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Previous Article | Next Article 
Molecular and Cellular Biology, June 2005, p. 4662-4675, Vol. 25, No. 11
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.11.4662-4675.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Molecular Biology and Functional Genomics, Stockholm University, S-10691 StockholmSweden
Received 31 March 2004/ Returned for modification 17 May 2004/ Accepted 28 February 2005
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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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Structurally, the 26S proteasome consists of a 20S catalytic core and a 19S regulatory complex that associates with the ends of the 20S proteasome in an ATP-dependent manner (3, 20, 46). The eukaryotic 20S proteasome is composed of 14 different subunits arranged in four stacked, seven-membered rings that form the barrel-shaped complex (18, 43). The 19S regulatory complex is itself composed of two distinct subcomplexes, the base and the lid (15). Six distinct ATPase subunits proposed to function in substrate unfolding and gating of the 20S pore have been localized to the base along with two additional subunits (7, 12, 16, 36). At least eight subunits form a lid subcomplex that is thought to be necessary for the processing of polyubiquitinated proteins and exhibit high similarities to the COP9/signalosome complex (15). An additional 19S subunit, Rpn10/S5a, has been observed to stabilize the interaction between the lid and the base subcomplexes. The Rpn10/S5a is a polyubiquitin binding protein (8, 44). Rpn10/S5a can be found both associated with the 26S proteasome and as a free form. For eukaryotic proteasomes, each subunit of the complex is coded by a unique gene, and the vast majority of these genes are essential for both proteasome activity and the survival of the organism (14, 17, 36, 37, 39).
Previously, we have observed that RNA interference (RNAi) depletion of the Rpn10/S5a subunit increases the level of 26S proteasome (28). This is in agreement with the observed overproduction of 26S proteasome observed in the Rpn10/S5a Drosophila melanogaster fly deletion which results in larval-pupal lethality (41). Drosophila cell lines lacking Rpn10/S5a show only minor increases in ubiquitin conjugate levels with no observable loss of in vivo proteasome activity (28). We sought to take advantage of this observation where knockdown of the Rpn10/S5a subunit apparently can induce proteasome levels without inducing substantial stress responses and thereby address the question of what genes are regulated components of the metazoan proteasome network. Transcriptional induction of the 26S proteasome mRNA levels has been recently observed for cells treated with proteasome inhibitors (32). To examine this in the Drosophila model system, we attempted to identify the pool of enriched mRNAs after two distinct treatments, MG132 inhibition of the 20S proteasome and knockdown of the Rpn10/S5a subunit by RNA interference. Treated Drosophila S2 cell lines were used to generate microarray probes to test for genome-wide expression pattern changes. The following set of RNAi-induced mRNAs was compared to the larger set of mRNAs that are induced by inhibition of the 20S proteasome. The combined results reveal a concise group consisting of known proteasome genes from both the 20S and 19S complexes, proteasome related genes, and a small number of novel genes.
A proteasome negative feedback circuit has been described in Saccharomyces cerevisiae that functions in maintaining proteasome levels through the selected degradation of the Rpn4 protein, a transcription factor that regulates both 26S proteasome and other genes through binding to a 9-bp upstream activating sequence termed PACE (30, 50). Metazoan genomes apparently lack an Rpn4 homolog, and neither a transcription factor nor a DNA regulatory element that regulates proteasome levels has been identified in higher eukaryotes. From the presented genome profiles, a list of Drosophila genes can be grouped as potential members of a proteasome regulatory network. The gene sequences and promoter regions for proposed genes that were found to be transcriptionally up-regulated were searched for regulatory elements. Promoter mapping and site-directed mutagenesis of a number of different Drosophila proteasome promoters indicate that an element within the 5' untranslated region (UTR) allows the proteasome regulatory network to sense the cellular level of proteasomes.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture. Schneider 2 (S2) cells were cultured in Schneider's Drosophila medium (Gibco) supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 µg/ml streptomycin and L-glutamine (2 mM). Cells were maintained at 24°C and passed every 7 days at a 1:4 dilution. Drosophila S2 stable cell lines expressing the green fluorescent protein (GFP) for the in vivo assay of the 26S proteasome were described previously (28). For constitutive expression in Drosophila S2 cells, the genes for the short-lived green fluorescent proteins UbG76VGFP and Ub-R-GFP (6) were previously subcloned into the pAct vectors (Invitrogen).
