This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Selitrennik, M.
Right arrow Articles by Choder, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Selitrennik, M.
Right arrow Articles by Choder, M.

 Previous Article  |  Next Article 

Eukaryotic Cell, December 2006, p. 2092-2103, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00288-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Nucleocytoplasmic Shuttling of the Rpb4p and Rpb7p Subunits of Saccharomyces cerevisiae RNA Polymerase II by Two Pathways{triangledown}

Michael Selitrennik, Lea Duek, Rona Lotan, and Mordechai Choder*

Department of Molecular Microbiology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel 31096

Received 7 September 2006/ Accepted 2 October 2006


arrow
ABSTRACT
 
Rpb4p and Rpb7p are subunits of the RNA polymerase II of Saccharomyces cerevisiae that form a dissociable heterodimeric complex. Whereas the only reported function of Rpb7p is related to transcription, Rpb4p has been found to also act in mRNA export and in the major mRNA decay pathway that operates in the cytoplasm, thus raising the possibility that Rpb4p links between the nuclear and cytoplasmic processes. Here we show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Shuttling kinetics of the two proteins are similar as long as their interaction is possible, suggesting that they shuttle as a heterodimer. Under normal conditions, shuttling of Rpb4p and Rpb7p depends on ongoing transcription. However, during severe stresses of heat shock, ethanol, and starvation, the two proteins shuttle via a transcription-independent pathway. Thus, Rpb4p and Rpb7p shuttle via two pathways, depending on environmental conditions.


arrow
INTRODUCTION
 
Cellular components function in the context of their location within the cell. Therefore, an important posttranslational control of many cellular proteins determines their precise localization. Coordination between the two main cellular compartments, the nucleus and the cytoplasm, is mediated by the nuclear pore complexes (NPCs) embedded within the nuclear envelope. NPCs are dynamic complexes composed of ~30 proteins called nucleoporins. They facilitate the bidirectional transport of macromolecules across the nuclear envelope (recently reviewed in references 31 and 36). Protein transport is also mediated by cargo-specific machineries that facilitate rapid, directional, and selective transport (40). Transport of various RNA molecules is mediated by transport machineries specific for RNP proteins whose translocation drives the RNA transport (6). The association of several known RNA binding proteins begins during transcription (24). Following mRNP export, or at a late stage during export, some proteins are removed from the mRNP, allowing them to return to the nucleus for an additional round of mRNA export (see references 18 and 25 and references therein). Thus, these proteins shuttle between the two compartments. Whereas the various classes of RNAs are exported by class-specific pathways, recent works have suggested that export of mRNAs is governed by several distinct pathways (12, 13). One distinct pathway is involved in the export of stress-induced mRNAs (29).

Relatively little is known about stress-specific transport pathways. The results of a few studies have suggested that NPC and nucleocytoplasmic transport are modified in response to stress. For example, the DEAD box protein Rat8p/Dbp5p, which plays a role in mRNP remodeling at the cytoplasmic side of the NPC (18), dissociates from the NPC following ethanol (EtOH) stress (25). Export of the bulk of the mRNAs of Saccharomyces cerevisiae is blocked during severe stress (e.g., upon exposure to 42°C or high concentrations of EtOH), whereas that of stress-induced mRNAs is carried out efficiently in a process that requires most of the factors essential for normal poly(A)+ RNA export (28, 29). One mRNA protein required for normal export, Npl3p, is not required for export of heat shock (HS) mRNA during HS (14). Conversely, export of HS mRNAs during HS is dependent on Nup42p/Rip1p, a nucleoporin dispensable for export under nonstress conditions (25, 29, 34). Nup42p and Rpb4p are the only proteins known to be essential for mRNA export only during stress (see below).

Rpb4p is a subunit of RNA polymerase II (Pol II), which is composed of 12 subunits (termed Rpb1p to Rpb12p) (43). Crystal structures of the 12 subunits of yeast Pol II have revealed that the 12-subunit enzyme consists of two distinct substructures (2, 3). The first is a 10-subunit core that constitutes the bulk of the Pol II structure and includes the catalytic active site. The major subunits of this core reside in the nucleus under all environmental conditions tested (8). The second part consists of two subunits, Rpb4p and Rpb7p, which form a dissociable heterodimeric complex (recently reviewed in references 5 and 30). Rpb4p and Rpb7p are present in excess over other Pol II subunit quantities (26), suggesting that they also play roles outside the context of the polymerase (5). Indeed, recent studies have demonstrated that Rpb4p can be localized in the cytoplasm and have assigned roles for this Pol II subunit in mRNA export during stress (8) and in mRNA degradation (17). In light of these observations, Rpb4p was proposed to be involved in the linkage between the nuclear and the cytoplasmic machineries that together determine the mRNA level within the cell (17). One prediction of the linkage hypothesis is that Rpb4p shuttles between the nucleus and the cytoplasm. Whether Rpb7p is involved in the posttranscriptional roles that Rpb4p mediates is presently unknown.

Here we show that Rpb7p localization, in similarity to that of Rpb4p, is responsive to the environment. We further show that both Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Interestingly, two distinct shuttling mechanisms transport the two proteins, depending on the environmental conditions. One is dependent on ongoing transcription, linking the functions of Rpb4 and Rbp7 in the nucleus with those in the cytoplasm. The other operates during severe stress conditions and is independent of transcription.


arrow
MATERIALS AND METHODS
 
Yeast strains and growth conditions. Yeast strains are listed in Table 1. Cells proliferated either in rich medium (2% glucose, 2% peptone, 1% yeast extract [YPD]) or, when selective conditions were required, in standard synthetic media.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Saccharomyces cerevisiae strains used in this study

Starvation was obtained by shifting the cells to synthetic medium lacking any sugar and lacking amino acids.

Plasmid construction. The plasmid carrying RPB4p-green fluorescent protein-RPB4 (RPB4p-GFP-RPB4) was described previously (8). Plasmids carrying RPB7-RFP or RPB4-RFP are derivatives of pRP1084 (pLSM1-RFP) (a generous gift of Roy Parker) (32). This plasmid was cut with HindIII and BamHI, and the vector that contained the RFP was ligated either to the RPB4p-RPB4-open reading frame (ORF) fragment (RPB4p-RPB4-ORF) or to the RP74p-RPB7-ORF fragment (both ORF fragments lacking the stop codon were inserted in frame with the RFP ORF). pMC116 is a pCM190 (9) derivative that contains 170 bp of an RPB7 5' noncoding region and an RPB7 open reading frame at the NotI-BamHI site. pMC257 is an HIS3 derivative of pCM189 (9). All plasmids are centromeric and carry a URA3, LEU2, or HIS3 selectable marker.

