The results show that Rvb2p has an essential role in snoRNP biogenesis and that this function most likely occurs at an early stage and in the nucleoplasm. Depletion or inactivation of the protein results in loss of both major classes of snoRNAs and impaired localization of core snoRNP proteins. Processing of rRNA is impaired as well, and these effects could also reflect loss of snoRNAs, since a few snoRNAs in each major family are needed for rRNA processing (35
). Two factors point to a nucleoplasmic role for Rvb2p. First, it is clear that the vast majority and possibly all of the protein is in the nucleoplasm (Fig. ) (40, 46). Second, results from a variety of other studies unrelated to snoRNAs have linked Rvb2p and its vertebrate counterparts with nucleoplasmic components (see below). On this basis, it seems most likely that Rvb2p functions in the nucleoplasm, although the possibility that it has a nucleolar phase is still formally open.
Several clues about the role of Rvb2p in snoRNA production are provided by studies of Rvb1p and Rvb2p orthologs (p50 and p55) in other organisms and in other contexts. The most definitive information available is that both proteins have been demonstrated to have DNA helicase activity in vitro (see below). Based on interactions with proteins of known function, the p50 and p55 proteins have been suggested to participate, variously, in transcription, chromatin remodeling, DNA recombination, signal transduction, and the cell cycle (9
). The potential for overlap among these processes is high, of course. It will be interesting to learn if some or all of the effects observed, including those described here, are linked to a single biochemical process or rather reflect similar DNA- or RNA-based roles in multiple processes. The key findings from these studies are discussed in the following sections, with a view to identifying common links and clues about snoRNA accumulation.
The earliest ties to transcription are based on observations that rat p55 was in immunoprecipitates of TBP prepared from nuclear extracts (32
) and, in a separate study, copurified with the RNA polymerase II (Pol II) holoenzyme through several chromatographic steps and density gradient centrifugation (55
). The interaction with the Pol II complex appears to be of high affinity. However, association of p55 with TBP and Pol II has not been determined to occur in the cell, and these effects may not extend to p50.
More recently, p50 and p55 have been implicated in transcription on other grounds. In a yeast study, Rvb2p (p50) was shown to be required for accumulation of mRNAs that specify ribosomal proteins (40
); in the present study we show that Rvb2p depletion affects snoRNAs before r-protein production. In rat cells,
p50 and p55 have been determined to influence oncogenic transformation in response to transcription factors c-Myc and H-Ras (82
). In the context of the present study, c-Myc may have limited relevance, since p50 is highly conserved and Myc-related factors are not. In other studies, human p50 and p55 were shown to interact biochemically with β-catenin and TBP and to serve as antagonistic regulators for activation of the Wnt signaling pathway (9
). All of these effects could be manifest at the level of transcription, but effects on downstream processes are also possible.
Intriguing new results appear to place the p50 and p55 proteins in position to influence transcription by remodeling chromatin. In one study Rvb2p and Rvb1p were both found to be present in a megadalton complex affinity purified from yeast using an epitope-tagged variant of chromatin-remodeling factor INO80 (61
). The complex contained 3′→5′ helicase activity and stimulated transcription from chromatin. In another study, human p50 and p55 were both identified in a complex isolated through binding to histone acetylase TIP60 (28
). The TIP60 complex acetylates nucleosomes in vitro and has ATPase and DNA helicase activities as well. Interestingly, the yeast and human complexes contain several common protein homologs (including p50 and p55) and are probably functionally equivalent in the two species. Notably, both Rvb2p and Rvb1p are essential for growth in yeast while INO80 is not.
More recently, a different affinity enrichment procedure was used to isolate a yeast complex containing Rvb1p and Rvb2p, and chromatin-remodeling activity was once again demonstrated (30
). In that study, it was shown that the two proteins are responsible for activation or repression of a subset of yeast genes that comprise up to 5% of the genome (30
). It would be interesting to learn the basis of these effects and the physiological significance.