dsRNA synthesis. For the RNAi experiments, double-stranded RNA (dsRNA) was synthesized with the aid of the MEGAscript kit (Ambion). Oligonucleotides were selected by searching the GadFly database. An approximately 700-bp fragment of exon sequence was amplified for GFP and Rpn10/S5a, respectively. The Rpn10/S5a T7-tailed DNA was produced with PCR amplification of a clone in a pET26b vector with the following primers: 5'-GAATTAATACGACTCACTATAGGGAGACGCGCTTAATGGCAAGGACG-3' and 5'-GAATTAATACGACTCACTATAGGGAGAGCGAGTCCTTGCCGTCGCTC-3'. GFP T7-tailed DNA was produced with PCR amplification from recombinant GFP with the following primers: 5'-GAATTAATACGACTCACTATAGGGAGAGGTGAGCAAGGGCGAGGAGCTGTTC-3' and 5'-GAATTAATACGACTCACTATAGGGAGAATGCCGAGAGTGATCCCGGCGGCGG-3'.
dsRNA synthesis to the new candidate targets was performed as described above with PCR amplification from genomic Drosophila DNA extracted from S2 cells with the QIAGEN DNeasy Tissue kit. The following primer sequences were used for the first PCR: for UMP1 CG9324, 5'-GTTGATTAGACGTGTTTTGCTTGAGGAATGTAATGCG-3' and 5'-GCAACTTCATGGACGATGTCCTGACTGGCC-3'; for TER94 CG2331, 5'-GGGCAGCCTCAGAGCAGAGTGAAGCC-3' and 5'-GCTGACCGACCCGGAGCCCTACTGC-3'; for Ufd-1 CG6233, 5'-GGTGGCTCCAGCGATTTCGCCGC-3' and 5'-ATCCATAGGCACGGAGAGCCCAGCACAC-3'; for sequestosome, ref(2)p CG10360, 5'-GGAGTAGTTGGCTGAGTGGAACGTGGAGTCG-3' and 5'-CAAGTGCGAGAGCAATATGCACG-3'; for CG5495, 5'-ATTCACGGGCACTCCACTCTCCAGCTCC-3' and 5'-CCTATCACCATGTCCGTGCGCGTGATC-3'; for CG12321, 5'-CCGAATAGTAGGTTCCAGGACTTGGGCACGG-3' and 5'-GGACAAGCGTACCACTCTGGACGTAGCAG-3'; for CG5039, 5'-GAATTAATACGACTCACTATAGGGAGACGTAGTTGTTGTGATTTCTTTGTACATCCCCTGAAAACG-3' and 5'-GAATTAATACGACTCACTATAGGGAGAGCGTTATTCGCTCCCTGTGGAAGAAAAAGGG-3'; for CG2046, 5'-GAATTAATACGACTCACTATAGGGAGACCTCGTCGCGTGCCTTCTGGGACG-3' and 5'-GAATTAATACGACTCACTATAGGGAGAGAGCTGCAGGCAACGCCCGCGGC-3'; for CG9828, 5'-GAATTAATACGACTCACTATAGGGAGACTGTCCGATCCCGAGAAGCGGCGC-3' and 5'-GAATTAATACGACTCACTATAGGGAGAGGGTACGTGCGCAGACAGACGTTTCG-3'; for S6 CG16916, 5'-CCGTCCATCTGGTTGAGCAGCTCGAGC-3' and 5'-GCAGGAGGAGTACATCAAGGACG-3'; for S6 homolog CG9475, 5'-CTGTCTGTGCATCAAAGCGCTTGGTGGC-3' and 5'-GGAGCTGGAGCTCATCCAGGTGCAGGAG-3'; for S8 CG1489, 5'-GTGCACTCGGTGTGATGGGCAACGG-3' and 5'-CGACATCTTGCATTGTGGTCCTTCAGTTCG-3'; for S8 homolog CG2241, 5'-CCAGTTCCAGGCGGTCCGTAGAGGAGC-3' and 5'-CAATAGCTCGGGAAGTTGTTGAGCGCTATACAAGG-3'.
