Structural differences between the closely related RNA helicases, UAP56 and URH49, fashion distinct functional apo-complexes

mRNA export is an essential pathway for the regulation of gene expression. In humans, closely related RNA helicases, UAP56 and URH49, shape selective mRNA export pathways through the formation of distinct complexes, known as apo-TREX and apo-AREX complexes, and their subsequent remodeling into similar ATP-bound complexes. Therefore, defining the unidentified components of the apo-AREX complex and elucidating the molecular mechanisms underlying the formation of distinct apo-complexes is key to understanding their functional divergence. In this study, we identify additional apo-AREX components physically and functionally associated with URH49. Furthermore, by comparing the structures of UAP56 and URH49 and performing an integrated analysis of their chimeric mutants, we exhibit unique structural features that would contribute to the formation of their respective complexes. This study provides insights into the specific structural and functional diversification of these two helicases that diverged from the common ancestral gene Sub2.

In the manuscript, Fujita et al. deal with the identification and characterization of the components of the apo-AREX complex and the mechanism of the different compositions between apo-TREX and apo-AREX by comparing the structures of UAP56 and URH49.The authors identified several apo-AREX components distinct from those of apo-TREX and showed that the newly identified apo-AREX components are indeed involved in the specific mRNA export.The author also showed that a single amino acid difference in the N-terminal domains of UAP56 and URH49 governs the composition of apo-TREX and the apo-AREX complexes.The author also showed that while the overall structures (in particular spatial arrangements of the N-and C-domains) of UAP56 and URH49 in their ATP-bound forms are homologous, the structures in their apo forms are different.These results imply that the specific single amino acid difference in their N-terminal domain of UAP56 and URH49 determines the export of a specific subset of mRNAs.
This study suggests that the formation of the two distinct mRNA export complexes depends on the distinct structural differences of homologous key helicases (UAP56 and URH49) in the complexes.This reviewer appreciates that the study presents mechanistic insights into the gene regulation by the distinct mRNA export complexes formation/composition (TREX and AREX).With that said, the rational explanation and discussion of the different structures of the apo UAP56 and URH49 are not provided.The structural differences between UAP56 and URH49 in their apo-forms are keys to the different mRNA exports.The structural comparison presented in the manuscript does not provide mechanistic insights into the distinct mRNA export complex formation (TREX or AREX).
The following points could be clarified before further consideration.
Major points 1)To enhance the accessibility of this study, the summary could be rewritten and reorganized.This reviewer thinks that the wording apo-complex in the abstract is confusing and that the potential readers would feel it difficult to follow the content because the description is too specific.
2) It is not clear why the spatial positions of the N-and C-domains of UAP56 and URH49 are different, even though the authors compare their structures.A single amino acid substitution in the N-terminal domains of UAP56 and URH49 can alter the composition of the TREX and AREX complex, and export a subset of mRNAs.This finding is one of the main ones in this manuscript.Thus, a rational explanation or pieces of evidence should be provided by the authors.
3) For the limited proteolysis of proteins (UAP56 and URH49) in the presence or absence of ADP.The authors assume that the ADP-bound and ATP-bound forms of UAP56 and URH49 are similar based on the data in Extended Data Fig. 1 (page 9 lines 247-250).It seems that there are differences in composition for AREX-complex formation in the presence of +ADP and +AMP-PNP.Need to explain.4) Related to 3).Did the authors do the limited proteolysis of proteins (UAP56 and URH49) in the presence of ATP?The same results could be obtained in the presence of ADP.(Is the remodeling ATP-hydrolysis dependent?) 5) Page 15: lines 414-416: "The structures of UAP56 and URH49 in apo-state showed the distinct configuration of the N-and C-domain due to different linker structures".Did the authors check the structure of UAP56 by themselves?The models in the public domain might not be well-refined, and not well-modeled.Are the amino sequences of the linkers conserved?Please show the sequence alignments in the supplementary figures.6) Page 15, lines 423-428."The loop structure within the C-domain of …, and prevent its own ATP binding" PAGE11: line 333.The data in Extended Data Fig8 G does not support the weaker ATP binding of URH49 than UAP56.The data presented show the difference is not significant.A quantitative kinetic analysis (ATP-hydrolysis) or affinity analysis would be required.The author could substitute amino acids in the loop and test the ATPase activity or ATP-binding.