Creating temperature-sensitive (ts) rpb7 mutants. RPB7 was mutagenized randomly, utilizing a PCR mutagenesis scheme as described previously (8), OMC367 (5'-GCATGCATGTGCTCTGTATG-3'), and OMC368 (5'-GTTACATGCGTACACGCG), with PMC116 as the template. The mutagenized rpb7-containing fragment was inserted into NotI-HapI-linearized pMC257 by cotransformation in yMC140. pRPB7::URA3 present in yMC140 was evicted by selecting cells that lose URA3 by using a 5-fluoroorotic acid (5-FOA) plate, resulting in cells that carry only the mutagenized rpb7. 5-FOA selection was done twice. 5-FOA-resistant strains were allowed to form colonies at 24°C and 37°C. Mutants that were unable to form colonies at 37°C were selected.

Fluorescent microscopy. Fluorescent microscopy was done as previously described (8, 17).

Two-hybrid assay. The bait RPB7-activating domain (AD) or AD-tagged rpb7 mutants were constructed as described previously (38). Two-hybrid interaction was determined by growth on plates lacking leucine, tryptophane, adenine, and histidine and supplemented with 5 mM 3-amino-1,2,4-triazole and by the ß-galactosidase liquid test, as previously reported (38).


arrow
RESULTS
 
Rpb7p is exported to the cytoplasm in response to various stress conditions. We have previously found that Rpb4 is exported to the cytoplasm in response to HS, EtOH stress, or starvation conditions (8). Since Rpb4p and Rpb7p are known to function in transcription as a heterodimer (5), we tested whether Rpb7p is also exported to the cytoplasm in response to these stress conditions. To this end, we created a strain that carries the RPB7-GFP fusion gene in place of RPB7 (yMC286). These cells proliferate similarly to wild-type (WT) cells, indicating that the fusion protein that replaces the essential Rpb7p is functionally similar to Rpb7p. As shown in Fig. 1, Rpb7p-GFP was localized to the nucleus during proliferation under optimal conditions, as expected from an RNA Pol II subunit. However, in response to HS, EtOH stress, or starvation, Rpb7p-GFP was relocated and found mainly in the cytoplasm. Addition of the translation inhibitor cycloheximide concomitant with a shift to the stress conditions did not affect Rpb7p-GFP relocation (Fig. 1). These results indicate that the Rpb7p-GFP molecules found in the cytoplasm were not newly synthesized and that no newly synthesized factors are required for relocation of Rpb7p-GFP. Thus, Rpb7p-GFP is exported from the nucleus to the cytoplasm in response to stresses in the form of HS, EtOH, or starvation. Since Rpb7p is required for transcription (5), some portion of it must remain in the nucleus. However, as Rpb7p molecules are present in excess over the Pol II complex quantities (5), the cytoplasmic fluorescence of Rpb7p-GFP masks its nuclear fluorescence. A similar rationale holds for Rpb4p-GFP localization during stress (8).


Figure 1
View larger version (48K):
[in this window]
[in a new window]

  		FIG. 1. Rpb7p-GFP localization is responsive to the environment. (A) Rpb7p is exported to the cytoplasm in response to various stress conditions. Cells carrying RPB7-GFP in place of RPB7 (yMC286) were allowed to proliferate for at least eight generations under optimal conditions in rich medium (YPD) at either 24°C (HS experiment) or 30°C (other experiments) until the mid-logarithmic phase (≤5 x 106 cells/ml). Cells were then treated with 50 µg/ml cycloheximide except for the starvation culture and were challenged with stress as indicated. The nonstressed samples were also treated with 50 µg/ml cycloheximide and were further incubated in parallel with the stress-treated cultures. "Starvation" denotes synthetic medium lacking sugar and amino acids. Rpb7p-GFP localization was examined by fluorescent microscopy 3 h after HS or 12 h after addition of EtOH or after 3 days of starvation. The middle panels of the HS experiment show the position of the nuclei stained by DAPI (4',6'-diamidino-2-phenylindole), and the bottom panels show bright-field (BF) images of the cells; the results shown demonstrate that Rpb7p localization during optimal proliferation is nuclear whereas after HS it is cytoplasmic. (B) Cytoplasmic Rpb7p-GFP in starved cells was reimported to the nucleus after cells were refed with glucose. Starved cells were refed with 2% glucose and divided into two cultures. One culture (designated CHX) was treated with 50 µg/ml cycloheximide, and the cells were examined microscopically 90 min after addition of glucose. In the absence of the drug, the kinetics of Rpb7p-GFP relocation was similar to that observed in its presence (results not shown). Note that in response to starvation (see panels A and B), Rpb7p-GFP was localized in discrete cytoplasmic foci that are smaller than nuclei. Some are denoted by arrows. These foci probably represent cytoplasmic P bodies that also accommodate Rpb4p (17). The bars represent 5µ.

The cytoplasmic Rpb7p in glucose-starved cells reentered the nucleus when glucose was reintroduced (Fig. 1B). Reentry was insensitive to cycloheximide after reentry, indicating that reentry does not require newly synthesized proteins. Thus, the subcellular localization of Rpb7p is responsive to the environment. In contrast with Rpb4p and Rpb7p, the core Pol II subunits Rpb2p and Rpb3p are localized constitutively in the nucleus during both optimal and stress conditions (8).

Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm. Our observations showing that the cellular localization of Rpb4p and Rpb7p is responsive to the environment raised the possibility that these proteins are capable of shuttling between the nucleus and the cytoplasm. To examine this possibility, we performed a standard nuclear export assay (15). This assay takes advantage of ts nucleoporin mutant nup49-313. At the restrictive temperature, import into the nucleus is blocked whereas export out of the nucleus is unaffected. Thus, if the fluorescent proteins normally shuttle between the nucleus and the cytoplasm, they should accumulate in the cytoplasm of the nup49-313 cells when these cells are shifted to 37°C, the nonpermissive temperature. Several shuttling factors have been identified using this assay (see, e.g., references 15 and 25). This assay was also useful in determining that several mRNA transport proteins, e.g., Rna14p, Rna15p, and Pcf11p, do not shuttle (11). RPB7-red fluorescent protein (RPB7-RFP) and GFP-RPB4 were coexpressed in the mutant cells carrying nup49-313. As shown in Fig. 2A, both proteins were found mainly in the nucleus upon incubation of nup49-313 cells at 24°C; however, they accumulated in the cytoplasm when the cells were shifted to the nonpermissive temperature (Fig. 2A and control results in 2D). Since cycloheximide was added before the increase in temperature, we concluded that the appearance of tagged Rpb4p and Rpb7p in the cytoplasm upon increasing the temperature was not due to accumulation of newly synthesized protein. In contrast with the rapid relocation of Rpb4p and Rpb7p in nup49-313 cells (Fig. 2A), incubation of WT cells at 37°C resulted in much slower relocation kinetics, so that by 5 h both Rpb4p and Rpb7p were still mainly in the nucleus (Fig. 2B). Immunoblotting of cell lysates made before and after the temperature increase confirmed that the Rpb4p and Rpb7p levels as well as integrity were not affected by incubating cells at 37°C (4, 8). Therefore, the cytoplasmic signal was due to presence of the intact fusion proteins. This localization pattern is indicative of shuttling activity and is characteristic of other shuttling proteins (see, e.g., references 11 and 15).


