Skirball Institute of Biomolecular Medicine, the Departments of Medicine, Cell Biology, and the Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA
*Author for correspondence (e-mail: ron{at}saturn.med.nyu.edu)
Accepted May 22, 2001
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SUMMARY |
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Key words: Endoplasmic reticulum, Protein kinase, Ribonuclease, RNA Processing, Signal transduction
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INTRODUCTION |
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It is not known how Ire1p kinase activity controls Ire1p-mediated splicing of HAC1 mRNA the regulated event in the yeast UPR. The bacterially expressed C-terminal effector domain of Ire1p is able to cleave the HAC1 intron in vitro; however, it is not known if this capacity of the bacterial protein requires kinase activity (Sidrauski and Walter, 1997). Recently two mammalian homologs of IRE1, IRE1 and IRE1ß were identified (Tirasophon et al., 1998; Wang et al., 1998). These homologs are implicated in activating target genes in the mammalian UPR and are capable of splicing the yeast HAC1 mRNA (Tirasophon et al., 1998; Niwa et al., 1999; Tirasophon et al., 2000); however, no mammalian mRNAs that are targets for splicing by IRE1 have been identified to date. The IRE1
mRNA may be cleaved by overexpression of IRE1
, but the mechanisms involved are not known (Tirasophon et al., 2000). Both IRE1
and IRE1ß possess kinase activity that is required for activating downstream signaling (Tirasophon et al., 1998), but it is not understood how this regulation is imposed.
It has recently been reported that mammalian IRE1 proteins undergo cleavage during their activation by the UPR. The cleaved C-terminal effector domain of IRE1 and IRE1ß were described to migrate to the nucleus, where it is proposed they engage their yet-to-be identified substrate mRNA and effect splicing (Niwa et al., 1999). These observations suggest that mammalian IRE1 activity may be controlled at the level of access to the substrate. However, mammalian IRE1s couple ER stress to stress-activated protein kinases (Urano et al., 2000) and have been reported to activate the membrane-bound form of the transcription factor ATF6 (Li et al., 2000), activities that are thought to take place in the cytoplasm and would probably not involve interactions with an mRNA substrate. We therefore sought to explore the interaction of mammalian IRE1s with RNA as an important step towards addressing the role of mRNA splicing in downstream signaling. We now report on the use of a sensitive in vivo method for detecting interactions between IRE1 and RNA, and show that these interactions are modulated by ER stress and are dependent on the kinase and endonuclease domains of IRE1.
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MATERIALS AND METHODS |
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In transfection experiments, two 10 cm dishes of 293T cells were used for each experimental point. Cells were transfected using the calcium chloride precipitation method with previously described expression plasmids (Wang et al., 1998): wild-type mIRE1ß, kinase inactive mIRE1ß K536A mutant, mIRE1ßnuc mutant truncated in the endonuclease domain (amino acids 1-820) and mIRE1ß
C mutant, which lacks most of the C-terminal domain (amino acids 1-518). Cells were processed for RNA immunoprecipitation and immunoblotting 36 hours post transfection.
u.v. crosslinking and RNA immunoprecipitations
Treated or untreated cells were placed on a flat bed of crushed ice (1.5 cm thick), washed twice with 5 ml ice-cold PBS and left in 2 ml PBS per 10 cm plate. Plates on their ice bed were placed in a u.v. Stratalinker 2400 oven (Stratagene) and irradiated with 900 mJ/cm2 (delivery of this amount of energy usually required 5 minutes of irradiation). Immediately thereafter, cells were scraped into ice-cold PBS, recovered by a 2000 g spin for 5 minutes and solubilized for 5 minutes on ice with 150 µl of 1% Triton buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrasodium pyrophosphate, 100 mM NaF, 17.5 mM ß-glycerophosphate, 1 mM phenylmethysulphonyl fluoride (PMSF), 4 mg ml1 aprotonin and 2 mg ml1 pepstatin A). Extracts were clarified by centrifugation for 10 minutes at 14,000 g 4°C. Lysates were then treated with 0.8 µg or 20 µg RNase A for 30 minutes at room temperature per AR42J cell extract or 293T cell extract respectively. SDS was added to the lysate to a final concentration of 1%, and the lysate was heated for 5 minutes at 90°C. The extracts were clarified by centrifugation for 30 minutes at 200,000 g in a TLA-100-2 rotor (Beckman).