T7 tails were added during a second PCR performed on purified products (PCR clean-up kit; QIAGEN). Primers for the second PCRs matched the 5' half of the original PCRs with the addition of T7 tail sequences (5'-GAATTAATACGACTCACTATAGGGAGA-3'). For some genes, the T7 tail was already present in the primer in the first round of PCR from genomic DNA. The T7-tailed dsRNA was purified with the RNeasy kit (QIAGEN) followed by annealing in 0.1 M NaCl plus 20 mM Na-Citrate, pH 6.8, buffer for 30 min at 65°C followed by slow cooling to room temperature in a water bath. The amount and quality of the produced dsRNA were determined by spectrophotometric analysis and agarose gel electrophoresis.
Double-stranded RNAi. RNAi treatment of Drosophila S2 cells was performed as previously described (4, 28). Briefly, Drosophila S2 cell cultures were diluted to a concentration of 1 x106 cells/ml in DES serum-free medium (Invitrogen). After cell attachment, the medium was replaced with 1 ml DES serum-free medium, and the cells were grown in the serum-free medium for 1 to 2 h. To initiate RNAi, 60 µg of dsRNA was directly added to the medium under constant agitation. After 5 h, 2 ml of Schneider's medium containing 10% fetal bovine serum was added, and the cells were cultured for 3 to 4 days prior to isolation of RNA, analysis by Western blot, reverse transcription (RT)-PCR, or 26S GFP in vivo assays.
MG132 treatment for proteasome induction studies. Approximately 12 million cells were treated with MG132 (affinity, dissolved in ethanol) at a final concentration of 10 µM for 5 h. In parallel, 12 million untreated cells were prepared and seeded under the same conditions. Total RNA was isolated as described below.
Isolation of total RNA and array probe synthesis. Total RNA was isolated using the RNeasy kit and QIAshredder (QIAGEN). Approximately 6 million RNAi-treated Drosophila S2 cells resulted in a total yield of 40 µg at a concentration of 2 µg/µl when eluted in 30 µl of RNase-free water. The quality of the RNA was analyzed on 1% agarose gel to visualize the two rRNA bands and by spectrophotometric determination of the value of the optical density at 260 nm/optical density at 280 nm. Ten micrograms of total RNA was used for synthesis of cDNA. Probe synthesis was carried out according to the Affymetrix Eukaryotic Target Preparation protocol. Enzymes, buffers, and additional reagents were purchased from Invitrogen. GeneChip T7-oligo(dT) 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3' was ordered and purified from Thermo Hybaid. Double-stranded cDNA and biotin-labeled cRNA were purified using a GeneChip Sample Cleanup Module (Affymetrix). Purified cRNA and fragmented cRNA were analyzed on a 1% agarose gel.
Analysis of mRNA expression using oligonucleotide arrays. The Drosophila microarray chips were purchased from Affymetrix. The chips were hybridized using the Affymetrix GeneChip Fluidic Station 400 and analyzed with the Hewlett Packard Gene Array scanner. Triplicate RNAi experiments for both GFP and Rpn10/S5a and duplicate experiments for both MG132-treated cells and untreated control cells were performed and compared using the Microarray Suite program. The processed microarray data are in the supplementary material, and raw data files are available on request.
Flow cytometry. For cell cycle analysis, Drosophila S2 stable cell line fluorescence was measured using a FACScan flow cytometer (Becton Dickinson), while data collection and analysis were performed using the CellQuest software.