Figure 2
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 2. GFP-Rpb4p and Rpb7p-RFP shuttle by a transcription-dependent mechanism. (A) Shuttling of both proteins was demonstrated using nup49-313(ts) mutant cells that are defective in protein import at elevated temperatures. Cells expressing both GFP-Rpb4p and Rpb7p-RFP (yMS3) were allowed to proliferate for at least eight generations under optimal conditions in selective media at 24°C. Both fluorescent proteins were then localized in the nucleus (results not shown). Cycloheximide (50 µg/ml) was then added, and the culture was divided into two samples. One was incubated for an additional 5 h at 24°C and the other for 5 h at 37°C. Images were then taken using green (GFP-Rpb4p), red (Rpb7p-RFP), or blue (to detect nuclei stained by DAPI) filters, as indicated. (B) Images of WT cells expressing Rpb4p-RFP and Rpb7p-GFP (yMS8) at 24°C or 5 h after incubation at 37°C in the presence of 50 µg/ml cycloheximide. (C) Export is blocked when transcription is arrested. Images of nup49-313(ts) rpb1-1(ts) cells expressing both GFP-Rpb4p and Rpb7p-RFP (yMS4) at 24°C or 5 h after incubation at 37°C are shown. Cycloheximide was added as described for panel A. (D) Export kinetics of Rpb7p-RFP after a shift from 24°C to 37°C in the absence (Cont.) or presence ({Phi}) of the transcription inhibitor 1,10-phenanthroline (100 µg/ml). Cycloheximide was added as described for panel A. To monitor export kinetics, samples of each culture were examined microscopically at the indicated time points and photographs of random fields were taken using both the green and red channels. More than 200 cells were classified into those exhibiting nuclear or whole-cell (i.e., cytoplasmic) localization of the respective fluorescent protein. The proportion of cells exhibiting cytoplasmic localization was plotted as a function of time. The merged images in panels A and C (created using Adobe PhotoShop) demonstrate that the distributions of the two fluorescent proteins were similar. The bars represent 5µ.

Rpb4p and Rpb7p shuttling is dependent on active transcription. One of the features that characterizes some shuttling factors involved in mRNA export is their dependency on transcription (1). Since they are Pol II subunits that also shuttle, it was important to determine whether their shuttling capacity was dependent upon ongoing transcription. To address this possibility, we performed the nuclear export assay using a strain carrying both nup49-313 and rpb1-1 encoding a ts mutant form of the Pol II subunit Rpb1p (22). Upon increase of the temperature of cells to 37°C, the products of both ts alleles became inactive, resulting in both import and transcription defects. In contrast to their capacity to exit the nucleus of nup49-313 cells upon heat inactivation of Nup49-313p, both GFP-Rpb4p and Rpb7p-RFP were unable to leave the nucleus of cells carrying both the nup49-313 and rpb1-1 alleles (Fig. 2C). These results indicate that efficient export of these proteins requires Rpb1p activity. To examine the dependency of GFP-Rpb4p and Rpb7p-RFP transport on transcription by other means, we used the drug 1,10-phenanthroline, which blocks transcription (see references 10 and 17 and references therein). Treating nup49-313 cells that carry WT RPB1 with this drug before the temperature shift upward blocked the export of both the tagged Rpb4p (data not shown) and the tagged Rpb7p (Fig. 2D). Taken together, the results lead us to conclude that ongoing transcription is necessary for export of Rpb4 and Rpb7.

In response to severe stresses, export of Rpb4p and Rpb7p is independent of transcription. It was previously found that, in response to severe HS (42°C) but not more moderate HS (e.g., 37°C), the export of most mRNAs is blocked and only HS mRNAs are exported (14, 28, 29, 34). Thus, different transport pathways are employed depending on the environment. We therefore compared the capacities of Rpb4p and Rpb7p to be exported under moderate or severe stress conditions. During moderate HS (37°C), GFP-Rpb4p and Rpb7p-RFP were retained in the nuclei in double-mutant nup49-313 rpb1-1 cells (Fig. 2C and 3A). In contrast, after these cells were shifted to severe HS (42°C), Rpb4p and Rpb7p were exported to the cytoplasm even under conditions that inactivate Rpb1-1p, i.e., in the absence of transcription (Fig. 3A). We next tested export of Rpb4p and Rpb7p during other severe stresses. Briefly, cells carrying both the nup49-313 and rpb1-1 alleles were shifted to 37°C to inactivate transcription and immediately thereafter challenged with either 6% EtOH or with starvation medium (see Materials and Methods). As in the response to severe HS, both Rpb4p and Rpb7p were also exported in response to EtOH and starvation, while transcription was blocked (Fig. 3A).


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		FIG. 3. Export of GFP-Rpb4p and Rpb7p-RFP during severe stresses is independent of transcription. (A) Images of nup49-313(ts) rpb1-1(ts) cells expressing both GFP-Rpb4p and Rpb7p-RFP (yMS4) in the mid-logarithmic phase at 24°C (left panels) or after 2 h under the indicated stress conditions in the presence of 50 µg/ml cycloheximide. The nonstressed sample was equally treated with cycloheximide and further incubated in parallel with the stress-treated cultures. The starved and EtOH-treated cultures were incubated at 37°C to inactivate transcription. The bars represent 5µ. (B) Export kinetics, determined as described for Fig. 2D, of Rpb7p-RFP after a shift from 24°C to 42°C in the absence (Cont.) or presence ({Phi}) of the transcription inhibitor 1,10-phenanthroline (100 µg/ml).

We next examined whether transcription-independent export of Rpb4p and Rpb7p can be observed by blocking the transcription of cells carrying WT Pol II by use of the drug 1,10-phenanthroline. Consistent with the results shown in Fig. 3A, Fig. 3B shows that the drug did not block export at 42°C, although it had a relatively minor inhibitory effect (compare Fig. 2D with 3B). These results indicate that during the tested severe stress conditions, export of Rpb4p and Rpb7p is largely independent of transcription (see Discussion).

Rpb4p and Rpb7p shuttle between the nucleus and the cytoplasm also during severe stresses. During severe stresses, when Rpb4p and Rpb7p are exported by a transcription-independent pathway and are found mainly in the cytoplasm (Fig. 1 and reference 8), a portion of these proteins must be located in the nucleus to enable transcription (5). This raises the question whether the stress-induced relocation reflects a one-way transport. Alternatively, these proteins also shuttle back and forth during severe stresses, but because their export is faster than their import, they gradually accumulate in the cytoplasm. In the latter case, accumulation of Rpb4p and Rpb7p in the cytoplasm under severe stress conditions is expected to be accelerated when their import is blocked due to Nup49-313p inactivation. To address the issue of shuttling during stress, we shifted nup49-313 cells producing either GFP-Rpb4p or Rpb7p-RFP to 37°C to inactivate protein import (see the results for nonpermissive temperature in Fig. 4A) and concomitantly challenged them with starvation or 6% EtOH or by further increasing the temperature to 42°C (severe HS). After a relatively short incubation period under these stress conditions, complete relocation was observed at the nonpermissive temperature, while relatively little relocation was observed at 24°C—a temperature that permits import (Fig. 4A). Although some relocation occurred at 37°C without additional stress, it was incomplete, as nuclear fluorescence was clearly detected (Fig. 4A). Moreover, little relocation was observed in WT cells exposed to even longer periods of the same stress conditions (Fig. 4B). These results indicate that, although Rpb4p (8) and Rpb7p (unpublished observation) are relocated very slowly in response to a temperature of 37°C, this temperature is not responsible per se for the rapid relocation in the mutant cells. Rather, the rapid relocation was due to inactivation of Nup49-313p. Taking these results together, we conclude that Rpb4p and Rpb7p shuttle under the examined stress conditions, as they do under more-hospitable conditions (see Fig. 2). We propose that under severe stress conditions Rpb4p and Rpb7p export is faster than their import and, hence, that they gradually accumulate in the cytoplasm.


