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  1. Author Response:

    Reviewer #1 (Public Review):

    The study visualizes the behavior of some of the components of a cell stress pathway in live cells. The study generates tools that may be of interest to cell biologists, though the claims of the study need to be tempered to better reflect what is actually observed and some of the reagents would benefit from additional characterization.

    Thank you for this input. We agree that some of our claims might have been badly phrased due to the fact that the manuscript had to be written quickly to coordinate initial submission with Belyy et al. (2021). We have now worked on the text to make the phrasing more accurate.

    We also agree that this manuscript may show limited information related to the characterization of the molecular tools used in this study. In the case of the imaging reporters, we provide evidence on their splicing and the proteins produced from both the unspliced or spliced versions of the mRNA, which in our opinion was the essential information needed to demonstrate/validate the capacity of these recombinant mRNAs to undergo splicing and be translated into the expected products. Apart from this basic set of data, we have characterized the abundance of these recombinant transcripts, their potential impact in endogenous UPR signaling, or the effect that MS2 tagging may have in their regulation.

    The authors have applied some live cell imaging tools to attempt to visualize the processing of XBP1 mRNA by IRE1a during the mammalian Unfolded Protein Response. Single particle tracking was combined with the MS2 tagging system to localize wt and mutant XBP1 mRNAs relative to the endoplasmic reticulum (ER). This is the first study to visualize XBP1 mRNA in a live cell and the information acquired supports existing models of XBP1 mRNA processing and potentially provides some clarity regarding spatial localization and rates of processing in live cells. The manuscript makes some claims that need to be modified as the data are sometimes more limited in terms of what is actually shown.

    We agree that some of the claims of our manuscript have to be rewritten for the sake of accuracy and to avoid overstatements. We have revised all the instances specifically mentioned by either of the reviewers.

    In addition, the authors perform some live cell imaging experiments with a tagged version of IRE1a, the stress sensor that cleaves XBP1 mRNA as part of its splicing process during stress. Previous studies have reported that IRE1a forms large visible clusters in response to ER stress. The authors have claimed that the clustering is an artifact of tagged IRE1a overexpression. More characterization of both the reporter and native untagged IRE1a are needed to make a stronger conclusion.

    To address this point, our study provides qPCR-based splicing assays, western blots of protein products, mutant analysis and smFISH for validation of mRNA expression/export/turnover. If additional characterization is needed, we ask for further clarification on the specific experiments, which would strengthen our conclusion.

    Of note, the IRE1a-GFP construct is identical to that established and characterized by Belyy et al. (2019). The only difference is that in our study we modified the promoter and 5’UTR to tune expression close to endogenous levels. We apologize for not making this clearer but believe that the functionality of the construct for analyzing IRE1a clustering was already demonstrated in that publication from the Walter lab.

    Overall, the study will be of interest to labs in the ER stress field and of potentially broader interest to groups studying mRNA trafficking and processing in live cells. With further characterization, the reagents may be useful for mechanistic studies of ER stress in single cells.

    Reviewer #2 (Public Review):

    This manuscript develops different reporters to monitor XBP1 targeting to the ER, which are used to confirm previous results showing that XBP1 is directed to the ER through a mechanism involving translation of the HR2 mRNA sequence. As indicated in the manuscript, this mechanism had been previously reported by Kohno, and, while the work presented here confirms this model, it does not extend it. The major advance from this manuscript, apart from the reporter development, relates to the fact that IRE1 clusters are not observed in cells expressing endogenous levels of IRE1-GFP and subjected to ER stress. This is in contrast to previous reports where IRE1 clusters were proposed to be the primary site of XBP1 splicing; however, IRE1 clustering from XBP1s splicing has been shown to been separable previously in Ricci et al (2019) FASEB J (where they showed that the flavinoid luteolin induces robust XBP1 splicing independent of clustering). Herein, the authors demonstrated that the clustering of IRE1-GFP is an artifact of overexpression, which is not observed upon expression of IRE1-GFP to endogenous levels.

    We agree with the reviewer that the results from Ricci et al. (2019) are consistent with our findings. Still, and beyond evidence based on the artificial activation of IRE1a by flavinoids, we provide evidence supporting that ER-stress induced XBP1 splicing does not occur at large, visible foci. In our opinion, our approach addresses this issue in a direct and conclusive manner and is the first to directly visualize the localization and translation of XBP1 mRNAs during ER stress in living cells.

    Ultimately, while the experiments appear well performed, the advance of this current manuscript is limited. The data included in Fig. 1-3 validate previous mechanisms proposed for XBP1 targeting to the ER using new approaches. While important to validate mechanisms using different approaches, there is no new insight included in this aspect of the work.