Minor points 1) TEXT: It is hard to follow the description.This reviewer suggests the authors rewrite and revise the text more clearly to enhance the accessibility to readers.

2) Figures
Extended Data Fig8G, H correct the labels of figures.
Reviewer #3 (Remarks to the Author): In Fujita et al., the authors examine differences between UAP56 and URH49 and uncover new differential binding partners, differential functional regions, and different structural aspects.Although it is unclear how all of these differences add up to explain how these two proteins regulate the nuclear export of different mRNAs, the paper is very well executed and of general interest to the mRNA nuclear export community.I am generally in favour of publication provided that the authors address the following points: 1) For Figure 2D, some indication of the purity of the cytoplasmic fractionation should be provided (i.e.distribution of nuclear and cytoplasmic RNAs/proteins)?2) When levels of a given mRNA decrease in the cytosol, does it go up in the nucleoplasm?3) eCLIP for ILF3 exists (https://www.nature.com/articles/s41586-020-2077-3)and the author should determine whether ILF3 associates with mRNAs that are disproportionately exported by URH49.4) An extended analysis of how distinct elements within URH49 and UAP56 are conserved (and how they differ between the two proteins) throughout vertebrates would be useful.5) Some of the immunofluorescent images that are pseudocolored red are very hard to see.I would recommend that the authors show all single channel immunofluorecent images as grey-scale images to help the readers clearly interpret the data.6) RUVBL1 and RUVBL2 are part of the R2TP complex which is thought to be involved in assembling multimeric complexes.This should be cited in the text.Are other components of the R2TP complex found in the URH49 precipitate?
Minor points: 1) UAP56 and URH49 are officially called DDX39B and DDX39A in humans -this should be stated at least once, just to help clarify confusions in the current literature.
2) Line 68: you may want to specify that "ZC11A" is an abbreviated form of "ZC3H11A" 3) Figure 3A some of the formatting of the numbers seems off (for example, the number "428" is split so that "42" and "8" are separated by a carriage return.)4) Lines 217-218: "Thus, the mechanism of mRNA export appears to be different from that of circular RNA export."Since the differential export of circular RNAs was not examined in this paper and there may be differences between cell lines, I would tone down this conclusion.Perhaps "how USP56 and URH49 differentially regulate the nuclear export of various circRNAs is not explained by differences in binding partners".5) Lines 225-226: "This result reflects that UAP56 and URH49 export distinct subsets of bulk mRNA substrates and do not the other."This sentence is hard to parse and should be rewritten.6) Line 387: "URH49 are required for…" should be "URH49 is required for…" 7) Line 404: a close bracket sign, ")", is missing.

Reviewer #1 (Remarks to the Author):
In this manuscript, using tandem-immunoprecipitation and mass spectrometry, Fujita et al. identified several novel key components together with URH49, a paralogue of helicase UAP56 in mammals, to form the apo-AREX complex.Although apo-AREX and apo-TREX complexes consist of distinct different components, they rebuilt into same ATP-complexes.The authors found that UAP56 and URH49 play key roles in dictating the formation of these two distinct apo-complexes.Surprisingly, only a single amino acid substitution in UAP56 or URH49 N-domain altered the formation and function of the apo-complexes.Further, using limited proteolysis assay, the authors claimed that UAP56 and URH49 exhibited different apo-structures, but with similar ADP binding conformation.They also solved the crystal structure of apo-URH49ΔN41, which showed three different features compared to the apo-UAP56ΔN42 structure.Based on these observations the authors proposed that UAP56 and URH49 form different complexes in the absence of ATP based on their diverse apo-conformation.This manuscript present mechanistic study that is important for understanding selective nuclear export of RNAs.The data are solid and the manuscript is overall clearly written.I have a couple of comments that the authors could consider to improve the study.
