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

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.

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.