The supernatant was diluted to 0.1% SDS in RIPA buffer without SDS (10 mM Tris HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% Triton, 0.5% sodium deoxycholate) and pre-cleared for 1 hour at 4°C on 20 µl protein A Sepharose (Zymed). Immunoprecipitations were carried out overnight at 4°C using 4 µl of anti-IRE1 or 6 µl of anti-IRE1ß polyclonal sera and 10 µl of protein A Sepharose. Immunoprecipitated material was washed three times for 5 minutes at room temperature in 1 ml RIPA buffer containing 1 M NaCl (10 mM Tris HCl pH 7.5, 1 M NaCl, 1 mM EDTA, 1% Triton, 0.5% sodium-deoxycholate, 0.1% SDS) and rinsed three times in kinase buffer (50 mM Tris HCl pH 7.5, 10 mM MgCl2 and 10 mM DTT). Residual buffer was then carefully removed using a syringe fitted with a 30 G needle. The beads were resuspended in 30 µl of kinase reaction mix (50 mM Tris HCl pH 7.5, 10 mM MgCl2 and 10 mM DTT, 100 µCi of [
-32P] ATP (7000Ci/mM) and 5 units of T4 polynucleotide kinase) and agitated at 1000 rpm on a Thermomixer (Eppendorf) at 37°C for 30 minutes. To minimize the autokinase activity of the immunopurified IRE1, MnCl2 was omitted from the kinase reaction buffer. Beads were washed three times for 5 minutes at room temperature in 1 ml RIPA buffer containing 1 M NaCl, and resuspended in 50 µl of SDS-PAGE loading dye. Samples were loaded on 8 or 10% SDS-PAGE, and gels were exposed to autoradiography or transferred to a nitrocellulose membrane, which was sequentially exposed to autoradiography and immunoblotted for mIRE1
, as described previously (Bertolotti et al., 2000). In Fig. 3, 90% of the immunoprecipitated material solubilized in SDS-PAGE loading dye was loaded on a SDS-PAGE followed by autoradiography and 10% was used for immunoblot with anti-IRE1ß antiserum (Bertolotti et al., 2000).
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RESULTS |
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To confirm the chemical nature of the IRE1-associated species that is labeled by T4 kinase, we treated the gel slices containing the labeled band with proteinase K, to digest the protein component and recovered the labeled species. On a denaturing acrylamide-urea gel, the major labeled species co-migrated with a polynucleotide
25 base in length. RNase A digestion of the labeled species confirmed that it had an RNA component (Fig. 1B). Given that proteinase K rarely digests proteins to completion, the species recovered from the gel may represent a peptide-RNA fragment, and consequently the size of the RNA component cannot be estimated with accuracy in this gel.
The radiolabeled crosslinked species was induced most strongly by DTT treatment, which is also the most potent activator of IRE1 in AR42J cells (Bertolotti et al., 2000; data not shown); however, other inducers of ER stress also increased complex formation (Fig. 2A). ER stress dependence of complex formation was also confirmed by treating cells with increasing concentrations of DTT (Fig. 2C). Additional evidence for the dynamic nature of IRE1
-RNA complex and for its modification by ER stress was provided by the observation that the size of the labeled species eluted from the complex was different in stressed and unstressed cells (Fig. 2B,D). The labeled species liberated from the IRE1
immunoprecipitate from unstressed cells was larger than that from stressed cells. Moreover, the intensity of the radiographic signal revealed by labeling with T4 kinase was greater for the smaller fragments recovered from stressed cells. After purification and elution from the gel, both the longer and shorter species were degraded by treatment with RNase, indicating their RNA content (Fig. 2D).
