribonucleoprotein particle. Apparently, at least in part, these
processes contribute to a quality control mechanism that helps ensure
that only functional RNA is incorporated into mature ribosomal
particles (8).
In search of structural features that underlie the interdependences as
well as possible roles of the transcribed spacers in rRNA maturation,
we initially looked for interactions between the spacer regions and
soluble cellular constituents (10, 11). Using gel retardation studies
we were able to demonstrate specific interactions between individual
transcribed spacer sequences and cellular proteins (10), and using ITS1
RNA we were able to isolate a complex of proteins that interacted
specifically with this spacer sequence (12). This complex, a ribosome
assembly chaperone (RAC), contained at least 20 proteins ranging in
size from 20 to 200 kDa. Mass spectroscopy and computer analyses
suggested that many of the proteins contained RNA binding motifs or
were nuclear in localization. Additional RNA components were not
evident. In the present study, proteins interacting with the ITS2
region also were isolated for further characterization. Subsequent
comparative analyses now indicate that a single protein complex has
independent binding sites for all the transcribed spacers in the
pre-rRNA.
 |
EXPERIMENTAL PROCEDURES |
Preparation of ITS RNA--
Schizosaccharomyces pombe
ITS1 and ITS2 RNA for ribonucleoprotein formation and affinity
chromatography were prepared by transcription using
T7RNA polymerase (13) as previously described (11,
12). For labeled molecules, the RNAs were labeled at the 5'-end using bacteriophage T4 polynucleotide kinase and
[
-32P]ATP after dephosphorylation with calf intestinal
alkaline phosphatase. All RNAs were purified on 6% denaturing
polyacrylamide gels.
Affinity Chromatography--
RNA binding proteins were purified
by affinity chromatography using ITS RNA bound to a poly(C)-agarose
support (Sigma) as recently described (12, 14). The RNA ligand was
immobilized on the column matrix using a poly(G) sequence at the 3'-end
which was initially inserted into the DNA template sequence. Protein was extracted from logarithmically growing S. pombe,
strain h
leu 1-32 ura 4-D18 and applied at a
concentration of 10 mg/ml in chromatography buffer (5 mM
MgCl2, 0.3 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol and 10 mM Tris-HCl, pH 7.5)
containing 5% glycerol, 0.01% Triton X-100 and 5-10 mg of unrelated
calf liver rRNA. The column was washed with chromatography buffer
containing 0.133 M KCl and then eluted in two steps using 1 ml of buffer containing either 0.3 M or 1 M KCl.
Electrophoretic Mobility Shift Assay--
Ribonucleoprotein
complexes were assayed by gel retardation as previously described (10,
11). Aliquots of in vitro transcribed and labeled spacer RNA
(0.3-2 ng/20,000-25,000 cpm were incubated with 5 µl of
protein extract in binding buffer (100 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol,
and 12 mM Tris-HCl, pH 8.0) containing 8% glycerol and
0.25 mg/ml calf liver rRNA to eliminate nonspecific interactions. For
competition analyses, ~0.3 ng of RNA was incubated with RAC protein
on ice for 10 min in 20 µl of buffer; the protein concentration was
adjusted to be limiting, allowing about 75-80% of the labeled RNA to
be incorporated into a ribonucleoprotein complex. For competition
experiments with unlabeled RNA, a 1-300-fold excess (0.03-0.1 µg)
was added to the incubation mixture. Complexes were fractionated from
free RNAs on 2% (w/v) agarose gels at 4 °C.
Electrophoretic Analysis of Protein Constituents--
For
analyses of column eluates, the proteins were fractionated directly on
10% SDS-polyacrylamide gels using a 5% stacking gel as described by
Laemmli (23). The bands were visualized using silver stain as
described by Merrill and co-workers (24). For ribonucleoprotein
constituents, sufficient protein was added to convert essentially all
the RNA (1-2 µg) into ribonucleoprotein. The complex was purified on
a 5% polyacrylamide gel using labeled RNA to identify its position,
and the excised band was polymerized in place of the normal 5%
stacking layer of the 10% SDS-polyacrylamide protein analysis gel,
described above. In some analyses, markers also were applied to gels to
estimate the molecular weights.
