Cellular organization depends on the ability of cells to recruit mRNA and protein molecules to precise subcellular localizations. In eukaryotic cells, mRNA transcripts that encode membrane and secreted proteins are targeted to the endoplasmic reticulum (ER) to facilitate the efficient and often co-translational delivery of their protein products to the ER lumen. mRNA targeting is mediated through the co-translational recognition of an N-terminal signal sequence by the signal-recognition particle (SRP; Walter et al., 1981). SRP-ribosome-nascent chain complexes are recruited to the surface of the ER by the SRP receptor (Gilmore et al., 1982), which channels the nascent polypeptide into the ER lumen through interaction with the Sec61 translocon (Görlich et al., 1992).

The unfolded protein response (UPR) acts as a combination of quality control pathways that monitor the folding status of proteins within the ER lumen and adjust the capacity of the ER’s folding machinery (Walter and Ron, 2011). IRE1α (inositol-requiring enzyme 1 alpha) triggers the most conserved branch of the UPR (Cox et al., 1993; Mori et al., 1993). It is an ER membrane resident stress sensor that is activated by the accumulation of misfolded proteins in the ER lumen and signals ER stress through the non-canonical splicing of X-box binding protein 1 mRNA (XBP1, HAC1 in yeast; Sidrauski and Walter, 1997; Tirasophon et al., 1998; Yoshida et al., 2001).

Processing of unspliced XBP1 (XBP1u) mRNA is initiated upon oligomerization and trans-autophosphorylation of IRE1α (Ali et al., 2011), which leads to the allosteric activation of its cytosolic kinase and RNAse domains (Korennykh et al., 2009). Once activated, IRE1α excises a highly conserved 26 nucleotide intron from the XBP1 coding sequence (Calfon et al., 2002; Yoshida et al., 2001) and the severed exons are rejoined by the tRNA ligase RtcB (Jurkin et al., 2014; Kosmaczewski et al., 2014; Lu et al., 2014). Intron excision causes a translational frameshift in the spliced XBP1 (XBP1s) transcript, which encodes a potent transcription factor that increases the folding capacity of the ER through a broad activation of stress response genes (Acosta-Alvear et al., 2007), including expression of ER-associated degradation factors (Brodsky, 2012). Beyond processing XBP1 mRNA, metazoan IRE1α is able to cleave a variety of mRNAs to initiate their rapid degradation in a pathway known as regulated IRE1-dependent decay (RIDD; Hollien et al., 2009; Hollien and Weissman, 2006). Even though RIDD has been found to play a key role in some pathological conditions, XBP1 splicing stands out as the main physiological output of IRE1 activation (Ishikawa et al., 2017).

To efficiently support rapid responses to ER stress, eukaryotic organisms display different strategies to ensure the timely encounter of IRE1α and its substrate mRNAs. In Saccharomyces cerevisiae, acute ER stress triggers the rapid oligomerization of IRE1 protein into a discrete number of foci (Aragón et al., 2009; Kimata et al., 2007). HAC1 mRNA, the yeast homolog of XBP1, is then recruited into these foci through a bipartite element that is located in the HAC1 3' untranslated region (UTR) while translational repression is imposed by the HAC1 intron itself (Aragón et al., 2009; Rüegsegger et al., 2001; van Anken et al., 2014). This swift targeting of HAC1 mRNA to pre-formed IRE1p clusters is essential to allow a timely response to ER stress and to sustain yeast proteostasis (Pincus et al., 2010).

The activation of metazoan IRE1α has been proposed to follow the same principles that were defined in yeast. Under ER stress, ectopic, fluorescently labeled IRE1α was found to cluster into large dynamic foci, and the kinetics of cluster assembly and disassembly approximately correlated with XBP1 splicing rates (Li et al., 2010). Yet, there is no direct evidence that the formation of large IRE1α clusters is required for splicing. Even though oligomerization of IRE1α has been proven to be the regulatory step that coordinates mRNA cleavage (Korennykh et al., 2009; Li et al., 2010) and the disruption of oligomerization interfaces has been shown to diminish RNAse activity (Karagöz et al., 2017; Sanches et al., 2014), the specific oligomeric state of splicing-competent IRE1α assemblies has not been precisely determined. In addition, only a minor fraction (~5%) of all cellular IRE1α protein concentrates in detectable foci (Belyy et al., 2020) and there is no direct evidence that they are indeed the sites of XBP1 processing at the ER.

