(Received for publication, January 2, 1996; and in revised form, February 6, 1996)
From the
Ribosomal protein L5, a 34-kDa large ribosomal subunit protein, binds to 5 S rRNA and has been implicated in the intracellular transport of 5 S rRNA. By immunofluorescence microscopy, L5 is detected mostly in the nucleolus with a fainter signal in the nucleoplasm, and it is known to also be a component of large ribosomal subunits in the cytoplasm. 5 S rRNA is transcribed in the nucleoplasm, and L5 is thought to play an important role in delivering 5 S rRNA to the nucleolus. Using RNA-binding assays and transfection experiments, we have delineated the domains within L5 that confer its 5 S rRNA binding activity and that localize it to the nucleolus. We found that the amino-terminal 93 amino acids are necessary and sufficient to bind 5 S rRNA in vitro, while the carboxyl-terminal half of the protein, comprising amino acids 151-296, serves to localize the protein to the nucleolus. L5, therefore, has a modular domain structure reminiscent of other RNA transport proteins where one region of the molecule serves to bind RNA while another determines subcellular localization.
Assembly of ribosomal subunits takes place in eukaryotic cell nucleoli and involves the coordination of several events prior to nuclear export of the mature subunit to the cytoplasm. These events include RNA polymerase I transcription and subsequent processing of 18, 28, and 5.8 S rRNAs, which occurs in nucleoli, the nuclear import, and nucleolar concentration of roughly 30 small subunit and 40 large subunit ribosomal proteins, as well as transcription by RNA polymerase III and nucleolar accumulation of 5 S rRNA (reviewed by Franke(1988), Gerbi et al.(1990), Warner(1990), Sollner-Webb and Mougey (1991), Scheer and Weisenberger(1994), and Melese and Xue(1995)). The components of nascent ribosomal subunits therefore originate in at least three different cellular compartments: the cytoplasm (ribosomal proteins), the nucleoplasm (5 S rRNA), and the nucleolus (18, 28, and 5.8 S rRNAs). This requires complex intracellular trafficking in order to ensure that all of the subunit components, in the proper stoichiometry, are present in the nucleoli so that efficient ribosome subunit assembly can occur.
One portion of this process, the
biogenesis of 5 S rRNA, is becoming better understood at the level of
mechanistic detail. In human somatic cells, transcription of 5 S rRNA
occurs mostly on genes clustered in repeats on the telomeric region of
the long arm of chromosome 1 (q42-q43, Steffensen et
al., 1974; Little and Braaten, 1989). Immediately after
transcription, 5 S rRNA is transiently associated with the La protein,
which functions in transcription termination of all polymerase III
transcripts (Gottlieb and Steitz, 1989). After association with La, 5 S
rRNA is bound by ribosomal protein L5 to form an RNP ()that
can be recognized by specific autoantibodies (Steitz et al.,
1988). Pulse-chase labeling, followed by immunoprecipitation
experiments with these autoantibodies, has demonstrated that the
L5-5 S RNP forms prior to, and is therefore a likely precursor
to, ribosome assembly. An intranuclear trafficking pathway has been
proposed whereby the L5-5 S RNP forms in the nucleoplasm and then
migrates to the nucleoli to participate in large ribosomal subunit
assembly, and a putative function of delivering 5 S rRNA to the
nucleolus was therefore assigned to L5 (Steitz et al., 1988).
The 5 S rRNA biogenesis pathway has been more extensively studied in Xenopus oocytes. In this system, because of the extraordinary
demands for ribosome production in the developing egg, the pathway is
far more complex. In previtellogenic oocytes, oocyte-type 5 S rRNA is
transcribed in large quantities prior to the production of other
ribosomal components and therefore is immediately exported to the
cytoplasm. While in the cytoplasm, it is complexed in one of two
different storage particles: the 7 S particle, which has transcription
factor IIIA (TFIIIA) as a protein component; or the 42 S particle,
which contains 5 S rRNA, tRNAs and other proteins (reviewed by Tafuri
and Wolffe(1993)). After synthesis of ribosomal proteins begins, during
vitellogenesis, 5 S rRNA is exchanged from the storage particles onto
L5 (Allison et al., 1991, 1993). The L5-5 S RNP migrates
back into the nucleus and then to the nucleoli where subunit assembly
occurs. Although this additional cytoplasmic phase of the 5 S rRNA
biosynthetic pathway is probably unique to oocytes and does not occur
in somatic cells (Allison et al., 1995), it is clear from
studies in both systems that L5 plays a significant role in the
intracellular trafficking of 5 S rRNA.
