|
|
|
|
|
|
|
||
Article |
Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2
Correspondence to Ulrike Kutay: ulrike.kutay{at}bc.biol.ethz.ch
During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation. Although hRio2 is not absolutely required for pre-40S export, deletion of its binding site for the export receptor CRM1 decelerated the kinetics of this process. Moreover, in the absence of hRio2, final cytoplasmic 40S maturation is blocked because the recycling of several trans-acting factors and cytoplasmic 18S-E precursor ribosomal RNA (rRNA [pre-rRNA]) processing are defective. Intriguingly, the physical presence of hRio2 but not its kinase activity is necessary for the release of hEnp1 from cytoplasmic 40S precursors. In contrast, hRio2 kinase activity is essential for the recycling of hDim2, hLtv1, and hNob1 as well as for 18S-E pre-rRNA processing. Thus, hRio2 is involved in late 40S maturation at several distinct steps.
© 2009 Zemp et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
 |
Introduction |
|---|
 TopAbstract Introduction ResultsDiscussionMaterials and methodsReferences |
|---|
Along their nuclear maturation pathway, early preribosomes are separated into pre-40S and -60S particles, which are exported to the cytoplasm as independent entities. Ribosomal subunit export involves a subset of nuclear pore complex components (Hurt et al., 1999; Moy and Silver, 1999, 2002; Stage-Zimmermann et al., 2000; Gleizes et al., 2001; Bernad et al., 2006) and relies on the RanGTPase system (Hurt et al., 1999; Moy and Silver, 1999, 2002; Stage-Zimmermann et al., 2000; Gleizes et al., 2001; Trotta et al., 2003).
Nuclear export of 60S subunits depends on the RanGTP-binding exportin Xpo1p/CRM1 (Ho et al., 2000; Gadal et al., 2001; Moy and Silver, 2002; Thomas and Kutay, 2003; Trotta et al., 2003), which recognizes a Leu-rich nuclear export sequence (NES; Fornerod et al., 1997; Stade et al., 1997) in the 60S export adapter Nmd3 (Ho et al., 2000; Gadal et al., 2001; Thomas and Kutay, 2003; Trotta et al., 2003). In yeast, additional factors support pre-60S export, including the mRNA export receptor Mex67p/Mtr2p, the pre-60S component Arx1p (Bradatsch et al., 2007; Hung et al., 2008; Yao et al., 2008), and the HEAT repeat protein Rrp12p, which, in addition, assists 40S biogenesis (Oeffinger et al., 2004).
Like pre-60S export, pre-40S export depends on a functional Xpo1p/CRM1 pathway in both yeast and vertebrate cells (Moy and Silver, 1999, 2002; Thomas and Kutay, 2003; Trotta et al., 2003). Two NES-containing trans-acting factors, Ltv1p and Dim2p, have been implicated as potential Xpo1p adapter proteins in pre-40S export in Saccharomyces cerevisiae (Seiser et al., 2006; Vanrobays et al., 2008), whereas export adapters serving 40S subunit export in vertebrate cells have not yet been identified.
After export to the cytoplasm, ribosomal subunit precursors need to be further matured before they can participate in translation. One important aspect in the cytoplasmic maturation of preribosomal subunits lies in the release of trans-acting factors from ribosomal precursor particles. Several such trans-acting factor recycling steps have been described for 60S biogenesis (for review see Zemp and Kutay, 2007), but how trans-acting factors are released from cytoplasmic pre-40S particles is largely unknown. In contrast to pre-60S particles, 40S precursors undergo a final cytoplasmic pre-rRNA processing event, which yields mature 18S rRNA from 20S pre-rRNA in yeast and from 18S-E pre-rRNA in human cells (Udem and Warner, 1973; Rouquette et al., 2005). It is currently not understood whether and how pre-rRNA processing is coordinated with the recycling of 40S biogenesis factors.
In yeast, Rio1p and Rio2p, two members of the RIO (right open reading frame) family of atypical protein kinases, were identified as trans-acting factors involved in late steps of 40S biogenesis (Vanrobays et al., 2001, 2003; Geerlings et al., 2003). Both proteins are essential, indicating that they do not have fully redundant functions. Still, depletion of either Rio1p or Rio2p results in the same pre-rRNA processing defect, i.e., the accumulation of 20S pre-rRNA. In contrast to Rio1p, Rio2p is a stable component of several distinct late pre-40S particles (Schafer et al., 2003). Because of conflicting data, it is not yet clear whether Rio2 is involved in pre-40S export in yeast. 20S pre-rRNA accumulated in the cytoplasm of Rio2p-depleted cells and in rio2-1 mutant cells (Vanrobays et al., 2003; Leger-Silvestre et al., 2004), whereas the 40S subunit reporter Rps2-GFP accumulated in the nucleus of rio2-1 mutant cells (Schafer et al., 2003).
Recently, human Rio2 (hRio2) has been shown to be required for cytoplasmic 18S-E to 18S rRNA processing, indicating that the role of Rio2 in final 18S rRNA maturation is conserved from yeast to vertebrates (Rouquette et al., 2005). In this study, we have further investigated the role of hRio2 in 40S subunit maturation in human cells. We could isolate and characterize an hRio2-containing 40S precursor, which contains 18S-E pre-rRNA and at least six trans-acting factors. Depletion of hRio2 from HeLa cells revealed that hRio2 is required for distinct steps of late 40S biogenesis. First, an NES in hRio2 plays a nonessential role in pre-40S export. Second, we demonstrate that hRio2 is required for the release of trans-acting factors from 40S precursors after export. Intriguingly, its physical presence but not its kinase activity is required for hEnp1 recycling to the nucleus. In contrast, hRio2 kinase activity is necessary for a second cytoplasmic maturation step during which hDim2, hLtv1, and hNob1 are released from 40S subunits and 18S-E to 18S rRNA processing occurs.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
|
|
10) relocalized hRio2 to the nucleus (Fig. 2 C), indicating that this sequence motif is a functional NES. Sequence alignment reveals that the NES in the C-terminal domain of hRio2 is well conserved in vertebrates (Fig. 2 B). The C-terminal domains of Rio2 sequences from lower eukaryotes like yeast are mostly much shorter than in human Rio2 and generally do not align well (Vanrobays et al., 2003; unpublished data). Nevertheless, a similar NES-like sequence can also be found, for instance, in yeast Rio2p (Fig. 2 B). To test whether the NES of hRio2 can mediate nuclear export of a reporter, we fused aa 388–410 of hRio2 to the C terminus of EGFP. Although EGFP displayed a predominantly nuclear localization, EGFP-NES(Rio2) localized to the cytoplasm (Fig. 2 D). Nuclear export of EGFP-NES(Rio2) was CRM1 dependent, as it relocalized to the nucleus upon LMB treatment. Similar results were obtained using the NES of hNmd3 as a positive control. Thus, the NES identified in hRio2 can mediate nuclear export in vivo.