    We agree with the reviewer but would like to raise two additional points:

    (i) While XBP1 targeting mechanisms have been proposed and discussed in the literature for a while, our study is the first to directly test and visualize them. Through this unbiased approach, we confirm previous models, but at the same time disproof others. We believe that our work will not only settle ongoing debates but also provides the foundation for many future studies.

    (ii) This confirmation of previously discussed targeting mechanisms also validates the functionality of our reporter transcripts and establishes them as useful tools for further investigation into XBP1 biology.

    The fact that IRE1 clustering results from an artifact resulting from overexpression of IRE1-GFP is important, although it is somewhat underdeveloped in this specific manuscript. However, this report does support findings in a recent preprint posted to bioRXIV (Belyy et al (2021)), similarly showing that IRE1 does not cluster, as previously, thought. Taken together, this work and the Belyy et al preprint does indicate that IRE1 clustering is not associated with activation, but instead represents an artifact of overexpression.

    We thank the reviewer for emphasizing this important finding. The IRE1a clustering experiments were not further pursued in our manuscript because we coordinated our study with the one from the Walter lab (Belyy et al., 2021).

    Reviewer #3 (Public Review):

    This manuscript applies single molecule imaging approaches to visualize the ER targeting of Xbp1 mRNA by the unfolded protein response and its processing by IRE1. The major conclusions are that translation of the hydrophobic HR2 domain localizes a portion of Xbp1 mRNA to the ER, that ER stress releases Xbp1 from the ER due to splicing action by IRE1, and that Xbp1 mRNA appears not to make stable associations with punctal clusters of IRE1 during ER stress, and that these clusters do not appear in this cell system at lower levels of ectopic IRE1 expression, potentially calling the role of these clusters in Xbp1 splicing. The strength of the work is in using single molecule imaging to test (and largely confirm) ideas that were previously advanced in the literature based on studies of lower resolution, although the apparent lack of a functional role for IRE1 clusters at least in this system addresses a point that remains unsettled in the field.

    The authors take advantage of tandem tagging to label both Xbp1 mRNA and the polypeptide product associated with translation of unspliced Xbp1 mRNA. The authors show convincingly that fluorescent dots corresponding to unspliced Xbp1 associate with the ER, to an extent greater than that achieved by Xbp1 in which the HR2 peptide cannot be translated, but to an extent less than that achieved by a conventional secretory protein. Why the Xbp1 mRNA achieves lower targeting efficiency is not specified. They also show that ER stress is associate with a loss of Xbp1 mRNA from the ER, and this is attributable to splicing by Xbp1, whereas unspliceable Xbp1 or wild-type Xbp1 when IRE1 is inhibited remains associated with the ER to an extent during stress that is not much lower than in the absence of stress. Conversely, constitutively spliced Xbp1 largely fails to associate with the ER. The last figure leverages the imaging of Xbp1 to show that Xbp1 mRNA appears not to associate with IRE1 clusters that are observed in a system similar (but not identical) to the cell system reported by the Walter lab in 2020.

    Overall, the experiments are intriguing and the quality of the data is high.

    We thank the reviewer for such a positive evaluation.

    The major novelty of the paper is its approach. The general findings (that the HR2 region of Xbp1 mRNA must be translated for Xbp1 mRNA to be targeted to the ER; and that splicing of Xbp1 mRNA, which shifts the reading frame of the HR2 region, causes Xbp1 mRNA to no longer be associated with the ER largely support/confirm the conclusions of the field arrived at through other methods. The last conclusion, about IRE1 clustering, is where new ground is tread. It is notable that IRE1 clusters are not observed when IRE1 is ectopically expressed to low levels. Therefore, phenomenologically at least, IRE1 clusters are not a prerequisite for at least some splicing of Xbp1 mRNA to occur.

    All that said, I have four substantive concerns about the manuscript:

    1. The conclusions with respect to Xbp1 mRNA (and also with respect to XBP1 translation) require that the visualized Xbp1 dots are indeed single molecules of Xbp1 mRNA, and that the process captures all Xbp1 mRNA molecules, rather than only a subpopulation. I am not so sure that either of these criteria is rigorously validated. In Supplementary Figure 2, it appears that MCP-Halo and scAB-GFP detect many spots that are either overlapping or immediately adjacent to each other - more than I would expect by chance given that these two sorts of spots arise necessarily from different RNAs. This raises the possibility that what is detected are not individual RNAs but clusters thereof.