In contrast to yeast HAC1, metazoan XBP1 mRNA is recruited to the ER surface through co-translational targeting that involves a peptide signal sequence and not a cis-acting localization element. Specifically, XBP1u transcripts encode a hydrophobic stretch (HR2) located at the C-terminal half of the protein that mimics a secretion signal (Yanagitani et al., 2009). On translation, this hydrophobic stretch is recognized by SRP, which delivers the nascent chain complex to the Sec61 translocon in the ER membrane (Plumb et al., 2015). Recognition of the HR2 peptide is aided by a translational pausing mechanism that has been proposed to stall the translating ribosome through high-affinity interactions with the peptide exit tunnel. This conveys stability to the mRNA-ribosome-nascent chain complex that facilitates its delivery to the ER membrane (Kanda et al., 2016; Yanagitani et al., 2011). Such a co-translational targeting mechanism suggests that IRE1α encounters XBP1u mRNA at the Sec61 translocon, where translating ribosomes would be poised. This notion is supported by the reported interaction of IRE1α with the translocon complex as well as by crosslinking data that find IRE1α in close contact with SRP, ribosomal RNAs (rRNAs), and a subset of ER-targeted mRNAs (Acosta-Alvear et al., 2018; Plumb et al., 2015). However, this model is difficult to reconcile with a situation where IRE1α molecules are recruited into large clusters with complex topologies that are not simply 2D patches in the ER membrane but have also been described to exclude the Sec61 translocon from specific regions within the clusters (Belyy et al., 2020).

Here, we have developed a single-molecule imaging approach that visualizes individual XBP1 mRNA transcripts and thus provides an important framework for the investigation of fundamental UPR biology principles and the recruitment of XBP1 mRNA to IRE1α and the ER surface. We image individual XBP1 transcripts that have been recruited for splicing on the ER and show that their recruitment is mediated by a translation-dependent targeting mechanism that involves SRP but functions through a non-canonical signal sequence. We demonstrate that XBP1 mRNAs are mobilized from the ER surface upon induction of ER stress by IRE1α-catalyzed splicing. Using a dual-color live imaging approach, we visualize individual XBP1 mRNA transcripts together with IRE1α-GFP (green fluorescent protein), which only assembles into clusters at increased expression levels and does not stably associate with XBP1 mRNA under splicing inhibition conditions. Instead, when expressed at endogenous levels, IRE1α-GFP simply outlines the ER and cleaves XBP1 mRNA in the absence of cluster formation during ER stress. This finding is further confirmed by a complimentary study that images single IRE1α molecules to characterize their oligomerization dynamics in response to ER stress and also demonstrates that large IRE1α clusters are not required for splicing activity (Belyy et al., 2021).

In order to directly visualize the recruitment of XBP1 mRNA to the ER, we developed a single-molecule imaging approach that takes advantage of the MS2 labeling system to detect individual reporter mRNAs in living cells (Bertrand et al., 1998). We generated an XBP1 wild-type (WT) reporter transcript that comprises the complete Mus musculus open reading frame (ORF) as well as its complete 3'UTR (Figure 1A, red; Calfon et al., 2002; Sugimoto et al., 2015). To enable the detection of single mRNA molecules at high signal-to-noise ratios, we further included 24 MS2 stem-loops in the 3'UTR of all reporter transcripts (Figure 1A) and made use of their specific recognition by fluorescently labeled synonymous tandem MS2 coat proteins (stdMCPs; Bertrand et al., 1998; Wu et al., 2015).

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To complement the XBP1 WT reporter, we introduced a frameshift mutation downstream of the ER intron (Figure 1A, yellow, HR2 mutant) to prevent synthesis of the HR2 peptide, which has been shown to be essential for non-canonical SRP-mediated translocation of XBP1u mRNA to the ER membrane (Kanda et al., 2016; Yanagitani et al., 2009; Yanagitani et al., 2011). In addition, we employed a previously characterized SRP-recruited reporter (Voigt et al., 2017) to benchmark ER association of XBP1 transcripts against this established reporter construct encoding a secreted Gaussia luciferase protein (Figure 1A, gray).

We next generated HeLa cell lines stably expressing these reporter transcripts under a doxycycline-inducible promoter and from single genomic loci (Weidenfeld et al., 2009). To allow detection of individual mRNA particles as diffraction limited spots in the cytoplasm of living cells, we co-expressed nuclear localization signal-encoding fluorescently labeled NLS-stdMCP-stdHalo fusion proteins, which recruit excess stdMCP to the nucleus and thereby increase the signal-over-noise ratio in the cytoplasm (Voigt et al., 2017).