To confirm that hRio2 is exported by CRM1, we performed in vitro binding assays using recombinant zz-hRio2 and CRM1 in the absence and presence of RanGTP, which increases the affinity of exportins for their substrates (Fornerod et al., 1997; Kutay et al., 1997). Indeed, binding of CRM1 to zz-hRio2 was observed in the presence but not in the absence of RanGTP (Fig. 2 E). In contrast, CRM1 did not associate with either zz-hEnp1 or the zz tag alone. Recombinant zz-hRio2(NESmut) and zz-hRio2(NES10) could be purified in similar purity and amounts as zz-hRio2(wild type [WT]) and displayed similar in vitro kinase activity (Fig. 2 F), indicating that the mutations did not grossly alter hRio2 structure. However, both zz-hRio2(NESmut) and zz-hRio2(NES10) displayed strongly reduced CRM1 binding (Fig. 2 E). Together, these results show that hRio2 directly binds CRM1 via an NES in its C-terminal domain and is an export substrate of CRM1. Because hRio2 is a component of late pre-40S particles (Fig. 1), it is a candidate adapter protein for CRM1 in pre-40S export.
hRio2 contributes to the efficient nuclear export of 40S subunit precursors
To study pre-40S export in human cells, we developed a novel reporter system based on a HeLaK cell line, which allows for the tetracycline (tet)-dependent induction of a YFP-tagged ribosomal protein of the 40S subunit, i.e., hRps2-YFP. Only low levels of hRps2-YFP were detected in these cells in the absence of tet induction (Fig. 3). However, tet addition led to a strong increase in hRps2-YFP signal, and the reporter localized predominantly to the cytoplasm. To determine whether hRps2-YFP becomes incorporated into translating ribosomes, we performed sucrose gradient analysis. The majority of hRps2-YFP was associated with 40S, 80S, and polysome fractions, indicating that it was indeed faithfully incorporated into functional 40S subunits (Fig. S1 D). A small pool of free hRps2-YFP was also observed, caused by the induction of newly synthesized, not yet incorporated protein upon tet addition (Fig. S1 E) and perhaps also partially by the increased total amount of hRps2 in these cells, endogenous and YFP tagged. Upon inhibition of pre-40S export by LMB in the reporter cell line, a nuclear accumulation of hRps2-YFP was observed (Fig. 3 A), demonstrating that our reporter allows for a sensitive readout of defects in 40S biogenesis and export and provides a sensitive assay to study the fate of newly assembled 40S subunits.
|
25% of cells displayed nuclear accumulation of the reporter protein, whereas in the others, hRps2-YFP was predominantly cytoplasmic or showed both nuclear and cytoplasmic staining. Moreover, we performed sucrose gradient analyses from extracts of hRio2-depleted as well as LMB-treated reporter cells and did not observe a significant change in the amount of hRps2-YFP detected in the free protein fractions, suggesting that the observed nuclear accumulation of hRps2-YFP in these cells is not caused by an assembly defect but by reduced pre-40S export (unpublished data). The pre-40S export defect upon hRio2 depletion was clearly not as prominent as after knockdown of CRM1, and not all hRio2-depleted cells showed a defect. Therefore, hRio2 appears not to be absolutely required for 40S export, but hRio2 knockdown may reduce pre-40S export efficiency. In line with this argument, hRps2-YFP accumulation in the nucleus of hRio2-depleted cells progressively disappeared when cells were released from tet treatment, demonstrating that export still takes place in hRio2-depleted cells, albeit at a slower rate (Fig. S2 B).
|
Similar results were obtained using an hRps3A-YFP reporter cell line (unpublished data), indicating that the observed pre-40S export defects are not specific to hRps2-YFP–expressing cells. Moreover, we investigated the effects of hRio2 depletion in a HeLa Yale (HeLaY) cell line that does not express a reporter protein using the localization of the trans-acting factors hNob1 (Fig. 4 A), hLtv1, hEnp1, and hDim2 (Fig. S3 B) as readouts for pre-40S maturation. Also, in HeLaY cells, RNAi against hRio2 reproducibly led to a mixed phenotype: for hNob1, 10% of cells displayed nuclear accumulation; the other cells showed little or no alteration of hNob1 localization (Fig. 4 A). In summary, we conclude that hRio2 contributes to pre-40S export because nuclear accumulation of 40S precursors can be observed in hRps2-YFP and hRps3A-YFP reporter cells as well as in HeLaY cells after hRio2 knockdown. Notably, there is no absolute requirement for hRio2 in pre-40S export, which may be explained by the presence of several redundant adapters for CRM1 in 40S precursors (see Discussion).
To ensure that the pre-40S export defects were indeed caused by hRio2 depletion, rescue experiments were performed using EGFP- or CFP-tagged hRio2, which allows for detection of cells in which the rescue construct was expressed. In addition to WT hRio2, we also used the NES mutant and deletion variants of hRio2. As expected, overexpression of CFP-hRio2(WT) rescued the mislocalization of hRps2-YFP in hRio2-depleted hRps2-YFP reporter cells (Fig. 4, B and C). In contrast, nuclear accumulation of hRps2-YFP was only partially rescued by CFP-hRio2(NESmut) and not rescued by CFP-hRio2(NES10), suggesting that the CRM1-binding site of hRio2 indeed contributes to pre-40S export.
