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Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana 70803
Received 7 March 2007/ Returned for modification 9 April 2007/ Accepted 11 April 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Long-term stalling of Pol II, either by DNA damage (1, 14, 42) or by other means of elongation pausing (42), induces polyubiquitylation and subsequent degradation of Rpb1, the largest subunit of Pol II. Rsp5 was initially identified as the E3 ubiquitin ligase that catalyzes Rpb1 polyubiquitylation (14, 42). Furthermore, it was shown that yeast extracts from rsp5-1 cells incubated at nonpermissive temperature could not ubiquitylate Rpb1 in vitro, even when a large amount of ubiquitin was added (34). A recent study showed that an E3 ubiquitin ligase, comprising elongin A, elongin C, cullin 3, and Rbx1 is also required for Rpb1 polyubiquitylation and degradation in yeast (35).
In human cells, DNA damage-induced ubiquitylation of Rpb1 is dependent on both CSA and CSB (3), indicating a connection between Rpb1 ubiquitylation and TCR. In yeast, it was found that ubiquitylation of Rpb1 is dependent on Def1, which is not required for TCR but forms a complex with Rad26 (56). However, Rad26 itself is not required for Rpb1 ubiquitylation. Furthermore, Def1 is dispensable for Rpb1 ubiquitylation and degradation in cells lacking Rad26 (56).
In addition to its TCR function, Rpb9 has multiple transcription-related functions, such as selection of correct transcription start site (8, 15, 58), transcription elongation (12, 49), and maintenance of transcription fidelity (29). In this paper, we present evidence that Rpb9 also plays an important role in Rpb1 ubiquitylation and degradation in response to UV radiation. This function of Rpb9 seems not to be related to any pathways of NER, including TCR mediated by Rpb9 itself and by Rad26.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ura3-52 his3-1 leu2-3 leu2-112 [cir0]) (24) or BJ5465 (MATa ura3-52 trp-1 leu2
1 his3200 pep4::HIS3 prb11.6R can1) (17) backgrounds. rad16, rpb9, and rad26 deletion mutants were created as described previously (24). Deletions of DEF1, ERG6, RAD2, RAD4, RAD7, and RAD14 were made by replacing sequences between nucleotides (with respect to the start codon ATG) +70 and +918, +311 and +1082, +129 and +2911, +75 and +2885, +214 and +1454, and +202 and +1114 with the yeast URA3 gene, respectively. Deletion of SPT4 was achieved by replacing the sequence between nucleotides +14 and +288 with the LEU2 gene. A strain containing RPB2 with three consecutive FLAG tags (FLAG3) was created using plasmid p3FLAG-KanMX (9).
Plasmids expressing truncated Rpb9 were constructed using vector pRS416 (41). A series of RPB9 fragments encompassing different truncated coding sequences, the promoter (a sequence of 
500 bp immediately upstream of the coding sequence), and the 3' mRNA processing sequence (a sequence of 300 bp immediately downstream of the coding sequence) were created by PCR and enzymatic ligation. These RPB9 fragments were inserted into the multiple cloning site of vector pRS416 (41).
Plasmids expressing truncated Rpb9 with three consecutive FLAG tags (6) at the N termini were created using vector pESC-URA (Stratagene). Two consecutive FLAG sequences were inserted downstream of the FLAG sequence in pESC-URA to create a vector with three consecutive FLAG sequences. RPB9 fragments encoding different truncated Rpb9 proteins were amplified by PCR and inserted downstream of the three FLAG tags in the vector.
UV irradiation, recovery incubation, and whole-cell extract preparation.
Unless otherwise described, yeast cells were grown at 30°C in minimal medium to late log phase (A600 1.0), harvested, and washed with ice-cold H2O. The washed cells were resuspended in ice-cold 2% glucose or galactose and irradiated with 240 J/m2 of 254-nm UV light. The irradiated-cell suspension was added to 1/10 volume of a stock solution containing 10% yeast extract and 20% peptone, and the mixture was incubated for various times in the dark at 30°C. Aliquots were removed from the cultures at different times of the incubation, and the cells in each aliquot were pelleted by centrifugation.
To examine the dependence of UV-induced Rpb1 degradation on the 26S proteasome, erg6 cells, which are permeable to the proteasome inhibitor MG132 (19, 20), were cultured in synthetic dextrose (SD) medium to late log phase. MG132 (dissolved in dimethyl sulfoxide) was added to the cultures to a final concentration of 50 µM. After 2 h of continued incubation, the cells were UV irradiated. The procedure for recovery incubation was the same as described above with the exception that 50 µM MG132 was included in the recovery medium.