To confirm that these reporter constructs were indeed splicing competent, we first performed qPCR-based splicing assays (Figure 1B). As expected, we detected an increase in the levels of spliced XBP1 WT mRNA (red), and a transient drop in the levels of unspliced WT mRNA in response to the induction of ER stress with thapsigargin (TG). Using these measurements, we calculated the splicing ratio (spliced/unspliced) as a quantitative readout of splicing efficiency. As expected, splicing ratios increased sharply upon induction of ER stress in WT reporter-expressing cells and were much lower in HR2 mutant-expressing cells (yellow). In agreement with the RNA analysis, we detected increased XBP1s protein levels in response to TG treatment in cells expressing WT reporter transcripts (black triangle, Figure 1C). HR2-mutant cells produced only residual levels of XBP1s in response to TG treatment (white triangle, Figure 1C) while the majority of their XBP1 protein products was still derived from unspliced HR2 mutant transcripts (black triangle, same size as WT XBP1s protein).

We validated that MS2 tagging of ectopic XBP1 mRNA did not compromise its capacity to undergo splicing when ER stress was induced with TG or tunicamycin (TM) and ensured it did not compromise the activation of other UPR signaling mechanisms, such as the one initiated by PERK (Figure 1—figure supplement 1).

To assess XBP1 mRNA mobility and investigate particle dynamics of individual transcripts, we acquired streaming movies at fast frame rates (20 Hz) that detected XBP1 mRNAs as Halo-labeled diffraction limited spots in the cytoplasm of individual HeLa cells (Figure 1D, red). We performed single-particle tracking (SPT) over 100 consecutive frames and used the resulting particle coordinates to determine instantaneous diffusion coefficients (IDCs) as a measure of particle mobility (Berg, 1993; Voigt et al., 2017).

According to current models, XBP1u WT mRNA (but not the HR2 mutant) should be constitutively recruited to the ER surface for IRE1α-mediated splicing during ER stress. To investigate XBP1 mRNA association with the ER, we therefore integrated a fluorescently labeled ER marker protein (Sec61b-SNAP) into the reporter cell lines introduced above (analogous to Belyy et al., 2020). We imaged dual-labeled cells using a fluorescence microscope equipped with two parallel light paths and registered cameras for simultaneous detection of mRNA and ER signal in independent channels (Video 1).

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Next, we quantified the mobility of individual particles with respect to their ER localization, which not only allowed us to visualize the recruitment of individual mRNAs to the ER surface (Figure 1D, upper panels; Video 2) but also enabled us to assess whether a particle is stably associated with the ER (Figure 1D, middle panels; Video 3). To this aim, we segmented the ER signal (Figure 1D, lower panels) and used it to generate distance maps that allowed us to correlate particle coordinates with ER boundaries. In these distance maps, positions on the ER were given positive values, and positions away from the ER were defined as negative. Based on particle trajectories, we determined the localization of individual transcripts throughout the entire image series and calculated cumulative ER localization indices that highlight robust localization phenotypes (Voigt et al., 2017). We combined the diffusion and ER colocalization analysis and employed it to benchmark the behavior of a Gaussia luciferase reporter transcript (Figure 1A) that encodes a secreted protein product and that we have previously shown to be predominantly localized to the ER (Voigt et al., 2017). Next, we performed the same analysis to quantify the mobility and ER association of XBP1 WT and HR2 mutant reporters (Figure 1E).

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The combined data show that a large fraction of XBP1 WT transcripts (Figure 1F, red dots) behaves similar to the secreted Gaussia mRNAs (Figure 1F, gray dots). Many XBP1 WT transcripts exhibit low mobility and colocalize with the ER. However, there is another population of WT reporter tracks not observed for the Gaussia reporter that is more mobile and tends to not localize to the ER. Interestingly, the behavior of this population is exactly matched by the XBP1 HR2 mutant tracks (Figure 1F, yellow dots). These reporter mRNAs seem to have lost their ability to be recruited to the ER surface and exhibit a generally higher degree of mobility that is also apparent upon visual inspection (Figure 1F, Video 4). We employed the correlated diffusion and ER colocalization analysis to quantify the fraction of ER-associated particles per cell. To this aim, we used the clearly ER-associated Gaussia cluster to define cut-offs (D<0.06 μm²s⁻¹ and positive ER localization index, dashed lines in Figure 1F) for identification of XBP1 mRNA particles that showed a similar behavior. Based on these parameters, we found an average (per cell) of 27.4 ± 19.4% (mean ± SD) of all XBP1 WT and 3.1 ± 6.2% of all HR2 mutant transcripts to be associated with the ER (Figure 1G).