    Previous publications have shown that MS2-labeled mRNAs are detected as diffraction-limited spots that correspond to single mRNA particles. Thus, we are using an established method that we have validated in many previous publications (Voigt et al., 2017, Cell Reports; Horvathova, Voigt et al., 2017, Mol Cell; Wilbertz et al., 2019, Mol Cell; Mateju et al., 2020, Cell).

    However, to address the reviewer’s concerns, we now provide additional results to control for the possibility that the point light sources we detect are not single molecules of Xbp1 mRNA but clusters of mRNAs:

    (i) We show spot intensity distributions from single molecule data of fixed (Figure 2-figure supplement 1D) and live (Figure 3-figure supplement 1E) cell imaging experiments. For both set-ups, these histogram plots exhibit a single defined peak, indicating that we do not detect higher order oligomeric species or clusters mRNA particles.

    (ii) To control for the spurious co-localization or unspecific interaction of MCP-Halo and scAB-GFP spots, we now provide an additional control experiment that is shown in Figure 2-figure supplement 2 and explained in more detail below.

    With respect to the colocalization of MCP-Halo and scAB-GFP spots that was mentioned by the reviewer: MCPHalo and scAB-GFP spots are meant to be overlapping or immediately adjacent to each other. They have to be since they originate from the same mRNP. Please take another look at Figure 2A (or Figure 2-figure supplement 2A). The XBP1u translation reporter includes both, the SM (translation imaging) and the MS2 (mRNA detection) tags. The fact that all scAB-GFP spots co-localize with MCP-Halo spots is precisely the validation that tells us that we are not looking at artifacts but indeed translation sites.

    If that is true (or even if it isn't), there also needs to be some way of validating that the technique is not biasing for only a certain population of Xbp1 mRNAs that behave in a certain way that is not necessarily representative of all Xbp1 mRNAs.

    We take great care not to bias our acquisition by choosing short frame rates (high temporal resolution) that allows detection of slow as well as fast particles. We sample all areas of the cells and only decide on which cells to image based on ER, not mRNA/translation site signal. Please elaborate on any additional sources of bias we might not be aware of.

    Indeed, the fact that FISH detects some Xbp1 mRNAs that scAB-GFP does not (Figure 2F) argues that scAB-GFP is not detecting everything, which raises the question of what features characterize mRNAs that it does not detect.

    scAB-GFP signal can only be detected for mRNAs that are actively translating. If some mRNAs have no such signal, it means that they are not being translated at the moment of cell fixation. Translation site imaging using scAB-GFP is an established method that was well characterized and successfully employed by us (Voigt et al,

    1. and others (Morisaki et al, 2016; Yan et al, 2016) in the past.
    1. I agree with the authors' interpretation that Xbp1 mRNA (or at least the Xbp1 mRNA that is being detected) does not stably associate with IRE1 clusters. However, it is not clear that one would expect a stable association. Rather, is it not possible that splicing might be by a "kiss-and-run" mechanism? To test/eliminate this possibility, the authors would need to show the fate of individual Xbp1 mRNAs before and after an IRE1 encounter and/or before and after leaving the ER. It would seem that the authors have the tools to accomplish this in their existing toolkit.

    We agree that splicing might be a “kiss-and-run” mechanism, which is why most of our XBP1 mRNA/IRE1a-GFP colocalization experiments were performed under IRE1a inhibition conditions or using unspliceable reporter transcripts. Both conditions should lead to the accumulation of XBP1 mRNA in IRE1a clusters (if those were the sites of splicing), the same way as they lead to an accumulation of XBP1 transcripts on the ER. And because we do not detect such an accumulation of XBP1 mRNAs under these conditions, we suggest that XBP1 mRNAs might not be recruited by IRE1 clusters in the first place. In addition, we now provide exemplary movies highlighting individual transcripts that are stably associated (Video

    1. or recently recruited before being stably associated (Video 2) with the ER. However, because the mRNA signal (MCP-Halo) is splicing independent, these observations allow no conclusions with respect to the “splice state” of a single mRNA transcript.

    Last, we are in the process of performing translation site imaging experiments in order to further characterize translation dynamics of XBP1u during ER association and are also setting up comparable experiments for an XBP1s translation site reporter, that will allow us to characterize translation dynamics after IRE1a processing.

    Unfortunately, the generation of such reporter cell lines takes months and was beyond the time frame of the coordinated re-submission that we agreed on with the authors of the complimentary manuscript by Belyy et al. (2021).

    1. The conclusion that splicing of Xbp1 mRNA causes its liberation from the ER membrane is largely inferential. I agree it is a reasonable conclusion, but, similarly to point 2 above, it requires tracking the mRNA before and after its cleavage and/or before and after its release from the ER to conclusively validate.