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Analysis of HAC1 splicing in a yeast strain with a conditional mutation in RNA polymerase II suggested that only newly synthesized HAC1 mRNA serves as a substrate for IRE1 (Sidrauski et al., 1996). To determine if the RNA species that interacts with mammalian IRE1 is newly synthesized or if IRE1
can associate with pre-existing RNA in a stress-dependent manner, we treated AR42J cells with Actinomycin D to block transcription and then exposed them to DTT. We have previously determined that induction of ER stress by DTT does not require new protein synthesis (Harding et al., 2000). Complexes between IRE1
and RNA were not influenced by Actinomycin D treatment (Fig. 4A). Furthermore, the characteristic shift in size of the RNA species recovered from the IRE1
-containing complexes also occurred independently of gene transcription (Fig. 4B). We conclude that the ER-stress mediated alteration in the IRE1
-RNA complex involves a pre-existing mRNA.
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DISCUSSION |
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Because the RNA species that associate(s) with IRE1 have not yet been identified, we can only speculate about the nature of the ER stress-induced alteration in the complex. This alteration may be a consequence of IRE1-mediated splicing of the bound RNA. The splicing event may diminish the length of the protected RNA fragment directly, or alter the conformation of the RNA affecting its accessibility to RNase A used in the assay. However, to the extent that the size of the RNA fragment is determined by its susceptibility to RNase A digestion, it is equally possible that activation-mediated conformational changes in IRE1 and/or the associated RNA that precede splicing alter the cleavage pattern of the RNA and account for the abrupt decrease in size of the peptide-RNA fragment.
The increase in radiolabeling of the RNA species recovered in complex with IRE1 from stressed cells might reflect activation-mediated increase in association of IRE1 with RNA. Alternatively, ER-stress-dependent changes in the bound RNA due to cleavage by IRE1 or altered digestion by RNase A may affect accessibility of the 5' OH group to T4 kinase used in the labeling reaction. This latter possibility is consistent with a model whereby IRE1 is pre-bound to its RNA target, facilitating rapid response to stress. It is noteworthy, in this regard, that the alterations in the IRE1-RNA complex are observed to occur in actinomycin D-treated stressed cells, indicating that the RNA species engaged by IRE1 is pre-existing in stressed cells.
Regardless of the precise identity of the RNA species crosslinked to IRE1, this assay correlated closely with IRE1 activation in the UPR. It is noteworthy in this regard that the changes in the complex occur in the absence of any evidence for IRE1 processing. At the very least, these observations indicate that substantial alterations can occur to IRE1 and its contingent molecules before the processing event that reportedly causes the effector domain of IRE1 to translocate to the nucleus (Niwa et al., 1999). If the RNA revealed by this assay represents the substrate for splicing by IRE1 and encodes effectors of the mammalian UPR, our observations would seriously question the role of nuclear translocation of the truncated effector domain of IRE1 as the proposed mechanism for effecting interactions with substrate mRNA (Niwa et al., 1999). It is also possible that activation of IRE1 by ER stress may mediate a general increase it is affinity towards RNA. However, this model too would call into question the role of IRE1 processing in mediating its effector functions, because we detect complexes only between full-length IRE1 and RNA.
The RNA detected by our assay could also represent a pre-existing structural component of the machinery that cleaves effector-encoding mRNAs in a stable complex with IRE1. Given the role of RNA in the regulation and catalysis of other RNA processing reactions in the cell, this possibility should be seriously considered. Accordingly, it is important to point out that the genetic screens for mutations that disrupt stress signaling in yeast have not been saturating and might not have uncovered an essential RNA component.