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To corroborate the findings from the single-particle imaging approach through an independent method, we performed membrane flotation assays that allow separation of membrane from cytosolic fractions (Figure 1—figure supplement 2A; Mechler and Rabbitts, 1981). As expected, we found that XBP1 WT reporter mRNAs were detected in the membrane fractions to a similar extent as the endogenous XBP1u mRNA. In contrast, XBP1u HR2 mutant mRNA lacked membrane association and behaved like endogenous XBP1s (Figure 1—figure supplement 2B). Upon reconstitution of the original ORF through integration of two additional nucleotides that restore the HR2 reading frame but not the upstream part of the ORF, membrane association was restored (Figure 1—figure supplement 2C, D). Association of XBP1 reporter transcripts with the ER is therefore unambiguously linked to the expression of the HR2 peptide.

Together, these findings indicate that XBP1 WT reporter mRNA is recruited to the ER surface albeit to a lesser extent than canonical secretion-signal encoding Gaussia transcripts. Recruitment depends on the expression of the HR2 peptide, since a reporter mRNA that does not produce HR2 failed to associate with the ER. Our results are consistent with the non-canonical mechanism of XBP1 delivery to the ER and confirm that HR2 expression conveys stable ER association in a co-translational manner.

To test if translation-dependent recruitment of XBP1 transcripts to the ER membrane is necessary to enable mRNA splicing, we generated an XBP1 translation site reporter that would allow us to directly monitor XBP1u translation on the ER (Figure 2A). Specifically, we used a nascent polypeptide imaging approach that relies on the co-expression of a well-folded protein scaffold (spaghetti monster, SM) containing nine GCN4 antigen repeats (Eichenberger et al., 2018; Morisaki et al., 2016; Yan et al., 2016) and the MS2 stem loop array introduced above. To quantify protein synthesis of XBP1u transcripts on the ER, we generated a XBP1u translation reporter construct that contains the GCN4-SM downstream of the UPR intron but in frame with the XBP1u ORF. Upon splicing and excision of the intron by IRE1α, the ORF changes to XBP1s and the GCN4-SM is no longer in frame. Thus, the translation site signal can only be detected prior to mRNA splicing.

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Quantitative real-time (RT)-PCR as well as western blot analysis confirmed that this construct was able to undergo splicing upon induction of ER stress (Figure 2B and C). To characterize the translational status of XBP1 mRNA in live imaging experiments, we employed GFP-fused single-chain antibodies (scAB-GFP; Voigt et al., 2017; Yan et al., 2016) that specifically recognize GCN4 peptides and allow for detection of individual translation sites as diffraction-limited spots in the cytoplasm of HeLa cells co-expressing the Sec61b-SNAP ER marker (Figure 2D, Figure 2—figure supplement 1A, Video 5). As a further characterization of this experimental set-up, we performed a similar dual-color live imaging experiment but this time focused on the simultaneous detection of mRNA and translation site signals. To test whether the bright GFP signal corresponded to individual translation sites, we first acquired the NLS-stdMCP-stdHalo mRNA in parallel with the scAB-GFP translation site signal (Figure 2—figure supplement 1B) and then treated the cells with puromycin (PUR) to inhibit translation (Figure 2—figure supplement 1C). Upon PUR treatment, all scAB-GFP spots disappeared, which led us to conclude that they represent active translation sites.

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We proceeded to quantify the degree of ER association observed for XBP1u translation sites in individual cells through the correlated diffusion and ER colocalization analysis introduced above (Figure 2E, Figure 2—figure supplement 1A). Interestingly, this analysis revealed that the majority of XBP1u translation sites colocalized with the ER (53.8 ± 22.1%, mean per cell ± SD) and exhibited a low mobility that is comparable to the behavior of predominantly ER-localized Gaussia transcripts (mean ER association = 57.3 ± 16.8%) but very different from the average degree of ER association assumed by XBP1 WT transcripts (mean ER association = 27.4 ± 19.4%). Thus, we conclude that XBP1u reporters are tethered to the ER surface in a translation-dependent manner.