As a step toward a more detailed understanding of the 5 S rRNA transport pathway, we were interested in the sequences within L5 that mediate its transport properties. In this report we delineate the domain in L5 that confers its ability to bind 5 S rRNA as well as the region that allows the protein to accumulate in the nucleolus, and find that these domains are separable. Additionally, we find that L5 mutants that maintain 5 S rRNA binding activity cannot localize to the nucleolus if they lack the carboxyl-terminal half of the protein, indicating that 5 S rRNA binding is neither necessary nor sufficient for nucleolar targeting. These results therefore strengthen the idea that L5 functions in part to target 5 S rRNA to the nucleolus.
To initiate our studies on the domain structure of L5, we
wanted to determine the sequences that contribute to its 5 S rRNA
binding activity. To do so, we used a rat L5 cDNA clone (Chan et
al., 1987), kindly provided by Dr. Ira Wool, to study binding to
human 5 S rRNA using a protein-RNA binding assay that is similar to
other published procedures (Boelens et al., 1993; Ashley et al., 1993). Briefly, a human 5 S rRNA gene (a kind gift of
Dr. Jim Sylvester) was cloned downstream of a T7 promoter to allow
production of the RNA by transcription in vitro. Biotin-UTP
was included in the transcription reaction to produce biotinylated 5 S
rRNA, which was then incubated in binding buffer with nonspecific
competitor RNA and S-labeled L5 protein derivatives made
by in vitro translation in reticulocyte lysate. The RNP
complexes where then selected by incubation with streptavidin coupled
to agarose beads followed by washing and bound proteins were analyzed
by SDS-PAGE. Under these assay conditions the full-length rat L5
protein bound tightly to 5 S rRNA (Fig. 1, lane 11),
whereas another RNA-binding protein, the human small nuclear RNP U1 A
protein, had no detectable binding activity (Fig. 1, lane
20), demonstrating the specificity of this assay. We next examined
the 5 S rRNA-binding capacity of a set of rat L5 deletion mutants and
found that deletion mutants that retained the amino-terminal half of
the protein maintained binding activity, while mutants lacking these
sequences failed to bind. The smallest fragment that retained binding
activity corresponds to amino acids 1-93 (Fig. 1, lane
15), while another fragment, comprising amino acids 101-296 (Fig. 1, lane 14), did not bind, indicating that the
first 100 amino acids of the L5 protein are both necessary and
sufficient for 5 S rRNA binding activity. Interestingly, the deletion
mutant 51-296 (Fig. 1, lane 12) also contains
binding activity, indicating the L5 RNA-binding domain may be
delineated even further within the first 100 amino acids.
Figure 1:
Delineation of
the 5 S rRNA-binding domain of L5. A, total S-labeled translation products of reactions programmed
with rat L5 (lane 1), rat L5 deletion mutants (lanes
2-8, named according to the L5 sequences encoded by that
particular plasmid), S. pombe L5 (lane 9), and human
U1-specific small nuclear RNP A protein (lane 10). Proteins
were fractionated on SDS-PAGE gels, and the gels were subsequently
fluorographed to enhance visualization of the proteins. B,
results of 5 S rRNA-binding reactions with the proteins displayed in A.
As an additional means to gain insight into the particular amino acids within L5 that contribute to 5 S rRNA binding activity, we cloned and sequenced the gene encoding L5 from the lower eukaryote S. pombe. An alignment between fission yeast L5 and the rat counterpart is presented in Fig. 2. The two proteins are 46% identical and 70% similar when conservative amino acid substitutions are considered. These numbers are consistent with the level of conservation between the fission yeast protein and the L5 homologues from chicken, frog, budding yeast, and rice (Kenmochi et al., 1991; Wormington, 1989; Tang and Nazar, 1991; Kim and Wu, 1993). When we tested the S. pombe L5 protein for binding, we found that, despite being only 46% conserved relative to the rat protein, the fission yeast L5 binds to human 5 S rRNA (Fig. 1, lane 19).
Figure 2: Amino acid alignment of the S. pombe and rat L5 proteins. Identical amino acids are highlighted in black boxes.
In order to understand the relationship between 5 S rRNA
binding and the intracellular localization of the rat L5 protein, we
transfected HeLa cells with epitope-tagged L5 derivatives and
determined their localization by immunofluorescence microscopy.
