Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR)1,2,3. ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5′ untranslated region4. After excision of the intron, transfer RNA ligase joins the severed exons5,6, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue4. Hac1 in turn drives the UPR gene expression program comprising 7–8% of the yeast genome7 to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3′ untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3′ untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.
In vitro studies indicate that the information required for HAC1 mRNA splicing is confined to the intron and the regions surrounding the splice junctions8. Surprisingly, in vivo splicing of HAC1 mRNA was greatly diminished when its 3′ untranslated region (3′ UTR) was replaced by the 3′ UTRs of other yeast mRNAs, such as that of actin (ACT1, Fig. 1b) or 3-phosphoglycerate kinase (PGK1, data not shown). Consistent with this finding, cells bearing a chimaeric HAC1 gene with the 3′ UTR of ACT1, HAC1-3′act1, expressed Hac1 protein at trace levels that were too low to mount a functional UPR and failed to grow in ER stress conditions (Fig. 1b). Thus, the HAC1 3′ UTR harbours an element important for HAC1 mRNA splicing in vivo.
Mutational probing experiments (not shown) indicate that the HAC1 3′ UTR contains a prominent, extended stem-loop (Fig. 1c). Interestingly, two short sequence motifs within the stem-loop are highly conserved among all HAC1 orthologues identified; eight representatives are shown in Fig. 1d. The sequence motifs map to opposite strands and are juxtaposed in the distal part of the stem, constituting a 3′ UTR bipartite element (3′ BE; Fig. 1c, 3′ BE in red).
To assess the importance of the 3′ BE for HAC1 mRNA splicing in vivo, we used a splicing reporter in which we replaced the first 648 nucleotides of the HAC1 coding sequence in the first exon with that of green fluorescent protein (GFP; Fig. 1a, green bar). This reporter allowed us to monitor the effect of 3′ UTR mutations on mRNA splicing in cells that can mount a functional UPR, sustained by endogenous HAC1 mRNA. The splicing reporter mRNA was efficiently spliced on UPR induction. In contrast, splicing was greatly diminished when the 3′ BE was deleted (Δ3′ BE, Fig. 1e). Consistent with these results, deletion of the 3′ BE in HAC1 severely reduced HAC1 mRNA splicing and impaired cell survival under ER stress conditions (Fig. 1e). Only residual splicing of endogenous HAC1 mRNA occurred in the absence of the 3′ BE, indicating that the 3′ BE accounts in large part for the contribution of the 3′ UTR to HAC1 mRNA splicing. Insertion of a 64-nucleotide 3′ UTR fragment containing the central portion of the stem including the 3′ BE (Fig. 1d, enlarged on right) into the splicing reporter bearing the ACT1 3′ UTR restored splicing greatly (Fig. 1f).
To test whether the 3′ UTR affects the ability of Ire1 endonuclease to bind or catalyse the cleavage of HAC1 mRNA, we reconstituted the intron excision reaction in vitro. Ire1 cleaved HAC1 mRNA with the same rate in the presence or absence of the 3′ BE (Fig. 1g). Thus, the 3′ BE is not required for splicing in vitro.
The importance of the 3′ BE for HAC1 mRNA splicing in vivo indicated that it may serve to target the mRNA to sites in the cell where splicing takes place. To test this notion, we visualized Ire1 protein and HAC1 mRNA in vivo, using the imaging constructs depicted in Fig. 2a. For Ire1, we inserted a GFP or mCherry into the cytosolic portion of Ire1 adjacent to its transmembrane region. For HAC1 mRNA, we inserted 16 copies of a U1A binding site into the 3′ UTR downstream of the 3′ BE. The mRNA can then be visualized by co-expression of a GFP-tagged U1A-RNA-binding protein that docks to the U1A binding sites9. Both Ire1 and HAC1 mRNA imaging constructs fully restored growth of ire1Δ and hac1Δ (Fig. 2b) cells under ER stress. In the absence of stress, Ire1–GFP co-localized with the ER marked by Sec63–mCherry (Fig. 2c). Most HAC1U1A mRNA displayed a grainy signal dispersed throughout the cytosol (Fig. 2d), with a fraction of HAC1 mRNA signal also found at the ER in agreement with previous observations10.