Figure 4
View larger version (63K):
[in this window]
[in a new window]

  		FIG. 4. Rpb4p and Rpb7p shuttle during severe stress conditions. (A) Relocation in nup49-313 cells is faster at the nonpermissive temperature. nup49-313(ts) cells expressing both GFP-Rpb4p and Rpb7p-RFP (yMS3) were allowed to proliferate for at least eight generations under optimal conditions in selective media at 24°C. Cells were then challenged with the indicated stress at 24°C (Permissive temp.) (left panels) or at 37°C, or 42°C when indicated (Non-permissive temp.) (right panels). Cycloheximide (50 µg/ml) was added to all samples as described for Fig. 2A. (B) Little relocation occurs in WT cells. Images of WT cells expressing both GFP-Rpb4p and Rpb7p-RFP (yMS8) similarly challenged with the indicated conditions in the presence of cycloheximide are shown. NA, not applicable. The bars represent 5µ.

Rpb4p and Rpb7p are exported at similar kinetics in response to stresses of HS, starvation, and EtOH. Rpb4p plays a role both in transcription within the nucleus and in the major mRNA decay pathway in the cytoplasm (17). An unresolved issue is whether Rpb4p, which functions in transcription together with Rpb7p, also functions in mRNA decay in the context of the Rpb4-Rbp7 heterodimer. If the two proteins function in the cytoplasm as a heterodimer, it is likely that they shuttle together. If this is the case, some predictions can be made, the first being that the export kinetics of the two proteins are similar. We also predict that disruption of Rpb4p and Rpb7p interaction, either by deleting the nonessential RPB4 or by mutating the essential RPB7, would have an effect on the kinetics.

As a first approach, we determined the export kinetics of the two fluorescent proteins in response to stresses of HS, EtOH, and starvation and found them to be identical (Fig. 5A, B, and C). To rule out the possibility that the fluorescent tag influenced the export kinetics, we exchanged the tags between the two proteins and found that the tags had no specific effect. To illustrate this observation, we show in Fig. 5A and C the results obtained using Rpb4p-RFP and Rpb7p-GFP and in Fig. 5B the results obtained using GFP-Rpb4p and Rpb7p-RFP. In both cases similar export kinetics results were observed. Moreover, the import kinetics results obtained with GFP-Rpb4p and Rpb7p-RFP in response to shifting starved cells to rich medium were also identical (Fig. 5D). Careful microscopic examination of these proteins under various environmental conditions indicated that their localization patterns were always similar (see, for example, the merge panels in Fig. 2A and C; other results not shown). Only in cases in which the heterodimer could not be formed did we observe differing results (see Discussion).


Figure 5
View larger version (36K):
[in this window]
[in a new window]

  		FIG. 5. In response to various stresses, Rpb4p and Rpb7p are exported to the cytoplasm at similar kinetics. (A to C) yMS4 (A), yMS18 (B), and yMS8 (C) cells expressing the indicated fluorescence-tagged proteins were allowed to proliferate for at least eight generations under optimal conditions in selective media at either 24°C (A) or 30°C (B, C, and D) until the mid-logarithmic phase (5 x 106 cells/ml). Cultures were then challenged under the indicated stress condition as described for Fig. 1. Export kinetics experiments were performed as described for Fig. 2D. (D) Import kinetics of GFP-Rpb4p and Rpb7p-RFP are comparable. yRL82 cells were starved until both fluorescent proteins were localized in the cytoplasm in 80% or 90% of the cells. Cells were then collected by centrifugation and resuspended in synthetic complete medium (containing glucose and amino acids) supplemented with 50 µg/ml cycloheximide (similar results were obtained when cycloheximide was not added). Import kinetics results were determined as described for panels A to C.

Note that stress-induced relocation of Rpb4p and Rpb7p in the WT cells (shown in Fig. 5) was slower than it was in nup49-313(ts) cells at 37°C (shown in Fig. 3A). For example, after 2 h at 37°C in starvation medium, Rpb4p and Rpb7p were localized mainly in the cytoplasm in all mutant cells (see results for starvation at 37°C in Fig. 3A). In contrast, relocation of these proteins after starving WT cells for 2 h was marginal, as only a small fraction of these cells exhibited complete relocation (Fig. 5B). These differences are in accord with our results presented in Fig. 4 that show that Rpb4p and Rpb7p shuttle under stress conditions such that by blocking import (as was the case in the experiment whose results are shown in Fig. 3A) their relocation is accelerated (see also legend to Fig. 8).


Figure 8
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 8. Model of the proposed shuttling pathways of the Rpb4-Rbp7 heterodimer. Rpb4p and Rpb7p shuttle as a heterodimer. Under conditions that support proliferation, export of the Rpb4-Rbp7 heterodimer is dependent on transcription. Import of the heterodimer (thick arrow) is faster than its export (thin arrow), resulting in mainly nuclear localization. In response to some severe stress conditions, a relatively robust transcription-independent mechanism is induced (thick dashed arrow) in addition to a relatively weak transcription-dependent mechanism (thin arrow). Export is then faster than import, and the general direction of the net flow is from the nucleus to the cytoplasm. Because the Rpb4-Rbp7 heterodimer must be located in the nucleus to enable transcription during stress (5), this flux of the Rpb4-Rbp7 heterodimer from the nucleus to the cytoplasm must be leveled off at some point during prolonge incubation under these stress conditions and balanced by the retrograde flux. While export of Rpb4p or Rpb7p does not seem to be inhibited by disruption of the heterodimer integrity, the import pathway is very sensitive to this integrity. Thus, import of Rpb4p or Rpb7p is severely compromised when the heterodimer is disrupted either by deleting RPB4 or by mutating RPB7. Import is also blocked by inactivating Nup49p. In all these cases the net flow of these molecules from the nucleus to the cytoplasm is faster than normal. N denotes the nucleus; C denotes the cytoplasm.

