Rrb1p is a member of a functionally diverse superfamily of WD-repeat-containing proteins (48
). It is present throughout the nucleus but is concentrated in the nucleolus. The nucleolar concentration of Rrb1p and the fact that patterns of RRB1
gene expression mirror those of the RP genes during diauxic shift (9
) suggested a possible involvement of Rrb1p in ribosome synthesis. To test this, we studied the effects of varying cellular levels of Rrb1p on the levels of ribosomes and ribosomal subunits. We observed that even moderate levels of Rrb1p depletion led to a decrease in the levels of 60S ribosomal subunits, 80S ribosomes, and polysomes (Fig. ). This was accompanied by a significant inhibition in the production of 25S rRNA. In contrast, Rrb1p depletion had little effect on the levels of 40S subunits and these subunits were capable of forming 43S preinitiation complexes (half-mers). Moreover, unlike that of 25S rRNA, the formation of 18S rRNA was unaffected by reduced levels of Rrb1p. Similar results have been documented in a variety of mutants that are defective in 60S subunit formation, including those containing mutations in genes encoding constitutive 60S subunit proteins and factors affecting large-subunit assembly and maturation (26
). On the basis of these results, we conclude that Rrb1p plays a specific role in the assembly of the 60S subunit.
The function of Rrb1p in ribosome biogenesis is likely linked to its physical association with the ribosomal protein rpL3. We showed by immunoprecipitation of Rrb1p from nuclear extracts that it specifically interacts with rpL3, with no other ribosomal proteins being visibly bound to Rrb1p. These results suggest that Rrb1p interacts with free rpL3 at a point prior to its incorporation into ribosomes. This conclusion is further supported by the observation that excess Rrb1p causes a specific accumulation of rpL3 (but not other ribosomal proteins such as rpL4A or rpL25) within the nucleus (Fig. B). At what point following its synthesis rpL3 binds to Rrb1p is unclear. The two proteins could associate with one another either after rpL3 enters the nucleus or in the cytoplasm with the resulting complex being imported into the nucleus. The latter scenario is possible since Rrb1p is capable of shuttling between the nucleus and the cytoplasm (Fig. ). Interestingly, we observed that Rrb1p's steady-state localization to the nucleus is dependent on ongoing protein translation. Both chemical inhibitors (cycloheximide and sodium fluoride) and mutations (prt1-1
) that block translation (19
) caused Rrb1p to be released into the cytoplasm. Upon reinitiation of translation, Rrb1p again concentrated in the nucleus. Moreover, inhibition of the protein synthesis also led to the coordinated transport of a Rrb1p-rpL3 pool (see below) out of the nucleus (Fig. ). Again, these proteins could be reimported into the nucleus after release of translational arrest, suggesting that a Rrb1p-rpL3 complex is capable of being imported into the nucleus. How the function of Rrb1p is linked to protein translation is yet to be investigated. However, the observation that a nucleolar protein's localization is dependent on translation is, to the best of our knowledge, unprecedented.
Consistent with their physical association, we also showed that the overproduction of Rrb1p leads to a disproportionate increase in steady-state levels of rpL3 relative to other ribosomal proteins (Fig. ). These data were surprising in light of numerous previous reports that the stoichiometric relationships between ribosomal proteins are tightly maintained and that excess unassembled ribosomal proteins are quickly degraded, having half-lives between 30 s and 3 min (66
). For example, it was shown that overproduced RPL3
mRNA accumulates in the cell (40
) and is efficiency translated but that excess rpL3 is rapidly degraded (30
). In contrast, in Rrb1p-overproducing cells, both following induction of the GAL1::RRB1
gene and in constitutively overexpressing cells, a surplus of rpL3 was detected (Fig. ). The excess pool of rpL3 may be explained in two ways. First, overexpression of RRB1
stimulates the expression of RPL3
(see below), thus likely increasing the production of rpL3. Second, the overproduced Rrb1p sequesters rpL3 within the nucleus, concentrating it within the nucleolus (Fig. ). Here it could directly protect rpL3 or segregate it from the proteolytic machinery that would normally degrade it. The latter mechanism is not unprecedented, as recent reports show that the sequestration of proteins within the nucleolus can protect them from degradation (50
) and regulate their activity (46
Within the nucleolus, the Rrb1p-rpL3 complex could act as a precursor from which rpL3 is recruited into newly forming preribosomes. Since we have not detected Rrb1p in association with precursor or mature 60S subunits (data not shown), the binding of rpL3 to the 90S precursor is likely accompanied by the dissociation of Rrb1p. Such a mechanism would suggest a role for Rrb1p in the deposition of rpL3 on the 90S precursor. Our observation that Rrb1p depletion decreases the rate of 25S rRNA formation (Fig. ) is consistent with a defect in rpL3 incorporation. Similar phenotypes have been observed when the incorporation of early assembly intermediates, including rpL3, onto pre-rRNA is altered (36