Induction of ER stress notably altered the localization of both Ire1 and HAC1 mRNA. Most Ire1 (82 ± 6%; see Methods) clustered into distinct foci localized both to the nuclear envelope and to the cortical ER (Fig. 2c–e), in agreement with recent observations11. HAC1 mRNA strongly co-localized (co-localization index (CI) of 56 ± 10; see Methods, Fig. 2e) with Ire1 in foci (Fig. 2d, arrowheads). This recruitment is specific, because control PGK1U1A mRNA remained dispersed in the cytosol under ER stress conditions (Fig. 2f).
Clustering of mRNAs in cytosolic foci is not unprecedented. Several stresses, such as nutrient starvation, cause aggregation of untranslated mRNAs into processing bodies (P-bodies) where they are stored and/or degraded12. The Ire1/HAC1 mRNA clusters, however, are distinct from P-bodies: on glucose depletion HAC1 mRNA did cluster into P-bodies marked by Lsm1–mCherry13. In contrast, under ER stress conditions Lsm1–mCherry did not co-localize with HAC1 mRNA foci but remained dispersed throughout the cytosol (Fig. 2g). Thus, the Ire1/HAC1 mRNA foci constitute previously unknown sites of mRNA clustering in the cytosol that are specific for the UPR.
We next determined the role of each of the three key UPR players—Ire1, HAC1 mRNA and tRNA ligase—in organizing the foci. In rlg1-100 cells bearing mutant tRNA ligase defective in UPR signalling5, co-clustering of Ire1 and HAC1 mRNA occurred normally (Fig. 2h). This result is consistent with the fact that cleavage of HAC1 mRNA by Ire1 is not dependent on the subsequent ligation step5. Likewise, HAC1 mRNA was not required for Ire1 clustering, because Ire1–GFP formed foci in hac1Δ cells (Fig. 2h and ref. 11). Conversely, HAC1 mRNA failed to form foci in ire1Δ cells (Fig. 2h). Thus, clustering of Ire1 in response to ER stress is epistatic to HAC1 mRNA clustering.
Having established that HAC1 mRNA is targeted to Ire1 foci in an ER-stress-driven manner, we assessed the role of the 3′ UTR of HAC1 mRNA in the process. To this end, we added the U1A visualization module to the splicing reporter used in Fig. 1e (SpRU1A). The SpRU1A mRNA containing a wild-type HAC1 3′ UTR co-localized with Ire1–mCherry in foci (CI: 64 ± 20; Fig. 2i). In contrast, co-localization with Ire1 foci of the SpRU1A mRNA lacking the 3′ BE was minimal (CI: 4 ± 6; Fig. 2i), at levels comparable to the control PGK1U1A mRNA (CI, 3 ± 4). Thus, the stem-loop structure in the 3′ UTR of HAC1 mRNA—with the 3′ BE at its core—indeed serves as a targeting element that guides HAC1 mRNA to Ire1 foci to allow splicing in vivo and cell survival under ER stress.
We next followed a time course of foci formation and downstream signalling on induction of ER stress. Clustering of Ire1 into foci and recruitment of HAC1 mRNA (Fig. 3a, b) or of SpRU1A mRNA (Supplementary Fig. 1) into these foci correlated well with the onset of HAC1 mRNA splicing and Hac1 protein production (Fig. 3c). These findings show that Ire1 and HAC1 mRNA clustering is geared to transduce ER stress rapidly. Under conditions in which ER stress builds up more gradually, the encounter of Ire1 and HAC1U1A mRNA in foci likewise paralleled the signalling response, but at a slower pace (Supplementary Fig. 2). The synchronicity of Ire1/HAC1 mRNA clustering and downstream signalling events underscores that the foci constitute functional mRNA-splicing centres.