To examine UV-induced Rpb1 degradation in the absence of protein synthesis, yeast cells were cultured in SD medium to late log phase and cycloheximide (CHX), a potent protein synthesis inhibitor (39), was added to the cultures to a final concentration of 500 µg/ml. We found that a concentration of 50 µg/ml of the chemical is sufficient to completely inhibit protein synthesis (not shown). After 40 min of continued incubation, the cultures were directly irradiated with 240 J/m2 of UV and incubated at 30°C. At different times during the recovery incubation, aliquots were taken and the cells in each aliquot were collected.
Whole-cell extract was made using a trichloroacetic acid (TCA) method. Pelleted cells were washed once with 20% TCA and resuspended in 10% TCA. The cells were broken by vortexing them with acid-washed glass beads (Sigma; G9268), and the cell debris was removed. The proteins in the cell lysates were pelleted by centrifugation at 20,000 x g for 15 min. The protein pellet was washed with ice-cold 80% acetone and dissolved in 2x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel loading buffer (38).
Immunoprecipitation. Yeast cells were cultured in yeast extract-peptone-dextrose or minimal medium containing the required amino acids to early log phase. Fifty milliliters of a cell culture was harvested, and cells were washed once with IP buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.4 mM Na4VO3, 10 mM Na4P2O7, 10 mM NaF, 0.5% NP-40, 1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, and protease inhibitors). The cell pellets were resuspended in 0.5 ml of IP buffer and disrupted by vortexing with acid-washed glass beads. The cell lysates were cleared by centrifugation twice at 20,000 x g for 10 min at 4°C. Fifteen micrograms of 8WG16 or 20 µg of anti-FLAG antibody M2 was added to a cell lysate, and the mixture was incubated at 4°C overnight with gentle rotation. Protein A-coated agarose beads (Sigma) were added to the mixture, and the mixture was incubated at 4°C for 3 h with gentle rotation. The beads were washed four times with IP buffer. Bound proteins were eluted by boiling the beads in 2x SDS-PAGE gel loading buffer (38).
Western blotting.
Proteins in whole-cell extracts or immunoprecipitated samples were resolved on an SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore). Rpb1 on the blots was probed with mouse monoclonal antibody 8WG16 (Neoclone), which specifically recognizes the C-terminal repeat domain (CTD) of Rpb1, or with H14 (Covance), which specifically recognizes serine 5-phosphorylated CTDs. FLAG-tagged proteins were probed with anti-FLAG antibody M2 (Sigma). Ubiquitylated proteins were probed with a rabbit polyclonal antiubiquitin antibody (Stressgen). As a loading control, -tubulin on the same blots was probed with rat anti--tubulin monoclonal antibody (GeneTex). Blots were incubated with SuperSignal West Femto maximum-sensitivity substrate (Pierce), and the protein bands were detected using a chemiluminescence scanner (Fluorchem 8800; Alpha Innotech). Band intensities on the Western blots were quantified using AlphaEaseFC 4.0 software.
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RESULTS |
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2 h after UV irradiation and starting to recover afterwards. However, the tagged Rpb2 and Rpb9 remained at a steady level (Fig. 1). These results indicate that Rpb1, but not the whole Pol II complex, is degraded in response to UV radiation.
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The impaired decrease of Rpb1 levels in rpb9 cells following UV irradiation could result from either less degradation or increased synthesis of Rpb1. To examine the two possibilities, we used the potent protein synthesis inhibitor CHX (39) to inhibit new protein synthesis during the period of incubation. In the absence of CHX, both Rpb1 and -tubulin (serving as an internal control) levels increased continuously in wild-type (WT) and rpb9 cells during the period of incubation (Fig. 3A), indicating that new proteins were synthesized. In contrast, in the presence of 50-µg/ml or higher concentrations of CHX, the levels of Rpb1 and -tubulin in WT and rpb9 cells were essentially not changed during the period of incubation (Fig. 3B and data not shown), indicating that protein synthesis was completely inhibited.