1.The authors performed immunoprecipitation with Flag-URH49 in 293 cells and detected many candidates for the apo-AREX components.Are the interactions of these new components with URH49 mediated by RNA?What is the function of the apo-AREX complex formed in the absence of ATP (or ADP).Considering that mRNA export requires ATP, it does not seem like it is important for this process.As this is the major finding of the work, the authors could discuss more, especially on the potential roles of this complex.
We appreciate your comment.
The RNase-treated nuclear extracts were used for immunoprecipitation.Thus, URH49 and the new AREX component can interact even in the absence of RNA.This point is clearly stated in the text (Result line 110 and Discussion line 402).We have described the potential role of the apo-TREX/AREX complex in mRNA processing, including splicing (Discussion line 428).
UAP56 is involved in splicing process by controlling spliceosome assembly through its ATPase and helicase activities, while whether URH49 is required for these activities remains unclear.Considering this, we propose the following model.UAP56 and URH49 function in the recognition and export of target mRNAs by forming their respective complexes.
Prior to splicing, UAP56 and URH49 are associated with their apo-complexes.The DEAD-box helicases to which UAP56 and URH49 belong generally bind to RNA independently of the RNA sequence.
Chimeric mutants with swapped complex formation abilities exhibited a swap in target specificity between UAP56 and URH49, raising the possibility that their apo-complex components other than UAP56 and URH49 specify the selective regulation of RNA binding.Based on these results, it is likely that each apocomplex binds to specific RNA sequences or is guided by upstream processes (such as chromatin regulation), leading to subsequent interactions with the target mRNA by UAP56 and URH49.
Subsequently, through the remodeling of each apo-complex into the ATP-TREX complex, the ATPase and helicase activities of UAP56 and URH49 are activated, leading to the splicing of the respective target pre-mRNA and, ultimately, mRNA export.Indeed, we also observed that UAP56 and URH49 function in the splicing process alongside their respective apo-complexes.
We analyzed the eCLIP data for ILF3 and HNRNPM (ILF3: GEO: GSE91760, HNRNPM: GEO: GSE91744) to determine whether ILF3 and HNRNPM associate with mRNAs that are disproportionately exported by URH49.For each STAR-mapped bam dataset, binding reads per gene were counted using Htseq count (doi:10.1093/bioinformatics/btu638)and normalized using Deseq2 (doi:10.18129/B9.bioc.D ESeq2).Next, we analyzed whether there were differences in the binding levels of ILF3 and HNRNPM between the target gene sets UAP56 and URH49.The target gene sets of UAP56 and URH49 were defined based on the information (mRNAs with their cytoplasmic expression level specifically decreased by 1.5 times or more by UAP56 and URH49 depletion) provided in the following paper (doi:10.1091/mbc.E09-10-0913).
The analysis revealed no significant differences in the degree of binding between the UAP56or URH49-targets of ILF3 and HNRNPM (For Reviewer's Figure (RFig).1, A-B).To elucidate the RNA binding sites of UAP56 and URH49, we performed Photo Activatable-Ribonucleoside-enhanced Cross Linking and Immuno Precipitation (PAR-CLIP) of both helicases (unpublished data) and analyzed in the same way.There was also no difference in the degree of UAP56 and URH49 binding between the two groups (RFig.1,C-D).
We similarly analyzed the respective eCLIP data for ILF3 and HNRNPM and PAR-CLIP data for UAP56 and URH49 for the newly defined target gene sets of UAP56 and URH49 using the following protocol (The cytoplasmic RNA from UAP56 and URH49 knockdown cells was analyzed by RNAseq.mRNAs with expression levels specifically decreased by two times or more by UAP56 and URH49 depletion were defined as new UAP56-or URH49-target mRNAs (unpublished data).The results obtained from these analyses are consistent with those mentioned previously (RFig.1, E-H).These results raise the possibility that UAP56 and URH49 (including the AREX complex) have the potential to bind globally to their respective target mRNAs when evaluated across the entire transcriptome.However, we observed that UAP56 and URH49 selectively bind to specific introns of their target mRNAs and regulate the splicing of this site (RFig.1,I, GTPBP2 mRNA; UAP56 preferentially binds to specific introns of this mRNA, as indicated by arrows, and UAP56 is required for splicing of this site. C1ORF63 mRNA; URH49 preferentially binds to specific introns in the C1ORF63 mRNA and regulates splicing at this site).
