Gle1 is required for tRNA to stimulate Dbp5 ATPase activity in vitro and promote Dbp5-mediated tRNA export in vivo in Saccharomyces cerevisiae

  1. Arvind Arul Nambi Rajan
  2. Ryuta Asada
  3. Ben Montpetit

  • Curated by eLife

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    eLife assessment

    The work is a valuable contribution to understanding the mechanism of nuclear export of tRNA in budding yeast. The authors present solid evidence that Dbp5 functions in parallel with Los1 and Msn5 in tRNA export, in a manner dependent on Gle1 for activation of its ATPase activity but independently of Mex67, Dbp5's partner in mRNA export. It further presents biochemical evidence that Dbp5 can bind tRNA but that Gle1 and InsP6 are required for activating ATP hydrolysis by the Dbp5-tRNA complex, suggesting a possible mechanism for tRNA export by Dbp5.

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Abstract

Cells must maintain a pool of processed and charged transfer RNAs (tRNA) to sustain translation capacity and efficiency. Numerous parallel pathways support the processing and directional movement of tRNA in and out of the nucleus to meet this cellular demand. Recently, several proteins known to control messenger RNA (mRNA) transport were implicated in tRNA export. The DEAD-box Protein 5, Dbp5, is one such example. In this study, genetic and molecular evidence demonstrates that Dbp5 functions parallel to the canonical tRNA export factor Los1. In vivo co-immunoprecipitation data further shows Dbp5 is recruited to tRNA independent of Los1, Msn5 (another tRNA export factor), or Mex67 (mRNA export adaptor), which contrasts with Dbp5 recruitment to mRNA that is abolished upon loss of Mex67 function. However, as with mRNA export, overexpression of Dbp5 dominant-negative mutants indicates a functional ATPase cycle and that binding of Dbp5 to Gle1 is required by Dbp5 to direct tRNA export. Biochemical characterization of the Dbp5 catalytic cycle demonstrates the direct interaction of Dbp5 with tRNA (or double-stranded RNA) does not activate Dbp5 ATPase activity, rather tRNA acts synergistically with Gle1 to fully activate Dbp5. These data suggest a model where Dbp5 directly binds tRNA to mediate export, which is spatially regulated via Dbp5 ATPase activation at nuclear pore complexes by Gle1.

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  1. Version published to 10.7554/elife.89835.4 on eLife
  2. Version published to 10.7554/elife.89835 on eLife
  3. Version published to 10.7554/elife.89835.3 on eLife
  4. eLife

    Author Response

    The following is the authors’ response to the previous reviews.

    We would like to thank the Editors and Reviewers for their additional comments and constructive feedback on our manuscript. We have made minor adjustments to the figures and texts based on their suggestions, including improved images in Figure 1 and correction of figure labels.

    Reviewer #1 (Public Review):

    In their previous paper (Lari et al, 2019; Azra Lari Arvind Arul Nambi Rajan Rima Sandhu Taylor Reiter Rachel Montpetit Barry P Young Chris JR Loewen Ben Montpetit (2019) A nuclear role for the DEAD-box protein Dbp5 in tRNA export eLife 8:e48410.) as well as in the current manuscript the authors states that Dbp5 is involved in the export of tRNA that is independent of and parallel to Los1. They state that Dbp5 binds to the tRNA independent of known tRNA export proteins. The obtained conclusion is both intriguing and innovative, since it suggests that there are other variables, beyond the ones previously identified as tRNA factors, that might interact with Dbp5 to facilitate the export process. In order to find out additional factors aiding this process the authors may employ total RNA-associated protein purification (TRAPP) experiments ( Shchepachevto et al., 2019; Shchepachev V, Bresson S, Spanos C, Petfalski E, Fischer L, Rappsilber J, Tollervey D. Defining the RNA interactome by total RNA-associated protein purification. Mol Syst Biol. 2019 Apr 8;15(4):e8689. doi: 10.15252/msb.20188689. PMID: 30962360; PMCID: PMC6452921) to identify extra factors involved in conjunction with Dbp5. The process elucidates hitherto uninvestigated tRNA export components that function in conjunction with Dbp5.

