Portal de Periódicos da CAPES

The Transcriptional Co-activator ADA5 Is Required for HAC1 mRNA Processing in Vivo

2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês

10.1074/jbc.275.5.3377

1083-351X

Ajith Welihinda, Witoon Tirasophon, Randal J. Kaufman,

Heat shock proteins research

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates signaling pathways to induce transcription of a number of genes encoding ER protein chaperones and-folding catalysts. In Saccharomyces cerevisiae this transcriptional induction is mediated by an increase in the synthesis of the transcription factor Hac1p. The transmembrane receptor Ire1p/Ern1p containing a Ser/Thr protein kinase and endoribonuclease activity transmits the unfolded protein response (UPR) from the ER to the nucleus. Activation of Ire1p kinase induces its endoribonuclease activity to cleave unspliced HAC1 mRNA and generate exon fragments that are subsequently ligated by tRNA ligase (RLG1). Whereas unspliced HAC1 mRNA is poorly translated, spliced HAC1 mRNA is efficiently translated. Subunits of the yeast transcriptional co-activator complex SAGA also play a role in the UPR. Deletion of GCN5,ADA2, or ADA3 reduces, and deletion ofADA5 completely abolishes, the UPR. AlthoughHAC1 mRNA requires only Ire1p and Rlg1p in vitro, we demonstrate that ADA5 is required for theIRE1/RLG1-dependent splicing reaction ofHAC1 mRNA in vivo. In addition, Ada5p interacts with Ire1p. These results suggest that subcomponents of transcriptional co-activator complexes may be involved in RNA processing events. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates signaling pathways to induce transcription of a number of genes encoding ER protein chaperones and-folding catalysts. In Saccharomyces cerevisiae this transcriptional induction is mediated by an increase in the synthesis of the transcription factor Hac1p. The transmembrane receptor Ire1p/Ern1p containing a Ser/Thr protein kinase and endoribonuclease activity transmits the unfolded protein response (UPR) from the ER to the nucleus. Activation of Ire1p kinase induces its endoribonuclease activity to cleave unspliced HAC1 mRNA and generate exon fragments that are subsequently ligated by tRNA ligase (RLG1). Whereas unspliced HAC1 mRNA is poorly translated, spliced HAC1 mRNA is efficiently translated. Subunits of the yeast transcriptional co-activator complex SAGA also play a role in the UPR. Deletion of GCN5,ADA2, or ADA3 reduces, and deletion ofADA5 completely abolishes, the UPR. AlthoughHAC1 mRNA requires only Ire1p and Rlg1p in vitro, we demonstrate that ADA5 is required for theIRE1/RLG1-dependent splicing reaction ofHAC1 mRNA in vivo. In addition, Ada5p interacts with Ire1p. These results suggest that subcomponents of transcriptional co-activator complexes may be involved in RNA processing events. endoplasmic reticulum unfolded protein response unfolded protein response element basic leucine zipper immunoglobulin binding protein glutathione S-transferase polymerase chain reaction SPT/ADA/GCN adapter complex green fluorescent protein tunicamycin The lumen of the endoplasmic reticulum (ER)1 is a highly specialized compartment in eukaryotic cells. It is the primary site for folding of secretory and transmembrane proteins that constitute about one-third of all cellular proteins. The ER lumen provides an oxidizing environment and contains a number of resident chaperones, such as BiP (GRP78), that facilitate protein folding. These chaperones are proposed to catalyze protein folding and/or prevent aggregation of folding intermediates. Perturbation of the protein folding machinery in the ER leads to an accumulation of unfolded proteins. Cells respond to unfolded protein in the ER by up-regulating the synthesis of resident chaperones, thereby increasing the folding capacity in the ER compartment. This cellular response is termed the unfolded protein response (UPR) and is conserved in all eukaryotic organisms (1.Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar). In the budding yeast Saccharomyces cerevisiae, genes that are transcriptionally activated in response to unfolded proteins in the ER contain a 22-base pair cis-acting promoter element, termed the UPRE, that is necessary and sufficient to mediate the transcriptional induction (2.Mori K. Sant A. Kohno K. Normington K. Gething M.J. Sambrook J.F. EMBO J. 1992; 11: 2583-2593Crossref PubMed Scopus (311) Google Scholar). The UPREs in these genes contain a partially palindromic sequence (CAGCGTG) with a spacer of one nucleotide that is required for the transcriptional induction (3.Mori K. Ogawa N. Kawahara T. Yanagi H. Yura T. J. Biol. Chem. 1998; 273: 9912-9920Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Thetrans-acting factor that binds the UPRE is a basic leucine zipper protein (bZIP) called Hac1p (4.Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar, 5.Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (306) Google Scholar). Recent studies demonstrated that activation of the UPR is dependent upon the cellular levels of Hac1p. HAC1 is constitutively transcribed independently of the protein folding status in the ER. In the absence of unfolded proteins in the ER, HAC1 mRNA is exported to the cytoplasm and engaged with ribosomes, but translation is stalled. A 252-nucleotide fragment near the 3′ end of HAC1 mRNA acts as a translational attenuator to limit Hac1p synthesis (6.Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar, 7.Chapman R.E. Walter P. Curr. Biol. 1997; 7: 850-859Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, in the presence of unfolded proteins in the ER, the translational attenuator in HAC1 mRNA is removed by an unconventional splicing reaction that does not utilize the cellular spliceasomal machinery. HAC1 mRNA splicing allows efficient translation of Hac1p and subsequent transcriptional activation of genes regulated by the UPR. Perhaps the most important component of the UPR pathway is Ire1p/Ern1p (8.Cox J.S. Shamu C.E. Walter P. Cell. 1993; 73: 1197-1206Abstract Full Text PDF PubMed Scopus (950) Google Scholar, 9.Mori K. Ma W. Gething M.-J. Sambrook J. Cell. 1993; 74: 743-756Abstract Full Text PDF PubMed Scopus (655) Google Scholar). Ire1p is a type 1 transmembrane protein of the ER that is a bifunctional enzyme. The cytosolic domain contains both a serine/threonine kinase (10.Welihinda A.A. Kaufman R.J. J. Biol. Chem. 1996; 271: 18181-18187Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11.Shamu C.E. Walter P. EMBO J. 1996; 15: 3028-3039Crossref PubMed Scopus (451) Google Scholar) and a site-specific endoribonuclease activity (12.Sidrauski C. Walter P. Cell. 1997; 90: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). In response to unfolded proteins in the ER, Ire1p forms oligomers that mediate trans-autophosphorylation to activate the site-specific endoribonuclease activity that cleaves at the splice site junctions within the 3′ end of HAC1 mRNA. The cleaved 5′ and 3′ exons of HAC1 mRNA are ligated together by the tRNA ligase RLG1, generating a new mRNA species that is efficiently translated (13.Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Because permanent activation of the UPR pathway is detrimental to cell growth (11.Shamu C.E. Walter P. EMBO J. 1996; 15: 3028-3039Crossref PubMed Scopus (451) Google Scholar, 14.Wang X.Z. Harding H.P. Zhang Y. Jolicoeur E.M. Kuroda M. Ron D. EMBO J. 1998; 17: 5708-5717Crossref PubMed Scopus (661) Google Scholar), the UPR needs to be tightly regulated. Upon phosphorylation, Ire1p recruits a serine/threonine phosphatase, PTC2, that dephosphorylates Ire1p and down-regulates the UPR (15.