Transcription reporter constructs. The transcription reporter vector used to analyze the promoter region was pBSlac20, previously described by Engstrom et al. (10). PCR products corresponding to the upstream promoter regions were generated from genomic DNA from wild-type flies. For each PCR, the 5' primer contained a SalI cleavage site, while the 3' primer contained a HindIII cleavage site. The resulting fragment was cloned and ligated into pBSlac20 that was previously digested with SalI and HindIII. The following set of oligonucleotides was used for the transcriptional mapping as shown in Fig. 6: beta 2 5' SalI, 5'-ACGCGTCGACGTTGACCACATGTGGCTCGGAAACATGG-3'; beta 2 3' HindIII, 5'-CCCAAGCTTCAAGGAAAACTAAGGTTTTAATTCTGCCGAGCGC-3'; S2 5' SalI, 5'-ACGCGTCGACGCCATGGATGGCCTTGCATCAGGCC-3'; S2 3' HindIII, 5'-CCCAAGCTTCTAGCTTGTCGGACTTAAGGGATTTAATTGGCACTACG-3'; S2 UTR, 5'-CCCAAGCTTGCAGTGTGACCGCGCGGCGAACG-3'; S2 #1, 5'-ACGCGTCGACCCACAATACCTCGGATCAAGAGG-3'; S2 #2, 5'-ACGCGTCGACGGAATTACAGCTAATACAACTGC-3'
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Northern slot blots to determine mRNA levels.
Total RNA was produced using an RNAeasy kit and QIAshredder (QIAGEN). Generally, different amounts of total RNA ranging from 5 to 1.25 µg were diluted in RNase-free water and blotted onto a nitrocellulose membrane, BrightStar-Plus (Ambion), using a Bio-Rad BioDot slot blot apparatus. After UV cross-linking, the membrane was labeled and washed using the buffers and instructions from the NorthernMax-Gly kit (Ambion). Specifically, the detection for each probe involved incubation overnight with a slot blot membrane in prehybridization buffer, ULTRAhyb (Ambion). For all experiments, the membranes were washed initially with a low-stringency buffer solution (Ambion) at room temperature for 10 min followed by two high-stringency washes (Ambion) at 68°C for 15 min. The slot blots were exposed and detected using a PhosphorImager. The radioactive RNA probes were produced using the Strip-EZ RNA StripAble RNA Probe Synthesis kit (Ambion). T7-tailed template DNA was produced with PCR from Drosophila genomic DNA using specific primers for each probe. [
-32P]UTP was incorporated according to the manufacturer's protocol. The different probes were approximately 700 bp in length. Individual membranes were used for multiple probings, and the previous probe was removed using the protocol and solutions provided in the Strip-EZ RNA StripAble RNA Probe Removal kit (Ambion).
Semiquantitative RT-PCR to confirm RNA interference. Total RNA from dsRNA-treated cells was isolated using the RNAeasy kit from QIAGEN. Specific primers covering intron sequence or the same primers used for amplification from genomic DNA were used to amplify mRNA levels using the Promega Access RT-PCR system. PCR products were separated onto agarose gels and compared to molecular weight markers to confirm that the product matched the expected size for spliced mRNA product (see Fig. S1 in the supplemental material).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Out of a possible 14,010 probe sets from the Drosophila Affymetrix Gene Chip, the RNA targets identified 5,500 genes (40%) from the S2 Drosophila cell line that gave signal hybridization strengths that were significant. Initially, the three similar triplicate samples were evaluated, and these comparisons showed that no significant differences were present. A three-by-three matrix evaluation was then carried out; each Rpn10/S5a RNAi treatment was compared to the three GFP controls, resulting in nine total comparisons. After comparing the GFP and the Rpn10/S5a, the absent and unchanged genes were subtracted, and the average severalfold changes from the nine comparisons were determined. The candidate up-regulated genes were ranked based on this average severalfold increase (Fig. 1A). The top 80 out of 99 genes with mRNA levels increased in a majority of comparisons, at least five of the nine comparisons with a P value of <0.0025, are listed in Tables 1 and 2. The complete processed data and the gene expression matrix are presented in the supplemental material. Overall, the mRNA expression profiles at 3 days posttreatment to knock down the proteasome subunit Rpn10/S5a led to a modest increase in the expression of the mRNAs required to assemble a 26S proteasome (Fig. 1A). Strikingly, proteasome genes represented the majority of mRNAs that showed any detectable increases after RNAi treatment.