; for a review, see reference 26
). Interestingly, two other yeast proteins that contain WD-repeats have also been suggested to assist in the incorporation of ribosomal proteins onto ribosomal subunits. The cytoplasmic protein Sqt1p may play a role in depositing Qst1p-rpL10 on the 60S subunit during a late assembly step in the cytoplasm (11
). In another example, Rrp7p, a protein presumed to be nuclear, is required for rRNA processing through a mechanism that is proposed to involve the addition of two proteins, rpS27A and rpS27B, to the 43S precursor (6
The coordinated increase in the levels of the rpL3 that accompanied Rrb1p overproduction prompted us to examine the effects of the RRB1
conditional allele on the expression of RPL3
and various other RP genes. A hallmark of RP gene transcription is that, under normal growth conditions as well as under conditions of stress including carbon source changes (20
), heat shock (21
), and alterations in protein secretion (28
), the expression of all RP genes is globally coordinated (9
). The mechanics of this process, however, are not well understood. The majority of RP genes contain upstream sequences that bind the protein Rap1p. Rap1p has been shown to act as a transcriptional activator of RP gene expression, and it plays a necessary role in the repression of transcription induced by amino acid starvation and defects in secretion (33
). A few RP genes, including RPL3
, lack the Rap1p-binding site and instead contain binding sites for the transcription factor Abf1p. Still, their expression is coordinated with the other RP genes under each of the various environmental conditions mentioned above (41
Interestingly, we showed that varying the levels of Rrb1p uncouples the regulation of RPL3 mRNA levels from the coordinated control of other RP mRNAs, potentially through the control of their transcription. The overexpression of RRB1 leads to a robust increase in the levels of rpL3 mRNA, while all of the other RP mRNAs examined remained at or near wild-type levels (Fig. ). In contrast, upon depletion of Rrb1p the levels of RPL3 mRNA appeared unaffected while mRNA levels of all other examined RP genes were increased. This includes RP genes whose promoters contain either Rap1p- or Abf1p-binding sites. Our analysis of the effects of RRB1 expression on the levels of RPL3 and RPL25 mRNAs (Fig. C) showed that the increase induced by overexpression (RPL3) or repression (RPL25) of RRB1 was dependent on the presence of RP's endogenous promoter. Thus, the increases in amounts of RP mRNAs that were detected upon depletion of Rrb1p may reflect an increase in transcription rather than a change in the half-life of these mRNAs. However, the latter possibility has not yet been tested.
The effects of Rrb1p on the levels of RPL3
mRNA may be linked to its physical association with rpL3. One scenario is that the state of Rrb1p, free versus bound to rpL3, would provide a means for Rrb1p to sense ongoing ribosome assembly and adjust RPL3
expression. For example, increased levels of free Rrb1p caused by a decrease in rpL3 could stimulate the expression of RPL3
. Rrb1p also appears to play a more global function in ribosome biogenesis by, directly or indirectly, suppressing the expression of other RP genes. How Rrb1p can act as a transcriptional activator in the context of the RPL3
gene and a repressor for other RP genes remains to be investigated. Of note, the surge in the expression of RP genes observed upon depletion of Rrb1p is similar to that previously reported in mutants expressing truncations of Rap1p, which lack domains implicated in transcriptional silencing or activation (18
). In both cases, the levels of RP mRNA significantly exceeded normal cellular levels detected in wild-type cells grown under the same conditions. This phenomenon is striking since normal levels of RP gene transcription already account for ~30% of RNA polymerase II-mediated transcription. The simplest explanation is that normal levels of transcription do not represent maximal levels.
While the functional links between the effects of Rrb1p and Rap1p are unclear, it is of interest that Rap1p appears to be functionally linked to a chromatin assembly complex that contains Msi1p (15
), a yeast protein exhibiting a high degree of sequence similarity to Rrb1p (data not shown). Both by its association with the chromatin assembly complex and, on a broader scale, as a consequence of its multiple effects on transcription regulation, Rap1p has been suggested to play a general role in chromatin remodeling (35
). If Rrb1p, like Msi1p, is functionally linked to Rap1p, it could function to modulate Rap1p activity at specific loci such as the RP genes.
Our results clearly indicate that Rrb1p plays a role both in the assembly of 60S ribosomal subunits and in the transcription of RP genes. Rrb1p is therefore positioned to link these two events and coordinate their activities. Moreover, the differential effects of Rrb1p on the expression of RPL3 as compared to other RP genes suggest that the coordinated regulation of RP gene expression is likely the result of independent, yet intertwined, regulatory pathways that together maintain similar levels of expression for all RP genes.