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Mol. Cell. Biol. 1998; 18: 1967-1977Crossref PubMed Scopus (97) Google Scholar). The transcriptional co-activator Gcn5p was isolated as a specific interactor with Ire1p in a yeast two-hybrid screen (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). In eukaryotes, transcriptional activation requires functional interaction between the activators that bind upstream activating sequences and the basal factors that occupy the TATA box. It is proposed that functional interaction between these two classes of transcription factors is mediated by transcriptional co-activators. Gcn5p along with other transcriptional co-activators, including Ada1, Ada2p, Ada3p, Spt3p, Spt7p, Spt8p, and Ada5p/Spt20p, constitute a 1.8-mDa SAGA complex that is responsible for histone acetylation during transcriptional activation (17.Georgakopoulos T. Thireos G. EMBO J. 1992; 11: 4145-4152Crossref PubMed Scopus (255) Google Scholar, 18.Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (887) Google Scholar, 19.Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar, 20.Horiuchi J. Silverman N. Pina B. Marcus G.A. Guarente L. Mol. Cell. Biol. 1997; 17: 3220-3228Crossref PubMed Scopus (99) Google Scholar, 21.Marcus G.A. Silverman N. Berger S.L. Horiuchi J. Guarente L. EMBO J. 1994; 13: 4807-4815Crossref PubMed Scopus (237) Google Scholar, 22.Marcus G.A. Horiuchi J. Silverman N. Guarente L. Mol. Cell. Biol. 1996; 16: 3197-3205Crossref PubMed Scopus (82) Google Scholar, 23.Roberts S.M. Winston F. Mol. Cell. Biol. 1996; 16: 3206-3213Crossref PubMed Scopus (104) Google Scholar, 24.Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Belotserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (291) Google Scholar). Mutations in the ADA genes were isolated as suppressors of the lethality induced by over-expression of the herpes simplex virus potent acid transcriptional activator VP-16 (19.Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar). The SPT genes were isolated as suppressors of Ty transposon insertions (23.Roberts S.M. Winston F. Mol. Cell. Biol. 1996; 16: 3206-3213Crossref PubMed Scopus (104) Google Scholar). Both sets of genes are required for maximal transcriptional activation from TATA-containing promoters. Yeast strains lacking Gcn5p, Ada2p, and Ada3p are partially defective in transcriptional activation of genes encoding ER protein chaperones in response to unfolded proteins in the ER. In contrast, cells lacking Ada5p are completely defective for the UPR but contain an intact heat shock response, demonstrating a specific requirement for Ada5p in the UPR (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). In the present study, we investigated the role of ADA5 in the UPR pathway. We found that apart from its function as a transcriptional co-activator in the UPR pathway, ADA5 is also required for HAC1mRNA processing, a hitherto novel function for a transcriptional co-activator. Moreover, our data reveal the molecular basis for the defective UPR in Δada5 cells. The Escherichia coli strain DH5α was used for the propagation of plasmids. The genotype of the S. cerevisiae strain used in this study was BWG1-7a, Matα leu2::3,112 his4–519 ade1–100 ura3–52 (19.Horiuchi J. Silverman N. Marcus G.A. Guarente L. Mol. Cell. Biol. 1995; 15: 1203-1209Crossref PubMed Google Scholar). The genetic methods and standard media were previously described (25.Sherman F. Fink G.R. Hicks J. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Plainview, NY1986Google Scholar). The construction of pAW65 (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar), pGEM4Z-ACT1 (15.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Mol. Cell. Biol. 1998; 18: 1967-1977Crossref PubMed Scopus (97) Google Scholar), and the fusion protein containing the Ire1p cytoplasmic (10.Welihinda A.A. Kaufman R.J. J. Biol. Chem. 1996; 271: 18181-18187Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) domain with glutathioneS-transferase (GST) were previously described. To construct GST-HAC1 fusion constructs, PCR amplified fragments from the wild-type HAC1 were subcloned into the BamHI andEcoRI sites of the bacterial expression vector, pGEX1λT (Amersham Pharmacia Biotech). The expression GST-Hac1p fusion product was confirmed by Western blotting with anti-GST antibodies. To construct pGEM4Z-HAC1, a 240-base pair fragment from HAC1was amplified by PCR using primers 5′-ccctctagacttcaagtcgactctgcctcc-3′ and 5′-tcaaagcttgcaacaaaagcgtcgtggc-3′ and subcloned into theXbaI and HindIII sites of pGEM-4Z. Similarly, a 206-base pair fragment from IRE1 was amplified by PCR using primers 5′-gagctgggaattcactcttatg-3′ and 5′-aataagcttggggaatgtcggagaaaccc-3′ and subcloned into theEcoRI and HindIII sites of pGEM-4Z to derive pGEM4Z-IRE1. Construction of plasmid pJC835 containingHAC1 i was described previously (4.Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). The yeast strain carrying the null allele of HAC1 was created by the method of one step gene disruption (26.Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1105) Google Scholar). BGW1-7a cells were transformed with BamHI digested pHAKO1 (4.Cox J.S. Walter P. Cell. 1996; 87: 391-404Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). Transformants containing the disrupted hac1 were selected for uracil prototrophy and the gene disruption was confirmed by PCR and Southern/Northern blot analyses. The construction of Δire1and Δada5 strains was described previously (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). Yeast cell lysates were prepared according to Williams et al. (27.Williams F.E. Varanasi U. Trumbly R.J. Mol. Cell. Biol. 1991; 11: 3307-3316Crossref PubMed Scopus (175) Google Scholar). Western blotting was performed by standard procedures (28.Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1988Google Scholar) using anti-Hac1p primary antibodies (generously provided by Dr. Peter Walter, University of California, San Francisco) and horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Life Technologies, Inc.). Bands were detected using the enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech) and quantified using NIH image software. Total protein and β-galactosidase activity were measured using commercially available kits from Bio-Rad and Promega (Madison, WI), respectively. Plasmids carrying GCN5, ADA2, ADA3, and ADA5 under the T7 promoter (generously provided by Dr. Leonard Guarente, Massachusetts Institute of Technology) were used in coupled in vitro transcription and translation assays to create [35S]methionine labeled products. Equal amounts of each recombinant protein were incubated with glutathione-Sepharose beads containing equal amounts of either GST-Ire1p cytoplasmic domain or GST-Hac1p fusion protein at 4 °C for 2 h in the binding buffer (phosphate-buffered saline, 10% glycerol, 2 mm EDTA). As a control, beads containing comparable amounts of GST were used. Beads were recovered, washed sequentially with phosphate-buffered saline containing 10% glycerol and 1, 0.5, and 0.05% Triton X-100, and boiled for 3 min in 1× Laemmli buffer (29.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). Extracts were electrophoresed on 10% SDS-polyacrylamide gels under reducing conditions, treated with En/Hance (NEN Life Science Products), and analyzed by autoradiography and PhosphorImager scanning (Molecular Dynamics, Sunnyvale, CA). Total RNA was isolated according to Schmitt et al. (30.Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1155) Google Scholar) from cells treated with or without Tm for 90 min for analysis using an RNase protection kit (Roche Molecular Biochemicals). pGEM-4ZHAC1, pGEM-4ZIRE1, and pGEM-4ZACT1 were linearized with XbaI, and antisense RNA probes were synthesized using T7 RNA polymerase (Roche Molecular Biochemicals) and [α32P]CTP (Amersham Pharmacia Biotech). A DNA sequencing ladder from a known template was used as size markers. GCN5, ADA2, ADA3, and ADA5 are required for the maximal transcriptional induction of genes encoding ER resident chaperones including KAR2/BiP in response to protein misfolding in the ER (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). In contrast, the SAGA complex is not required for the heat-mediated transcriptional activation of KAR2 (16), suggesting that these co-activators play a specific