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Rpb9 plays an important role in ubiquitylation of Rpb1 in response to UV radiation. Few substrates are known to be recognized directly by the 26S proteasome. Most are thought to be delivered to the proteasome via the degradation mark of a covalently attached polyubiquitin chain (27). We wondered whether the impaired degradation of Rpb1 in rpb9 cells is due to a role for Rpb9 in promoting ubiquitylation of Rpb1. Rpb1 in whole-cell extracts from UV-irradiated WT and rpb9 cells was probed with H14 antibody on a Western blot. The H14 antibody, which recognizes serine 5-phosphorylated CTD of Rpb1, has been repeatedly used to detect ubiquitylation of Rpb1 (3, 18, 21, 32, 35, 36). Indeed, apparent smearing bands can be seen above the major Rpb1 band for cell extracts prepared from UV-irradiated WT cells, but not rpb9 cells (Fig. 4A). The pattern of these smearing bands is very similar to that of the yeast ubiquitylated Rpb1 reported previously (1, 35, 36). The hypo- and hyperphosphorylated forms of yeast Rpb1 migrate as a very closely spaced doublet and are not easily distinguished by SDS-PAGE (1). It is therefore likely that these smearing bands reflect ubiquitylated Rpb1 molecules that are also phosphorylated at serine 5 of the CTDs. To confirm that the smearing bands are indeed ubiquitylated, Rpb1, the largest Pol II subunit, was immunoprecipitated with anti-Rpb1 antibody 8WG16. The immunoprecipitated Rpb1 was then probed with antiubiquitin antibody on a Western blot. As shown in Fig. 4B, a stronger signal representing ubiquitylated Rpb1 can be observed in WT cells but not rpb9 cells following irradiation. To examine if rpb9 cells have an overall deficiency in protein ubiquitylation, the whole-cell extracts from WT and rpb9 cells were subjected to Western blotting and probed with antiubiquitin antibody. As can be seen in Fig. 4C, rpb9 cells did not show an overall deficiency in protein ubiquitylation. These results indicate that Rpb9 specifically promotes ubiquitylation of Rpb1 in response to UV radiation.
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Almost the full length of Rpb9 is required for a strong interaction with core Pol II. One question which arose was how different truncated forms of Rpb9 interact with core Pol II and how the interaction may affect the degradation of Rpb1 in response to UV radiation. To address this question, a series of plasmids expressing truncated Rpb9 with FLAG3 tags at their N termini were created. These plasmids were transformed into rpb9 cells, and the interaction between the tagged Rpb9 fragments and core Pol II was analyzed by coimmunoprecipitation. We observed that the FLAG3 tags did not affect the functions of Rpb9 for TCR and for promoting UV-induced Rpb1 degradation (not shown). Protein complexes containing the FLAG3-tagged Rpb9 fragments were immunoprecipitated using anti-FLAG antibody. The core Pol II that coimmunoprecipitated with the tagged Rpb9 was examined by Western blotting using anti-Rpb1 antibody 8WG16. As shown in Fig. 6, FLAG3-tagged full-length Rpb9 (residues 1 to 122) and Rbp9 with three amino acids deleted at the N terminus (residues 4 to 122 remaining), which are fully functional for transcription elongation, TCR (22), and UV-induced Rpb1 degradation (Fig. 5), could efficiently coimmunoprecipitate Rpb1. Deletion of 16 amino acids from the N terminus (residues 17 to 122 remaining) compromised the coimmunoprecipitation to some extent (Fig. 6). However, deletions of 30 or 49 amino acids (residues 31 to 122 or 50 to 122 remaining) from the N terminus resulted in undetectable coimmunoprecipitation of Rpb1 (Fig. 6). Deletions from the C terminus also dramatically compromised coimmunoprecipitation of Rpb1. Rpb9 fragments containing Zn1 and the linker (residues 1 to 53) could coimmunoprecipitate a small amount of Rpb1. However, a deletion from the C terminus down to amino acid 45, which abolishes the functions of transcription elongation and TCR (22) and promotion of UV-induced Rpb1 degradation (Fig. 5), resulted in undetectable coimmunoprecipitation of Rpb1 (Fig. 6).
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To rule out the possibility that the different forms of FLAG3-tagged Rpb9 affect the constitutive level of Rpb1, the whole-cell extracts (i.e., the immunoprecipitation input) were probed with 8WG16 on a Western blot. As can be seen in Fig. 6, the cellular Rpb1 levels are similar among the strains expressing the different forms of FLAG3-tagged Rpb9.