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Up-regulation of proteasome-associated genes and novel candidates. For the Rpn10/S5a RNAi-treated cells, proteasome genes rank from 1 for the proposed Rpt6/S8 homolog to 66 for the Rpn7/S10a subunit. Within this range of 66 genes, a number of genes with similarity to proteasome-associated proteins are present (Table 2). An additional three genes that have recently been proposed to be additional subunits of the 19S complex, Uch-L3, p37A, and Sem1p, were also up-regulated (13, 19).
A Drosophila candidate with 22% similarity to the yeast 20S maturation factor UMP1p and 40% similarity to the human UMP1 homolog was identified (34, 47). The valosin-containing protein (VCP) ATPase (TER94, cdc48) and three gene products known to associate with this ATPase, Ufd1, Npl4, and p47 (2), were also found to be up-regulated, and this complex, sometimes termed the segregase, has been implicated in the recognition and presentation of ubiquitinated proteins to the proteasome (1). Recently, the ubiquitin-binding protein sequestosome 1 (SQSTM1/p62) was reported to serve a central role in polyubiquitin chain binding and proteasomal degradation in mammalian cells and Saccharomyces (38, 40). No homolog to this gene has been described in Drosophila. Our results show induction of a Drosophila gene that has similarity (27%) to the sequestosome protein sequence CG10360. This gene contains the conserved ubiquitin-associated (UBA) domains and a specific zinc-binding domain found in the mammalian form. In general, heat shock proteins were not identified as up-regulated genes after Rpn10/S5a RNAi. Drosophila HSP23 and HSP27 were exceptions, with significant increases in mRNA levels for six of nine comparisons. Recently, HSP27 has been identified to bind ubiquitin and interact directly with the 26S proteasome (33). A number of genes with RNA binding domains were identified as having increased mRNA levels and ranked high on the overall comparisons (Table 2). Approximately a dozen proteins within the top 80 up-regulated genes have no described function and were classified as novel. The majority of the novel Drosophila genes identified show high similarity to an uncharacterized mammalian gene.
RNAi-mediated knockdown of proteasome pathway candidates. To test if the genes that showed concerted up-regulation with the known proteasome genes had essential proteasome functions, RNAi was carried out on a set of these candidates. A total of 15 Drosophila genes were selected for RNAi experiments (Tables 1 and 2). To confirm that RNAi of the targeted gene had occurred, semiquantitative RT-PCRs were carried out on the treated cells, and the data are presented in the supplemental material. The majority of genes tested showed a clear reduction of the targeted mRNA. However, repeated attempts for RNAi against CG4673 (NPL4) and CG6697, a gene containing a Ub-like motif and phosphatase domain, failed to decrease their mRNA levels. The RNAi treatments were specific for decreasing the target mRNA while maintaining the levels of other proteasome genes.
To test the potential functions of the candidate up-regulated genes, immunoblots to examine overall ubiquitin conjugate levels and in vivo proteasome assays using stable cell lines expressing short-lived GFP proteasome substrates were carried out on the RNAi-treated cells (Fig. 2 and 3). Immunoblots from whole-cell lysates were carried out from treated and control cells and probed using ubiquitin antibodies. The in vivo 26S proteasome assay measures the ability of the cells to degrade GFP that has been engineered to act as an N-end rule or ubiquitin fusion degradation (UFD) substrate and was originally tested and described in mammalian cell lines (6). We have recently used the GFP substrates in Drosophila stable S2 cell lines to measure in vivo proteasome activity (28). The short-lived GFP reporters used in those studies lack endoplasmic reticulum signal sequences and were observed to be localized to the cytoplasm and nucleus when stabilized through proteasome inhibitor treatment.