role in the UPR as opposed to global transcriptional induction. Among these co-activators, Ada5p is the most interesting because cells lacking Ada5p are completely defective in responding to unfolded proteins in the ER and are inositol auxotrophs, the two phenotypes associated withΔire1 and Δhac1 cells. To elucidate the mechanistic role of the SAGA complex in the UPR pathway, we asked whether the defective UPR in cells lacking these co-activators results from inefficient HAC1 transcription. HAC1 encodes the bZIP transcription factor Hac1p, which is essential for transcriptional induction of genes responding to the UPR. The levels ofHAC1 mRNA were measured in Δada5 cells by an RNase protection assay. In this assay, the presence ofHAC1 mRNA (both processed and unprocessed) should protect a 240-base pair nucleotide fragment from the internal labeled probe. ACT1 mRNA served as an internal control for the amount of RNA in the reaction. The results show that HAC1was transcribed in Δada5 as well as in Δire1cells. The level of HAC1 transcription in these cells was comparable with that in the wild-type cells with or without tunicamycin (Tm) treatment, a drug that inhibits N-linked glycosylation and therefore disrupts protein folding in the ER (Fig.1 A, lanes 1–4 and6–7). These results demonstrate that Δada5cells efficiently transcribe HAC1. Therefore, theΔada5 cells are similar to Δire1 cells where the abrogated UPR does not arise from defective HAC1transcription. The cellular level of Hac1p regulates the UPR. Although HAC1is constitutively transcribed, only the processed transcripts are translated. HAC1 mRNA processing requires the activity of the site-specific endoribonuclease, Ire1p. We therefore asked whether IRE1 transcription is reduced in Δada5cells. An RNase protection assay demonstrated that both wild-type andΔada5 cells had comparable levels of IRE1mRNA, indicating that IRE1 is efficiently transcribed inΔada5 cells (Fig. 1 B, lanes 1–4). Therefore, the defective UPR in these cells is not due to reducedIRE1 transcription. Gcn5p was identified as a component in the UPR by its interaction with Ire1p, detected by the yeast two-hybrid system and by co-immunoprecipitation from cells co-expressing Gcn5p and Ire1p (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). To identify the component(s) of this complex that interact directly with Ire1p, in vitro affinity adsorbption experiments were performed with [35S]methionine-labeled in vitro transcription-translation products of GCN5,ADA2, ADA3, and ADA5. Hac1p and Ire1p were produced as soluble GST fusion proteins (GST-Hac1p, GST-Ire1p) inE. coli and purified by adsorption to glutathione-Sepharose beads. Beads containing either GST-Hac1p or GST-Ire1p fusion proteins were used to capture the Gcn5p, Ada2p, Ada3p, or Ada5p translation products from reticulocyte lysates. As a control, Sepharose beads bound to equimolar amounts of GST were used. In these in vitropull-down assays, the amounts of Ada2p, Ada3p, and Ada5p bound to GST-Hac1p were not significantly different from that obtained with control GST beads (Fig. 2, lanes 5–8 and 10–12). In contrast, GST-Hac1p brought down a 4.5-fold greater amount of Gcn5p than the GST control (Fig. 2,lane 5 versus 9), indicating a direct interaction between the two proteins. GST-Ire1p, on the other hand, interacted with Ada2p and Ada5p (4.1- and 3.1-fold more than the GST control, respectively) (Fig. 2, lanes 14 and 16) but not with Gcn5p or Ada3p (Fig. 2, lanes 13 and15). The specificity for the interaction with Ada5p was further demonstrated by the enrichment of the full-length Ada5p translation products by GST-Ire1p. These results indicate that Ire1p directly interacts with Ada2p and Ada5p and suggest that the original yeast two-hybrid interaction detected between Ire1p and the SAGA complex was mediated through Ada2p and Ada5p but not through Gcn5p. Therefore, the original interaction between Gcn5p and Ire1p detected in yeast was likely indirect and due to