With the exception of certain introns, both UAP56 and URH49 commonly bind to many introns.This suggests that UAP56 and URH49 bind to numerous mRNAs regardless of their respective targets.
When UAP56 or URH49 is depleted, the splicing of the respective target mRNAs is aberrant; therefore, the target mRNAs are expected to remain in the nucleus.
The insights obtained from the eCLIP of ILF3 and HNRNPM are consistent with this model and are also relevant to what we described as the potential role of the apo-TREX/AREX complex in mRNA splicing (Discussion line 435).Because this aspect is beyond the scope of this study, it is pursued as part of our future research endeavors.
2. FISH assay was performed to detected the nuclear poly(A) RNA accumulation in apo-AREX components depleted U2OS cells.What is the reason that the authors selected the U2OS cells in the FISH assay?They performed the immunoprecipitation in the 293 cells, dose the FISH assay can get the same result as that in the U2OS cells?
We appreciate your comment.There were two reasons for selecting U2OS cells for the RNA-FISH assay.First, compared to U2OS cells, 293 cells require a larger quantity of siRNA and transfection reagents for siRNA-mediated knockdown (please visit the information about the lipofectamine 2000 mediated siRNA transfection the manufacture's web site below and in the site, you can easily see that 293 cells require more siRNA and transfection than other cells in general.https://www.thermofisher.com/jp/ja/home/life-science/cell-culture/transfection/rnai-transfection/rnai-transfection-protocols.html).Second, in addition to higher siRNA and transfection reagent requirements, the RNA-FISH assay is challenging in 293 cells compared to U2OS cells due to the following reason.We investigated the transfection conditions in 293 cells and performed UAP56 knockdown.The RNA-FISH assay showed that, compared to the control knockdown, poly(A) + RNA (red signal) appeared to accumulate in the nuclei of UAP56 knockdown cells.
However, UAP56 knockdown cells shrank, making it difficult to distinguish between nuclear and cytoplasmic poly(A) + RNA (RFig.2).Therefore, we used U2OS cells to observe the observation of poly(A) + RNA localization.
3. The digested fragments of URH49ΔN41 and full length seems obtain different patterns, which indicates the depleted N-terminal region may change the behaviour of URH49 against trypsin digestion.
The R1 and R4 bands are very week, and the A1, A3 R3 include two bands, maybe the limited proteolysis assay should be further optimized, such as incubating with less enzyme or shorter time.
Besides, the limited proteolysis assay is not the best way to detect the protein conformation in solution, more evidence should be provided to confirm this observation.
We appreciate your comment.
We have realigned the positions of the marker molecular weights and revised the presentation of Fig. 4A-D.In addition, to objectively demonstrate the similarity between URH49 WT and URH49ΔN41 in terms of the digested fragments (R2-like band and R2 band), we measured the mobility of the digested fragments in Fig. 4A-D in comparison to the marker (RFig.3,RFig.3.xlsx).For comparison, we also measured the similarity between A1-like band and A1 band of UAP56 WT and UAP56ΔN42, respectively.
The results indicate that the positions of the bands (R2-like band and R2 band) derived from URH49 WT and URH49ΔN41 are similar.We appreciate this comment, which has helped us rectify any potentially misleading expressions.
