Here I describe that Rrp12, a protein involved in ribosome subunit maturation and export, plays critical roles in cell cycle-related processes. Such roles have been unveiled through two independent approaches. First, by promoting the degradation of this protein in a degron strain, I have shown that the loss of Rrp12 leads to severe defects in S-phase entry and progression, inefficient DNA damage responses, and a milder delay in the M/G1 transition. Those dysfunctions were clearly dissociated from any defect in ribosomal production. Second, I found serendipitously that the DHFRts-Rrp12 protein expressed by the rrp12-td degron strain had some intrinsic structural defects that impaired its cell cycle/DNA damage response-related functions while keeping intact its ribosomal biosynthesis-linked roles. This property made it possible to dissociate the functional roles of Rrp12 in vivo by studying the rrp12-td strain under permissive conditions.
Interestingly, I observed that Rrp12 has unique functions during the cell cycle that are not shared by other proteins involved in ribosome biosynthesis. Thus, unlike results for other preribosomal components previously linked to cell cycle events, I could not detect any major defect in the Start cell cycle transition, in the loading of prereplicative complexes onto chromatin during the G1
phase, or in cytokinesis. Consistent with this idiosyncratic role, our protein complex purification experiments and coimmunoprecipitation experiments with epitope-tagged proteins (unpublished results) did not detect an interaction of Rrp12 with prereplicative components as previously reported for the pre-60S factors Noc3 and Noc7 (9
). Instead, I have found that the influence of Rrp12 on the activation of the DNA damage response and to some extent on S-phase progression is due to its implication in a very specific process that requires Kap121-dependent transport of proteins from the cytosol to the nucleus. Such a process favors the proper sequestration of the small Rnr2 and Rnr4 subunits of ribonucleotide reductase in the nucleus, thus contributing to the controlled production of dNTPs during specific cell cycle stages. In agreement with such a role, I have observed that yeast strains expressing defective versions of Kap121 and Rrp12 do not properly localize Rnr2 and Rnr4 inside the nucleus. Instead, these RNR subunits accumulate in the cytosol, leading to an abnormal production of dNTPs in both asynchronous and G1
-arrested cultures, in the case of DHFRts
What is the role of Rrp12 in the context of Kap121-mediated transport routes? Prima facie
, two possible activities could be envisioned. One of them is that Rrp12 is just another Kap121 cargo that, when malfunctioning, leads to a dominant negative effect on the nuclear import machinery. I believe that such a scenario does not fit the experimental data, because I have observed no changes in the subcellular localization of Rrp12 in cells expressing nonfunctional versions of Kap121. Furthermore, although a dominant negative effect could be justified in the case of cells expressing the DHFRts
-Rrp12 protein, it is not compatible with the data obtained when the Rrp12 protein is degraded in the degron strain at the nonpermissive temperature. Another possibility is that Rrp12 is in fact a functional element of the Kap121 transport machinery. Several independent observations support this model. First, I observed that these two proteins form stable associations that survive the rather stringent conditions of the complex purification experiments. Indeed, I have detected Kap121, but not Kap121-dependent cargos, bound to Rrp12 and DHFRts
-Rrp12 proteins. Likewise, Rrp12, but not Kap121 cargos, was detected in immunotrapped GFP-Kap121 complexes. Second, and perhaps more importantly, I have found that the overexpression of wild-type Rrp12 can rescue some of the nuclear import defects present in yeast cells expressing a Kap121ts
mutant, including the transport of histone H3 and Rrn4. These results suggest that the Rrp12/Kap121 interaction might help the Kap121ts
mutant to overcome its intrinsic structural defects. Third, I have observed that wild-type Kap121 is less stable and becomes mislocalized to the cytosol and vacuoles in cells expressing the DHFRts
-tagged version of Rrp12, further indicating that Kap121 requires the presence of wild-type Rrp12 to ensure full functionality. Given that Rrp12 is a shuttling HEAT repeat-containing protein that can bind directly to nucleoporins (30
) and, in addition, that Kap121 requires interactions with the nuclear pore components for its association to the nuclear envelope (25
), Rrp12 may be also important to ensure the tethering of Kap121 to the nuclear pore. This last possibility remains to be determined. However, given that the DHFRts
-Rrp12 maintains its functionality in nucleolus-dependent functions, such as its assembly with preribosomes and the subsequent nuclear-to-cytoplasmic export of ribosomal subunits, I surmise that the basis of the Kap121 defect found in rrp12-td
cells must be prior to any alteration linked to nuclear pore interaction or the ensuing mobilization into the nucleus.
