Our work addresses the complex roles of the Imp3, Mpp10, and Imp4 proteins in ribosome biogenesis. The three proteins are functionally dependent on one another and are involved in key steps of SSU processome activity. Imp3p is essential for the interaction of the Mpp10 and Imp4 proteins with the U3 snoRNA, while Mpp10p is essential for the stability of both Imp3p and Imp4p. To more fully understand the mechanism by which Mpp10p, an essential component of the SSU processome, functions within the cell, we also examined the genetic interaction between base-pairing-defective U3 snoRNAs and the carboxy-terminal truncated Mpp10p. These analyses revealed that while mutation of either processome component results in modest defects in cleavage at sites A0, A1, and A2, combination of these mutations results in a strikingly drastic reduction in cleavage at site A2 only. Additionally, it appears that sequences in the carboxy terminus of Mpp10p function in a species-specific manner to achieve maturation of the small subunit rRNA.
The carboxy-terminal truncation of Mpp10p, which confers cold sensitivity, results in defective processing at sites A1
Mpp10, when expressed in yeast, also results in cold-sensitive growth and defects in processing at sites A1
. Interestingly, the growth and processing defects attributed to dMpp10 expression were suppressed by swapping the fly and yeast carboxy termini. Since this portion of Mpp10p contains a coiled-coil domain, it is possible that a specific protein interacts with this region and is involved in processing at these sites. Because depletion of Utp16p or Dhr1p also results in the loss of cleavage at sites A1
; B. Mitchell and S. Baserga, unpublished data), their ability to interact with Mpp10p was examined in the two-hybrid system. Unfortunately, neither protein was found to interact with Mpp10p (data not shown). To date, Mpp10p has been shown to interact only with Imp3p and Imp4p (18
Considerable evidence suggests that the Imp3, Imp4, and Mpp10 proteins exist as an interdependent unit within the cell (Fig. ). The three proteins copurify in the absence of other SSU processome components (F. Dragon, S. Wormsley, and S. Baserga, unpublished data). By binding to (18
) and stabilizing Imp3p and Imp4p, Mpp10p may function as the key regulatory subunit within this complex. In human cells, Mpp10 was first identified as a protein phosphorylated at specific sites during mitosis (34
). As is the case with other types of cell cycle-dependent protein-protein interactions (reviewed in reference 1
), it may be that phosphorylation of Mpp10 renders it defective for interaction with Imp3p and Imp4p. This, in turn, may result in their degradation. Since the Imp proteins are essential for SSU processome function (18
), we propose that their degradation ultimately leads to the rapid inhibition of pre-rRNA processing that is observed at the onset of mitosis in vertebrate cells (27
FIG. 8. A model for the role of Mpp10p, Imp3p, and Imp4p in SSU processome activity. Thin gray lines, thick gray lines, and thin black lines indicate the transcribed spacers, the 18S rRNA, and the U3 snoRNA, respectively. The Imp4, Mpp10, and Imp3 proteins are (more ...)
The first 70 nucleotides of the U3 snoRNA are essential for SSU processome function (22
) and contain sequences that base pair with the pre-rRNA (4
). Recent evidence has demonstrated that this region of the U3 snoRNA is also required for Mpp10p association (36
). Our results indicate, however, that it is the putative RNA binding protein Imp3p (18
) that is required for Mpp10p and Imp4p to associate with the U3 snoRNA. Therefore, we suggest that it is the Imp3 protein that directly associates with the first 70 nucleotides of the U3 snoRNA and thereby directs the preassembled Imp3p/Mpp10p/Imp4p complex to the U3 preprocessome. Since Imp3p bears the rRNA binding domain of the S4 family of ribosomal proteins (18
) and probably associates with the region of the U3 snoRNA containing homology to the 5′ ETS, it is the best candidate to guide or facilitate binding of the U3 snoRNA to the pre-rRNA (Fig. ).
The cold-sensitive growth and defects in RNA processing conferred by the mutations in the U3 snoRNA and Mpp10p (13
) suggested that they may be involved in an identical step: for example, in pre-rRNA-U3 snoRNA base pairing. If this were indeed the case, combining the mutations should not have an additive effect on either growth or pre-rRNA processing defects. In contrast, our results suggest that the two mutations together do indeed cause increased growth defects at low temperatures. Surprisingly, increased defects in pre-rRNA processing were observed specifically at the A2
cleavage site in the pre-rRNA but not at A1
. Complete loss of the 27SA2
precursor in the presence of both the U3 and Mpp10p mutations demonstrates a strong combinatorial defect in cleavage at site A2
and is consistent with a model in which U3 and Mpp10p are involved at different steps required for this cleavage event.
Imp4p is a member of a superfamily of RNA binding proteins and coimmunoprecipitates all of the pre-rRNA precursors that contain SSU processome cleavage sites (33
). Since Mpp10p interacts with Imp4p (18
) and mediates its interaction with the U3 snoRNA, it seems probable that Imp4p uses its RNA binding domain to contact the pre-rRNA directly. In light of these results, it is tempting to speculate that the 3′ portion of box A does in fact base pair with nucleotides 1139 to 1143 of the 18S rRNA, as previously proposed (13
), and that Imp4p is responsible for bringing the two RNA molecules into contact (Fig. ). Previous reports of the inability to suppress the 3′ box A mutations by the creation of compensatory mutations in the rRNA (24
) can thus be explained: mutation of the rRNA disrupts the Imp4p binding site. Therefore, we propose that in the double-mutant strains, the nature of the truncated Mpp10p alters the ability of the Imp4 protein to interact with the pre-rRNA and mutations in the U3 snoRNA prevent RNA-RNA base pairing.
While the results discussed above suggest a model in which cleavage at A2
is tightly linked to the formation of the central pseudoknot of the 18S rRNA, they do not address the process by which A2
is brought into the active site of the SSU processome. Recent evidence has revealed that components of the SSU processome are required for formation of the “terminal knobs” that were first described by Oscar Miller over 30 years ago (20
). It may be that the pre-rRNA is wound around the SSU processome in a fashion similar to the way the DNA is wound around the histones (Fig. ). Such a process may explain how the 3′ portion of the central pseudoknot is first brought into position for Imp4p binding (Fig. ) and how subsequent cleavage at A2
is dependent on formation of the pseudoknot. It is possible that following pseudoknot formation, the pre-rRNA continues to wrap around the processome until the A2
cleavage site is precisely positioned at the active site (Fig. ). In this model, positioning of the cleavage sites is partially dependent on pseudoknot formation. Thus, it appears that the cleavages may act, in part, as a checkpoint, signaling through the release of the rRNA that the chaperone role of the SSU processome has been accomplished and that the pseudoknot has been appropriately formed.