When analyzing hNob1 localization in these rescue experiments (Fig. 4, B and C), CFP-hRio2(WT) partially relieved nuclear accumulation of hNob1, whereas CFP-hRio2(NESmut) and CFP-hRio2(NES10) did not rescue hNob1 localization but rather increased the number of cells displaying nuclear hNob1 compared with hRio2-depleted cells. The same rescue experiments were also performed in HeLaY cells and analyzed by hNob1 immunostaining (Fig. 4 D). hNob1 mislocalization caused by hRio2 knockdown was almost completely rescued by EGFP-hRio2. In contrast, EGFP-hRio2(NESmut) or EGFP-hRio2(NES10) did not rescue but instead led to a further increase in nuclear accumulation of hNob1 compared with hRio2-depleted cells. Collectively, our data in both HeLaY and hRps2-YFP cells support a role for hRio2 in pre-40S export that is dependent on its NES. Therefore, hRio2 might serve as an adapter protein for CRM1 in 40S subunit export (see Discussion).
Cytoplasmic maturation of pre-40S particles is stalled in the absence of hRio2
One interesting observation consistent in all hRio2 knockdown experiments in the different cell lines was an obvious cytoplasmic accumulation of 40S trans-acting factors in many cells (Fig. 4 and Fig. S3). This observation was particularly striking in HeLaK cells, in which we did not even observe nucleoplasmic accumulation of these proteins. Thus, HeLaK cells turned out to be a suitable model to further study these effects independently of nuclear export defects. Indeed, immunofluorescence analysis of hRio2-depleted HeLaK cells revealed that 90% of cells displayed a partial cytoplasmic accumulation of hEnp1 and hDim2, which are restricted to the nucleus in control cells (Fig. 5 A). Moreover, both hNob1 and hLtv1 appeared to be more strongly cytoplasmic after hRio2 knockdown (Fig. 5 A). Together, these results suggest that in the absence of hRio2, these four trans-acting factors are found on cytoplasmic 40S precursors that are stalled in their maturation.
|
hRio2 kinase activity is required for the release of hDim2, hLtv1, and hNob1 but not hEnp1 from cytoplasmic 40S precursors
To determine whether the kinase activity of hRio2 is required for its function in cytoplasmic 40S maturation, we generated a kinase-dead (KD) variant of hRio2. Based on a sequence alignment of the kinase domain of Rio2 from several species with protein kinase A (unpublished data), we identified Lys123 and Asp246 as two conserved residues in the kinase domain of hRio2 that are likely essential for its kinase activity. Indeed, mutation of these residues to Ala led to loss of autophosphorylation activity, and thus, hRio2(K123A,D246A) represents a KD variant, hRio2(KD) (Fig. 6 A). We then used EGFP-hRio2(WT) or EGFP-hRio2(KD) in rescue experiments and analyzed cells for the localization of trans-acting factors. The aberrant partial relocalization of hEnp1 to the cytoplasm upon hRio2 depletion could be rescued by overexpression of either WT or KD EGFP-hRio2 (Fig. 6, B–D). In contrast, only EGFP-hRio2(WT) but not EGFP-hRio2(KD) was able to restore hDim2 localization to nucleoli after hRio2 knockdown (Fig. 6, B–D). For the cytoplasmic proteins hLtv1 and hNob1, rescue experiments were combined with LMB treatment to check whether or not their shuttling behavior could be rescued. EGFP-hRio2(WT) indeed almost fully restored rapid shuttling of both factors (Fig. 6, E and F). However, EGFP-tagged KD hRio2 not only failed to rescue the fast LMB-dependent relocalization of hLtv1 and hNob1 but even exacerbated the retention of both proteins in the cytoplasm compared with hRio2-depleted cells. In summary, the kinase activity of hRio2 appears to be required for final 40S maturation in the cytoplasm to occur and for the release of hDim2, hLtv1, and hNob1 from 40S precursors for the next round of 40S biogenesis. Surprisingly, hEnp1 recycling only required the physical presence of hRio2 but not its kinase activity, suggesting that hEnp1 is released from 40S precursors at an earlier cytoplasmic maturation step for which the presence of inactive hRio2 is sufficient.
|
35% in the whole cell population, as expected based on the 39% transfection efficiency obtained for this construct (Fig. 7, C and D). Thus, although the physical presence of hRio2 is sufficient for the release of hEnp1 from cytoplasmic 40S precursors, hRio2 kinase activity is necessary for the recycling of hDim2, hLtv1, and hNob1 as well as 18S-E pre-rRNA cleavage. Therefore, cytoplasmic 40S maturation consists of at least two distinct steps, both of which depend on hRio2.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The isolated human pre-40S particle contains homologues to most trans-acting factors found in late 40S precursors in yeast (Schafer et al., 2003), namely hTsr1, hLtv1, hRio2, hEnp1, hNob1, and hDim2. In addition, trans-acting factors identified on yeast late 40S preribosomes included the 18S rRNA dimethylase Dim1p and the CK1 (casein kinase 1) isoform Hrr25p. Homologues to these two factors were not present in our MS analysis. However, low levels of hDim1 can be detected in the hRio2 immunoprecipitate by Western blotting (unpublished data). Possibly, hDim1 is only weakly associated with hRio2-containing 40S precursors in human cells. It also remains to be seen whether a CK1 isoform is associated with these particles but escaped MS detection. Clearly though, most if not all pre-40S components found in yeast are also present in human 40S precursors. Conversely, we did not detect 40S-associated trans-acting factors that are specific to human cells and absent in yeast pre-40S particles. Thus, the composition of late 40S preribosomes seems to be highly conserved from yeast to human.
A Leu-rich NES in hRio2 contributes to efficient pre-40S export
hRio2 localizes to the cytoplasm at steady state but rapidly accumulates in the nucleus upon inhibition of CRM1 using LMB (Rouquette et al., 2005; Bonazzi et al., 2007; this study). We could show that hRio2 contains a functional NES located in its C-terminal domain and that hRio2 directly interacts with CRM1 via this NES. As a component of 40S precursors that directly binds CRM1, hRio2 could therefore serve as an adapter for CRM1 in pre-40S export.