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RESULTS AND DISCUSSION |
In a previous study (11), gel retardation analyses indicated that
the extended hairpin in the S. pombe ITS2
sequence interacted specifically with one or more soluble proteins. In
the present study, the ITS2 region of the S. pombe pre-rRNA
was synthesized using T7 RNA polymerase (13) and used as a
ligand in affinity chromatography (12) to isolate and further
characterize the ITS2-specific spacer binding proteins from a cellular
extract. The ITS2 sequence first was fused to a polyguanylic acid
cluster that permitted the preparation of RNA with a poly(G) tail,
which subsequently was used to immobilize the RNA on a poly(C)-agarose matrix (12). Protein was applied in low salt buffer (14) with calf
liver RNA to eliminate nonspecific binding. After an extensive wash
with 0.133 M KCl, the bound protein was eluted sequentially with buffer containing 0.3 and 1.0 M KCl. As shown in Fig.
1 (left), the 0.3 M fraction contained the majority of protein that
effectively formed a stable ribonucleoprotein complex with ITS2 RNA
(+). When this band was excised and polymerized into the stacking layer of a standard SDS-acrylamide protein analysis gel and the protein components were electrophoretically fractionated, a diverse group of
proteins was evident (Fig. 1, right). Despite the
surprisingly large number of proteins, most bands were present in
similar amounts, consistent with a homogeneous protein complex
(RAC).

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Fig. 1.
Protein constituents of gel-purified
ribonucleoprotein. A, S. pombe ITS2 RNA,
transcribed in vitro and labeled at the 5'-end ( ), was
incubated with affinity-purified protein (0.3 M fraction)
and unrelated carrier RNA (+) before fractionation on a 6%
nondenaturing polyacrylamide gel (left). The
ribonucleoprotein (RNP) band (indicated on the
left) was excised and applied to a 10% SDS/polyacrylamide
gel (right) together with the initial cellular protein
extract (Protein) for further electrophoretic fractionation.
The proteins were visualized by silver stain. The positions of
molecular weight standards, as separately fractionated with the initial
cellular protein extract, are indicated at the left and were
used to estimate the molecular weight of each band as indicated in
parentheses.
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Because an equally large number of proteins also was isolated in the
earlier study using ITS1 RNA (12), the ITS1- and ITS2-associated proteins were compared directly. In each case the fraction containing the majority of the spacer binding protein was used, the 1.0 M fraction for ITS1 and the 0.3 M fraction for
ITS2. As shown in Fig. 2, despite
differences in the RNA ligands and in the elution conditions, the
protein components that formed a ribonucleoprotein complex were found
to be largely or completely identical. The fact that the same protein
complex was eluted at different salt concentrations (1.0 versus 0.3 M KCl) raised the possibility that each ITS region interacted with a separate binding site in the protein
complex.

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Fig. 2.
Comparison of RAC complex proteins purified
with alternate ITS sequence ligands. Ribonucleoprotein was
prepared with labeled S. pombe ITS1 and ITS2 RNA, and the
protein constituents were fractionated on an SDS-polyacrylamide gel as
described in Fig. 1.
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Earlier studies already had suggested limited sequence equivalence in
the ITS sequences, but some competition for protein was evident when
large excesses of competing RNA were present (10, 11). This raised the
alternate possibility that the two internal spacers competed for a
common binding site, a possibility that also is consistent with the
similarities in the protein components (Fig. 2). To evaluate these
possibilities further, a complex formation was examined under more
competitive conditions. As illustrated in Fig.
3A (lane b) with
ITS2 RNA, in these studies the amount of affinity-purified protein was
limited to allow ~80% of the labeled RNA to be incorporated into a
ribonucleoprotein complex. As also shown in Fig. 3A, when an
equal amount of ITS1 RNA was included (lane c),
the amount of ITS2 RNA in the complex was not reduced and ~80% of
the ITS1 molecules were incorporated into the ribonucleoprotein complex
as well (lane c). This observation indicated that
the two sequences were not directly competing for the same site;
rather, they were equally bound in two separate sites. Such a model was
further supported when an equal amount of ITS2 sequence was added
instead of ITS1. As shown in Fig. 3B, under these conditions
ITS1 was efficiently incorporated into the complex (lane
c), but further ITS2 RNA was not incorporated (lane d) and ~60% of the total or all of the
extra ITS2 RNA remained unbound.