    Author Response: We greatly appreciate this suggestion and agree with the reviewer that identification of the composition of the export competent Dbp5 containing tRNA complex is a critical next step for understanding the mechanism of Dbp5 mediated tRNA export, which will form the foundation of a future investigation in the laboratory and warrants its own study.

    Reviewer #1 (Public Review):

    Various reports suggest that eukaryotic translation elongation factor 1 eEF1A is involved tRNA export Bohnsack et al., 2002 (Bohnsack MT, Regener K, Schwappach B, Saffrich R, Paraskeva E, Hartmann E, Görlich D. Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 2002 Nov 15;21(22):620515. doi: 10.1093/emboj/cdf613. PMID: 12426392; PMCID: PMC137205), Grosshans etal., 2002; Grosshans H, Hurt E, Simos G. An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev. 2000 Apr 1;14(7):830-40. PMID: 10766739; PMCID: PMC316491). The presence of mutations in eEF1A has been seen to hinder the nuclear export process of all transfer RNAs (tRNAs). eEF1A has been shown to interact with Los1 aiding in tRNA export. The authors can also explore the crosstalk between Dbp5 and eEF1A in this study. Additionally, suppressor screening analysis in dbp5R423A , los1∆dbp5R423A los1∆msn∆dbp5R423A could shed more light on this.

    Author Response: Thank you for this suggestion and raising an important possible role for Dbp5 in eEF1A mediated tRNA export. Based on more recent investigation of eEF1A function in tRNA export (PMID: 25838545), it is likely that eEF1A functions in re-export of charged tRNAs specifically (likely in conjunction with Msn5). The current manuscript has largely focused on the role of Dbp5 in pre-tRNA export, but a more careful mechanistic characterization of Dbp5 and re-export will be conducted in follow-up studies given the physical interaction between Dbp5 and spliced tRNAs we previously reported. Similarly, suppressor screens with the Dbp5 and los1Δmsn5Δ mutants will likely be a useful tool in identifying additional tRNA export factors and we thank the reviewer for this suggestion.

    Reviewer #1 (Public Review):

    The addition of Gle1 is potentially novel but it's unclear why the authors didn't address the potential involvement of IP6.

    Author Response: The text has been revised to highlight the importance of InsP6 in Gle1 mediated activation of Dbp5. This includes referencing InsP6 throughout the manuscript during discussions of Gle1 activation of Dbp5 and lines 401-404 discussing the potential role for the small molecule in regulating mRNA and tRNA export in different cellular contexts (e.g., stress and disease).

  5. eLife

    eLife assessment

    The work is a valuable contribution to understanding the mechanism of nuclear export of tRNA in budding yeast. The authors present solid evidence that Dbp5 functions in parallel with Los1 and Msn5 in tRNA export, in a manner dependent on Gle1 for activation of its ATPase activity but independently of Mex67, Dbp5's partner in mRNA export. It further presents biochemical evidence that Dbp5 can bind tRNA but that Gle1 and InsP6 are required for activating ATP hydrolysis by the Dbp5-tRNA complex, suggesting a possible mechanism for tRNA export by Dbp5.

  6. eLife

    Reviewer #1 (Public Review):

    This study focuses on the defining cellular pathways critical for tRNA export from the nucleus. While a number of these pathways have been identified, the observation that the primary transport receptors identified thus far (Los1 and Msn5) are not essential and that cells are viable even when both the genes are deleted supports the idea that there are as yet unidentified mediators of tRNA export from the nucleus. This study implicates the helicase Dbp5 in one of these parallel pathways arguing that Dbp5 works in a pathway that is independent of Los1 and/or Msn5. The authors present genetic data to support this conclusion. At least one results suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathways to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway. The authors provide solid evidence that Dbp5 interacts with tRNA directly and that addition of the factor Gle1 together with the previously identified co-factor InsP6 can trigger helicase activity and release of tRNA. The combination of in vivo studies and biochemistry provide evidence to consider how Dbp5 contributes to export of tRNA and more broadly adds to the conversation about how coding and non-coding RNA export from the nucleus might be coordinated to control cell physiology.