To study nuclear export of 40S subunits, we have developed a new experimental approach that allows for a very sensitive readout for defects in 40S biogenesis and export. Using a tet-inducible Rps2-YFP reporter, we are able to specifically detect preribosomes that are newly assembled during the time of tet induction. In comparison with our previous assays relying on rRNA localization by FISH or on noninducible Rps (ribosomal protein of the small subunit) reporters (Thomas and Kutay, 2003), much clearer effects of CRM1 inhibition on 40S export can be observed (Fig. 3).
In this reporter cell line, depletion of hRio2 by RNAi caused nuclear accumulation of hRps2-YFP in 25% of cells, indicating a role for hRio2 in pre-40S export. Moreover, in support of the conclusion that the efficiency of pre-40S export is reduced in the absence of hRio2, several trans-acting factors like hNob1 accumulated to some degree in the nucleus both in hRps2-YFP reporter cells and HeLaY cells. hNob1 is a pre-40S component, which is predominantly cytoplasmic at steady state and redistributes to the nucleus upon inhibition of 40S export (Fig. 4). As hNob1 is a component of the 40S biogenesis machinery but not part of the end product, the translating 40S subunit, its localization allows for a kinetic readout on how 40S maturation progresses. Using a series of rescue constructs, we could show that the role of hRio2 in pre-40S export depends on its CRM1-binding site. Upon mutation or deletion of its NES, hRio2 lost the ability to rescue the pre-40S export defects observed in hRio2-depleted hRps2-YFP and HeLaY cells. These NES-deficient rescue mutants of hRio2 even increased the nuclear accumulation of hNob1.
Analogous to the role of hNmd3 in 60S export, the simplest model for the involvement of hRio2, and particularly its NES, in nuclear export of 40S precursors is that hRio2 contributes to efficient pre-40S export as an adapter protein for CRM1. Yet, we have not been able to conclusively demonstrate CRM1 recruitment to 40S precursors via hRio2 (unpublished data). Interestingly, when blocking pre-40S export by CRM1-independent means, namely by RNAi-mediated depletion of hRps15 (Rouquette et al., 2005), hRio2 showed only a slight nuclear accumulation, whereas the majority of the protein remained cytoplasmic (Fig. S4), which is similar to results in yeast (Leger-Silvestre et al., 2004). This might be interpreted such that either hRio2 does not enter the nucleus to participate in the translocation of 40S precursors through nuclear pore complexes or it joins the particle after association of hRps15. Our observation of hRio2 being associated with 40S precursors in LMB-treated cells (Fig. S1, A–C) argues for a nuclear function for hRio2 and supports the conclusion that hRio2 directly acts in pre-40S export. One model reconciling these data would predict that hRio2 association with pre-40S particles is hRps15 dependent and represents one of the very last steps of nuclear 40S maturation, followed by 40S export. In the absence of hRps15, hRio2 would remain a free protein that can directly bind CRM1 and rapidly be exported to the cytoplasm.
Surprisingly, although depletion of hRio2 caused nuclear accumulation of hRps2-YFP as well as several trans-acting factors in our reporter cells, no such defects were observed in HeLaK cells, from which the reporter system was developed over several steps of clonal selection. However, upon RNAi against hRio2 in the cell line bearing the flippase recognition target (FRT) integration site and expressing the tet repressor (TetR), but not yet stably transfected with hRps2-YFP, nuclear accumulation of hNob1 could also be observed (Fig. S2 C). These results indicate that the pre-40S export defects observed in the reporter cells do not represent an artifact caused by hRps2-YFP overexpression. Importantly, pre-40S export defects after knockdown of hRio2 were also found in HeLaY cells.
Clearly though, based on our data as well as published work on hRio2 (Rouquette et al., 2005), there is no absolute requirement for hRio2 in 40S export. One possible explanation for this observation is that 40S precursors may contain several CRM1-binding proteins. Indeed, two other pre-40S components have been suggested to act as adapter proteins for Xpo1p in yeast, namely Dim2p and the nonessential protein Ltv1p (Seiser et al., 2006; Vanrobays et al., 2008). However, evidence for a direct binding of Xpo1p to Ltv1p or Dim2p is lacking. Moreover, as for hRio2, no conclusive evidence has been provided in these previous studies to show that either of these proteins recruits Xpo1p to 40S precursors. Yet, it seems possible that recruitment of CRM1 to export-competent pre-40S particles is a redundant process relying on several CRM1-binding adapter proteins. Indeed, considering that pre-60S export involves at least three export receptors in yeast, it is not surprising that pre-40S export may require more than one export adapter to recruit multiple copies of CRM1.
Therefore, we have also investigated the potential of hLtv1 and hDim2 to serve as CRM1 adapter proteins to the small subunit. hLtv1 directly binds CRM1 via a C-terminal Leu-rich NES (unpublished data). However, cells depleted for both hRio2 and hLtv1 do not display an export defect stronger than that caused by hRio2 depletion alone, and overexpression of an NES-deficient mutant of hLtv1 in human cells does not cause a pre-40S export defect (unpublished data). Collectively, our experiments so far failed to provide positive evidence for a contribution of hLtv1 to 40S export. Recently, a conserved NES in yeast Dim2p has been implicated in 40S biogenesis and export (Vanrobays et al., 2008). Notably, we can indeed show that hDim2 directly binds CRM1 (Fig. S5); however, mutation of the CRM1-binding site proposed in hDim2 (Vanrobays et al., 2008) did not abolish in vitro CRM1 binding of hDim2 (Fig. S5). For these reasons, the elucidation of the potential roles of hDim2 and hLtv1 in pre-40S export remains a challenging subject for future studies.
hRio2 is required for distinct steps of cytoplasmic 40S maturation
In contrast to the nonessential role of hRio2 in pre-40S export, cytoplasmic 40S maturation strongly depends on hRio2. Our RNAi analysis revealed that hRio2 is necessary for the recycling of four 40S-associated trans-acting factors. Furthermore, by performing rescue experiments with kinase-active and KD hRio2, we could separate two events in cytoplasmic 40S biogenesis.
