Genetic depletion of Rrp5p blocks synthesis of both 18S rRNA, requiring the snoRNP-dependent cleavages at A0–A2, as well as 5.8SS
rRNA, which depends upon the RNaseMRP-directed cleavage at A3. This led to the hypothesis that the major role of Rrp5p is to ensure functional integration of the many trans
-acting factors required for accurate and efficient pre-rRNA processing at these sites (30
). Subsequent mutational analysis (31
) demonstrated that the C-terminal region of the protein, containing seven TPR motifs, is specifically involved in 18S rRNA synthesis, whereas the N-terminal region, encompassing 12 S1 RNA-binding motifs, is crucial for production of the 5.8SS
rRNA. Thus, the C-terminal domain might interact with the snoRNP complex that carries out the early processing cleavages, while the N-terminal domain assists RNase MRP. The data presented in this paper constitute direct experimental proof for the latter suggestion as they demonstrate that removal of either S1 motifs 3–5 (rrp5
or 5–8 (rrp5
) leads to a complete block in the RNase MRP-directed cleavage at site A3 (Fig. ).
Interestingly, our experiments reveal a second effect of the rrp5-Δ3 and rrp-Δ4 mutations, namely the occurrence of a hitherto unobserved processing event in ITS1 at a site about midway between A2 and A3 which we have designated A4 (Fig. ). The simultaneous presence of substantial amounts of an A4-E fragment (Fig. D, E and F), normal amounts of the D-A2 fragment (Fig. H) and, in particular, the detection of the A4 site in primer extension experiments using probes 6 and 7 complementary to different regions of ITS2 clearly support the conclusion that the rrp5-Δ3 and rrp-Δ4 mutations cause the processing machinery to bypass A3 and instead to convert the 27SA2 pre-rRNA into an alternative, novel 27SA4 precursor species.
The currently available data do not allow us to decide whether formation of the 27SA4
species is an endo- or an exonucleolytic event. However, we favor the former possibility for the following reasons. First, the processing intermediate having its 5′-end at A4 accumulates in the presence of LiCl, which strongly inhibits the major 5′→3′ exonucleases Xrn1p and Rat1p known to be involved in pre-rRNA processing (Figs and ). Second, the 27SA2
pre-rRNA, the probable immediate precursor of the 27SA4
species, appears to be a poor substrate for these exonucleases, in any case, at least under normal conditions (13
). Finally, as shown in Figure , the A4 site is located in a region of ITS1 that has been relatively well conserved with respect to both primary and secondary structure over a wide spectrum of yeast species, suggesting that it could be the recognition site for an endonuclease, the nature of which remains to be identified. There is no obvious structural similarity of this region with those containing other known processing sites within the pre-rRNA. It should also be noted that processing at A4 might be less precise than the standard processing events. In all cases we observe a set of three bands corresponding to consecutive nucleotides, whose relative intensity varies somewhat from one experiment to another (Figs and ).
Although the rrp5-Δ3 and rrp-Δ4 deletions cause identical changes in ITS1 processing the fate of the resulting precursors differs in the two mutants. In the rrp5-Δ3 strain subsequent processing almost exclusively follows the ‘long’ pathway leading to 5.8SL rRNA. In the rrp5-Δ4 mutant, on the other hand, substantial processing still occurs via the ‘short’ pathway, presumably by exonucleolytic degradation of the 27SA4 precursor, resulting in ~40% 5.8SS rRNA. We propose that the presence of mutant Rrp5p protein causes a structural alteration in the processing complex that reduces either the rate of entry of the exonucleases or the rate of exonucleolytic digestion, allowing processing at site B1L to get the upper hand. Clearly, the extent of this negative effect on B1S processing is different for the two mutant proteins, being largest for the rrp5-Δ3 mutation.
A synthetic lethality screen using the rrp5
mutation resulted in the isolation of the REX4
gene, which encodes a protein belonging to a family of non-essential 3′→5′ exonucleases. Although other members of this family were shown to be involved in pre-rRNA processing, no evidence for such a role was found in the case of the Rex4p protein (19
). The data presented in Figures and , however, clearly demonstrate that inactivation of the REX4
gene does have a rather surprising effect on pre-rRNA processing in strains expressing either of the mutant Rrp5pΔ proteins. Both the rrp5
double mutants show normal ITS1 processing as well as a wild-type 5.8SS
ratio. We conclude that the change in ITS1 processing induced by the rrp5
mutations requires intact Rex4p and that in the absence of Rex4p the mutant processing complex containing either mutant Rrp5p protein regains the ability to direct RNaseMRP to the A3 site. The molecular basis for this phenomenon remains a matter of speculation. Rex4p could participate directly in ribosome biogenesis, a conceivable hypothesis in view of the fact that its human homolog resides in the nucleolus (39
). Another possibility is that Rex4p plays a role in the formation of an as yet unidentified trans
-acting factor that forms part of the processing complex containing Rrp5p. In wild-type cells, however, the role of Rex4p is not critical.
The lack of any detectable abnormalities in pre-rRNA processing in the rrp5
double mutants left us without an obvious explanation for the sl
phenotype of these mutants. Therefore, we considered the possibility of a defect in ribosome assembly, which for obvious reasons we studied in strains in which the wild-type RRP5
gene is conditionally expressed. Sucrose gradient analysis demonstrated that in fact the rrp5
mutations by themselves already cause a significant defect in 60S subunit assembly. Extracts from cells expressing the Rrp5pΔ3 protein show a clear deficit in the large, relative to the small, subunits as well as the characteristic presence of halfmer polyribosomes (Fig. ). This could be a consequence of the almost exclusive presence of 5.8SL
rRNA in these cells which might be assembled less efficiently than its smaller counterpart, similar to 3′-extended 5.8S rRNA (17
). This idea is supported by the fact that the deficit in 60S subunits, in particular as judged from the absence of detectable amounts of halfmers, is less severe in the rrp5
mutant, which contains a higher proportion of 5.8SS
; this paper). On the other hand, the structural abnormality of the Rrp5p protein might also directly affect 60S subunit assembly in the mutant cells.
Sucrose gradient analysis of extracts prepared from the rrp5-Δ3/rex4– and rrp5-Δ3/rex4– double mutants revealed a very low amount of 80S ribosomes (Fig. ). The combination of mutant Rrp5p and the absence of Rex4p, therefore, appears to cause a severe defect in ribosome assembly, which would explain the sl phenotype of the double mutants. This finding further supports the hypothesis that Rex4p is involved in ribosome biogenesis, although its role only becomes manifest in the presence of the mutant Rrp5p proteins. We are presently studying the role of Rex4p in ribosome biogenesis further using both genetic and biochemical approaches.
In summary, the data presented in this paper firmly establish the role of the S1 domain of Rrp5p in assisting RNaseMRP in its cleavage at site A3. The involvement of Rrp5p in this processing step seems to be rather complex, however, because the requirement for the S1 motifs in question can be abrogated by removing Rex4p. The data also indicate a further role for Rrp5p, either directly or indirectly, in the subsequent exonucleolytic processing to site B1S and demonstrate a negative effect of the two deletion mutations on 60S subunit biogenesis. Furthermore, both genetic and biochemical evidence clearly indicates the involvement of Rex4p in the assembly of yeast ribosomes, albeit in an unusual manner.