  7. eLife

    Reviewer #2 (Public Review):

    In the manuscript by Rajan et al., the authors have highlighted the direct interaction between Dbp5 and tRNA, wherein Dbp5 serves as a mediator for tRNA export. This export process is subject to spatial regulation, as Dbp5 ATPase activation occurs specifically at nuclear pore complexes. Notably, this regulation is independent of the Los1-mediated pre-tRNA export route and instead relies on Gle1. The manuscript is well constructed and nicely written.

  8. Version published to 10.7554/elife.89835.2 on eLife
  9. eLife

    Author Response

    The following is the authors’ response to the original reviews.

    Thank you again to the reviewers and editors for all constructive feedback. We have made several edits to the manuscript and data to address concerns raised during the initial review and strengthen the completeness of this study. Please find below our response to each, with referee comments in black and our responses in blue.

    eLIFE Assessment:

    The authors report that Dbp5 functions in parallel with Los1 in tRNA export, in a manner dependent on Gle1 and requiring the ATPase cycle of Dbp5, but independent of Mex67, Dbp5's partner in mRNA export. The evidence for this conclusion is still incomplete, as is the biochemical evidence that Dbp5 interacts directly with tRNA in vitro with Gle1 and co-factor InsP6 triggering Dbp5 ATPase activity in the Dbp5-tRNA complex. The evidence that Dbp5 interacts with tRNA in cells independently of Los1, Msn5 and Mex67 is, however, solid.”

    Thank you for the constructive feedback and assessment of our article. We have made several improvements to the quality of data (Figure 1E, Figure 3C, Figure 4), added additional tRNA Northern Blot/FISH targets to further generalize observed phenotypes beyond pre-tRNAIleUAU (Supplement 1C/D/E/F), provided growth assays for los1Δ/msn5 Δ/dbp5R423A (Supplement 1B), add added data showing gle1-4/los1Δ double mutants phenocopy los1Δ/dbp5R423A to further support the involvement of Gle1 and the Dbp5 ATPase cycle in tRNA export (Figure 5D).

    Additionally, we added quantification to assess the extent of overexpression of Dbp5 mutants in Figure 3 and a discussion of how these mutants alter the localization of the protein to better assess how they may impact tRNA export (lines 211-226). Furthermore, several minor edits to the text/figures have been made to remove typos and improve readability (e.g., labels of FISH/Northern data in Figure 1). Additional edits include adjusting the text and the model presented in Figure 6 to improve conclusions drawn from our data. This includes lines 106-107 and lines 366-371 which clarifies that the Dbp5 mediated tRNA export pathway may not be entirely independent of Mex67.

    Reviewer #1 (Public Review):

    "At least one result suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathway to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway…. The obvious missing experiment here with respect to genetics is the test of whether deletion of the MSN5 gene in the cells, which combines deletion of LOS1 and the dbp5_R423A allele, shown in Figure 1D would be lethal…. The authors provide evidence of a model where the helicase Dbp5 plays a role in tRNA export from the nucleus. Further evidence is required to determine whether Dbp5 could function in the same pathway as the previously defined tRNA export receptors, Los1 and Msn5. There are genetic tests that could be performed to explore this question. Some of the biochemistry presented would show when Los1 is absent that the interaction of Dbp5 with tRNA decreases, which could support a model where Dbp5 plays a role in coordination with Los1”

    Author Response: We thank the reviewers for this suggestion and consideration. We have added data showing growth phenotypes for the los1Δ/msn5Δ/dbp5R423A triple mutants. We discuss possible explanations and alternative hypothesis for why these triple mutants are viable and the observed reduction in Dbp5-pre-tRNA interaction in the context of los1Δ (lines 128131; lines 172-174).