It has been proposed that the p50 and p55 orthologs are involved in other aspects of DNA function as well. Based on modest sequence similarity with the bacterial RuvB protein, which participates in homologous recombination and repair of double-stranded breaks, it was suggested that p55 has a similar function(s) in eukaryotic cells (55
). That case is somewhat weakened, however, by the fact that the RuvB-like Walker A and B domains in p50 and p55 are much further apart than in RuvB. A role in recombination was also suggested by a positive two-hybrid interaction of human p55 with a subunit of human replication protein A (hsRPA3), but there is no other experimental evidence supporting this suggestion.
Results from two other studies are also interesting and difficult to interpret. In one, p55 was reported to occur in nuclear matrix preparations from human and rat cells. Specifically, p55 was enriched in an insoluble pellet following treatment of nuclei with detergent, buffer of high ionic strength, and DNase I (26
). A different study, with human erythrocytes, found that p50 and p55 (named ECP-51 and ECP-54) bind a fragment of the membrane protein stomatin in vitro (57
). These results could reflect important subcellular associations for p50- and p55-containing complexes, although the erythrocyte situation may not be relevant to snoRNP synthesis, since these cells do not have a nucleus.
In light of the complexity and number of processes in which Rvb2p is involved, it is possible that the effects on snoRNA accumulation and snoRNP protein localization could arise from alterations in more than one nuclear process. An important goal for the future will be to carry out systematic studies on the order and kinetics of the various snoRNP-related events that occur during Rvb2p depletion and inactivation. These results should yield valuable insights into the precise cause-and-effect relationships between Rvb2p function and the biogenesis of snoRNPs and ribosomes.
The results from our point mutation studies show that the Walker A and B domains are essential for cell growth (see also reference 40
) and for accumulation of snoRNAs. The growth phenotypes (Table and Fig. ) would be predicted in some cases, based on effects of mutations in known ATPases such as eIF-4A and RuvB (43
). For example, G75 and K81 in yeast Rvb2p (Walker A) are vital for function and correspond to residues required for ATP binding and/or ATPase activity in eIF-4A (51
). Similarly, the D296N mutation blocks function. Overexpression of the latter protein causes a dominant lethal growth phenotype in cells with a wild-type gene (Fig. C). This situation is analogous to that observed for Escherichia coli
RuvB, where the equivalent mutation (D113N) causes a loss of ATPase activity and overexpression of the allele in vivo results in a dominant-negative UV-sensitive phenotype (43
). These results strongly suggest that Rvb2p has ATPase activity in vivo and are consistent with reports showing that p50 has ATPase and ATP-dependent DNA helicase activities in vitro (28
). DNA helicase activity can be imagined to be important in gene transcription, for example, in remodeling chromatin structure or influencing the binding of transcription factors. Our present results argue that Rvb2p does not influence snoRNA accumulation by altering transcription of host mRNA genes. Thus, for these and perhaps the other snoRNAs, the effect of Rvb2p is apparently downstream of transcription.
In principle, it is possible that Rvb2p can alter helices in RNA as well as DNA. Several proteins with DNA helicase activity can also unwind RNA:RNA and RNA:DNA helices in vitro (reference 60
and citations therein). One protein of this class, yeast Sen1p, is particularly relevant to the present study, as it also affects snoRNP biogenesis, with similar consequences. Sen1p from Schizosaccharomyces pombe
has helicase activity with both RNA and DNA in vitro, and the S. cerevisiae
ortholog affects the processing of snoRNAs and localization of the snoRNP proteins Nop1 (C/D class) and Sbp1 (H/ACA class) (56
). RNA helicases can be imagined to function at different stages of snoRNP synthesis, including dissociation of snoRNA transcripts from DNA, remodeling of RNPs during pre-snoRNA processing or snoRNP assembly, and perhaps interactions with the nucleolar trafficking machinery.