As the translational frameshift induced by IRE1α-mediated splicing should abolish translation of the GCN4 repeats, we assessed the fraction of translating XBP1u transcripts in response to induction of ER stress. We treated the cells with TM and quantified the degree of colocalization for XBP1u mRNA and translation site spots. To maximize detection efficiency and more accurately estimate particle numbers per cell, we performed a combined single-molecule fluorescence in-situ hybridization (smFISH) and immunofluorescence (IF) experiment in fixed cells (Figure 2F, Figure 2—figure supplement 1D). Specifically, we used smFISH probes against the 5'-end of the M. musculus XBP1 ORF and an anti-GFP antibody that allowed for detection of the scAB-GFP labeled nascent polypeptides by IF. We confirmed that XBP1 mRNA and scAB-GFP translation site spots corresponded to single translation sites that only colocalized when expressed from the same mRNA transcripts (Figure 2—figure supplement 2).

Next, we investigated how ER stress affects the association of XBP1 mRNA with the ER and set out to determine how XBP1u molecules encounter IRE1α. In order to distinguish between the behavior of unspliced and spliced mRNA transcripts, we generated a XBP1 reporter variant with point mutations in the 5' and 3' splice sites of the UPR intron that maintain its stem-loop structure but render the substrate cleavage incompetent (unspliceable, dark blue, Figure 3A, Figure 3—figure supplement 1A, B; Calfon et al., 2002; Gonzalez et al., 1999). In addition, we also generated a variant lacking the intron and constitutively expressing the XBP1s protein (spliced, light blue, Figure 3A, Figure 3—figure supplement 1A, B). We performed dual-color live imaging experiments (Figure 3B, Videos 6 and 7) and quantified reporter mobility and their degree of colocalization with the ER through correlated diffusion and ER colocalization analysis under non-stress conditions (Figure 3—figure supplement 1C, D). To further characterize particle behavior and control against potential higher-order oligomeric assemblies of reporter transcripts, we assessed the mean spot intensities detected for unspliceable and spliced reporter transcripts. They were comparable to the mean intensities of XBP1 WT and HR2 mutant spots that were collected in the same imaging experiment (Figure 3—figure supplement 1E) and exhibited a single defined peak confirming that the spot signal originated from single mRNA transcripts that did not associate in larger oligomeric assemblies.

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As expected, unspliceable reporter transcripts often exhibited a lower mobility (Figure 3—figure supplement 1C) and associated with the ER to a degree that is comparable to WT transcripts (Figure 3C) while spliced reporter mRNAs tended to diffuse at higher mobilities (Figure 3—figure supplement 1D) and displayed a lower degree of ER association (Figure 3C) comparable to cytoplasmic protein-encoding mRNAs (Voigt et al., 2017).

To determine the effect of ER stress on the ER association of WT, unspliceable and spliced reporter transcripts, we induced ER stress with TM (5 µg/ml) at least 3 hours before the start of the imaging session (Video 8). In addition, and to specifically assess the involvement of IRE1α in such association, we performed the same imaging experiments including 50 µM 4µ8C, a small molecule inhibitor that blocks substrate access to the active site of the IRE1α RNase domain and thereby selectively inactivates XBP1 cleavage (Video 9; Cross et al., 2012). As anticipated, ER stress-induced processing of WT reporters caused a strong decrease of their mean ER association from 27.4 ± 19.4% (mean ± SD) in the untreated condition to 10.1 ± 9.3% under TM treatment (Figure 3C, red). This result supports the idea that, upon completion of the splicing reaction, WT mRNAs are released from the ER membrane and behave like intron-free transcripts (Figure 3C, light blue) in the absence of ER stress (10.0 ± 9.1%). ER stress-induced mobilization of spliced WT reporter transcripts was a genuine consequence of IRE1α catalysis, since addition of 4µ8C to the TM condition restored ER association of WT reporters back to 33.2 ± 15.6% (Figure 3C, red). Taken together, these findings suggest that IRE1α-mediated splicing drives the release of translationally active, ER-tethered mRNAs.