    Reviewer #1 (Public Review):

    “While some of the binding assays show rather modest band shifts (Figure 4B for example), the data in Figure 4A showing that there is no binding detected unless a non-hydrolyzable ATP analogue is employed, argues for specificity in nucleic acid binding. The question that does arise is whether the binding is specific for tRNA.”

    Author Response: We have adjusted brightness/contrast of the EMSAs in Figure 4 to allow for better visualization of band shifts. Additionally, a discussion of the specificity of Dbp5-nucleic acid binding and the observed tRNA binding has been added (lines 313-322)

    Reviewer #1 (Public Review):

    “With the exception of the binding studies, which also employ a mixture of yeast tRNAs, this study relies primarily on a single tRNA species to come to the conclusions drawn. Many other studies have used multiple tRNAs to explore whether pathways characterized are generalizable to other tRNAs.“

    Author Response: We have added additional tRNA targets for FISH/Northerns in Supplement 1C/D/E/F)

    Reviewer #2 (Public Review):

    “There are some pieces of data that are misinterpreted. (Figure 1A and B look the same; in Fig 1E, the DAPI staining is abnormal; in Fig 4 the bands can't be seen.)”

    Author Response: Thank you for your constructive feedback. We have replaced FISH images to improve DAPI staining (Figure 1E), adjusted EMSAs to allow for better visualization of band shifts. (Figure 4), improved Northern Blots for quality (Figure 3C), and rearranged Figure 1A/B for readability. We maintain that the results from Figure 1A/B are not misinterpreted but agree that the readability of the figure was poor and have adjusted labels/formatting accordingly. The results of these experiments show that the deletion of Los1 does not alter Dbp5 localization and conversely loss of Dbp5 does not alter Los1 localization. As such the localization patterns under loss-of-function conditions look the same as wild-type for each protein respectively.

  10. eLife

    eLife assessment

    The work is a valuable contribution to understanding the mechanism of nuclear export of tRNA in budding yeast. The authors present solid evidence that Dbp5 functions in parallel with Los1 and Msn5 in tRNA export, in a manner dependent on Gle1 for activation of its ATPase activity but independently of Mex67, Dbp5's partner in mRNA export. It further presents solid biochemical evidence that Dbp5 can bind tRNA but that Gle1 and InsP6 are required for activating ATP hydrolysis by the Dbp5-tRNA complex, suggesting a possible mechanism for tRNA export by Dbp5.

  11. eLife

    Joint Public Review:

    In the manuscript by Rajan et al., the authors have highlighted the direct interaction between Dbp5 and tRNA, wherein Dbp5 serves as a mediator for tRNA export. This export process is subject to spatial regulation, as Dbp5 ATPase activation occurs specifically at nuclear pore complexes. Notably, this regulation is independent of the Los1-mediated pre-tRNA export route and instead relies on Gle1.

    The manuscript is well constructed and nicely written. The authors have addressed the concerns as raised by the previous reviewers and added additional experiments.

    I have a few comments for polishing the manuscript.