Taken together, our results indicate that almost the full length of Rpb9 is required for a strong interaction with core Pol II, although a weakened interaction exists when Zn2 is deleted. However, as described above, the Zn2 domain is essential for Rpb9 to promote UV-induced Rpb1 degradation, whereas the Zn1 and linker may play a subsidiary role in the process. Therefore, the subsidiary role of Zn1 and the linker in promoting UV-induced Rpb1 degradation may be achieved simply by enhancing the association of Rpb9 with the core Pol II.
The role of Rpb9 in promoting UV-induced degradation of Rpb1 is not affected by different NER pathways. It has been shown that UV-induced degradation of Rpb1 is impaired in def1 cells (56). However, normal UV-induced degradation of Rpb1 occurs in rad26 and def1 rad26 cells (56). Based on these and other findings, it was proposed that the TCR factor CSB or the yeast homolog Rad26 uses DNA translocase activity to remodel the Pol II-DNA interface, so that repair can take place if the obstacle is a DNA lesion. However, when this action is not possible and Pol II is left irreversibly trapped on DNA, the complex is instead rescued by ubiquitylation and subsequent degradation (44). We wished to examine if a NER pathway, especially the TCR pathway, affects the role of Rpb9 in promoting UV-induced Rpb1 degradation.
Rad7 and Rad16 are required for GGR but are dispensable for TCR (53). In contrast, Rad26 (47) and Rpb9 (24) mediate two subpathways of TCR but are not required for GGR. Rad2, Rad4, and Rad14 are essential for both TCR and GGR (31). As shown in Fig. 7A, following UV irradiation, the Rpb1 level decreased dramatically in all the rad mutant cells analyzed. In WT (Fig. 1A, top) and rad7 and rad16 cells (Fig. 7A), the cellular Rpb1 level was lowest 2 h following UV irradiation and started to recover afterwards. However, in rad26, rad2, rad4, and rad14 mutant cells, the Rpb1 level decreased continuously throughout the entire recovery period of 4 h (Fig. 7A). This is in agreement with previous reports indicating that rad26 (56) and rad14 (36) cells degrade Rpb1 more rapidly and to a greater extent than WT cells in response to UV damage. Taken together, these results indicate that UV-induced degradation of Rpb1 is independent of Rad26-mediated TCR, Rad7/Rad16-mediated GGR, or the entire NER process. However, TCR activity may play a role in the recovery of the Rpb1 level following UV damage.
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In agreement with a previous report (56), UV-induced degradation of Rpb1 was impaired in def1 cells (Fig. 7C) but occurred more rapidly in rad26 cells (Fig. 7A). Furthermore, deletion of RAD26 reactivated Rpb1 degradation in def1 (56) and rad16 def1 cells (Fig. 7C). These results indicate that Def1 plays a role in Rpb1 degradation only in the presence of its partner Rad26 and that a mechanism of Rpb1 degradation that is independent of Def1 exists in the cells. In contrast to Def1, Rpb9 seems to play a role in promoting UV-induced Rpb1 degradation regardless of Rad26, as the degradation is equally impaired in rpb9 (RAD26+) and rad26 rpb9 cells (Fig. 2B and 7B). We also found that def1 rpb9 double-deletion mutants are inviable (not shown), indicating that Def1 and Rpb9 may play overlapping roles in some essential cellular process(es).
Reinstatement of TCR in otherwise TCR-deficient cells does not affect UV-induced Rpb1 degradation. Our experiments described above suggest that TCR and UV-induced Rpb1 degradation are mutually independent processes. If this is the case, reinstatement of TCR in otherwise TCR-deficient cells may not affect UV-induced Rpb1 degradation. To test this idea, we examined the degradation in cells lacking Spt4, a regulator of transcription repression and elongation (11, 37, 45, 54, 57). It has been shown that deletion of SPT4 reinstates TCR in rad26 cells (16). We recently found that the deletion also reinstates TCR in rad26 rpb9 cells (22). As shown in Fig. 8, similar levels of UV-induced Rpb1 degradation occurred in rad7 rad26 and rad7 rad26 spt4 cells. The degradation was similarly impaired in rad7 rad26 rpb9 and rad7 rad26 rpb9 spt4 cells. These results further indicate that UV-induced Rpb1 degradation is unrelated to TCR but requires Rpb9.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The detailed biochemical mechanism as to how Rpb9 promotes Rpb1 ubiquitylation and degradation waits to be investigated. It seems that Pol II stalled at a DNA lesion or at a natural pause site may elicit a signal for ubiquitylation and degradation (42). We show that the Zn2 domain is essential for Rpb9 to promote Rpb1 degradation, whereas the Zn1 and linker domains play a subsidiary role. One possibility is that the Zn2 domain is involved in the formation of a conformational structure of Pol II. This conformational structure may be required for "sensing" a transcription arrest or for signaling ubiquitylation and degradation of Rpb1 upon transcription arrest. Alternatively, the Zn2 domain may be involved in recruiting factors for ubiquitylation of Rpb1. The subsidiary role of the Zn1 and linker domains may be achieved simply by enhancing the association of Rpb9 with the core Pol II. The crystal structure of Pol II indicates that almost the entire Rpb9 protein interacts with the core Pol II: Zn1 contacts the Rpb2 lobe, the linker forms a strong ß-addition motif with ß28 on the Rpb1, and Zn2 interacts with the Rpb1 funnel (5). It is therefore not surprising that almost the full length of Rpb9 seems to be required for a strong interaction with the core Pol II (Fig. 6).