Ire1 clusters in only ∼3–10 foci per cell. Because yeast contains ∼200–300 molecules of Ire1 per cell14, the foci are composed of a few tens of Ire1 molecules each, indicating that the foci harbour higher-order oligomers of Ire1. From the crystal structure of the Ire1 ER-luminal domain, we identified two separate dimerization interfaces, both of which are essential for optimal UPR signalling, indicating that oligomerization is important for cells to mount a robust UPR15 (Fig. 3d). Accordingly, simultaneous disruption of both interfaces notably reduced HAC1 mRNA splicing and cell growth under ER stress conditions, whereas the single-interface disruptions, which still can form Ire1 dimers by means of one interface, displayed intermediate splicing and growth phenotypes (Fig. 3e). Disruption of either interface prevented foci formation (Fig. 3f, Supplementary Fig. 3 and ref. 11), indicating that Ire1 oligomerization is the organizing principle for UPR signalling foci. Importantly, the inability of Ire1 to form foci impaired HAC1U1A mRNA recruitment (Fig. 3f and Supplementary Fig. 3). Thus, when Ire1 fails to oligomerize, HAC1 mRNA recruitment becomes rate limiting. In agreement, we found that artificially induced dimerization16 of Ire1 supported HAC1 mRNA splicing and cell survival under ER stress conditions only to the level of the single-interface mutants and did not support Ire1 foci formation (Supplementary Fig. 4). We conclude that robust Ire1 oligomerization and HAC1 mRNA targeting serve to concentrate both key UPR components into foci to ensure efficient RNA processing and ER stress signalling.
HAC1 mRNA is no longer a substrate for Ire1 after removal of its intron, indicating that the spliced HAC1 mRNA should disengage from Ire1 foci and not be recruited again. Accordingly, SpRU1A mRNA lacking the intron displayed reduced targeting to foci (CI: 17 ± 9) compared to wild-type SpRU1A mRNA (Fig. 4a, b), although targeting was not as markedly reduced as when the 3′ BE was deleted (Figs 2i and 4b). In further support, overexpression of SpRU1A mRNA containing the intron reduced splicing of endogenous HAC1 mRNA, presumably by competitively saturating Ire1 after being targeted there, but did not do so when SpRU1A mRNA lacked either the 3′ BE or the intron (Fig. 4c). These observations indicate that the 3′ BE alone is not sufficient for efficient targeting. In agreement, insertion of the 3′ UTR stem of HAC1 (Fig. 1c) into the 3′ UTR of PGK1 could not facilitate recruitment of this heterologous mRNA to Ire1 foci (Fig. 4d). Thus, the intron and 3′ BE cooperate to effect HAC1 mRNA targeting.
The intron keeps HAC1 mRNA translationally silent (Fig. 1a), indicating that translational repression may be key to HAC1 targeting similar to the situation in other mRNA targeting mechanisms, as observed for ASH1 mRNA17. To test this hypothesis, we inserted a small stem-loop into the 5′ UTR of the PGK1U1A mRNA to repress its translation (ref. 17 and Fig. 4e). When we expressed PGK1U1A mRNA containing both the small stem-loop in the 5′ UTR and the HAC1 3′ BE-containing stem in the 3′ UTR, we found that this mRNA efficiently targeted to Ire1 foci (CI: 60 ± 19, Fig. 4f, h). Conversely, the corresponding mRNA lacking the 3′ BE was not targeted (Fig. 4g, h). We conclude that the 3′ BE-containing stem is both necessary and sufficient to target a heterologous mRNA to UPR-induced Ire1 foci, provided that its translation is on hold. Translational repression, therefore, is not only key to facilitate timely synthesis of Hac1 protein on induction of the UPR, but is also integral to the targeting of HAC1 mRNA to ER stress signalling centres.