endogenous levels of Ada2p and Ada5p that could bridge Gcn5p with Ire1p. The site-specific endoribonuclease activity of Ire1p is required for theHAC1 mRNA processing event that leads to the generation of a translatable form of HAC1 mRNA and subsequent activation of the UPR. The direct interaction between Ire1p and some components of the SAGA complex suggested that the SAGA complex might play a role in HAC1 mRNA processing. To test this hypothesis, we used a HAC1 probe to perform Northern blot analysis on RNA isolated from different yeast strains. In wild-type cells, upon Tm treatment, the majority of the HAC1 mRNA was processed to a smaller RNA species (HAC1 i, Fig.3A, lanes 1 and 2). In contrast, upon Tm treatment of Δire1 cells, processedHAC1 mRNA was not generated, indicating a complete absence of UPR-activated Ire1p-dependent HAC1mRNA processing in these cells (Fig. 3 A, lanes 3 and 4). Deletion of GCN5, ADA2, and ADA3 had no effect on the Ire1p-dependentHAC1 mRNA processing, as similar amounts ofHAC1 i mRNA were detected after ER stress compared with the wild-type strain (Fig. 3 A, lane 2 versus lanes 6, 8, and 10). Like theΔire1 cells, Δada5 cells were completely defective in HAC1 mRNA processing upon activation of the UPR (Fig. 3 A, lanes 11 and 12), demonstrating that Ada5p plays a critical role in this mRNA processing reaction. To substantiate the evidence for defective HAC1 mRNA processing in Δada5 cells, Western blot analysis was performed to measure the steady state levels of Hac1p before and after ER stress. An antibody that specifically recognizes the carboxyl terminus of Hac1ip can distinguish Hac1up from Hac1ip because they differ in the carboxyl-terminal amino acids due to the Ire1p-mediated splicing reaction. Upon Tm treatment, Hac1ip was increased 5-fold in wild-type cells (Fig.3 B, lanes 4 and 8) but not inΔada5 cells (Fig. 3 B, lanes 1 and5) or Δire1 cells (Fig. 3 B,lanes 3 and 7). In this blot, a co-migrating background band was observed in all strains includingΔhac1 without Tm treatment (Fig. 3 B,lanes 2 and 6). These results are consistent with the observation that Δada5 cells are defective inHAC1 u mRNA processing and are therefore incapable of generating a translatable form of HAC1 i mRNA. The data presented thus far suggest that the abrogated UPR in Δada5 cells result from defective HAC1 mRNA processing. We therefore asked whether the UPR could be restored upon expression ofHAC1 i in Δada5 cells. To evaluate the UPR, cells were transformed with a centromere-containing reporter plasmid harboring a lacZ reporter gene under the control of a single 22-base pair UPRE. The resultant transformed wild-type cells turned blue on plates containing X-gal and Tm, indicating an intact UPR pathway. In contrast, Δada5 strains harboring the UPRE-lacZ vector remained white, demonstrating a defective UPR pathway. However, after the introduction ofHAC1 i, both the wild-type and Δada5strains turned blue and light blue, respectively, on media containing X-gal plus Tm (data not shown). The amount of HAC1 mRNA detected in Δada5 cells harboring theHAC1 i plasmid was greater than in the wild-type cells (Fig. 1 A, lanes 1 and 2 and9 and 10). This observation further strengthens the notion that the defective UPR in Δada5 cells is not the result of abrogated HAC1 transcription. Quantification of the UPR by liquid assay, revealed that the basal level of β-galactosidase expression from the 22-base pair UPRE was reduced 3.5-fold in the Δada5 strain compared with the wild-type strain (Fig. 4, lanes 1 and5). Upon Tm treatment, induction of β-galactosidase was reduced 20-fold compared with wild-type cells (Fig. 4, lanes 2 and 6). Expression of HAC1 i increased the basal of β-galactosidase activity by 10- and 6-fold in the wild-type andΔada5 strains, respectively (Fig. 4,lanes 1, 3, 5, and 7). However, β-galactosidase expression in HAC1 i -expressingΔada5 cells was reduced 6-fold compared withHAC1 i -expressing wild-type cells (Fig. 4,lanes 3 and 7). On