    We agree that the conclusion is inferential (even though we validate the mechanism by using both, mutant reporters and small molecule inhibitors). What remains unclear to us is, how such tracking data (of which we have a lot) would allow us to draw conclusions with respect to splicing. In other words, how would we differentiate between a spliced mRNA leaving the ER and an unspliced transcript (that might be released for other reasons).

    At the moment, the only approach that could realize such an experiment would involve tracking individual IRE1a protein molecules in combination with tracking individual XBP1 mRNA transcripts and then assessing mRNA mobility immediately before and after the encounter. We have refrained from performing single-IRE1a imaging experiments in the past due to our coordination with the Walter lab but will set them up for future studies

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  2. Evaluation Summary:

    This study takes on a lingering question in the study of the endoplasmic reticulum unfolded protein response (UPR), namely the relationship between the oligomeric state and activity of the UPR transducer IRE1. Applying modern imaging tools to cultured mammalian cells the authors conclude that much of IRE1's effector function (the unconventional 'splicing' of the XBP1 mRNA) is carried out by finely dispersed IRE1 molecules and not by large clusters. Whilst some of the analytical tools used here remain to be fully validated, the study is likely to be of interest to students of the UPR and its timeliness is highlighted by a recent posting on BioRxiv addressing the same question (Belyy et al., 2021 DOI: 10.1101/2021.09.29.462487)

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2021.09.29.462487v1

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  3. Reviewer #1 (Public Review):

    The study visualizes the behavior of some of the components of a cell stress pathway in live cells. The study generates tools that may be of interest to cell biologists, though the claims of the study need to be tempered to better reflect what is actually observed and some of the reagents would benefit from additional characterization.

    The authors have applied some live cell imaging tools to attempt to visualize the processing of XBP1 mRNA by IRE1a during the mammalian Unfolded Protein Response. Single particle tracking was combined with the MS2 tagging system to localize wt and mutant XBP1 mRNAs relative to the endoplasmic reticulum (ER). This is the first study to visualize XBP1 mRNA in a live cell and the information acquired supports existing models of XBP1 mRNA processing and potentially provides some clarity regarding spatial localization and rates of processing in live cells. The manuscript makes some claims that need to be modified as the data are sometimes more limited in terms of what is actually shown. In addition, the authors perform some live cell imaging experiments with a tagged version of IRE1a, the stress sensor that cleaves XBP1 mRNA as part of its splicing process during stress. Previous studies have reported that IRE1a forms large visible clusters in response to ER stress. The authors have claimed that the clustering is an artifact of tagged IRE1a overexpression. More characterization of both the reporter and native untagged IRE1a are needed to make a stronger conclusion. Overall, the study will be of interest to labs in the ER stress field and of potentially broader interest to groups studying mRNA trafficking and processing in live cells. With further characterization, the reagents may be useful for mechanistic studies of ER stress in single cells.

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  4. Reviewer #2 (Public Review):

    This manuscript develops different reporters to monitor XBP1 targeting to the ER, which are used to confirm previous results showing that XBP1 is directed to the ER through a mechanism involving translation of the HR2 mRNA sequence. As indicated in the manuscript, this mechanism had been previously reported by Kohno, and, while the work presented here confirms this model, it does not extend it. The major advance from this manuscript, apart from the reporter development, relates to the fact that IRE1 clusters are not observed in cells expressing endogenous levels of IRE1-GFP and subjected to ER stress. This is in contrast to previous reports where IRE1 clusters were proposed to be the primary site of XBP1 splicing; however, IRE1 clustering from XBP1s splicing has been shown to been separable previously in Ricci et al (2019) FASEB J (where they showed that the flavinoid luteolin induces robust XBP1 splicing independent of clustering). Herein, the authors demonstrated that the clustering of IRE1-GFP is an artifact of overexpression, which is not observed upon expression of IRE1-GFP to endogenous levels.

    Ultimately, while the experiments appear well performed, the advance of this current manuscript is limited. The data included in Fig. 1-3 validate previous mechanisms proposed for XBP1 targeting to the ER using new approaches. While important to validate mechanisms using different approaches, there is no new insight included in this aspect of the work. The fact that IRE1 clustering results from an artifact resulting from overexpression of IRE1-GFP is important, although it is somewhat underdeveloped in this specific manuscript. However, this report does support findings in a recent preprint posted to bioRXIV (Belyy et al (2021)), similarly showing that IRE1 does not cluster, as previously, thought. Taken together, this work and the Belyy et al preprint does indicate that IRE1 clustering is not associated with activation, but instead represents an artifact of overexpression.