The fact that p50 and p55 were affinity selected in vitro with a model C/D snoRNA is consistent with a role for one or both proteins at the RNA or RNP level. Specificity is suggested by the finding that complex formation required an intact C/D motif, as mutations in box D blocked assembly (46
). The two C/D snoRNP core proteins, Nop56p and Nop58p, were present in the complex in nearly stoichiometric amounts, as seen in natural complexes from yeast (21
). The p50 and p55 proteins occurred at similar levels (21
). Attempts to immunoprecipitate snoRNAs from mouse extracts with rabbit antibodies to mouse p50 were negative (46
), and in the present study we were unable to detect snoRNAs in immunoprecipitates of protein A-tagged yeast Rvb2p (data not shown). These results suggest that p50 may not be present in mature snoRNP particles. To date, neither p50 nor p55 has been detected in preparations of natural C/D (and H/ACA) snoRNPs enriched from yeast or human extracts by affinity enrichment schemes. However, the complexes analyzed thus far were incomplete. Because p50 and p55 exhibited stable, stoichiometry-like binding in the initial isolation strategy, it seems likely that their association with snoRNAs is relevant and that this association probably occurs in vivo as well, in the nucleoplasm.
The p50 and p55 proteins could bind directly to snoRNA or indirectly through protein-protein interactions. The observation that loss or mutation of Rvb2p impairs the accumulation of H/ACA snoRNAs implies that the association may not be limited to C/D snoRNAs. In the context of snoRNA binding, this result has several possible interpretations. One possibility is that RNA binding is direct and occurs through RNA structure elements common to both H/ACA and C/D snoRNAs. Simple sequence elements are not likely to be involved here, as the only conserved sequences known are the family-specific box elements. Alternatively, direct binding to RNA could involve secondary- or tertiary-structure domains common to both major classes of snoRNAs. Arguing against this possibility is the belief that the C/D snoRNAs do not have conserved secondary structures beyond the C/D motif.
More likely is the possibility that p50 and p55 associate with both major classes of snoRNAs through common elements that occur in the snoRNP proteins. The signals could be present in a protein common to all C/D and H/ACA snoRNAs, although none has yet been identified. Alternatively, p50 could bind to different family-specific proteins with common motifs. The latter possibility is especially attractive at present, as the two sets of unique core proteins each include one member with striking sequence relatedness to the other. One of these corresponds to the Snu13p/15.5 kDa protein, which binds directly to the C/D motif and which is present in natural C/D snoRNPs (80
). The related H/ACA protein is Nhp2p in yeast (hNHP2 in humans) (54, 65). Yeast Snu13p and Nhp2p exhibit an overall relatedness of 33% identity and 44% similarity. The related sequences are concentrated in the middle of the protein, where a stretch of 22 amino acids is 55% identical and 82% similar. These core proteins provide a common link between the two major classes of snoRNAs and are excellent candidates for the binding of p50 and p55. We note that Snu13p was not observed in the affinity isolation procedure that identified p50 and p55; however, it might have been overlooked in the gel analysis because of its small size (46
The fact that Snu13p is also present in the U4 snRNP raises the interesting possibility that mRNA splicing and snoRNA synthesis may be linked. It is possible that Snu13p has separate, unrelated functions in splicing and snoRNP production. However, the processes are clearly coupled, and Snu13p is a common link. Similarly, the ties between p50 and chromatin remodeling and gene expression suggest that p50 (and p55) might participate in coupling the processes that coordinate ribosome and protein synthesis. Relevant to this, the Snu13p and Nhp2p sequences have some similarity to ribosomal protein L30 (RPL30) in S. cerevisiae,
and corresponding proteins in a wide range of organisms from the Archaea
to humans (80
). The sequence relatedness, 24% identity and 33% similarity, between Snu13p or Nhp2 and RPL30 is modest; however, these similarities raise the possibility of yet other links between splicing, snoRNP production, and ribosomes. Sorting out these relationships promises to be both interesting and challenging.