Additionally, we conducted experiments involving varying enzyme concentrations and shorter incubation times but were unable to observe limited digestion products other than A1-A3 and R1-R4 from UAP56ΔN42 and URH49ΔN41 (RFig.4).Although a limited proteolysis assay may not be the best method to detect protein conformation in solution, in this study, we believe it is sufficient to demonstrate the following three points: 1) Under the conditions with the absence of ATP, UAP56ΔN42 and URH49ΔN41 indeed exhibit structural differences in solution.2) URH49 C223VΔN41 shows UAP56ΔN42-like structural features indicating the significance of the differences between UAP56 224V and URH49 223C in the structural variances observed in the solution under ATP deficient conditions.3) Under ATP loaded conditions, UAP56 and URH49 adopted a UAP56-like structure in solution.In the main text, we have also mentioned the need for future analyses of the protein conformations of UAP56 and URH49 in solution (Discussion line 494).In addition to the above analysis, we also performed a Small Angle X-ray Scattering analysis (SEC-SAXS), to detect the protein conformation of UAP56 and URH49 in solution.
However, obtaining sufficiently precise data is challenging.This is because this analysis requires highly purified UAP56ΔN42 and URH49ΔN41 in large quantities, along with extensive optimization of conditions for the analysis.
4. In Extended Data Fig. 7A, the authors showed the URH49ΔN41(-ADP) was digested into two separate fragments R1 and R2, which was different from the other three samples.There seems no solid evidence to support this model.
We appreciate your comment.
As suggested, we eliminated the model (Extended Data Fig. 7A in original paper) because there was no experimental evidence for the order in which the degradation products occurred, as in this model.5.The two RecA domains within UAP56 do not adopt fixed relative orientation in solution, and the crystal structure reported here reflects just one state of URH49 in solution.So, more evidences are required to support the conclusion that the apo-structures of UAP56 and URH49 make them to integrate into separate complexes.
We appreciate your comment.
As pointed out, the crystal structure generally reflects one of the possible structures in solution.We found that the RecA1 and RecA2 domains of UAP56 and URH49 differ by 32 °in their crystal structures (Fig.

5A) but
have not yet verified the extent to which the two domains are different in solution.Further analysis is required to verify this issue, which has been added to the revised text (Discussion line 494).We have also added that unidentified factors other than the observed differences in the crystal structures of UAP56 and URH49 may affect the difference in complex formation between UAP56 and URH49 (Discussion line 497).
To show that UAP56 and URH49 predominantly adopt different conformations in solution, we performed limited digestion of purified UAP56 and URH49 in solution and found that the products of their limited digestion differed under apo-conditions in which no ADP was added (Fig. 4C).This suggests that a significant portion of the apo-structures of UAP56 and URH49 may differ in solution.In addition, the limited digestion products of the URH49 C223 mutant, which exhibited a switch in complex formation from the apo-AREX to the apo-TREX complex, showed highly similar patterns to those of the limited digestion products of UAP56 under apo-conditions (Fig. 4C).These observations suggest that the difference in the apo-structures of UAP56 and URH49 in solution is a major determinant of complex formation.
To verify whether the apo-structures of UAP56 and URH49 are crucial for complex formation, it is necessary to examine the structures of UAP56 and URH49 in solution within the complex (e.g., cryo-EM analysis of the complex) and to observe how complex formation occurs.These analyses are important for understanding the mechanism of complex formation between UAP56 and URH49.This has been clearly stated in the text (Discussion line 499).Based on this, since the current study focused on the identification and functional analysis of novel components of the AREX complex, in addition to the possible major factors of complex differentiation, we would like to proceed with that as a separate study.6.It is intriguing that no structural difference was observed with the key amino acid (URH49 C223 and UAP56 V224) that determines the different apo-complex of UAP56 and URH49.The author proposed that the difference in the loop structure might be caused by this amino acid.It would be great if they could use molecular modelling to examine this possibility.
We appreciate your comment.
As you mentioned, we created a model based on homology modeling using Modeller (https://salilab.org/modeller/)(RFig.5).To estimate the plausibility of the structural model obtained by the analysis, a structural model of URH49 was created based on the UAP56 crystal structure (1XTI) (upper panel, URH49Δ41 model).The resulting URH49 structure model is almost identical to the UAP56 crystal structure (1XTI) (RMSD = 0.22 Å), unlike the structure model (Fr48) derived from the actual URH49 crystal structure.This result suggests that in UAP56 and URH49, where the total amino acid homology is high, is unable to build a structural model from the template by molecular modeling; even if the URH49Δ41 C223V model is created from the URH49 structural model (Fr48), the structure of the URH49Δ41 C223V model was similar to that of the URH49 structural model (Fr48) (lower panel).