Several groups have reported recently on the use of microarray technology to identify polynucleotide species recovered by purification after crosslinking to their protein ligands (Ren et al., 2000; Takizawa et al., 2000). To adapt the cross-linking procedure described here to that application, we would need to overcome two main obstacles. Isolation of the IRE1-RNA complex and its purification from other RNAs currently requires extensive digestion of the RNA before immunoprecipitation. This severely limits the size of the RNA remnant associated with the complex and reduces its utility as a template for producing a hybridization probe in any subsequent application. Irreversible crosslinking, as effected by u.v. treatment, produces a covalent modification of the RNA; this modification may inhibit the reverse-transcriptase step that forms the basis of any procedure that seeks to display or amplify cDNA to the RNA. These difficulties not withstanding, the next step is clearly to identify or clone the RNA species complexed with IRE1 in vivo.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Bertolotti, A., Zhang, Y., Hendershot, L., Harding, H. and Ron, D. (2000). Dynamic interaction of BiP and the ER stress transducers in the unfolded protein response. Nat. Cell Biol. 2, 326-332.[Medline]
Chapman, R. E. and Walter, P. (1997). Translational attenuation mediated by an mRNA intron. Curr. Biol. 7, 850-859.[Medline]
Cox, J. S., Shamu, C. E. and Walter, P. (1993). Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell 73, 1197-1206.[Medline]
Cox, J. S. and Walter, P. (1996). A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391-404.[Medline]
Harding, H., Novoa, I., Zhang, Y., Zeng, H., Schapira, M. and Ron, D. (2000). Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099-1108.[Medline]
Kaufman, R. J. (1999). Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211-1233.
Kawahara, T., Yanagi, H., Yura, T. and Mori, K. (1997). Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response [In Process Citation]. Mol. Biol. Cell 8, 1845-1862.
Li, M., Baumeister, P., Roy, B., Phan, T., Foti, D., Luo, S., Lee, A. S., Wang, Y., Shen, J., Arenzana, N. et al. (2000). ATF6 as a transcription activator of the endoplasmic reticulum stress element: thapsigargin stress-induced changes and synergistic interactions with NF-Y and YY1. Mol. Cell. Biol. 20, 5096-5106.
Mori, K. (2000). Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell 101, 451-454.[Medline]
Mori, K., Ma, W., Gething, M. J. and Sambrook, J. (1993). A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell 74, 743-756.[Medline]
Niwa, M., Sidrauski, C., Kaufman, R. J. and Walter, P. (1999). A role for presenilin-1 in nuclear accumulation of Ire1 fragments and induction of the mammalian unfolded protein response. Cell 99, 691-702.[Medline]
Ren, B., Robert, F., Wyrick, J. J., Aparicio, O., Jennings, E. G., Simon, I., Zeitlinger, J., Schreiber, J., Hannett, N., Kanin, E. et al. (2000). Genome-wide location and function of DNA binding proteins. Science 290, 2306-2309.
Sidrauski, C., Cox, J. S. and Walter, P. (1996). tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell 87, 405-413.[Medline]
Sidrauski, C. and Walter, P. (1997). The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90, 1031-1039.[Medline]
Takizawa, P. A., DeRisi, J. L., Wilhelm, J. E. and Vale, R. D. (2000). Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier. Science 290, 341-344.
Tirasophon, W., Lee, K., Callaghan, B., Welihinda, A. and Kaufman, R. J. (2000). The endoribonuclease activity of mammalian IRE1 autoregulates its mRNA and is required for the unfolded protein response. Genes Dev. 14, 2725-2736.
Tirasophon, W., Welihinda, A. A. and Kaufman, R. J. (1998). A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev. 12, 1812-1824.
Urano, F., Wang, X., Bertolotti, A., Zhang, Y., Chung, P., Harding, H. and Ron, D. (2000). Coupling of stress in the endoplasmic reticulum to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science 287, 664-666.
Wang, X. Z., Harding, H. P., Zhang, Y., Jolicoeur, E. M., Kuroda, M. and Ron, D. (1998). Cloning of mammalian Ire1 reveals diversity in the ER stress responses. EMBO J. 17, 5708-5717.