    Major comments:
    1. In their previous paper (Lari et al, 2019; Azra Lari Arvind Arul Nambi Rajan Rima Sandhu Taylor Reiter Rachel Montpetit Barry P Young Chris JR Loewen Ben Montpetit (2019) A nuclear role for the DEAD-box protein Dbp5 in tRNA export eLife 8:e48410.) as well as in the current manuscript the authors states that Dbp5 is involved in the export of tRNA that is independent of and parallel to Los1. They state that Dbp5 binds to the tRNA independent of known tRNA export proteins. The obtained conclusion is both intriguing and innovative, since it suggests that there are other variables, beyond the ones previously identified as tRNA factors, that might interact with Dbp5 to facilitate the export process. In order to find out additional factors aiding this process the authors may employ total RNA‐associated protein purification (TRAPP) experiments ( Shchepachevto et al., 2019; Shchepachev V, Bresson S, Spanos C, Petfalski E, Fischer L, Rappsilber J, Tollervey D. Defining the RNA interactome by total RNA-associated protein purification. Mol Syst Biol. 2019 Apr 8;15(4):e8689. doi: 10.15252/msb.20188689. PMID: 30962360; PMCID: PMC6452921) to identify extra factors involved in conjunction with Dbp5. The process elucidates hitherto uninvestigated tRNA export components that function in conjunction with Dbp5.

    2. Various reports suggest that eukaryotic translation elongation factor 1 eEF1A is involved tRNA export Bohnsack et al., 2002 (Bohnsack MT, Regener K, Schwappach B, Saffrich R, Paraskeva E, Hartmann E, Görlich D. Exp5 exports eEF1A via tRNA from nuclei and synergizes with other transport pathways to confine translation to the cytoplasm. EMBO J. 2002 Nov 15;21(22):6205-15. doi: 10.1093/emboj/cdf613. PMID: 12426392; PMCID: PMC137205), Grosshans etal., 2002; Grosshans H, Hurt E, Simos G. An aminoacylation-dependent nuclear tRNA export pathway in yeast. Genes Dev. 2000 Apr 1;14(7):830-40. PMID: 10766739; PMCID: PMC316491). The presence of mutations in eEF1A has been seen to hinder the nuclear export process of all transfer RNAs (tRNAs). eEF1A has been shown to interact with Los1 aiding in tRNA export. The authors can also explore the crosstalk between Dbp5 and eEF1A in this study. Additionally, suppressor screening analysis in dbp5R423A , los1∆dbp5R423A los1∆msn∆dbp5R423A could shed more light on this.

    3. Unfortunately, this article is not significantly different from that published in eLife in 2018. In fact, it raises more questions than it brings answers by not identifying a transporter for export and not identifying a role for the helicase activity of Dbp5. The addition of Gle1 is potentially novel but it's unclear why the authors didn't address the potential involvement of IP6.

  12. Version published to 10.1101/2023.06.29.547072v4 on bioRxiv
  13. Version published to 10.1101/2023.06.29.547072v3 on bioRxiv
  14. Version published to 10.1101/2023.06.29.547072v2 on bioRxiv
  15. Version published to 10.7554/elife.89835.1 on eLife
  16. eLife

    Author Response

    We would like to thank reviewers and editors for their thoughtful and constructive review of our manuscript. Below we have provided responses to specific points in the reviewers’ comments and eLIFE assessment, highlighting areas of the manuscript that will be edited for clarity and where efforts will be made to provide data to address reviewer concerns upon a future resubmission.

    eLife assesment:

    The authors report that Dbp5 functions in parallel with Los1 in tRNA export, in a manner dependent on Gle1 and requiring the ATPase cycle of Dbp5, but independent of Mex67, Dbp5's partner in mRNA export. The evidence for this conclusion is still incomplete, as is the biochemical evidence that Dbp5 interacts directly with tRNA in vitro with Gle1 and co-factor InsP6 triggering Dbp5 ATPase activity in the Dbp5-tRNA complex. The evidence that Dbp5 interacts with tRNA in cells independently of Los1, Msn5 and Mex67 is, however, solid.

    We intend to edit the text to make clear our conclusions and accommodate clarifications on a few details of this assessment.

    (1) We would clarify that our data supports a model in which Dbp5 recruitment to tRNA is independent of Mex67 as an adapter in cells; however, this does not mean that Mex67 and Dbp5 do not still co-function in tRNA export. For example, it is possible Dbp5 and Mex67 could still co-function in the same pathway, but instead of Dbp5 working down stream of Mex67, Dbp5 may in fact work upstream as an adapter for Mex67. Edits to the text will be made to ensure this distinction is clear and highlight the possibility for future investigation to elucidate this relationship.