Our results describe the first example, to our knowledge, of mRNA targeting as a central feature in a signalling pathway. HAC1 mRNA is delivered to the site where it is processed as part of the main switch regulating the UPR. The mRNA guidance mechanisms characterized so far serve other goals, such as delivery of mRNA to sites of storage or degradation18,19, or restricted distribution of the proteins they encode20,21,22. HAC1 mRNA delivery to Ire1 foci has in common with other mRNA-targeting mechanisms that it depends on a signal in the 3′ UTR and on translational repression of the mRNA23. The mechanism of translational control of HAC1 mRNA serves both to prevent translation of a functional transcription factor when the UPR is off, and to allow the mRNA access to the splicing machine, which removes the intron to allow its translation, when the UPR is on. In this way, the targeting signal is inactivated when translation of HAC1 mRNA resumes, even though the 3′ BE remains present in the spliced mRNA.
The translational block in Saccharomyces cerevisiae is exerted by means of a 16-base-pairing interaction between sequences in the 252-nucleotide-long intron and the 5′ UTR4. Most HAC1 or XBP1 orthologues bear introns that are shorter (∼20–26 nucleotides) and show no sequence complementarity to support 5′ UTR/intron-based translational blocks. It is conceivable that other means of translational repression come into play. For instance, the general translational attenuation in response to ER stress as mediated by the ER-resident transmembrane eIF2α kinase PERK24 could serve a functionally similar role in XBP1 mRNA targeting in metazoans.
Our findings emphasize the role of Ire1 oligomers, rather than dimers, in UPR signalling. Early co-immunoprecipitation studies already provided evidence for oligomerization25, and the identification of two functionally important interfaces that link Ire1 luminal domains into linear filaments in the crystal lattice supports an attractive model by which neighbouring Ire1 molecules are ‘stitched’ together by the binding of unfolded proteins in the ER lumen15. This model and the epistasis data in Fig. 2h indicate that Ire1 foci formation is governed by self-organization. Overexpression of Ire1 caused an enlargement of the foci, but did not increase their number (not shown), indicating that there is a limited number of nucleation sites per cell and that foci may arise at such predisposed sites at the ER membrane. Because HAC1 mRNA recruitment occurs with amazing speed and efficiency (for example, Fig. 3), one can further speculate that the 3′ BE-containing targeting signal may allow HAC1 mRNA to travel actively along cytoskeletal filaments to these pre-disposed sites, where Ire1 concentrates.
Clustering of activated signalling receptors occurs in many systems, such as in the immunological synapse26 and in bacterial chemotaxis27, and the resulting local concentration of the signalling machinery can greatly enhance the efficiency of signal transduction. Interestingly, we found that on oligomerization in vitro the nuclease activity of the Ire1 kinase/nuclease domains vastly increases28 over the activity observed for Ire1 dimers29. Thus, by clustering into oligomers, Ire1 acquires enhanced avidity towards its substrate HAC1 mRNA and reaches full enzymatic activation at the same time. These mechanistic features converge into a signalling relay that provides the efficiency and timeliness required to combat ER stress.
Microscopy data acquisition and analysis
Cells were visualized on a Yokogawa CSU-22 spinning disc confocal on a Nikon TE2000 microscope. Images of Ire1–mCherry and U1A–GFP-decorated HAC1U1A, SpRU1A and PGK1U1A mRNAs and variants thereof were analysed using a customized MatLab script to determine the fraction of Ire1–mCherry in foci and to score the recruitment of U1A–GFP-decorated mRNA in Ire1 foci. The annotated MatLab script is available in the Supplementary Information. In brief, after background subtraction we defined the fraction of Ire1–mCherry in foci as the ratio between the integrated fluorescence intensity of pixels with a signal greater than a threshold value and the total integrated fluorescence intensity. The threshold was empirically defined such that under non-stress conditions no signal was scored as ‘foci’. Similarly, RNA foci were defined as pixels exceeding by twofold the mean intensity in the RNA channel. A ‘co-localization index’ was then defined as the integrated intensity of the pixels within the RNA foci that had pixels in common with Ire1 foci divided by the total RNA intensity, and expressed in arbitrary units in a range from 0 to 100. For each condition, the percentage of Ire1–mCherry in foci and the co-localization index for the mRNA recruited to the foci was determined for 5–9 individual cells. Values and the standard error of the mean are given in histograms in Figs 2–4. Because, in contrast to the covalently fluorescently tagged Ire1, we do not know what fraction of U1A–GFP is bound to mRNAs containing U1A-binding sites, background subtraction for U1A–GFP was arbitrary. Therefore, we quantified the data by the co-localization index rather an absolute percentage co-localization measure. The co-localization index robustly scores the differences in mRNA recruitment we observed qualitatively in the fluorescent micrographs.