Full-length L5 protein localizes predominantly to the nucleoli and also
exhibits a fainter nucleoplasmic staining pattern (Fig. 3). This
staining pattern is in good agreement with that observed using an
antiserum that recognizes the L5-5 S RNP particle (Steitz et
al., 1988), suggesting that uncomplexed L5, if it exists in
sufficient quantity to be detected, co-localizes with the RNA-bound
form. We next examined two deletion mutants, L5 1-150 and L5
151-296 (Fig. 3). Interestingly, we found that the
1-150 fragment, which binds 5 S rRNA in vitro, can enter
the nucleus but does not localize to the nucleolus. Conversely, the
151-296 fragment localizes to both the nucleus and nucleolus with
wild type efficiency yet cannot bind 5 S rRNA. These results
demonstrate that the 5 S rRNA-binding domain is not required for the
nuclear import or intranuclear transport of the L5 protein and that the
signal(s) which mediates nucleolar localization resides in the
carboxyl-terminal 150 amino acids. In order to further delineate the
sequences within amino acids 151-296 that confer nucleolar
localization, we wanted to first transfer the region onto a
heterologous, non-nuclear/nucleolar protein and confer nucleolar
localization onto that fusion protein. Unfortunately, repeated attempts
with either full-length L5 or L5 151-296 fused to a number of
different reporters, including the bacterial proteins
-galactosidase and maltose-binding protein as well as chicken
pyruvate kinase, resulted in a nuclear, but not nucleolar localization
of these proteins (data not shown). The reason for this is unknown, but
the inability to transfer a nucleolar localization sequence onto a
reporter protein has been previously noted for other nucleolar proteins
(Peculis and Gall, 1992; Schmidt-Zachmann and Nigg, 1993).
Interestingly, in the case of the
-galactosidase and
maltose-binding protein full-length L5 fusion proteins, the ability to
bind 5 S rRNA in vitro is maintained (data not shown), whereas
the nucleolar localization properties are not, which further supports
the conclusion that 5 S rRNA binding activity alone is insufficient for
nucleolar localization.
Figure 3: Intracellular localization of rat L5 (L5) and deletion mutants comprising amino acids 1-150 (L5 1-150) and 151-296 (L5-151-296). Plasmids encoding these proteins were transfected into HeLa cells. Forty h post-transfection, the cells were fixed and processed for immunostaining with monoclonal antibody 9E10, which recognizes the Myc tag (panel 9E10). The phase contrast image is depicted in panel (Phase).
Figure 4: Amino acid alignment of the defined 5 S rRNA-binding domain of rat L5 to the corresponding regions in S. pombe (yeast) and rice (rice) L5 proteins and the archebacterial M. vannielii L18 protein (ArBa). The residues which are absolutely conserved or highly similar between all four proteins are indicated beneath the alignment in bold lettering. The conserved proline at rat L5 position 57 is boxed in the consensus, and the regions within the domain flanking the proline are designated I and II.
The idea that nucleolar proteins require specific targeting signals to localize to the nucleolus has been challenged recently due to the lack of a clear consensus motif within the several proteins for which this type of signal has been delineated. Rather, it appears plausible that nucleolar localization is a retention-driven process whereby proteins accumulate within nucleoli by virtue of binding to nucleolar components (see Yan and Melese(1993)). For instance, in the case of the abundant nucleolar protein nucleolin, the domains that are necessary for nucleolar accumulation map to the RNA-binding domains of the protein, which indicates that interaction with pre-rRNA is the driving force for nucleolar localization of this protein (Schmidt-Zachmann and Nigg, 1993). Our results with L5 demonstrate that the carboxyl half of the protein contains the sequences required for nucleolar localization, that 5 S rRNA binding activity is neither necessary nor sufficient for L5 nucleolar localization and consequently that 5 S rRNA does not contain the information required to properly localize the L5-5 S RNP to the nucleolus. Therefore, if the retention-driven model is correct, 5 S rRNA does not act as the anchor for L5 nucleolar localization. Additionally, nascent large ribosomal subunits seem to be unlikely candidates as the L5-5 S RNP is present in large excess over nascent subunits within nucleoli (Phillips and McConkey, 1976). One possibility is that the L5-5 S RNP first localizes to the nucleolus by virtue of an interaction between the carboxyl terminus of L5 and some as yet unknown nucleolar component and then serves to nucleate large subunit assembly. This would help explain why the L5-5 S RNP is present in excess over other ribosomal proteins within nucleoli.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U48270[GenBank].