    (2) We would like to highlight that based on structural and biochemical data detailing synergistic activation of Dbp5 ATPase cycle by Gle1/InsP6 and single stranded RNA, it is difficult to imagine a scenario where the apparent synergistic activation of Dbp5 ATPase cycle by tRNA and Gle1/InsP6 (Figure 5) is achieved independent of direct RNA binding. For this reason, we still support the claim that the observed synergistic activation, in combination with other in-vivo and in-vitro data provided in the manuscript, support a model where Dbp5 directly binds tRNA. However, we intend to edit the text to highlight this nuance and potential alternative conclusions based on reviewer feedback.

    Reviewer #1 (Public Review):

    “At least one result suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathway to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway…. The obvious missing experiment here with respect to genetics is the test of whether deletion of the MSN5 gene in the cells, which combines deletion of LOS1 and the dbp5_R423A allele, shown in Figure 1D would be lethal…. The authors provide evidence of a model where the helicase Dbp5 plays a role in tRNA export from the nucleus. Further evidence is required to determine whether Dbp5 could function in the same pathway as the previously defined tRNA export receptors, Los1 and Msn5. There are genetic tests that could be performed to explore this question. Some of the biochemistry presented would show when Los1 is absent that the interaction of Dbp5 with tRNA decreases, which could support a model where Dbp5 plays a role in coordination with Los1”

    We agree that this is an important point that should be made clear and discussed in the text. We also agree that further experiments would be needed to be to confirm Dbp5 functions broadly in tRNA export in parallel to both Msn5 and Los1. We will aim to address these points in resubmission and discuss possible alternative conclusions of the presented results.

    Reviewer #1 (Public Review):

    “While some of the binding assays show rather modest band shifts (Figure 4B for example), the data in Figure 4A showing that there is no binding detected unless a non-hydrolyzable ATP analogue is employed, argues for specificity in nucleic acid binding. The question that does arise is whether the binding is specific for tRNA.”

    The specificity of the in-vitro interactions of Dbp5 are an important point of discussion. We will work to expand the topic of specificity of the in-vitro experiments during resubmission.

    Reviewer #1 (Public Review):

    “With the exception of the binding studies, which also employ a mixture of yeast tRNAs, this study relies primarily on a single tRNA species to come to the conclusions drawn. Many other studies have used multiple tRNAs to explore whether pathways characterized are generalizable to other tRNAs.“

    It was previously shown that Dbp5 functions to support the export of multiple tRNA species (https://doi.org/10.7554/eLife.48410). As such, we agree that additional tRNAs should be tested to explore whether phenotypes reported here are also generalizable to other tRNAs. We will add data targeting additional tRNAs during resubmission.

    Reviewer #2 (Public Review):

    “there are some pieces of data that are misinterpreted. (Figure 1A and B look the same; in Fig 1E, the DAPI staining is abnormal; in Fig 4 the bands can't be seen.)”

    Figure 1A and B represent separate experiments, showing that deletion of Los1 does not alter Dbp5 localization and conversely loss of Dbp5 does not alter Los1 localization. As such localization patterns under loss-of-function conditions look the same as wild-type localization for each protein respectively as noted. We believe that we have come to the same conclusion as the reviewer on Figure 1A and B (and this data is not misinterpreted), but also understand this panel will need to be adjusted for clarity and readability. We will make efforts to edit this figure and accompanying text make the data and conclusions clearer, including addressing the EMSAs in figure 4 and associated text for clarity.