In a first step, which depends on the physical presence of hRio2 but not on its kinase activity, hEnp1 is released from 40S precursors and reimported into the nucleus. Several models can be put up to explain the requirement for hRio2 in this process. For example, a so far unknown hEnp1 recycling factor may depend on hRio2 to be activated or to associate with 40S precursors. Alternatively, the absence of hRio2 may alter the structure of pre-40S particles such that hEnp1 release is no longer possible. As the kinase activity of hRio2 is not required for this recycling step, it appears that phosphorylation of hEnp1 by hRio2 is not involved in hEnp1 release from the subunit. Interestingly, phosphorylation of yeast Enp1p is known to promote its dissociation from 40S precursors in the course of the formation of the 40S "beak" structure (Schafer et al., 2006). In agreement with our results, this previous study demonstrated a requirement for the casein kinase isoform Hrr25p but not Rio2p in Enp1p phosphorylation. However, the recycling of yeast 40S trans-acting factors from the cytoplasm to the nucleus has not yet been addressed, and it remains to be seen whether Hrr25p-mediated phosphorylation of Enp1p contributes to its recycling to the nucleus. Our results identify an important, kinase-independent role for hRio2 in hEnp1 release from 40S precursors.
In contrast to its function in hEnp1 recycling, hRio2 kinase activity is essential for a second phase of cytoplasmic 40S maturation, in which cleavage of 18S-E pre-rRNA takes place and hDim2, hLtv1, and hNob1 are released from maturing 40S preribosomes. It is still unclear how the kinase activity of hRio2 relates to its function in pre-rRNA processing. hRio2 may phosphorylate and thereby activate the endonuclease itself or a cofactor of the endonuclease. Alternatively, hRio2 kinase activity could be required for a conformational change in the 40S precursor that is a prerequisite for endonuclease activity, or for the removal of an inhibitory factor. Indeed, it is tempting to speculate that hRio2 triggers the release of hDim2, hLtv1, and/or hNob1 by phosphorylating one or several of these factors, which, in turn, allows pre-rRNA processing to occur. Unraveling the molecular mechanism of hRio2 function will likely require knowledge of the substrate of hRio2 kinase activity, but despite our attempts to identify hRio2 phosphorylation targets, they remain elusive to date.
In summary, we could show that late maturation of 40S preribosomes depends on hRio2 at several distinct steps. hRio2 supports pre-40S export efficiency and may act as an adapter protein for CRM1. After nuclear export, hRio2 but not its kinase activity is required for release of hEnp1 from 40S precursors. Then, phosphorylation of an unknown target by hRio2 leads to the recycling of hDim2, hLtv1, and hNob1 and to 18S-E pre-rRNA processing. Therefore, cytoplasmic 40S biogenesis consists of at least two steps, both of which depend on hRio2. Further analysis of other trans-acting factors involved in this process may lead to the identification of additional maturation events, which collectively allow 40S subunits to reach translation competence.
|
Materials and methods |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Antibodies
Antibodies were raised against purified recombinant His-tagged proteins and affinity purified using the respective antigens coupled to SulfoLink (Thermo Fisher Scientific). Peptide-specific antibodies against a C-terminal peptide of hNmd3 (C-DLHISQDATGEEGASMLT) and a C-terminal peptide of hLtv1 (C-RQEKELLNLKKNVEGLKL) were also raised in rabbits and purified as described above. Peptide-specific 
-hLtv1 antibody was used for Western blotting, and anti–recombinant hLtv1 was used for immunofluorescence. -CRM1 (Lund et al., 2004) and -Rpl23A (Pool et al., 2002; Thomas and Kutay, 2003) antibodies have been previously described. Anti–β-actin was purchased from Sigma-Aldrich, anti-hRpl10 was purchased from Santa Cruz Biotechnology, Inc., and secondary antibodies for immunofluorescence were purchased from Invitrogen.
Molecular cloning, protein expression, and purification
Coding regions of hDim2, hEnp1, hLtv1, hNob1, hRio2, hRps2, and hRps3 were amplified by PCR from HeLaY cell cDNA. For expression in Escherichia coli, fragments were cloned into pQE (QIAGEN) or pET (EMD) derivatives. For transient transfection in HeLa cells, hRio2 was cloned into the XhoI–BamHI sites of pEGFP-C1 (Clontech Laboratories, Inc.) or an analogous pCFP vector. Note that three individual cDNA clones of hRio2 contained the same three nucleotide changes, leading to one amino acid change compared with the GenBank/EMBL/DDBJ database sequence (NM_018343; from Arg to Gly at position 349).
Site-directed mutagenesis of hRio2 (L400A and I403A [NESmut] or K123A and D246A [KD hRio2]) was performed using the QuikChange kit (Agilent Technologies). Deletion of the NES of hRio2 (NES10 construct) was performed using PCR fragments amplified from the full-length hRio2 construct. aa 309–408 of hRio2 were replaced by a Gly-Thr sequence based on a KpnI site used for cloning. Coding sequences for aa 388–410 of hRio2 and 475–493 of hNmd3 were inserted into the XhoI–BamHI sites of pEGFP-C1.
zz-hRio2, zz-hRio2 mutants, and zz-hEnp1 were expressed in BLR(pRep4) cells at 25°C. Cells were harvested by centrifugation and lysed by sonication in 50 mM Tris/HCl, pH 7.6, 700 mM NaCl, 5 mM MgCl2, and 5% (vol/vol) glycerol. The lysate was ultracentrifuged and passed over nickel–nitrilotriacetic acid agarose (QIAGEN), and the protein was eluted with 400 mM imidazole in lysis buffer. Purified proteins were rebuffered to 50 mM Tris/HCl, pH 7.6, 200 mM NaCl, and 5 mM MgCl2. Recombinant hRanQ69L-GTP and CRM1 were prepared as previously described (Muhlhausser et al., 2001).