Interestingly, Rpb9 fragments that contain Zn1 and most of the linker but have the entire Zn2 domain deleted are almost fully functional for transcription elongation (22, 49) and TCR (22). However, our coimmunoprecipitation assay suggests that the interaction between these fragments and the core Pol II is dramatically weakened (Fig. 6). Therefore, a weak interaction between the Rpb9 fragments and the core Pol II may be enough for transcription elongation and TCR functions. However, although less likely, it is also possible that these fragments may exert transcription elongation and TCR functions without physically interacting with the core Pol II.
Our data, together with those of others (e.g., reference 56) suggest that Rpb1 degradation is unrelated to TCR in yeast. First, TCR mediated by Rad26 or the entire NER activity is not required for Rpb1 degradation (Fig. 7) (56). Second, the Zn2 domain is essential for Rpb9 to promote Rpb1 degradation but is dispensable for Rpb9-mediated TCR (22). Third, reactivation of TCR activity in rad26 or rad26 rpb9 cells (by deleting SPT4) does not affect UV-induced Rpb1 degradation (Fig. 8). However, it has been clearly shown that the two TCR factors CSA and CSB are required for DNA damage-induced Rpb1 ubiquitylation and degradation in human cells (3). The discrepancy between the yeast and human data indicates that these processes may not be conserved between the two eukaryotic systems. However, it has been shown that some essential NER proteins, such as XPA, XPD, and XPG, are not required for ubiquitylation and degradation of Rpb1 in human cells (32), indicating that TCR per se may not be required for the ubiquitylation and degradation processes. Therefore, the human CSA and CSB may be similar to the yeast Rpb9 in that their function for TCR is unrelated to their function for DNA damage-induced ubiquitylation and degradation of Rpb1.
It has been shown that lysine 695 of Rpb1 is spontaneously ubiquitylated in vivo (13). In vitro reconstitution of Pol II ubiquitylation also identified lysine 695 of Rpb1 as a ubiquitylation site (42). However, replacement of the lysine with arginine in Rpb1 did not significantly affect degradation of Rpb1 upon UV irradiation (not shown). Therefore, in response to DNA damage, lysine 695 of Rpb1 may not be the major ubiquitylation site that leads to Rpb1 degradation in the cell. It has also been shown that the ubiquitylation site is not located in the CTD of Rpb1 in human cells (32). Thus, the ubiquitylation site(s) of Rpb1 that leads to degradation in the cell in response to DNA damage remains to be identified.
Although dramatically impaired, degradation of Rpb1 in response to UV radiation is not completely absent in either rpb9 (Fig. 2B and C) or def1 (Fig. 7C) cells. It is therefore likely that another minor Rpb1 degradation mechanism(s) also exists in the cells. Upon UV irradiation, Rpb1 can be degraded by apoptotic enzymes (caspases) in human xeroderma pigmentosum cells (26). Although yeast cells lack some elements of the complex apoptotic machinery of metazoan cells, recent studies showed that many features of apoptosis, including a caspase-like activity, can be induced in yeast by DNA damage (55). The cause of the residual UV-induced Rpb1 degradation in rpb9 and def1 cells remains to be understood.
Finally, our data show that Rpb1, instead of the whole Pol II complex, is ubiquitylated and degraded in response to DNA damage. The selective degradation of Rpb1 in response to DNA damage may offer an economical way for the cell to recycle other Pol II subunits.
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ACKNOWLEDGMENTS |
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This study was supported by NIH grant ES012718 from the National Institute of Environmental Health Sciences.
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FOOTNOTES |
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Published ahead of print on 23 April 2007.
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