Rpb4p is required for efficient import of Rpb7p from the cytoplasm to the nucleus. To further examine whether Rpb4p and Rpb7p shuttle independently or as a heterodimer, we determined whether disrupting the heterodimer affected their export or import kinetics. We first deleted RPB4 and found that the stress-induced relocation of Rpb7p-GFP was substantially faster in the absence of Rpb4p than it was in its presence. This can be demonstrated by the cytoplasmic localization of Rpb7p-GFP in the rpb4{Delta} cells after a relatively short exposure to stress while Rpb7p-GFP localization in the WT cells was still mainly nuclear (Fig. 6A). The rapid relocation may be due to accelerated export or, since Rpb7p-GFP shuttles (Fig. 3C), may be due to slow import (or to both). We therefore determined whether import of Rpb7p-GFP was affected by Rpb4p by refeeding starved cells whose Rpb7p-GFP was localized in the cytoplasm with fresh medium that contained all the necessary nutrients. As shown in Fig. 6B, Rpb7p-GFP was imported to the nucleus more slowly in the absence of Rpb4p than in its presence, suggesting that the rapid relocation of Rpb7p-GFP when rpb4{Delta} cells were exposed to stress was due at least in part to slow import.


Figure 6
View larger version (44K):
[in this window]
[in a new window]

  		FIG. 6. Transport of Rpb7p-GFP during severe stresses is affected by RPB4. (A) Stress-induced relocation of Rpb7p-GFP is accelerated in the absence of RPB4. Images of WT cells carrying RPB7-GFP in place of RPB7 (yMC286) and isogenic rpb4{Delta}-derivative cells (yMC310) that were challenged with various stresses for relatively short periods of time are shown. The kinds of stresses and their durations are indicated on the right side of each panel. Starvation conditions were simulated by use of synthetic medium lacking sugar and amino acids. Note that after a brief period of stress, relatively little export was detected in the WT cells (left panels) whereas export in the mutant was more advanced (right panels). (B) Efficient import of Rpb7p-GFP is dependent on Rpb4p. The strains described for panel A were incubated in starvation medium for 72 h to permit relocation of Rpb7p-GFP in 77% of WT cells and 100% of the mutant cells. Cells were then collected by centrifugation and resuspended in synthetic complete medium (containing glucose and amino acids) supplemented with 50 µg/ml cycloheximide. Import kinetics results were determined as described for Fig. 2. Note that in response to starvation or HS, Rpb7p-GFP can be localized in discrete cytoplasmic foci that are smaller than nuclei. We suspect that these foci represent cytoplasmic P bodies (17).

WT Rpb7p is required for efficient import of Rpb4p from the cytoplasm to the nucleus. To determine whether the export and import of Rpb4p are dependent upon its capacity to interact with Rpb7p, we randomly mutagenized the essential RPB7 and selected for ts mutants, a phenotype characteristic of rpb4{Delta} cells (see Materials and Methods). As was reported previously, single-point mutations in Rpb7p in highly conserved residues are incapable of compromising Rpb7p function (44). Consistently, all our ts mutants carried more than one mutation; for example, Rpb7-29p carried five point mutations (see Table 1). We then selected mutants that were defective in their capacity to form two-hybrid interactions with Rpb4p. The following experiments were performed with three mutants that exhibited defective interaction with Rpb4 and gave very similar results. Only one mutant, Rpb7-29p, which binds Rpb4p poorly even at the permissive temperature (Fig. 7A), is described hereafter. Rpb7-29p was tagged with RFP, and its relocation in response to stress was examined. Consistent with its defective interaction with Rpb4p, Rpb7-29p-RPF was relocated faster than WT Rpb7p-RFP during starvation (Fig. 7B) or during HS (results not shown), much like its accelerated relocation in cells lacking RPB4 (see results for Rpb7p-RFP in Fig. 6A). We then examined stress-induced relocation of WT GFP-Rpb4p in rpb7-29 cells and found it to be faster than it was in WT cells (Fig. 7B). Again, this rapid relocation might be due either to accelerated export or, since Rpb4p shuttles (Fig. 2 and 4), to slow import. To determine whether GFP-Rpb4p import was affected by the mutations in Rpb7p, we refed starved cells whose GFP-Rpb4p was localized in the cytoplasm with fresh medium that contained all the necessary nutrients. Import of WT GFP-Rpb4p was much slower in rpb7-29 mutant cells than it was in the parental RPB7 cells (Fig. 7C). In contrast, the import kinetics results obtained with WT GFP-Rpb4p and WT Rpb7p-RFP in WT cells, in which the heterodimer can be formed, were identical (Fig. 5D).


Figure 7
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 7. Transport of GFP-Rpb4p during starvation is affected by RPB7. (A) Two-hybrid interaction between Rpb7p-DBD and Rpb4-AD (upper row) and between Rpb7-29p-DBD and Rpb4p-AD (lower row). Cells were spotted in threefold serial dilutions starting with 106 cells/spot on an indicator plate (Selective conditions) as described in Materials and Methods. Growth on this plate indicates interaction (38). To demonstrate spotting of equal amounts of cells, cells were spotted on a nonselective plate that allowed growth of cells that carry both the bait and prey plasmids independently of the two two-hybrid interactions (using medium lacking only leucine and tryptophane). Beta-galactosidase (ß-Gal) values, quantitative means used to determine interactions (38), are indicated at the right. They reflect the average values determined for three independent experiments. Background values obtained with cells expressing only the bait plasmid were subtracted. (B) Starvation-induced relocation of GFP-Rpb4p and Rpb7p-RFP is faster in rpb7-29 cells than in WT cells. Images of WT cells carrying RPB7-RFP in place of RPB7 and GFP-RPB4 (yRL82) or cells carrying a rpb7-29-RFP derivative thereof (yRL81) that were challenged with starvation (using a synthetic medium lacking sugar and amino acids) at 37°C for a relatively short period of time (45 min) are shown. The same field was photographed using either the green (for detection of GFP-Rpb4p) or red (for detection of Rpb7p-RFP) channel, as indicated. Note that after a short period of starvation, relatively little export was detected in the WT cells (upper right panels) whereas export of both fluorescent proteins in the mutant was more advanced (lower right panels). (C) Import of GFP-Rpb4p is impaired in rpb7-29 mutant cells. WT cells (yRL82) or rpb7-29 mutant cells (yRL81) expressing GFP-RPB4 were starved until GFP-Rpb4p was localized in the cytoplasm in ~90% of the cells. Cells were then collected by centrifugation and resuspended in synthetic complete medium (containing glucose and amino acids) supplemented with 50 µg/ml cycloheximide (similar results were obtained when cycloheximide was not added). Import kinetics results were determined as described for Fig. 5D. Examples of photographs of cells taken at the indicated time points after refeeding are shown below the graph.

The results shown in Fig. 6 and 7 indicate that the import kinetics of both proteins are severely impaired when the heterodimer integrity is compromised either by deleting RPB4 or by mutating RPB7. Thus, disruption of the heterodimer should affect GFP-Rpb4p or Rpb7p-GFP localization much as inactivation of Nup49-313p does, because in both cases import is compromised. Indeed, in both cases the net relocation of the tagged Rpb4p and Rpb7p from the nucleus to the cytoplasm in response to stress was faster than it was in WT cells. Thus, these results and those shown in Fig. 3C indicate that both Rpb4p and Rpb7p shuttle under the examined stress conditions. Moreover, the results shown in Fig. 5, 6, and 7 suggest that both proteins normally shuttle as a heterodimer.


arrow
DISCUSSION
 
Rpb4p is unusual among Pol II subunits in that it couples between transcription and mRNA transport (8). Moreover, Rpb4p is the only factor of the basal transcription apparatus that is known to also control mRNA decay in the cytoplasm, thus regulating mRNAs at both their synthesis and decay levels (17). Therefore, unlike some other export factors that are reimported immediately following mRNA export (18, 25, 33), Rpb4p remains associated with the mRNA in the cytoplasm until its degradation in the P bodies (17). It was not known whether Rpb4p is degraded after its role in the cytoplasmic P bodies is completed or is imported back to the nucleus for recycling.