Yeast strains and plasmids
Standard cloning and yeast techniques were used for construction, transformation and integration of plasmids30,31,32. HA-tagged versions of HAC1 with either its own 3′ UTR or that of ACT1 or PGK1 were integrated as a genomic copy, replacing endogeneous HAC1. The splicing reporter construct was generated by replacing positions 1 to 648 of the HAC1 coding sequence in exon 1 with the GFP ORF. In the Δ3′ BE mutants, positions 176–182 and 212–218 of the 3′ UTR of HAC1 were deleted. The 3′ BE stem that was placed between the stop codon and the ACT1 3′ UTR of the splicing reporter comprised positions 155–187 and 207–236 of the 3′ UTR of HAC1. The mRNA visualization constructs were created by inserting into the pRS426 vector33 the sequences of PGK1, HAC1 or a non-fluorescent GFP–R96A mutant of the splicing reporter ending at position 280 of the 3′ UTR of HAC1, followed by 16 tandem repeats of the U1A binding sequence and the PGK1 terminator, derived from pPS2037 (a gift from R. Parker), and a polyA signal. A copy of the U1A RNA-binding domain fused to GFP was integrated into the genome from plasmid pRP1187 (a gift from R. Parker). Surprisingly, the key to the low noise in the imaging lies in the curious fact that in pRP1187 the U1A–GFP ORF is inserted backwards, so that its expression is driven by a cryptic, uncharacterized promoter element within the (reverse) PGK1 transcription terminator. The low levels of U1A–GFP expression derived from this construct prove ideal for mRNA imaging. By PCR, a previously described17 5′ stem-loop structure was introduced 26-nucleotides upstream of the start codon of PGK1, and nucleotides 108–280 of the HAC1 3′ UTR, comprising the entire stem, were inserted after the PGK1 stop codon, where indicated. A monomeric (A206R), yeast-codon-adapted version of GFP, derived from pKT12734, or mCherry was placed into Ire1 between residues I571 and G572, and the FKBP-derived Fv2E domain (Ariad) between R112 and Y449, replacing the core ER-stress-sensing domain15. Ire1 luminal interface mutants are: IF1L (T226W/F247A), IF2L (W426A) and IF1/2L (T226W/F247A/W426A)15. Ire1 variants in all assays were expressed at near-endogenous levels from centromeric pRS315.
RNA and protein analysis
RNA preparation, electrophoresis, labelling of probes for northern blot analysis and quantification of splicing efficiencies were performed as described4. Protein extraction, electrophoresis and transfer to nitrocellulose for immunoblot analysis with anti-HA antibody were performed as described4.
All samples were taken from yeast cells that were kept in early log phase for at least 24 h in synthetic media containing excess amounts of adenine and tryptophan before imaging. Light microscopy was done with a Yokogawa CSU-22 spinning disc confocal on a Nikon TE2000 microscope. GFP was excited with the 488 nm Ar-ion laser line and mCherry with the 568 nm Ar-Kr laser line. Images were recorded with a ×100/1.4 NA Plan Apo objective on a Cascade II EMCCD. The sample magnification at the camera was 60 nm per pixel. The microscope was controlled with µManager and ImageJ. Images were selected for analysis and for display in figures to contain no saturated pixels (in case of the RNA imaging) and a signal substantially above background (in case of Ire1–mCherry imaging). We excluded images of cells with strong vacuolar autofluorescence. Images were processed in ImageJ and Adobe Photoshop such that the linear range of the signal was comparable between images.