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TER94, the Drosophila VCP ATPase, and CG6233, a Ufd1 gene, showed strong GFP stabilization for both the UFD and N-end rule substrates (Fig. 2C). The knockdown of TER94 yielded the highest percentage of GFP-stabilized cells and the highest levels of fluorescence per cell, even higher than at 7 h of 50 µM MG132 treatment. Cells lacking TER94 were generally found to have GFP stabilized throughout the cell with the greatest accumulations in the nucleus and were also found to contain cytoplasmic vacuoles containing stabilized GFP (Fig. 3).
RNAi against CG10360, which has similarity to sequestosome 1 binding protein, showed no increase in ubiquitin chain levels or GFP stabilization. The other tested candidates (underlined in Table 2) also showed no significant increases in ubiquitin conjugate pools or GFP stabilization after RNAi knockdown (data not shown).
Mapping of Drosophila proteasome transcription response elements. The availability of complete sequenced genomes from different Drosophila species allowed us to search for conserved regions in proteasome promoters among the different fly species. No obvious conserved motifs were found upstream of the proteasome genes, but almost all genes were found to contain 5' conserved UTRs. These sites are well conserved within individual proteasome genes for different species. However, only limited conservation exists between different proteasome genes. The conserved areas typically resided directly downstream of the proposed start site of transcriptional initiation. Through the use of plasmid-based transcriptional reporter assays, we have examined the importance of these conserved regions. Due to the difficulty in carrying out transcriptional reporter assays on RNAi-treated cells, we chose to focus on the mechanism of proteasome mRNA induction after MG132 treatment. Initially, treatment of Drosophila cells with 10 µM MG132 or 5 µM epoxomicin revealed increased levels of mRNA for proteasome genes and TER94, with a maximum increase at 5 h (Fig. 4A and B), and these conditions were used for the transcriptional assays.
As an initial step to understand the mechanism of transcriptional induction of proteasome genes, the promoter regions of 19S and 20S Drosophila proteasome genes were cloned into lacZ transcriptional promoter construct pBSlac20 (10) and transfected into S2 cells. Depending upon the distance between the proteasome gene and the preceding upstream gene, the transcriptional promoter constructs contained from 200 to 1,000 bp of upstream sequence and included the 5' UTRs of each gene tested. Previously, 5' rapid amplification of cDNA ends and Northern blots showed that proteasome inhibition did not change the transcriptional start points or overall messenger sizes for proteasome mRNA after inhibitor treatment (data not shown). As a control, the promoter region of the Drosophila proteasome activator REG was used. The Drosophila PA28 proteasome activator is under the transcriptional control of the DNA replication elements and is not transcriptionally up-regulated in the current microarray experiments. LacZ mRNA was measured using Northern slot blot experiments after 3 days posttransfection in combination with 5 h of treatment with proteasome inhibitor MG132. The promoter constructs contained 5' UTRs, and all proteasome subunits revealed a transcriptional increase in lacZ mRNA levels, while the REG promoter showed no change or a minor decrease (Fig. 4C). These inductions also correspond to increases in protein levels as determined by beta-galactosidase activity (data not shown).
Deletion mapping of upstream promoter regions alpha-2 and Rpn1/S2 was unable to identify MG132 response elements. All deletions in the upstream promoter regions that had remaining basal transcriptional activity were also responsive to MG132 treatment as long as the 5' UTRs were intact (data not shown). However, site-directed mutations in the conserved region of the 5' UTR of a beta-2 20S proteasome reporter construct revealed that the conserved region is required for the induction of mRNA (Fig. 5). Placement of a NotI site within the conserved region did not abolish basal transcription levels but did prevent the induction after proteasome inhibition. Mutations in the conserved 5' UTRs for beta-3 and S14 also inhibited induction of reporter mRNAs (data not shown).