Transient transfections and RNAis
Transient transfections were performed using FuGENE reagent (Roche). After 24 h, cells were fixed in 4% PFA in PBS, washed, and mounted for confocal microscopy. For RNAi experiments, the following siRNA oligonucleotides (Microsynth) were used (sense sequence): si-CRM1-1 (5'-UGUGGUGAAUUGCUUAUAC-3') and si-Nmd3-a (5'-GAAUGGUGCUAUCCUUCAA-3') at 35 nM concentration and si-Rio2-d (5'-GGAUCUUGGAUAUGUUUAA-3') at 17.5 nM. As a negative control, AllStars (QIAGEN) was used at 35 nM. Note that si-Rio2-d targets the 3' untranslated region of hRio2 mRNA, allowing rescue experiments with WT hRio2. siRNAs were transfected using Oligofectamine (Invitrogen). After 48 h of RNAi, cells were fixed for immunofluorescence analysis or harvested for Western blotting, extract preparation, or RNA isolation. In rescue experiments, cells were transfected with rescue constructs 24 h before harvest/fixation. In hRps2-YFP cells, reporter protein expression was induced with 0.125 µg/ml tet for 13 h before harvest/fixation.
Immunofluorescence analysis
Cells were fixed in 4% PFA and permeabilized in 0.1% Triton X-100 and 0.02% SDS in PBS for 5 min. After blocking with 10% goat serum in 2% BSA/PBS for 30 min, primary antibodies diluted in blocking solution were added for 1 h. Cells were washed three times for 4 min in 2% BSA/PBS and incubated with secondary antibody (Alexa Fluor 488– or Alexa Fluor 633–labeled goat anti–rabbit antibody) diluted in blocking solution for 30 min. Cells were again washed three times, incubated briefly in 0.1% Triton X-100 and 0.02% SDS in PBS, and fixed with 4% PFA. After washing with PBS, coverslips were mounted in VectaShield (Vector Laboratories) for confocal microscopy.
Confocal microscopy
Images from fixed transfected or immunostained cells were taken at RT with a confocal scanning system (SP2; Leica) using a 63x NA 1.4 differential interference contrast oil HCX Plan-Apochromat objective and the confocal software (Leica). Excitation at 405 nm was used for CFP fluorescence, 488 nm for EGFP, YFP, or Alexa Fluor 488, and 633 nm for Alexa Fluor 633. 
correction for FISH pictures was performed using Photoshop (Adobe).
CRM1-binding assay
200 pmol of purified bait proteins was immobilized on IgG Sepharose (GE Healthcare) in 50 mM Tris/HCl, pH 7.6, 175 mM KCH3CO2, 2 mM MgCl2, and 0.001% (vol/vol) Triton X-100. Beads were incubated with 1 µM CRM1 and 0.16 mg/ml BSA in a volume of 100 µl in the presence or absence of 3 µM hRanQ69L-GTP. Binding reactions were incubated on ice for 2 h. After washing three times, beads were transferred to spin columns (Mobicol; Life Systems Design), washed once with 50 mM Tris/HCl, pH 7.6, and eluted in SDS-PAGE sample buffer.
Kinase assays
10-µl reactions containing 1 µg of purified protein and 1 µCi -[32P]ATP in 50 mM Tris/HCl, pH 7.6, 200 mM NaCl, and 5 mM MgCl2 were incubated at 30°C for 30 min. Reactions were stopped by the addition of 30 µl of SDS sample buffer and analyzed by SDS-PAGE, and the gels were dried and analyzed by phosphoimaging.
Extract preparation
For extract preparation, cells were incubated for 10 min with 100 µg/ml cycloheximide (Sigma-Aldrich), washed once with PBS, detached with PBS containing 0.5 mM EDTA and 100 µg/ml cycloheximide, and centrifuged (2000 g for 5 min at 4°C). Cells were washed with 10 mM Tris/HCl, pH 7.4, 10 mM KCl, 2 mM MgCl2, and 50 µg/ml cycloheximide, centrifuged again, and resuspended in the same buffer containing 1 mM EGTA, 1 mM DTT, 0.05% Triton X-100, and protease inhibitors. After incubation on ice for 20 min, lysis was performed by passing cells through a 27-gauge needle. After centrifugation (1000 g for 5 min at 4°C), the supernatant was used as cell extract. For Fig. S1 (A–C), the extraction of cells was performed with 50 mM Tris/HCl, pH 7.4, 100 mM KCl, 2 mM MgCl2, 0.5% NP-40, 50 µg/ml cycloheximide, and protease inhibitors.
Sucrose gradient analysis
For analysis of RNAi experiments, cell extracts (400 µg of total protein) were loaded onto linear 10–45% sucrose gradients in 50 mM Hepes/KOH, pH 7.5, 100 mM KCl, and 3 mM MgCl2 and centrifuged for 2 h at 55,000 rpm at 4°C in a TLS55 rotor (Beckman Coulter). 160-µl fractions were collected, precipitated with TCA, and analyzed by Western blotting. To analyze the association of hRio2 and hEnp1 with ribosomal particles, cell extract (1 mg total protein) was loaded on 10–45% sucrose gradients and centrifuged for 3 h at 40,000 rpm at 4°C in an SW41 rotor (Beckman Coulter). Fractions were collected and precipitated with TCA for Western blotting. To prepare pooled 40S fractions for immunoprecipitation, HeLa cell low salt extract (Kutay et al., 1998) was fractionated on 10–45% sucrose gradients by centrifugation for 13 h and 50 min at 18,600 rpm at 4°C in an SW32 rotor (Beckman Coulter). 40S-containing fractions were pooled.
Immunoprecipitation
hRio2- and hNmd3-specific antibodies coupled to a 9:1 mixture of protein A/protein G–Sepharose beads (GE Healthcare) were incubated with pooled 40S fractions in 50 mM Tris/HCl, pH 7.6, 225 mM KCH3CO2, 2 mM MgCl2, and 0.001% (vol/vol) Triton X-100. After washing, beads were eluted with 0.2 M Gly/HCl, pH 2.2. Eluates were precipitated with TCA and analyzed by SDS-PAGE. Bands were cut from the gel, digested with trypsin, and analyzed by MS at the Functional Genomics Center Zurich.