the other hand, Tm induction of β-galactosidase activity was comparable in both wild-type andΔada5 strains expressing HAC1 i, at 6- and 8-fold respectively (Fig. 4, lanes 3, 4,7, and 8). Therefore, expression of Hac1ip restored induction, although the basal expression level was reduced. Taken together, these results suggest that both transcriptional co-activator and RNA processing functions ofADA5 are essential for maximal transcriptional activation from the UPRE. Upon accumulation of unfolded proteins in the lumen of the ER, Ire1p initiates a novel mRNA splicing reaction. The endoribonuclease activity of Ire1p cleaves the 5′ and 3′ splice site junctions within HAC1 mRNA. Each splice site junction is composed of a simple structure, a stem with a seven-member loop. Only 4 bases within the loop are apparently required for specificity of the cleavage reaction (31.Gonzalez T.N. Sidrauski C. Dorfler Walter P. EMBO J. 1999; 18: 3119-3132Crossref PubMed Scopus (180) Google Scholar). The 5′ and 3′ exons of HAC1 mRNA are tethered together by base pairing and joined by tRNA ligase (RLG1). Whereas precursor HAC1 u mRNA is not translated well, the product HAC1 i mRNA is efficiently translated. This splicing reaction was reconstitutedin vitro with only two components, Ire1p and Rlg1p (12.Sidrauski C. Walter P. Cell. 1997; 90: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). However, data suggest that the HAC1 u mRNA processing reaction is more complex in vivo. First, the low degree of specificity for the stem-loop structure inHAC1 u mRNA would indicate that other cellular mRNAs may be nonspecifically cleaved by the Ire1p endoribonuclease. This notion is consistent with the observation that oligonucleotides consisting of the stem-loop structures are less efficient substrates than a 600-base pair substrate that contains both 5′ and 3′ splice site junctions (12.Sidrauski C. Walter P. Cell. 1997; 90: 1031-1039Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Second, when the intron within the 3′ end ofHAC1 u mRNA was placed into the 3′ untranslated region of a green fluorescent protein (GFP) marker gene, GFP was not translated (7.Chapman R.E. Walter P. Curr. Biol. 1997; 7: 850-859Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). This result is consistent with the proposed role of the intron as a translational attenuator (6.Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). However, upon induction of the UPR, the HAC1 intron within the GFP mRNA was not removed, indicating that Ire1p does not simply recognize the stem-loop and intron sequence within HAC1 u mRNA. Fourth, although human Ire1αp can cleave the 5′ splice site of yeastHAC1 mRNA in vitro, yeast HAC1mRNA was not cleaved when expressed in mammalian cells (32.Foti D.M. Welihinda A.J. Kaufman R.J. Lee A.S. J. Biol. Chem. 1999; 274: 30402-30409Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Finally, using a temperature-sensitive mutant of RNA polymerase, it was shown that only newly transcribed HAC1 mRNA is processed by Ire1p (13.Sidrauski C. Cox J.S. Walter P. Cell. 1996; 87: 405-413Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Although the majority of human Ire1p was localized within the ER membrane, a subpopulation preferentially associated with the nuclear envelope and possibly the nuclear pore complex (33.Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (751) Google Scholar), suggesting both cytoplasmic and nucleoplasmic localization of the carboxyl terminus. It was proposed that Ire1p cleaves HAC1mRNA at the nuclear envelope as it is transported to the cytoplasm for translation. This hypothesis is consistent with detection of Rlg1p at the nuclear pore complex (34.Clark M.W. Abelson J. J. Cell Biol. 1987; 105: 1515-1526Crossref PubMed Scopus (83) Google Scholar). In this report, we provide data that demonstrate a requirement forADA5 in the Ire1p-mediated cleavage of HAC1mRNA in S. cerevisiae. First, whereas the UPR transcriptional induction is reduced severalfold