    Read the original source
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  5. Reviewer #3 (Public Review):

    This manuscript applies single molecule imaging approaches to visualize the ER targeting of Xbp1 mRNA by the unfolded protein response and its processing by IRE1. The major conclusions are that translation of the hydrophobic HR2 domain localizes a portion of Xbp1 mRNA to the ER, that ER stress releases Xbp1 from the ER due to splicing action by IRE1, and that Xbp1 mRNA appears not to make stable associations with punctal clusters of IRE1 during ER stress, and that these clusters do not appear in this cell system at lower levels of ectopic IRE1 expression, potentially calling the role of these clusters in Xbp1 splicing. The strength of the work is in using single molecule imaging to test (and largely confirm) ideas that were previously advanced in the literature based on studies of lower resolution, although the apparent lack of a functional role for IRE1 clusters at least in this system addresses a point that remains unsettled in the field.

    The authors take advantage of tandem tagging to label both Xbp1 mRNA and the polypeptide product associated with translation of unspliced Xbp1 mRNA. The authors show convincingly that fluorescent dots corresponding to unspliced Xbp1 associate with the ER, to an extent greater than that achieved by Xbp1 in which the HR2 peptide cannot be translated, but to an extent less than that achieved by a conventional secretory protein. Why the Xbp1 mRNA achieves lower targeting efficiency is not specified. They also show that ER stress is associate with a loss of Xbp1 mRNA from the ER, and this is attributable to splicing by Xbp1, whereas unspliceable Xbp1 or wild-type Xbp1 when IRE1 is inhibited remains associated with the ER to an extent during stress that is not much lower than in the absence of stress. Conversely, constitutively spliced Xbp1 largely fails to associate with the ER. The last figure leverages the imaging of Xbp1 to show that Xbp1 mRNA appears not to associate with IRE1 clusters that are observed in a system similar (but not identical) to the cell system reported by the Walter lab in 2020.
    Overall, the experiments are intriguing and the quality of the data is high. The major novelty of the paper is its approach. The general findings (that the HR2 region of Xbp1 mRNA must be translated for Xbp1 mRNA to be targeted to the ER; and that splicing of Xbp1 mRNA, which shifts the reading frame of the HR2 region, causes Xbp1 mRNA to no longer be associated with the ER largely support/confirm the conclusions of the field arrived at through other methods. The last conclusion, about IRE1 clustering, is where new ground is tread. It is notable that IRE1 clusters are not observed when IRE1 is ectopically expressed to low levels. Therefore, phenomenologically at least, IRE1 clusters are not a prerequisite for at least some splicing of Xbp1 mRNA to occur.

    All that said, I have four substantive concerns about the manuscript:

    1. The conclusions with respect to Xbp1 mRNA (and also with respect to XBP1 translation) require that the visualized Xbp1 dots are indeed single molecules of Xbp1 mRNA, and that the process captures all Xbp1 mRNA molecules, rather than only a subpopulation. I am not so sure that either of these criteria is rigorously validated. In Supplementary Figure 2, it appears that MCP-Halo and scAB-GFP detect many spots that are either overlapping or immediately adjacent to each other - more than I would expect by chance given that these two sorts of spots arise necessarily from different RNAs. This raises the possibility that what is detected are not individual RNAs but clusters thereof. If that is true (or even if it isn't), there also needs to be some way of validating that the technique is not biasing for only a certain population of Xbp1 mRNAs that behave in a certain way that is not necessarily representative of all Xbp1 mRNAs. Indeed, the fact that FISH detects some Xbp1 mRNAs that scAB-GFP does not (Figure 2F) argues that scAB-GFP is not detecting everything, which raises the question of what features characterize mRNAs that it does not detect.

    2. I agree with the authors' interpretation that Xbp1 mRNA (or at least the Xbp1 mRNA that is being detected) does not stably associate with IRE1 clusters. However, it is not clear that one would expect a stable association. Rather, is it not possible that splicing might be by a "kiss-and-run" mechanism? To test/eliminate this possibility, the authors would need to show the fate of individual Xbp1 mRNAs before and after an IRE1 encounter and/or before and after leaving the ER. It would seem that the authors have the tools to accomplish this in their existing toolkit.

    3. The conclusion that splicing of Xbp1 mRNA causes its liberation from the ER membrane is largely inferential. I agree it is a reasonable conclusion, but, similarly to point 2 above, it requires tracking the mRNA before and after its cleavage and/or before and after its release from the ER to conclusively validate.

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