Additional observations highlight the specificity of Rrp12 toward Kap121. Thus, I have observed that Rrp12 does not interact with other karyopherins and that its deficiency/malfunction does not disrupt the transport of other proteins to the nucleus. Furthermore, Rrp12 is required for the efficient action of Kap121 on nuclearly localized cargos (i.e., Rnr2, Rnr4, and histone H3) but not on a cargo that is delivered to the nuclear envelope (Ulp1). The latter result is interesting, because it suggests that Kap121 utilizes different structural determinants or associates with different cotransporters to achieve its specificity toward different cargos. The results indicating that other proteins involved in ribosomal biogenesis (Tsr1 and Nop1) and nuclear trafficking events (Kap123) do not rescue the functionality of the Kap121ts
mutant protein further underscore the unique role of Rrp12 in nuclear import-dependent events. Interestingly, it has been reported recently that eIF3, a translation initiation factor that participates in the maturation of pre-40S and pre-60S subunits, cooperates with Kap121 in transporting the proteasome into the nucleus in Schizosaccharomyces pombe
). Thus, it is possible that the Rrp12/Kap121 functional interaction described here is part of a more general regulatory theme in which Kap121 cooperates with preribosome-associated factors to promote the nuclear import of different protein complexes. This network could potentially contribute to the proper coupling of ribosomal biogenesis and parallel biological routes required for optimal cell proliferation or growth. In this context, it is interesting to point out that our experiments have revealed that Kap121 can also associate with the preribosome export factor Arx1 that, intriguingly, is known to be required for a Kap121-mediated mechanism that recycles some ribosome synthesis factors from the cytosol back into the nucleus (20
I demonstrated that the artificial enhancement of ribonucleotide reductase activity could restore normal DNA damage responses in rrp12-td cells, providing genetic proof for the idea that the defects in the regulation of that enzyme are the main cause for the DNA damage sensitivity present in the rrp12-td strain. Such a strategy, however, did not suppress the delay in S-phase progression exhibited by rrp12-td cells, suggesting that there must be other events mediated by Rrp12 at this stage of the cell cycle. Whether such events occur through Kap121-dependent nuclear import routes or through Kap121-independent mechanisms remains to be determined.
Although the sml1Δ
rescue experiments pinpoint a clear implication of ribonucleotide reductase in the cell cycle/DNA damage response defects present in rrp12-td
cells, they also raise some interesting questions pertaining to the role of dNTP levels in such processes. Thus, it is known that increased synthesis of dNTP via the activation of the cytosolic ribonucleotide reductase holoenzyme is required for proper S-phase progression and DNA damage checkpoint activation. In this context, it would be expected that rrp12-td
cells would show efficient S-phase progression and reduced sensitivity to DNA damage since they contain higher levels of dNTPs than control cells do. Given that rrp12-td
cells display instead a slow S phase and hypersensitivity to DNA damage, the results indicate that the elevation of dNTPs is not a benefit for the cell. This is in contrast with previous findings showing that a moderate elevation of dNTPs does not alter cell cycle progression and confers increased viability to DNA damage (4
). Therefore, it seems that it is the specific defect in the regulation of RNR exhibited by rrp12-td
cells that is particularly negative. One possibility is that a constant presence of Rnr2/Rnr4 in the cytoplasm interferes with the timing or duration of other known regulatory mechanisms that control RNR activity during the cell cycle (i.e., Sml1 degradation, dATP feedback inhibition, and transcriptional regulation of RNR genes) (4
). In favor of this possibility, time course experiments have shown that the dNTP production kinetics along the cell cycle is delayed in rrp12-td
cells compared to control cells. Therefore, although they contain higher-than-normal dNTP levels in G1
cells reach S phase without having an optimal concentration of dNTPs. This defect may cause inefficient initiation of DNA replication and defective sensing of DNA damage.
The discovery that Rrp12 participates in nuclear import events raises the question of how this function is related to the previously described roles for this protein in ribosome biogenesis. I have shown that the overexpression of Rrp12 promotes the import of ribosome-unrelated proteins and that the partially defective DHFRts-rrp12
mutant exhibits defects in Kap121-mediated functions not implicated in ribosome maturation or export. Therefore, Rrp12 participates in nuclear import activities unrelated to ribosome synthesis. Whether Rrp12 is also involved in the nuclear import of ribosome biogenesis factors is presently unknown, but this is an interesting possibility to be addressed in the future. Rrp12 was proposed to have a role in ribosome subunit export based on its structural similarity with β-karyopherins, its ability to interact with nucleoporins and with Ran, and the fact that its depletion causes the nuclear accumulation of pre-40S and pre-60S particles (30
). However, it is generally difficult to distinguish whether a particular factor is required for the actual export process or whether it is required to achieve export competence. In fact, the finding that Rrp12 depletion causes defects in pre-rRNA processing and preribosome maturation has been taken to suggest that its involvement in ribosome subunit export might be indirect (51
). The role of Rrp12 in nuclear import described here opens the possibility that the defects in ribosome maturation and export of Rrp12-depleted cells could be caused by a deficient import of essential ribosome biogenesis factors, some of which are already known to be Kap121 cargos.
Although I have focused the present work on the elucidation of the extraribosomal functions of Rrp12, functional screening also revealed that Utp5, a 90S preribosome component essential for ribosome biogenesis, is important for cell cycle progression. However, unlike the case of Rrp12, the elimination of that protein elicits only defects in S-phase entry, indicating that Rrp12 and Utp5 affect different processes during the cell cycle. The G1
-S delay of Utp5-depleted cells is accompanied by abnormal pre-rRNA synthesis and processing, but this is not necessarily the cause of their cell cycle phenotype. My finding of a degron strain (the utp18-td
mutant) that produces abnormal pre-rRNA levels but displays normal cell cycle progression indicates that an alteration of the pre-rRNAs contents per se
does not cause a slow G1
-S transition. This is at odds with previously published data showing that defects in pre-rRNA processing are sensed at the Start checkpoint to delay the entry into S phase (2
). My results suggest that such a sensing mechanism might become activated only when major disruptions in preribosome formation or nucleolar structure take place or when the accumulation of unprocessed pre-rRNAs reaches some threshold level.