in the Δada2, Δada3, and Δgcn5 strains, it is completely absent in the Δada5 strain. Second, upon activation of the UPR, HAC1 i mRNA was produced in the Δada2, Δada3, and Δgcn5strains, but not in the Δada5 strain. As previously demonstrated, cleaved HAC1 mRNA products that are not ligated by the tRNA ligase accumulate in the cytoplasm (35.Kawahara T. Yanagi H. Yura T. Mori K. J. Biol. Chem. 1998; 273: 1802-1807Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, these products were not detected in the Δada5 strain, supporting a role for Ada5p in HAC1 u mRNA cleavage and not in Rlg1p-mediated exon-exon ligation. Third, upon activation of the UPR, the levels of IRE1 mRNA andHAC1 mRNA were not altered, and Hac1ip was not detected. Finally, an in vitro pull-down assay detected an interaction between Ire1p and both Ada5p and Ada2p, but not Ada3p and Gcn5p, consistent with a role of Ada5p in Ire1p-mediated cleavage. The requirement for ADA5 in the HAC1 mRNA cleavage reaction can explain both the UPR defect and the inositol requirement observed for the Δada5 strain. These two phenotypes are identical to those observed in the Δire1, Δhac1, and rlg1–100 strains (36.Cox J.S. Chapman R.E. Walter P. Mol. Biol. Cell. 1997; 8: 1805-1814Crossref PubMed Scopus (314) Google Scholar). These results implicate a novel mRNA processing role for Ada5p, a subunit of the transcriptional co-activator SAGA. Our experimental data support the proposed notion that Gcn5p interacts with the basic leucine zipper protein, Hac1p, to maximally activate the UPR (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). In vitro pull-down assays described here suggest that Gcn5p directly interacts with Hac1p. These findings are not surprising because another bZIP protein, Gcn4p, interacts with Gcn5p for maximal activation of amino acid biosynthetic genes under conditions of amino acid starvation (17.Georgakopoulos T. Thireos G. EMBO J. 1992; 11: 4145-4152Crossref PubMed Scopus (255) Google Scholar, 21.Marcus G.A. Silverman N. Berger S.L. Horiuchi J. Guarente L. EMBO J. 1994; 13: 4807-4815Crossref PubMed Scopus (237) Google Scholar). We have incorporated the results of our study into a model for SAGA function in activation of the UPR (Fig.5). Within the SAGA complex, Gcn5p has histone acetyltransferase activity that is required for opening chromatic structures for transcriptional activation. Disruption ofADA2, ADA3, or GCN5 destroys the histone acetyltransferase activity of the complex and reduces transcriptional activation of UPR-responsive genes by severalfold (16.Welihinda A.A. Tirasophon W. Green S.R. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4289-4294Crossref PubMed Scopus (53) Google Scholar). Because Δada2 cells, Δada3 cells, andΔgcn5 cells grow without inositol and can spliceHAC1 mRNA to activate the UPR, they are not required for the UPR. Gcn5p interacts with the bZIP transcription factor Hac1p to enhance transcriptional induction of ER stress-responsive genes.ADA5/SPT20 and SPT7 are required to maintain the integrity of the SAGA complex (24.Sterner D.E. Grant P.A. Roberts S.M. Duggan L.J. Belotserkovskaya R. Pacella L.A. Winston F. Workman J.L. Berger S.L. Mol. Cell. Biol. 1999; 19: 86-98Crossref PubMed Scopus (291) Google Scholar). Ire1p is localized to the inner leaflet of the nuclear envelope, adjacent to the nuclear pore. Both Ada2p and Ada5p interact with Ire1p, although only Ada5p function is required to promote Ire1p-mediated cleavage of HAC1mRNA. Although we have not demonstrated that Ada5p acts directly in this reaction, the finding that Ada5p interacts with Ire1p would suggest that Ada5p may function to increase the efficiency ofHAC1 mRNA processing by Ire1p. We thank Dr. Leonard Guarente for providing yeast strain BWG1-7a and plasmids containing GCN5, ADA2, ADA3, and ADA5 and Dr. Peter Walter for plasmid pHAKO1 and anti Hac1ip antibodies. We also thank Dr. Kotlo Kumar for helpful criticism and Dr. Kyungho Lee for the careful review of this paper.