Northern blotting
RNA was isolated from HeLaK cells using the RNeasy Mini kit (QIAGEN). 2 µg RNA was separated on a 1% agarose gel in 30 mM triethanolamine, 30 mM tricine, and 1.25% formaldehyde, transferred to a Hybond-N+ membrane (GE Healthcare) in 0.5x TBE buffer (45 mM Tris base, 44 mM boric acid, and 1 mM EDTA) by electrotransfer, and UV cross-linked to the membrane. Equal loading of the samples was analyzed by ethidium bromide staining. The membrane was prehybridized in 0.5 M Na phosphate, pH 7.2, 1 mM EDTA, 7% SDS, and 1% BSA for 30 min and hybridized with a radiolabeled 5' ITS1 probe (5'-CCTCGCCCTCCGGGCTCCGTTAATGATC-3') in the same buffer at 45°C overnight. After rinsing twice with 2x SSC/0.1% SDS and washing once for 10 min with 1x SSC/0.1% SDS, the membrane was exposed for phosphoimaging.
FISH
FISH was performed as described previously (Rouquette et al., 2005) using a 5' Cy5–labeled, 5' ITS1–specific probe (5'-CCTCGCCCTCCGGGCTCCGTTAATGATC-3'; Microsynth).
Online supplemental material
Fig. S1 shows sucrose gradient analyses to reveal pre-40S association of hRio2 and hEnp1 in LMB-treated cells as well as incorporation of hRps2-YFP into ribosomes in the reporter cell line. Fig. S2 shows Western blot analysis for the experiments of Fig. 4 A and a tet release experiment in the reporter cell line as well as the analysis of pre-40S export defects upon hRio2 depletion in HeLaK FRT TetR cells. Fig. S3 demonstrates that hRio2-depleted hRps2-YFP and HeLaY cells display recycling defects. Fig. S4 shows the effect of hRps15-depletion on hRio2 localization in HeLaK cells. Fig. S5 demonstrates that mutation of Ile153A, Leu155, and Ile158 of hDim2 to Ala does not abolish its CRM1-binding activity. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200904048/DC1.
|
Acknowledgments |
|---|
This work was supported by grants from the Swiss National Science Foundation, the ETH Zurich (both to U. Kutay), and the French Agence Nationale de la Recherche (Ribosome biogenesis in eukaryotes project; to P.-E. Gleizes).
Submitted: 8 April 2009
Accepted: 2 June 2009
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Bernad, R., D. Engelsma, H. Sanderson, H. Pickersgill, and M. Fornerod. 2006. Nup214-Nup88 nucleoporin subcomplex is required for CRM1-mediated 60 S preribosomal nuclear export. J. Biol. Chem. 281:19378–19386.
Bonazzi, S., S. Güttinger, I. Zemp, U. Kutay, and K. Gademann. 2007. Total synthesis, configuration, and biological evaluation of anguinomycin C. Angew. Chem. Int. Ed. Engl. 46:8707–8710.[CrossRef][Medline]
Bradatsch, B., J. Katahira, E. Kowalinski, G. Bange, W. Yao, T. Sekimoto, V. Baumgartel, G. Boese, J. Bassler, K. Wild, et al. 2007. Arx1 functions as an unorthodox nuclear export receptor for the 60S preribosomal subunit. Mol. Cell. 27:767–779.[CrossRef][Medline]
Fornerod, M., M. Ohno, M. Yoshida, and I.W. Mattaj. 1997. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell. 90:1051–1060.[CrossRef][Medline]
Fromont-Racine, M., B. Senger, C. Saveanu, and F. Fasiolo. 2003. Ribosome assembly in eukaryotes. Gene. 313:17–42.[CrossRef][Medline]
Fujiyama, S., M. Yanagida, T. Hayano, Y. Miura, T. Isobe, F. Fujimori, T. Uchida, and N. Takahashi. 2002. Isolation and proteomic characterization of human Parvulin-associating preribosomal ribonucleoprotein complexes. J. Biol. Chem. 277:23773–23780.
Gadal, O., D. Strauss, J. Kessl, B. Trumpower, D. Tollervey, and E. Hurt. 2001. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol. Cell. Biol. 21:3405–3415.
Geerlings, T.H., A.W. Faber, M.D. Bister, J.C. Vos, and H.A. Raue. 2003. Rio2p, an evolutionarily conserved, low abundant protein kinase essential for processing of 20 S Pre-rRNA in Saccharomyces cerevisiae. J. Biol. Chem. 278:22537–22545.
Gleizes, P.E., J. Noaillac-Depeyre, I. Leger-Silvestre, F. Teulieres, J.Y. Dauxois, D. Pommet, M.C. Azum-Gelade, and N. Gas. 2001. Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex. J. Cell Biol. 155:923–936.
Granneman, S., and S.J. Baserga. 2004. Ribosome biogenesis: of knobs and RNA processing. Exp. Cell Res. 296:43–50.[CrossRef][Medline]
Hayano, T., M. Yanagida, Y. Yamauchi, T. Shinkawa, T. Isobe, and N. Takahashi. 2003. Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome. J. Biol. Chem. 278:34309–34319.
Henras, A.K., J. Soudet, M. Gerus, S. Lebaron, M. Caizergues-Ferrer, A. Mougin, and Y. Henry. 2008. The post-transcriptional steps of eukaryotic ribosome biogenesis. Cell. Mol. Life Sci. 65:2334–2359.[CrossRef][Medline]
Ho, J.H., G. Kallstrom, and A.W. Johnson. 2000. Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J. Cell Biol. 151:1057–1066.
Hung, N.J., K.Y. Lo, S.S. Patel, K. Helmke, and A.W. Johnson. 2008. Arx1 is a nuclear export receptor for the 60S ribosomal subunit in yeast. Mol. Biol. Cell. 19:735–744.
Hurt, E., S. Hannus, B. Schmelzl, D. Lau, D. Tollervey, and G. Simos. 1999. A novel in vivo assay reveals inhibition of ribosomal nuclear export in ran-cycle and nucleoporin mutants. J. Cell Biol. 144:389–401.
Kudo, N., N. Matsumori, H. Taoka, D. Fujiwara, E.P. Schreiner, B. Wolff, M. Yoshida, and S. Horinouchi. 1999. Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. Proc. Natl. Acad. Sci. USA. 96:9112–9117.