Here we show that Rpb4p shuttles between the nucleus and cytoplasm by a cycloheximide-insensitive pathway. Thus, a likely scenario is that Rpb4p is imported back to the nucleus to start a new cycle of transcription, mRNA transport, and degradation only after its role in mRNA decay is completed. Rpb4p may represent a new kind of shuttling protein that shuttles between the nucleus and P bodies. It is possible that this shuttling contributes to the proposed role of Rpb4p in communicating between transcription in the nucleus and mRNA decay in the cytoplasm. In addition, we found that Rpb7p, the close partner of Rpb4p, also shuttles between the two compartments. Moreover, Rpb7p's cellular location is responsive to the environment in a cycloheximide-insensitive manner, most likely due to environmentally regulated changes in the balance between its import and export rates (see Fig. 8).

Although Rpb4p and Rpb7p function as a heterodimer in transcription, it was not known whether they also function as a heterodimer outside this context. In fact, whereas Rpb4p was known to carry out functions outside the Pol II context, there were no indications that this is also the case for Rpb7p. Moreover, because RPB4 and RPB7 are found as two separate genes in archeal organisms and in all eukaryotes whose genomes have been sequenced (5), it is likely that the two proteins would physically separate at some point. Nevertheless, in spite of our efforts to find such a separation in WT cells, our current results do not support this possibility. Instead, it seems that Rpb4p and Rpb7p shuttle as a heterodimer under the studied conditions. First, Rpb4p import was compromised when Rpb7p was defective (Fig. 7). Likewise, in the absence of Rpb4p, Rpb7p import was compromised (Fig. 6). Second, in response to severe stresses, the two proteins were exported to the cytoplasm with similar kinetics results. Importantly, export kinetics was not influenced significantly by the nature of the tag (see text discussing Fig. 5). Only in mutant cells whose Rpb4-Rbp7 heterodimer cannot be formed normally did we find different relocation kinetics or different localization patterns. For example, in response to challenging rpb7-29 cells (yRL82) with starvation for 72 h, GFP-Rpb4p exhibited whole-cell localization in 90% of the cells, whereas Rpb7-29p-RFP exhibited similarly distributed localization in only 50% of the cells while in the other 50% of the cells it was mainly in the nucleus. This is consistent with our observation that Rpb4p import is more sensitive than Rpb7p import to disruption of the heterodimer integrity (compare the very slow import of Rpb4p shown in Fig. 7C with the faster import kinetics of Rpb7p shown in Fig. 6B). The mechanistic basis for these differences remains to be determined. Third, both Rpb4p and Rpb7p are constituents of two other complexes, i.e., cytoplasmic P bodies (17; R. Lotan and M. Choder, unpublished observation) and Npl3p-containing RNAs (17). Taken together, all these observations suggest that the role of Rpb4p within the cytoplasm is carried out in the context of the Rpb4-Rbp7 heterodimer. Although our current results do not support separate functions, we still believe that under yet-unexplored conditions the two proteins would function independently of each other.

Whether or not Rpb4p and Rpb7p have nuclear localization and nuclear export sequences remains to be determined. In this regard, it should be mentioned that studies that have dissected the transport signals responsible for localization of multiprotein complexes have uncovered numerous examples of a single subunit providing the transport signal for the entire complex (16, 19, 23, 35, 41). Therefore, it is possible that the Rpb4-Rbp7 heterodimer does not carry functional nuclear localization and nuclear export sequences of its own but instead shuttles as a component of larger complexes.

Recently it was shown that the Rpb4-Rbp7 heterodimer interacts in vitro with the transcript as soon as it emerges from Pol II (39). This interaction is mediated by at least one of the two RNA-binding domains of Rpb7p (21, 37). Thus, the Rpb4-Rbp7 heterodimer is probably the first protein that assembles with the transcript and is probably involved in recruiting other RNA-interacting proteins (39). We provided an in vivo demonstration that the Rpb4-Rbp7 heterodimer interacts with the Npl3p-containing mRNP (17). These observations and our observation that the two partners seem to export as a heterodimer in a transcription-dependent manner provoked us to propose that the Rpb4-Rbp7 heterodimer is exported together with the transcript that it helps synthesize, as is the case for other shuttling factors, e.g., Npl3p (15). Consistently, Rpb4p is essential for exporting the transcript (8).

Rpb4p's role in the export of poly(A)+ mRNA is detected under conditions of mild temperature stress (37°C), as is consistent with its capacity to shuttle under these conditions. However, at a moderate temperature of 22°C, Rpb4p is not essential for mRNA export (8). Since Rpb4p's role in mRNA decay is evident under optimal conditions at 24°C (17), we suspect that Rpb4p is also involved in mRNA export at 22°C but that under these conditions it does not play a critical role. Consistent with this possibility is the observation that nucleocytoplasmic transport is relatively resilient to perturbations when cells are incubated under optimal conditions but that it is very sensitive to the environmental temperature. Specifically, yeast strains lacking any one of approximately a third of their nucleoporins are able to grow at 22°C but die at elevated temperatures. Regarding another third of the essential nucleoporins, deletion of substantial portions thereof results in temperature-sensitive proliferation (27, 36).

Unexpectedly, we found that while Rpb4p and Rpb7p shuttle under all environmental conditions tested, including severe stress (Fig. 4, 6, and 7), in response to severe stress in the form of HS (42°C), EtOH (≥6%), or starvation, these proteins are exported by a transcription-independent mechanism. We suspect that during severe stress, the Rpb4-Rbp7 heterodimer is also exported by the transcription-dependent pathway because (i) export at 42°C is partially inhibited by the transcription inhibitor 1,10-phenanthroline (Fig. 3B) and (ii) Rpb4p is required for the export of HS mRNAs at 42°C, thus suggesting that the Rpb4-Rbp7 heterodimer is exported together with these mRNAs (8). We therefore propose that the transcription-dependent pathway is constitutive. The simplest scenario explaining all our current and previous observations suggests the existence of two shuttling mechanisms, which are likely to employ common factors, as schematized in Fig. 8. While Rpb4p and Rpb7p always shuttle, the balance between these two pathways determines the localization patterns of these proteins. This balance is responsive to the environment. It is likely that this balance affects transcription and mRNA decay. There has been another report of two pathways that control transport of an RNA binding protein, Pab1p. More specifically, Pab1p can exit the nucleus by transcription-dependent and transcription-independent pathways (7). However, it is not known how the two pathways of Pab1p export respond to the environment. Our results show that the two export pathways can regulate the transport of a given factor in an environmentally responsive manner.