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To confirm these results, the Rpn1/S2 5' UTR was mapped by the insertion of HindIII restriction sites throughout the 5' UTR (Fig. 7A). Transcriptional assays were initially carried out on the original HindIII site-directed mutants (Fig. 7B). The reporter constructs were then digested with HindIII enzyme and religated, giving a series of deletion constructs. These deletions were also assayed for their ability to respond to proteasome inhibition by MG132 (Fig. 7C). A 67-nucleotide region of the S2 5' UTR was sufficient to respond to proteasome inhibitor treatment. This area corresponds to a highly conserved region directly downstream of the site of transcriptional initiation.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we carried out RNAi against two Drosophila 19S proteasome subunits, Rpn10/S5a and Rpn11/S13 (28). Loss of either subunit results in the induction of 20S proteasomes. Other groups have reported similar findings with the loss of other 19S subunits and demonstrated that the loss of a specific 19S subunit increased the mRNA levels for fellow subunits (48). While the Rpn10/S5a subunit has recently been shown to be essential for the development of Drosophila (41), our work on Drosophila cells lacking Rpn10/S5a revealed almost no defects except for a modest increase in ubiquitin conjugate chains. In contrast, knockdown of essential proteasome subunits markedly affects proteasome activity and leads to a wide range of cellular responses.
The loss of the proteasome Rpn10/S5a subunit results in increased levels of both 20S and 19S subunits. The observed increased levels were modest, with less than a twofold increase for the majority of proteasome genes. Yet the increased levels are significant and result in a list of induced genes that is abundant in proteasome subunit genes. As a comparison, a previous study that examined proteasome inhibitor induction of subunit concentrations revealed a two- to threefold increase at the protein level (32). The induction levels observed likely underestimate the actual severalfold increase of mRNA induction possible after the complete loss of the Rpn10/S5a subunit. Unlike MG132 treatments, the RNAi effect on Drosophila cells occurs over a long time period, 3 days, and therefore, it is likely that a proportion of the Rpn10/S5a RNAi-treated cells had proteasome induction occur prior to the harvesting of mRNAs. We consider that the very high density of known proteasome genes present on the microarray ranking list (Fig. 1A) is the best evidence that knockdown of Rpn10/S5a provides valuable data to identify new members. The obtained low increases do make it likely that a number of genes that are in fact coregulated with the proteasome will be missed in this survey due to the lack of sensitivity in detecting up-regulation. As an example, the Ecm29 gene has been characterized to have a role in proteasome-dependent degradation (22). However, we were unable to find any change in Ecm29 mRNA levels after S5a RNAi treatment even though up-regulation (3.6-fold) was observed after proteasome inhibition with MG132.
During the preparation of the manuscript, a number of coregulated genes (Table 2) have shown relevance for the proteasome pathway in other model systems. The Drosophila gene CG13779 has 35% identity to the small protein Sem1p, a recently proposed 26S proteasome subunit from Saccharomyces cerevisiae (40), and has 60% identity to the proposed human protein homolog DSS1. Surprisingly, MG132-treated cells showed no observed increases in the Drosophila Sem1p mRNA. Another candidate, CG10360, shows similarity to a human protein that has recently been found to bind polyubiquitin and contribute to proteasome degradation, the sequestosome 1/p62 (38). While lower in overall identity (27%), both proteins share positionally conserved dystrophin-like zinc-binding domains and UBA domains. A recent study using RT-PCR quantification of proteasome inhibitor-treated cells revealed that the mammalian version of the 20S proteasome maturase was transcriptionally induced after inhibitor treatment (32). Our results confirm a metazoan coregulation between proteasome genes and the 20S maturase (CG9324). In addition, the concerted up-regulation of p37A/Uch-L3 (CG3431) and a gene with high sequence similarity to Ubp6p (CG5384) lends support to the idea that these two conserved proteins are actual proteasome subunits that easily dissociate from the complex during purification (27).