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Fig. 3.
Distinct ITS binding sites in the S. pombe RAC complex. A, in vitro
synthesized and labeled ITS2 RNA (a) was incubated with
affinity-purified protein (RAC) in the absence (b) or
presence (c) of an equal amount of labeled ITS1 RNA
(d). B, in vitro synthesized and
labeled ITS2 RNA (a) was incubated with affinity-purified
protein (RAC) in the absence (b) or presence (c)
of an equal amount of labeled ITS1 RNA or an equal amount (total = 2 ×) of labeled ITS2 RNA (d). Ribonucleoprotein
(RNP) formation was assayed by agarose gel
electrophoresis. Equal amounts of ITS1 and ITS2 RNA are bound in two
distinct sites (c); extra ITS1 is not bound in the ITS2 site
(B, lane d).
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Previous studies on the 3'-ETS region in the S. pombe
pre-rRNA based on gel retardation analyses have indicated that a
conserved, extended hairpin structure in this transcribed spacer also
forms a stable ribonucleoprotein complex with cellular protein, a
complex that has been experimentally linked with rRNA maturation (15). Studies on the 5'-ETS (16) again have identified an extended hairpin
structure that is critical to rRNA maturation. Competition studies
between the 3'-ETS and either unlabeled internal transcribed spacer did
not show a relationship in protein-binding sites, although a limited
sequence homology appeared present and raised the possibility of a
structural equivalence (11). To explore the possibility that the RAC
complex also contained binding sites for the external spacers,
ribonucleoprotein formation again was examined using the
affinity-purified protein. As shown in Fig.
4A, the results with the
3'-ETS were strikingly similar to those observed with the two internal
spacers (Fig. 2). The 3'-ETS-derived RNA (lane e)
was able to form a stable complex (lane d). More
importantly, in the presence of ITS1-derived RNA (lane
c) both RNAs were incorporated equally into the complex
whereas the amount of complex-bound 3'-ETS remained constant. This
observation again supported comparable but distinct binding sites for
both spacer regions. As shown in Fig. 4B, the 5'-ETS-derived
RNA also formed a stable ribonucleoprotein complex (lane
d) with ITS-purified protein. Again, in the presence of
ITS1-derived RNA (lane c) both RNAs were equally
incorporated and no 5'-ETS was displaced.

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Fig. 4.
Distinct ETS binding sites in the S. pombe RAC complex. A, in vitro
synthesized and labeled ITS1 (a) or 3'-ETS (e)
RNA was incubated with affinity-purified protein (RAC) individually
(b and d, respectively) or together
(c). B, in vitro synthesized and
labeled ITS1 (a) or 5'-ETS (e) RNA was incubated
with affinity-purified protein (RAC) individually (b and
d, respectively) or together (c).
Ribonucleoprotein (RNP) formation was assayed by agarose gel
electrophoresis. The protein binds an equal amount of each spacer in
distinct sites.
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The specific nature of this interaction was further demonstrated when
complex formation was reexamined in the presence of excess unlabeled
spacer RNA (Fig. 5). When ITS1 binding
was compared with the 5'-ETS (Fig. 5A) in the presence of
unlabeled ITS1-derived RNA (lane d), only labeled
ITS1-derived RNA was displaced and in the presence of unlabeled
5'-ETS-derived RNA (lane e), only labeled 5'-ETS-derived RNA
was displaced. These observations again were entirely consistent with
separate and specific spacer binding sites. Similarly, when ITS1
binding was compared with ITS2 (Fig. 5B), equivalent results
were obtained. In the presence of unlabeled ITS1-derived RNA
(lane c), only labeled ITS1 RNA was displaced and
in the presence of unlabeled ITS2-derived RNA (lane
d), only labeled ITS2 RNA was displaced. Once more these
results strongly supported separate and specific binding sites.

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Fig. 5.