Why have cells evolved a transcription-independent export pathway during stress? A normal response to stress involves not only transcriptional induction of stress-induced genes but also repression of many other genes whose expression is unfavorable under these conditions (see reference 17 and references therein). We surmise that one mechanism for rapid repression of the stress-repressed genes is exportation of their transcription factors from the nucleus. The Rpb4-Rbp7 heterodimer is a transcription factor that is required for the transcription of stress-induced and stress-repressed genes. Because Rpb4-Rbp7p's level in the nucleus exceeds that of other Pol II subunits (26), we speculate that it would be beneficial to export a surplus of but not all of the molecules of the Rpb4-Rbp7 heterodimer. As a result, a nuclear environment with limiting levels of Rpb4-Rbp7 heterodimer-containing Pol II will be created. This will permit promoter competition on Rpb4-Rbp7 heterodimer-containing Pol II, resulting in transcriptional induction of stress-induced genes and repression of others. An alternative explanation for the accelerated export of the Rpb4-Rbp7 heterodimer during severe stresses is that surplus of the Rpb4-Rbp7 heterodimer is harmful because it titrates some of the transcription factors necessary for the normal response to the stress. It remains to be determined whether eviction of other transcription factors from the nucleus is a normal response to severe stress.


arrow
ACKNOWLEDGMENTS
 
We thank Amnon Harel for critically reading the manuscript, Roy Parker for plasmids, and Nancy Woychik, Pamela Silver, Charles Coles, and Fred Winston for yeast strains.

This work was supported by a grant from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Department of Molecular Microbiology, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel 31096. Phone: (972) 4 829-5423. Fax: (972) 4 829-5225. E-mail: choder{at}technion.ac.il. Back

{triangledown} Published ahead of print on 20 October 2006. Back


arrow
REFERENCES
 
    1
  1. Aguilera, A. 2005. Cotranscriptional mRNP assembly: from the DNA to the nuclear pore. Curr. Opin. Cell Biol. 17:242-250.[CrossRef][Medline]
    2. 2
  2. Armache, K. J., H. Kettenberger, and P. Cramer. 2003. Architecture of initiation-competent 12-subunit RNA polymerase II. Proc. Natl. Acad. Sci. USA 100:6964-6968.[Abstract/Free Full Text]
    3. 3
  3. Bushnell, D. A., and R. D. Kornberg. 2003. Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription. Proc. Natl. Acad. Sci. USA 100:6969-6973.[Abstract/Free Full Text]
    4. 4
  4. Choder, M. 1993. A growth rate-limiting process in the last growth phase of the yeast life cycle involves RPB4, a subunit of RNA polymerase II. J. Bacteriol. 175:6358-6363.[Abstract/Free Full Text]
    5. 5
  5. Choder, M. 2004. Rpb4 and Rpb7: subunits of RNA polymerase II and beyond. Trends Biochem. Sci. 29:674-681.[CrossRef][Medline]
    6. 6
  6. Dreyfuss, G., V. N. Kim, and N. Kataoka. 2002. Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell Biol. 3:195-205.[CrossRef][Medline]
    7. 7
  7. Dunn, E. F., C. M. Hammell, C. A. Hodge, and C. N. Cole. 2005. Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export. Genes Dev. 19:90-103.[Abstract/Free Full Text]
    8. 8
  8. Farago, M., T. Nahari, C. Hammel, C. N. Cole, and M. Choder. 2003. Rpb4p, a subunit of RNA polymerase II, mediates mRNA export during stress. Mol. Biol. Cell 14:2744-2755.[Abstract/Free Full Text]
    9. 9
  9. Garí, E., L. Piedrafita, M. Aldea, and E. Herrero. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13:837-848.[CrossRef][Medline]
    10. 10
  10. Grigull, J., S. Mnaimneh, J. Pootoolal, M. D. Robinson, and T. R. Hughes. 2004. Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol. Cell. Biol. 24:5534-5547.[Abstract/Free Full Text]
    11. 11
  11. Hammell, C. M., S. Gross, D. Zenklusen, C. V. Heath, F. Stutz, C. Moore, and C. N. Cole. 2002. Coupling of termination, 3' processing, and mRNA export. Mol. Cell. Biol. 22:6441-6457.[Abstract/Free Full Text]
    12. 12
  12. Hieronymus, H., and P. A. Silver. 2003. Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery. Nat. Genet. 33:155-161.[CrossRef][Medline]
    13. 13
  13. Kim Guisbert, K., K. Duncan, H. Li, and C. Guthrie. 2005. Functional specificity of shuttling hnRNPs revealed by genome-wide analysis of their RNA binding profiles. RNA 11:383-393.[Abstract/Free Full Text]
    14. 14
  14. Krebber, H., T. Taura, M. S. Lee, and P. A. Silver. 1999. Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export. Genes Dev. 13:1994-2004.[Abstract/Free Full Text]
    15. 15
  15. Lee, M. S., M. Henry, and P. A. Silver. 1996. A protein that shuttles between the nucleus and the cytoplasm is an important mediator of RNA export. Genes Dev. 10:1233-1246.[Abstract/Free Full Text]
    16. 16
  16. Leslie, D. M., W. Zhang, B. L. Timney, B. T. Chait, M. P. Rout, R. W. Wozniak, and J. D. Aitchison. 2004. Characterization of karyopherin cargoes reveals unique mechanisms of Kap121p-mediated nuclear import. Mol. Cell. Biol. 24:8487-8503.[Abstract/Free Full Text]
    17. 17
  17. Lotan, R., V. G. Bar-On, L. Harel-Sharvit, L. Duek, D. Melamed, and M. Choder. 2005. The RNA polymerase II subunit Rpb4p mediates decay of a specific class of mRNAs. Genes Dev. 19:3004-3016.[Abstract/Free Full Text]
    18. 18
  18. Lund, M. K., and C. Guthrie. 2005. The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim. Mol. Cell 20:645-651.[CrossRef][Medline]
    19. 19
  19. Maridor, G., P. Gallant, R. Golsteyn, and E. A. Nigg. 1993. Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits. J. Cell Sci. 106(Pt. 2):535-544.[Abstract]
    20. 20
  20. McKune, K., K. L. Richards, A. M. Edwards, R. A. Young, and N. A. Woychik. 1993. RPB7, one of two dissociable subunits of yeast RNA polymerase II, is essential for cell viability. Yeast 9:295-299.[CrossRef][Medline]
    21. 21
  21. Meka, H., F. Werner, S. C. Cordell, S. Onesti, and P. Brick. 2005. Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II. Nucleic Acids Res. 33:6435-6444.[Abstract/Free Full Text]
    22. 22
  22. Nonet, M., C. Scafe, J. Sexton, and R. Young. 1987. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis. Mol. Cell. Biol. 7:1602-1611.[Abstract/Free Full Text]
    23. 23
  23. Pereira, G., M. Knop, and E. Schiebel. 1998. Spc98p directs the yeast gamma-tubulin complex into the nucleus and is subject to cell cycle-dependent phosphorylation on the nuclear side of the spindle pole body. Mol. Biol. Cell 9:775-793.[Abstract/Free Full Text]
    24. 24
  24. Rodriguez, M. S., C. Dargemont, and F. Stutz. 2004. Nuclear export of RNA. Biol. Cell 96:639-655.[CrossRef][Medline]
    25. 25
  25. Rollenhagen, C., C. A. Hodge, and C. N. Cole. 2004. The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock. Mol. Cell. Biol. 24:4869-4879.[Abstract/Free Full Text]
    26. 26
  26. Rosenheck, S., and M. Choder. 1998. Rpb4, a subunit of RNA polymerase II, enables the enzyme to transcribe at temperature extremes in vitro. J. Bacteriol. 180:6187-6192.[Abstract/Free Full Text]
    27. 27
  27. Ryan, K. J., and S. R. Wente. 2000. The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. Curr. Opin. Cell Biol. 12:361-371.[CrossRef][Medline]
    28. 28
  28. Saavedra, C., K. S. Tung, D. C. Amberg, A. K. Hopper, and C. N. Cole. 1996. Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. Genes Dev. 10:1608-1620.[Abstract/Free Full Text]
    29. 29
  29. Saavedra, C. A., C. M. Hammell, C. V. Heath, and C. N. Cole. 1997. Yeast heat shock mRNAs are exported through a distinct pathway defined by Rip1p. Genes Dev. 11:2845-2856.[Abstract/Free Full Text]
    30. 30
  30. Sampath, V., and P. Sadhale. 2005. Rpb4 and Rpb7: a sub-complex integral to multi-subunit RNA polymerases performs a multitude of functions. IUBMB Life 57:93-102.[Medline]
    31. 31
  31. Schwartz, T. U. 2005. Modularity within the architecture of the nuclear pore complex. Curr. Opin. Struct. Biol. 15:221-226.[CrossRef][Medline]
    32. 32
  32. Sheth, U., and R. Parker. 2003. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300:805-808.[Abstract/Free Full Text]
    33. 33
  33. Sommer, P., and U. Nehrbass. 2005. Quality control of messenger ribonucleoprotein particles in the nucleus and at the pore. Curr. Opin. Cell Biol. 17:294-301.[CrossRef][Medline]
    34. 34
  34. Stutz, F., J. Kantor, D. Zhang, T. McCarthy, M. Neville, and M. Rosbash. 1997. The yeast nucleoporin rip1p contributes to multiple export pathways with no essential role for its FG-repeat region. Genes Dev. 11:2857-2868.[Abstract/Free Full Text]
    35. 35
  35. Subramaniam, P. S., and H. M. Johnson. 2004. The IFNAR1 subunit of the type I IFN receptor complex contains a functional nuclear localization sequence. FEBS Lett. 578:207-210.[CrossRef][Medline]
    36. 36
  36. Suntharalingam, M., and S. R. Wente. 2003. Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell 4:775-789.[CrossRef][Medline]
    37. 37
  37. Todone, F., P. Brick, F. Werner, R. O. Weinzierl, and S. Onesti. 2001. Structure of an archaeal homolog of the eukaryotic RNA polymerase II RPB4/RPB7 complex. Mol. Cell 8:1137-1143.[CrossRef][Medline]
    38. 38
  38. Uetz, P., L. Giot, G. Cagney, T. A. Mansfield, R. S. Judson, J. R. Knight, D. Lockshon, V. Narayan, M. Srinivasan, P. Pochart, A. Qureshi-Emili, Y. Li, B. Godwin, D. Conover, T. Kalbfleisch, G. Vijayadamodar, M. Yang, M. Johnston, S. Fields, and J. M. Rothberg. 2000. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623-627.[CrossRef][Medline]
    39. 39
  39. Ujvári, A., and D. S. Luse. 2006. RNA emerging from the active site of RNA polymerase II interacts with the Rpb7 subunit. Nat. Struct. Mol. Biol. 13:49-54.[CrossRef][Medline]
    40. 40
  40. Weis, K. 2003. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 112:441-451.[CrossRef][Medline]
    41. 41
  41. Wendler, P., A. Lehmann, K. Janek, S. Baumgart, and C. Enenkel. 2004. The bipartite nuclear localization sequence of Rpn2 is required for nuclear import of proteasomal base complexes via karyopherin alphabeta and proteasome functions. J. Biol. Chem. 279:37751-37762.[Abstract/Free Full Text]
    42. 42
  42. Winston, F., C. Dollard, and S. L. Ricupero-Hovasse. 1995. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11:53-55.[CrossRef][Medline]
    43. 43
  43. Young, R. A. 1991. RNA polymerase II. Annu. Rev. Biochem. 60:689-715.[CrossRef][Medline]
    44. 44
  44. Zaros, C., and P. Thuriaux. 2005. Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation. Mol. Microbiol. 55:104-114.[CrossRef][Medline]