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In line with this notion, unspliceable reporter transcripts (Figure 3C, dark blue) not only associate with the ER to a high level (32.4 ± 13.5%) but also fail to show a similar reduction in ER association upon treatment with TM (23.7 ± 18.4%) and TM + 4µ8C (20.3 ± 12.6%) (Figure 3C). The same holds true for the spliced reporter construct. Since it does not encode the HR2 peptide and can therefore not be delivered to the translocon, it only associates with the ER to a limited extent (10.0 ± 9.1%). Upon induction of ER stress, its ER association rate is further reduced and similar to the unspliceable reporter, we do not observe significant changes in ER association between ER stress conditions in the absence (4.8 ± 5.0%) and presence (4.6 ± 4.4%) of 4µ8C.

We noticed that, for unspliceable and spliced reporters, ER stress caused a slight reduction of ER association when compared to untreated conditions. This effect may result from the general inhibition of cellular translation initiation triggered by the eIF2α kinase PERK, that promotes the UPR branch of the integrated stress response (Pakos-Zebrucka et al., 2016). It is plausible that the slightly reduced levels of ER association under ER stress conditions are due to the decreased recruitment of translating mRNPs to the ER surface, affecting all mRNAs to a limited extent (Voigt et al., 2017). This effect is not observed for the XBP1 WT reporter, where conversion of unspliced into spliced molecules is the major driver of mobilization from the ER. In summary, these experiments demonstrate that IRE1α activity is not required for ER association of XBP1 reporter mRNAs but suggest that IRE1α-mediated catalysis might contribute to the release of spliced mRNA molecules to the cytosol.

In combination, our findings support a model where IRE1α-mediated splicing is instrumental for the mobilization of XBP1 transcripts that are anchored to the ER in a translation-dependent manner. Based on this hypothesis, we sought to further investigate and visualize the sites of XBP1 processing on the ER membrane. To this aim, we developed an approach that allowed us to detect IRE1α in the reporter transcript-expressing HeLa cell lines introduced above and that was complementary to the single-molecule imaging approach, which detects endogenously tagged IRE1α protein molecules and is published in parallel to this study (Belyy et al., 2021). We knocked out the endogenous IRE1α using CRISPR/Cas9 and reconstituted its expression with a GFP-tagged IRE1α protein (Figure 4A). Identical to the previously published design of a splicing competent IRE1α-GFP construct, we introduced a GFP moiety in between the lumenal and kinase/RNase domains on the cytoplasmic site of the transmembrane protein (Belyy et al., 2020; Li et al., 2010).

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IRE1α has been shown to form large oligomeric assemblies and microscopically visible clusters upon induction of ER stress in a number of studies (Belyy et al., 2020; Kimata et al., 2007; Li et al., 2010; Tran et al., 2021). Yet, the physiological relevance of these clusters remains unclear. To determine if the extent of IRE1α-GFP expression could artificially affect IRE1α clustering, we generated lentiviral constructs that induced different IRE1α-GFP expression levels. Specifically, we took advantage of the previously characterized Emi1 5'UTR that has been shown to downregulate translation approximately 40-fold (Yan et al., 2016). Since this 5'UTR was derived from the cell cycle protein Emi1, we termed the construct Emi1-IRE1α-GFP. For comparison, we also generated an IRE1α-GFP expression construct that was lacking the Emi1 5'UTR and expressed the IRE1α-GFP at higher levels.

As anticipated, western blot analysis confirmed that reconstituted IRE1α at low (Emi1-IRE1α-GFP) as well as at high levels (IRE1α-GFP) restored the functionality of IRE1α in KO cells, albeit to different extents (Figure 4B). While Emi1-IRE1α-GFP levels were similar to those of endogenous IRE1α, IRE1α-GFP expression was approximately 10-fold higher (Figure 4C). In both cell lines, ectopic IRE1α-GFP expression rescued XBP1 mRNA splicing under ER stress conditions, as determined by quantitative RT-PCR (Figure 4D) and by western blot detection of the resulting XBP1s protein (Figure 4B). At the same time, we noticed that GFP tagging slightly reduced XBP1 splicing levels both in the endogenous XBP1 mRNA and the WT reporter transcript (Figure 4—figure supplement 1A-C and Figure 4D). Other than that, IRE1α-GFP expressed from the Emi1 promoter supported transcription of XBP1-regulated genes, like ErdJ4, and did not significantly affect other signaling branches (Figure 4—figure supplement 1D). In line with previous reports (Li et al., 2010), strong overexpression of IRE1α-GFP triggered XBP1 mRNA splicing (and XBP1s synthesis) even in the absence of ER stress, underscoring the importance of adequate IRE1α expression levels for fine-tuning of the UPR.