Quantitative analysis of Ire1 foci and co-localization of mRNA in foci
Images of Ire1–mCherry and U1A–GFP-decorated HAC1U1A, SpRU1A and PGK1U1A mRNAs and variants thereof were analysed using a customized MatLab script to determine the fraction of Ire1–mCherry in foci and to score the recruitment of U1A–GFP-decorated mRNA in Ire1 foci. The annotated MatLab script is available (Supplementary Information). In brief, the mean pixel intensity of a background area (∼20% of section area) was defined in an intracellular area excluding ER. Ire1 was defined as all signal exceeding the mean background by 1.1-fold. Under non-stress conditions, we never observed this signal to exceed a 1.5-fold background threshold. We thus defined the fraction of Ire1 in foci as the ratio of Ire1–Cherry fluorescence intensity above threshold divided by the total Ire1–Cherry fluorescence intensity. The threshold was empirically defined such that under non-stress conditions no signal was scored as ‘foci’. Similarly, RNA foci were defined as pixels exceeding by twofold the mean intensity in the RNA channel. A ‘co-localization index’ was then defined as the integrated intensity of the pixels within the RNA foci that had pixels in common with Ire1 foci divided by the total RNA intensity, and expressed in arbitrary units in a range of 0 to 100. For each condition, the percentage of Ire1–mCherry in foci and the co-localization index for the mRNA recruited to the foci was determined for 5–9 individual cells. Values and the standard error of the mean are given in histograms in Figs 2–4. Because, in contrast to the covalently fluorescently tagged Ire1, we do not know what fraction of the fluorescent reporter U1A–GFP in cells is bound to mRNAs containing U1A binding sites, background subtraction for U1A–GFP was arbitrary. Therefore, we report co-localization by this ‘co-localization index’ rather than by an absolute percentage co-localization measure. The co-localization index robustly scores the differences in mRNA recruitment we observed in the fluorescent micrographs.
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We thank M. Jonikas and B. Kornmann for their help with the MatLab scripts; R. Parker for the pPS2037 and pRP1187 plasmids; K. Thorn for the pKT127 plasmid and for his assistance with microscopy at the Nikon Imaging Center at UCSF; and C. Guthrie, R. Andino, J. Gross and members of the Walter laboratory for discussion and comments on the manuscript. T.A. was supported by the Basque Foundation for Science and the Howard Hughes Medical Institute; E.v.A. by the Netherlands Organization for Scientific Research (NWO); D.P. and C.A.R. by the National Science Foundation; C.A.R. by the President’s Dissertation Year Fellowship; and A.V.K. by the Jane Childs Memorial Fund for Medical Research. P.W. is an Investigator of the Howard Hughes Medical Institute.
Author Contributions T.A. and E.v.A. wrote the manuscript, conceived the experiments and together with D.P. carried out most of the experimental work. I.M.S. and E.v.A. observed Ire1 foci, and C.A.R. performed all experiments concerning tRNA ligase localization. A.V.K. carried out kinetic analyses. P.W. directed the research programme and writing of the manuscript.
This file contains Supplementary Figures S1-S4 with Legends. (PDF 8494 kb)
This file contains the script which calculates (1) The fraction of Ire1 concentrated in foci and (2) The colocalization index (CI), that scores for the fraction of RNA foci colocalizing with Ire1. (TXT 2 kb)
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Aragón, T., van Anken, E., Pincus, D. et al. Messenger RNA targeting to endoplasmic reticulum stress signalling sites. Nature 457, 736–740 (2009). https://doi.org/10.1038/nature07641
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