Recent interest has focused on proteins that assist in the transfer of ubiquitinated proteins to the 26S proteasome (1). Our results support a central role for the segregase complex (24) in proteasome-dependent degradation. These results indicate conserved regulation between metazoan and Saccharomyces cerevisiae. The yeast homolog of VCP, cdc48, is transcriptionally coregulated with proteasome subunits and has a PACE element that interacts with the yeast Rpn4 transcription factor (23). Past studies have implicated VCP in an extremely wide range of cellular functions, from fusion of the endoplasmic reticulum to degradation of I
B (5, 26). For the current microarray data, the VCP ATPase (CG2331), Npl4 (CG4673), and Ufd-1 (CG6233) genes of the segregase complex (2) were found to be consistently up-regulated. RNA interference was successful against two of the genes, the VCP ATPase and Ufd-1, resulting in stabilization of overall ubiquitin conjugates and in vivo GFP substrates. This confirms recent results showing that RNAi knockdown of the VCP ATPase stabilized ubiquitin conjugates in both Drosophila and HeLa cell lines (49). RNAi against the Drosophila VCP and Ufd-1 shows surprisingly strong stabilizations of both UFD and N-end rule substrates throughout the cell. This is in contrast to the yeast Ufd1 that is required only for UFD substrate degradation and was found to be nonessential for N-end rule substrate proteolysis (21).
The microarray profiling revealed that two homologs of ATPase subunits, Rpt6/S8 and Rpt3/S6, were strongly up-regulated. Both ATPase homologs have been previously proposed to be male-specific expressed homologs; however, little is known about their potential function (29). Previous 19S Drosophila proteasome purification studies did not identify these homologs as significant components of the 19S regulatory complex (19). In this study, RNAi treatment against the homologs showed no increase in overall ubiquitin chain levels or loss of proteasome activity (data not shown). In comparison, RNAi knockdown of either bona fide ATPase subunit resulted in increased ubiquitin conjugates and stabilization of 26 proteasome subunits in vivo. Currently, it is unclear how the ATPase homologs participate and function within the proteasomal pathway, but it is clear that they are coregulated with other proteasome genes and expressed in Drosophila cell lines.
Little is known about potential downstream members of the proteasome pathway. The 26S proteasome degrades proteins preferentially to small peptides (9), and additional proteases are required for the complete hydrolysis of substrates to individual amino acids. A number of peptidases were found to be up-regulated in our microarray analysis. A proposed carboxypeptidase (CG4572), a leucyl-aminopeptidase (CG7340), and dipeptidyl-peptidase III (CG7415) were all found to be up-regulated and may indicate a functional interaction with the proteasome in the downstream processing of short peptides to amino acids.
To start addressing the mechanism of proteasome mRNA induction after a loss of proteasome activity, we have tested promoter reporter plasmids using sequences directly upstream of 20S and 19S proteasome open reading frames. As expected, induction of the reporters was observed after proteasome inhibition. Alignments of upstream promoter regions have so far failed to reveal conserved DNA sequence motifs. However, when individual proteasome genes are compared across different Drosophila species, conserved regions are present in the vast majority of genes just downstream of the site of transcriptional initiation in the 5' UTRs. Removal of 5' UTRs from promoter constructs eliminates the previously observed induction after proteasome inhibition. Site-directed mutagenesis mapping of the Rpn1/S2 5' UTR confirms that the conserved region in the 5' UTR is essential for up-regulation of Drosophila proteasome genes. Finally, transfer of the proteasome subunit Rpn1/S2 5' UTR to a noninducible control promoter was sufficient to confer inducibility after proteasome inhibition of the reporter plasmid. These initial results indicate that an element(s) within the 5' UTR of Drosophila proteasome genes is essential for the cell to regulate proteasome levels. Sequence comparisons of the 5' UTRs show no obvious consensus sequence for all regulated genes. This suggests that either a number of distinct factors interact with a variety of conserved sites or the sites reflect a folded element on the 5' end of the newly synthesized mRNA. Future studies to determine what factor(s) is interacting with the 5' UTRs at either the DNA or RNA level should greatly aid in understanding metazoan proteasome regulation. Our results demonstrate that the concerted expression of proteasome genes includes both 26S proteasome subunit genes and functionally related genes. Future use of bioinformatic tools and promoter mapping studies should allow further characterization of the system that globally regulates proteasome levels in higher eukaryotes.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Swedish Research Council and the Carl Tryggers Stiftelse.
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FOOTNOTES |
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Supplemental material for this article may be found at http://mcb.asm.org/.
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