In order to investigate if IRE1α clusters were the sites of XBP1 mRNA splicing on the ER, we imaged IRE1α-GFP in the HeLa cell lines stably expressing XBP1 reporter transcripts along with NLS-stdMCP-stdHalo and Sec61b-SNAP introduced above (Figure 4E). In agreement with earlier reports (Li et al., 2010) as well as a parallel study (Belyy et al., 2021), we detected IRE1α-GFP foci (defined as a ≥fivefold enrichment of GFP signal over background) in 21.89 ± 7.31% of cells expressing high levels of the fusion protein already after relatively short induction of ER stress with TM (5 µg/ml) for 2 hours (Figure 4F). Surprisingly, this was not the case for cells expressing Emi1-IRE1α-GFP at low levels, where we were unable to detect IRE1α-GFP clusters even after prolonged exposure to TM (5 µg/ml) for up to 7 hours. To make sure that we were not missing IRE1α clusters due to imaging conditions optimized for detection of fast-moving mRNA particles (e.g. short 50 ms exposure times), we acquired IRE1α-GFP signal from the same cells in the presence and absence of ER stress but this time using longer exposures (2000 ms) and maximum laser intensities. Under such conditions, we were able to detect IRE1α-GFP signal, which exhibited a characteristic ER-like distribution pattern but no IRE1α clusters (Figure 4E, right panel).

This observation suggested that IRE1α clusters are not necessary for the production of XBP1s, which we were able to detect in the absence of cluster formation (Figure 4D). To ensure that we were not missing a potential function of the previously observed IRE1α foci, we proceeded to image XBP1 WT mRNA recruitment to these oligomeric assemblies at high temporal and spatial resolution (Figure 4F, Video 10). Interestingly, we did not find XBP1 WT transcripts accumulating in IRE1α-GFP clusters even after prolonged TM treatment (5 µg/ml for up to 4 hours) and inhibition of IRE1α cleavage activity. XBP1 particles freely diffuse around IRE1α-GFP foci and only very rarely colocalize with the IRE1α-GFP signal (Video 11). This observation was true for both XBP1 WT (in the presence of 4µ8C) as well as unspliceable reporter transcripts (Video 12).

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Taken together, our data indicate that Emi1-IRE1α-GFP supports splicing in the absence of foci formation. This suggests that XBP1 mRNA is spliced by lower oligomeric assemblies of IRE1α molecules, which can easily contact ER-associated ribosome-mRNPs, while IRE1α foci or large oligomeric clusters are not the sites of XBP1 mRNA processing during the UPR.

In this study, we present a single-molecule imaging approach that allows visualization of individual XBP1 transcripts and use it to investigate the recruitment of XBP1 mRNA to ER-localized IRE1α, which is a fundamental step of the XBP1 splicing mechanism. Based on previous yeast studies and the visualization of overexpressed IRE1α, splicing of XBP1 mRNA has been suggested to take place in large clusters of IRE1α oligomers that form during the UPR and could function as ER stress response centers (Li et al., 2010). Our findings challenge this view and suggest a different model for mammalian cells, where IRE1α might be recruited to the XBP1 mRNA and not vice versa.

Direct visualization of the recruitment of XBP1 mRNAs to the ER surface using single-molecule imaging revealed that XBP1 molecules become ER-associated in an HR2-dependent manner that is consistent with the targeting model proposed previously (Kanda et al., 2016; Yanagitani et al., 2009; Yanagitani et al., 2011). Furthermore, assessment of the translational status of single mRNA particles demonstrated that their ER association depends on interactions with the ribosome-nascent chain complex. Co-translational membrane tethering therefore immobilizes XBP1 transcripts on the ER surface and hints at a substrate recruitment mechanism where IRE1α diffuses through the ER membrane until it encounters XBP1u mRNAs at the Sec61 translocon. Direct interactions that have been reported for IRE1α and the translocon, SRP, as well as RNAs (Acosta-Alvear et al., 2018; Plumb et al., 2015) further increase the affinity of the interaction and underline the potential significance of such a recruitment mechanism.

Upon induction of ER stress, XBP1 transcripts are spliced and released from the ER surface. However, even though we can derive from our data that ER association correlates with IRE1α cleavage activity, we did not find XBP1 mRNAs colocalizing with IRE1α clusters. Moreover, large, microscopy-visible clusters were only detected when IRE1α-GFP was overexpressed at high levels. Thus, IRE1α foci are not the primary sites of XBP1 splicing. Instead, our findings support a model where ER-associated XBP1 transcripts are processed by small IRE1α oligomers that could dynamically assemble throughout the ER membrane.