Spacer-specific binding sites in the S. pombe RAC complex. A, in vitro
synthesized and labeled ITS1 (a) and 5'-ETS (b)
RNA were incubated together with affinity-purified protein (RAC)
individually (b and d, respectively) or together
(c-e) in the presence of a 100-fold excess of
unlabeled ITS1 (d) or 5'-ETS (e) RNA.
B, in vitro synthesized and labeled ITS1 and ITS2
RNAs (a) were incubated together with affinity-purified
protein (RAC) (c and d) in the presence of a
100-fold excess of unlabeled ITS1 (c) or ITS2 (d)
RNA. Excess unlabeled RNA displaces only equivalent labeled RNA.
RNP, ribonucleoprotein.
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Taken together, the results of this study provided experimental
evidence for a model of rRNA maturation (Fig.
6) that we first suggested to
explain distant effects in rRNA processing (17). The model proposes
that as ribosomal proteins assemble on the mature ribosomal RNA
sequences, nucleolar constituents simultaneously assemble on the spacer
regions basically forming a nucleolar pre-rRNA particle consisting of
three domains; two of those domains correspond with the ribosomal
subunits and one domain is composed of spacers and nucleolar proteins
or RNAs, comprising a common processing domain. The RAC complex
described in this study would represent the core of this domain with
spacer sequences and transacting factors appropriately organized on
this core. Normally this particle fully forms before rRNA cleavages are
initiated (9) and appears to be essential for an efficient maturation
of the ribosomal RNA (7, 9, 15, 16). Our past studies on the RNA spacer
sequences are entirely consistent with such a model. For example,
mutations in the known protein-binding site of the 3'-ETS (15) or ITS1 RNA (12) also have been shown to dramatically affect RNA maturation with comparable effects on protein binding. Furthermore, mutations in
the protein-binding regions in the ITS2 (11) and 5'-ETS (16) sequences
have been shown to critically affect RNA maturation, and equivalent
regions in the internal spacers of the preribosomal RNA of
Saccharomyces cerevisiae also have been shown to be
essential for rRNA processing (18).

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Fig. 6.
A model for the assembly of a nucleolar
80-90 S preribosomal particle. Ribosomal proteins assemble on the
rRNA sequences to form the ribosomal subunits whereas the nucleolar RAC
proteins assemble on the spacer sequences to form a common processing
domain that is acted on by nucleases and other nucleolar factors or
RNAs.
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As noted earlier, in many ways the particle described in this study
resembles the spliceosomal complexes associated with mRNA processing. As in the case of spliceosomes, the roles of individual proteins in the present complex remain unclear. Our attempts to demonstrate specific cleavages with the affinity-purified RAC protein
have failed (results not shown), but various alternate enzymatic
activities associated with rRNA maturation previously have been
reported (19). These observations suggest that the affinity-purified
protein acts not to cleave the RNA but to organize it in a way that
allows its precise and efficient cleavage by other peptides such as the
RNase III-like enzymes (20, 21), the Rrp4p protein, and the related
"exosome" (22). In this respect the new complex would act as
a ribosome assembly chaperone or a kind of "rack" on which the
maturing structures are organized. The protein complex may even act by
sterically hindering the processing enzymes, allowing complexes such as
the "exosome" to act only in a limited and defined fashion.
Whatever the case, the new particle (RAC) raises a very different
picture of ribosome maturation and provides an explanation for the
previously described distant interdependences in rRNA maturation that,
at least in part, serve as a "quality control" mechanism for
ribosome biogenesis (8, 17).
 |
FOOTNOTES |
*
This study was supported by the Natural Sciences and
Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Axelrod Bldg.,
University of Guelph, Guelph, Ontario N1G2W1, Canada. Tel.:
519-824-4120 (ext. 3004); Fax: 519-837-2075; E-mail:
rnnazar@UoGuelph.CA.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M101157200
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ABBREVIATIONS |
The abbreviations used are:
snRNA, small nuclear
RnA;
snoRNA, small nucleolar RNA;
ETS, external transcribed spacer;
ITS, internal transcribed spacer;
RAC, ribosome assembly chaperone..
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.