  17. eLife

    eLife assessment

    The work is a useful contribution to understanding the mechanism of nuclear export of tRNA in budding yeast. The authors report that Dbp5 functions in parallel with Los1 in tRNA export, in a manner dependent on Gle1 and requiring the ATPase cycle of Dbp5, but independent of Mex67, Dbp5's partner in mRNA export. The evidence for this conclusion is still incomplete, as is the biochemical evidence that Dbp5 interacts directly with tRNA in vitro with Gle1 and co-factor InsP6 triggering Dbp5 ATPase activity in the Dbp5-tRNA complex. The evidence that Dbp5 interacts with tRNA in cells independently of Los1, Msn5 and Mex67 is, however, solid.

  18. eLife

    Reviewer #1 (Public Review):

    Summary:
    This study focuses on the defining cellular pathways critical for tRNA export from the nucleus. While a number of these pathways have been identified, the observation that the primary transport receptors identified thus far (Los1 and Msn5) are not essential and that cells are viable even when both the genes are deleted supports the idea that there are as yet unidentified mediators of tRNA export from the nucleus. This study implicates the helicase Dbp5 in one of these parallel pathways arguing that Dbp5 works in a pathway that is independent of Los1 and/or Msn5. The authors present genetic data to support this conclusion. At least one result suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathway to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway. The authors provide solid evidence that Dbp5 interacts with tRNA directly and that the addition of the factor Gle1 together with the previously identified co-factor InsP6 can trigger helicase activity and release of tRNA. The combination of in vivo studies and biochemistry provides evidence to consider how Dbp5 contributes to the export of tRNA and more broadly adds to the conversation about how coding and non-coding RNA export from the nucleus might be coordinated to control cell physiology.

    Strengths and weaknesses:
    A major strength of this manuscript is the multi-pronged approach to explore a potential role for the helicase Dbp5 in one of the multiple export pathways for tRNA from the nucleus.

    The obvious missing experiment here with respect to genetics is the test of whether deletion of the MSN5 gene in the cells, which combines deletion of LOS1 and the dbp5_R423A allele, shown in Figure 1D would be lethal. This key experiment would lend substance to the argument that Dbp5 functions in a tRNA export pathway that is parallel to the Los1 and Msn5 pathways.

    While some of the binding assays show rather modest band shifts (Figure 4B for example), the data in Figure 4A showing that there is no binding detected unless a non-hydrolyzable ATP analogue is employed, argues for specificity in nucleic acid binding. The question that does arise is whether the binding is specific for tRNA.

    With the exception of the binding studies, which also employ a mixture of yeast tRNAs, this study relies primarily on a single tRNA species to come to the conclusions drawn. Many other studies have used multiple tRNAs to explore whether pathways characterized are generalizable to other tRNAs.

    The authors provide evidence of a model where the helicase Dbp5 plays a role in tRNA export from the nucleus. Further evidence is required to determine whether Dbp5 could function in the same pathway as the previously defined tRNA export receptors, Los1 and Msn5. There are genetic tests that could be performed to explore this question. Some of the biochemistry presented would show when Los1 is absent that the interaction of Dbp5 with tRNA decreases, which could support a model where Dbp5 plays a role in coordination with Los1.

    This work allows insight into key questions which still remain about the multiple pathways that are required for tRNA trafficking as well as how transport pathways for coding and non-coding RNAs might be coordinated. These questions are important as many of these pathways may be regulated in response to cellular conditions or during development and defining the fundamental pathways will be critical to understanding these dynamic processes.

  19. eLife

    Reviewer #2 (Public Review):

    This submission is about the role of Dbp5/Gle1 in tRNA export. The manuscript provides data showing that Dbp5/Gle1 are involved in tRNA export from the nucleus which is an essential process critical to translation. The authors provide data that largely supports conclusions, however, there are some pieces of data that are misinterpreted. (Figure 1A and B look the same; in Fig 1E, the DAPI staining is abnormal; in Fig 4 the bands can't be seen.)

    Additionally, the methods used are fairly standard so the article does not contain any new technical achievements.

  20. Version published to 10.1101/2023.06.29.547072v1 on bioRxiv