Eukaryotic Cell, December 2006, p. 2092-2103, Vol. 5, No. 12
1535-9778/06/$08.00+0     doi:10.1128/EC.00288-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Belanger, K. D., Walter, D., Henderson, T. A., Yelton, A. L., O'Brien, T. G., Belanger, K. G., Geier, S. J., Fahrenkrog, B. (2009). Nuclear localisation is crucial for the proapoptotic activity of the HtrA-like serine protease Nma111p. J. Cell Sci. 122: 3931-3941 [Abstract] [Full Text]  
  • Jasiak, A. J., Hartmann, H., Karakasili, E., Kalocsay, M., Flatley, A., Kremmer, E., Strasser, K., Martin, D. E., Soding, J., Cramer, P. (2008). Genome-associated RNA Polymerase II Includes the Dissociable Rpb4/7 Subcomplex. J. Biol. Chem. 283: 26423-26427 [Abstract] [Full Text]  
  • Goler-Baron, V., Selitrennik, M., Barkai, O., Haimovich, G., Lotan, R., Choder, M. (2008). Transcription in the nucleus and mRNA decay in the cytoplasm are coupled processes. Genes Dev. 22: 2022-2027 [Abstract] [Full Text]  
  • Sampath, V., Balakrishnan, B., Verma-Gaur, J., Onesti, S., Sadhale, P. P. (2008). Unstructured N Terminus of the RNA Polymerase II Subunit Rpb4 Contributes to the Interaction of Rpb4{middle dot}Rpb7 Subcomplex with the Core RNA Polymerase II of Saccharomyces cerevisiae. J. Biol. Chem. 283: 3923-3931 [Abstract] [Full Text]  
  • Lotan, R., Goler-Baron, V., Duek, L., Haimovich, G., Choder, M. (2007). The Rpb7p subunit of yeast RNA polymerase II plays roles in the two major cytoplasmic mRNA decay mechanisms. JCB 178: 1133-1143 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow E-mail this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Selitrennik, M.
Right arrow Articles by Choder, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Selitrennik, M.
Right arrow Articles by Choder, M.

Copyright © 2010 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»