Kutay, U., and S. Güttinger. 2005. Leucine-rich nuclear-export signals: born to be weak. Trends Cell Biol. 15:121–124.[CrossRef][Medline]
Kutay, U., F.R. Bischoff, S. Kostka, R. Kraft, and D. Gorlich. 1997. Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor. Cell. 90:1061–1071.[CrossRef][Medline]
Kutay, U., G. Lipowsky, E. Izaurralde, F.R. Bischoff, P. Schwarzmaier, E. Hartmann, and D. Gorlich. 1998. Identification of a tRNA-specific nuclear export receptor. Mol. Cell. 1:359–369.[CrossRef][Medline]
Leger-Silvestre, I., P. Milkereit, S. Ferreira-Cerca, C. Saveanu, J.C. Rousselle, V. Choesmel, C. Guinefoleau, N. Gas, and P.E. Gleizes. 2004. The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast. EMBO J. 23:2336–2347.[CrossRef][Medline]
Lund, E., S. Güttinger, A. Calado, J.E. Dahlberg, and U. Kutay. 2004. Nuclear export of microRNA precursors. Science. 303:95–98.
Moy, T.I., and P.A. Silver. 1999. Nuclear export of the small ribosomal subunit requires the ran-GTPase cycle and certain nucleoporins. Genes Dev. 13:2118–2133.
Moy, T.I., and P.A. Silver. 2002. Requirements for the nuclear export of the small ribosomal subunit. J. Cell Sci. 115:2985–2995.
Muhlhausser, P., E.C. Muller, A. Otto, and U. Kutay. 2001. Multiple pathways contribute to nuclear import of core histones. EMBO Rep. 2:690–696.[CrossRef][Medline]
Oeffinger, M., M. Dlakic, and D. Tollervey. 2004. A pre-ribosome-associated HEAT-repeat protein is required for export of both ribosomal subunits. Genes Dev. 18:196–209.
Pool, M.R., J. Stumm, T.A. Fulga, I. Sinning, and B. Dobberstein. 2002. Distinct modes of signal recognition particle interaction with the ribosome. Science. 297:1345–1348.
Rouquette, J., V. Choesmel, and P.E. Gleizes. 2005. Nuclear export and cytoplasmic processing of precursors to the 40S ribosomal subunits in mammalian cells. EMBO J. 24:2862–2872.[CrossRef][Medline]
Schafer, T., D. Strauss, E. Petfalski, D. Tollervey, and E. Hurt. 2003. The path from nucleolar 90S to cytoplasmic 40S pre-ribosomes. EMBO J. 22:1370–1380.[CrossRef][Medline]
Schafer, T., B. Maco, E. Petfalski, D. Tollervey, B. Bottcher, U. Aebi, and E. Hurt. 2006. Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit. Nature. 441:651–655.[CrossRef][Medline]
Seiser, R.M., A.E. Sundberg, B.J. Wollam, P. Zobel-Thropp, K. Baldwin, M.D. Spector, and D.E. Lycan. 2006. Ltv1 is required for efficient nuclear export of the ribosomal small subunit in Saccharomyces cerevisiae. Genetics. 174:679–691.
Stade, K., C.S. Ford, C. Guthrie, and K. Weis. 1997. Exportin 1 (Crm1p) is an essential nuclear export factor. Cell. 90:1041–1050.[CrossRef][Medline]
Stage-Zimmermann, T., U. Schmidt, and P.A. Silver. 2000. Factors affecting nuclear export of the 60S ribosomal subunit in vivo. Mol. Biol. Cell. 11:3777–3789.
Thomas, F., and U. Kutay. 2003. Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway. J. Cell Sci. 116:2409–2419.
Trotta, C.R., E. Lund, L. Kahan, A.W. Johnson, and J.E. Dahlberg. 2003. Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates. EMBO J. 22:2841–2851.[CrossRef][Medline]
Tschochner, H., and E. Hurt. 2003. Pre-ribosomes on the road from the nucleolus to the cytoplasm. Trends Cell Biol. 13:255–263.[CrossRef][Medline]
Udem, S.A., and J.R. Warner. 1973. The cytoplasmic maturation of a ribosomal precursor ribonucleic acid in yeast. J. Biol. Chem. 248:1412–1416.
Vanrobays, E., P.E. Gleizes, C. Bousquet-Antonelli, J. Noaillac-Depeyre, M. Caizergues-Ferrer, and J.P. Gelugne. 2001. Processing of 20S pre-rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non-ribosomal cytoplasmic protein. EMBO J. 20:4204–4213.[CrossRef][Medline]
Vanrobays, E., J.P. Gelugne, P.E. Gleizes, and M. Caizergues-Ferrer. 2003. Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae. Mol. Cell. Biol. 23:2083–2095.
Vanrobays, E., A. Leplus, Y.N. Osheim, A.L. Beyer, L. Wacheul, and D.L. Lafontaine. 2008. TOR regulates the subcellular distribution of DIM2, a KH domain protein required for cotranscriptional ribosome assembly and pre-40S ribosome export. RNA. 14:2061–2073.
Yanagida, M., A. Shimamoto, K. Nishikawa, Y. Furuichi, T. Isobe, and N. Takahashi. 2001. Isolation and proteomic characterization of the major proteins of the nucleolin-binding ribonucleoprotein complexes. Proteomics. 1:1390–1404.[CrossRef][Medline]
Yao, W., M. Lutzmann, and E. Hurt. 2008. A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export. EMBO J. 27:6–16.[CrossRef][Medline]
Zemp, I., and U. Kutay. 2007. Nuclear export and cytoplasmic maturation of ribosomal subunits. FEBS Lett. 581:2783–2793.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
, large, hydrophobic amino acid (underlined and bold in Rio2 sequences); x, any amino acid. Leu400 and Ile403, the two residues mutated in hRio2(NESmut), are depicted in red. (C) Mutation of the NES of hRio2 leads to its nuclear accumulation. HeLaK cells were transiently transfected with EGFP-hRio2, EGFP-hRio2(NESmut) or EGFP-hRio2(NES