These observations are in good agreement with an earlier study, in which the pharmacological activation of IRE1α with the flavonol luteolin promoted strong splicing of XBP1 in the absence of IRE1α clustering (Ricci et al., 2019). In addition, and directly related to our work, a parallel study shows that endogenously tagged IRE1α also fails to assemble into large clusters upon induction of ER stress (Belyy et al., 2021). In this work, the authors characterize IRE1α oligomerization during ER stress and find that the resting pool of IRE1α in the ER membrane is dimeric, while in response to stress transient IRE1α tetramers are assembled as the functional subunits that are required for trans-autophosphorylation and XBP1 splicing. Most likely, such a dynamic equilibrium between dimers and small oligomers allows cells to build timely, fine-tuned responses to local or transient perturbations in ER protein folding.

In combination, our findings suggest a novel mechanism for XBP1 recruitment to functional, dynamic IRE1α assemblies that continuously patrol the ER membrane to encounter substrates that are targeted there.

Following a different strategy, yeast IRE1p foci arrange the recruitment of unspliced HAC1 mRNA to the ER membrane and efficiently localize the mRNA for splicing (Aragón et al., 2009; van Anken et al., 2014). Given the strong conservation of most UPR principles, upon visualization of large IRE1α clusters in human cells (Li et al., 2010; Tran et al., 2021) it was plausible to speculate that polarization of IRE1α might build splicing centers. However, these studies mostly relied on overexpression of ectopic IRE1α, which likely contributed to the perception that clusters were required for XBP1 splicing and explains the discrepancies between this and previous reports.

Our findings shed light on a fundamental step of the XBP1 splicing mechanism, which is the recruitment of IRE1α to ER-localized XBP1 transcripts. Yet, several open questions remain:

1. Are XBP1 mRNAs that are tethered to the ER surface as part of ribosome-nascent chain complexes continuously translated? Or is translation stably stalled while the mRNA remains poised for recruitment by IRE1α? And if so, how is translation resumed? And how relevant is the translational status of XBP1 mRNA for splicing?

2. Does IRE1α also bind XBP1 transcripts in the absence of the ribosome/translocon interaction? Since we observe a low degree of splicing for HR2-mutant reporters, we speculate that translation-independent recruitment of XBP1 transcripts and recognition through IRE1α might also be possible.

3. How does IRE1α discriminate between its distinct substrates? Beyond XBP1 splicing, IRE1α processes a broad range of substrates including RIDD mRNAs (Hollien et al., 2009; Hollien and Weissman, 2006) and a recently described, larger group of mRNAs that are processed through an unanticipated mode of cleavage with looser specificity (RIDDLE) (Le Thomas et al., 2021). Most of these mRNA substrates encode signal sequence-containing proteins and are delivered to the ER by SRP. While we know that activation of the IRE1α RNAse domain requires IRE1α dimerization/oligomerization as well as trans-autophosphorylation, the specific role and substrate specificity of the distinct assemblies remain unclear. It is tempting to speculate that the interplay of different oligomeric IRE1α assemblies with the translocon/ribosome/SRP environment may define the code for selective processing of distinct IRE1α substrates, avoiding the detrimental cleavage that might result from unrestrained RNA degradation.

In summary, our data have allowed us to uncover unanticipated features of one of the key steps of UPR initiation, the encounter of XBP1 mRNA with IRE1α to undergo splicing. Additional studies will be needed to further dissect the underlying mechanisms behind the regulation of IRE1α activity in homeostasis and disease.

All imaging data generated in this study have been uploaded to Zenodo.

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Author details

1. Silvia Gómez-Puerta

   Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

   Contribution

   Investigation

   Competing interests

   No competing interests declared

2. Roberto Ferrero

   Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Tobias Hochstoeger

   1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland
   2. University of Basel, Basel, Switzerland

   Contribution

   Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8061-7857

4. Ivan Zubiri

   Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Jeffrey Chao

   Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

   Contribution

   Funding acquisition

   Competing interests

   No competing interests declared

6. Tomás Aragón

   Department of Gene Therapy and Regulation of Gene Expression, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   taragon@unav.es

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1700-2729

7. Franka Voigt

   Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review and editing

   For correspondence

   franka.voigt@fmi.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9515-0367

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