Therefore, molecular modeling could not validate whether UAP56 V224 and URH49 C223 were key to the structural differences between the two.Therefore, to identify the residues that could potentially cause global structural differences between UAP56 and URH49, we exhaustively calculated the alteration of all backbone dihedral angles between the two molecules and detected the critical residues whose local conformations differed significantly (dihedral angles greater than 120°, as shown in magenta in Fig. 5C, see also Table.S3).Some residues were located in the linker region of UAP56 and URH49 (in the linker region: UAP56-254E, -257 L, URH49-253E, and -256 L).This suggests that the differences in the arrangement of these residues may be the cause of the structural differences between UAP56 and URH49 in linker region.Among these amino acid residues, the amino acids closest to UAP56 V224 and URH49 C223 were UAP56 D245 and URH49 D244, located at the UAP56 243-245 linker and URH49 242-244 linker, respectively (Fig. 5C).In addition, linker sites are generally susceptible to structural changes.We examined the amino acid residues that interact with UAP56 V224 and URH49 C223 on each linker and found UAP56 M243 and URH49 M242 residues (Fig. 5D).We observed that UAP56 M243 and URH49 M242 directly interacted with UAP56 V224 and URH49 C223, respectively, altering the spatial arrangement of UAP56 M243 and URH49 M242, which in turn altered the arrangement of UAP56 D245 and URH49 D244 (Fig. 5D).These spatial arrangements influence subsequent linkers (UAP56 243-245 linker and URH49 242-244 linker) and interdomain linkers and are expected to eventually contribute to the C-domain arrangement.Thus, URH49 C223 and UAP56 V224 may be the key amino acids responsible for their structural differences.This point was stated in the text (Result line 344).
7. It would be important for the authors to at least discuss how the difference in their apo-complexes determines the substrate specificity of UAP56 and URH49.
We appreciate your comment.
As answered to comment 1, we added to the potential role of the apo-AREX complex in the discussion section (Discussion line 428).
8. In Extended Data Fig. 7C, I guess the sample on the right side was treated with ADP, the label should be corrected.
We appreciate your comment.

In Table
We appreciate your comment.
The notation in this part was incorrect and has been corrected (Table S2-2).
TREX (ATP-dependent Transcription-Export) complex is a regulator of mRNA export.One of the TREX complex components, UAP56 helicase, is a key factor in the assembly of the TREX complex.
Upon ATP-binding to UAP56, TREX (ATP-TREX) recruit additional factors, thereby, facilitating mRNA export.In humans, a paralogue of UAP56, URH49, forms a distinct complex from the TREX complex, termed as AREX (Alternative-mRNA-export) complex.The composition of ATP-unbound AREX (apo-AREX) is different from that of TREX (apo-TREX).However, upon ATP-binding to URH49 of AREX, the AREX is remodeled and the component of ATP-bound AREX becomes homologous to those of ATP-bound TREX.UAP56 and URH49 selectively regulate the export of a specific subset of mRNAs, thus, the diversified mRNA export pathway by UAP56 and URH49 regulates the gene expression.
In the manuscript, Fujita et al. deal with the identification and characterization of the components of the apo-AREX complex and the mechanism of the different compositions between apo-TREX and apo-AREX by comparing the structures of UAP56 and URH49.The authors identified several apo-AREX components distinct from those of apo-TREX and showed that the newly identified apo-AREX components are indeed involved in the specific mRNA export.The author also showed that a single amino acid difference in the N-terminal domains of UAP56 and URH49 governs the composition of apo-TREX and the apo-AREX complexes.The author also showed that while the overall structures (in particular spatial arrangements of the N-and C-domains) of UAP56 and URH49 in their ATP-bound forms are homologous, the structures in their apo forms are different.These results imply that the specific single amino acid difference in their N-terminal domain of UAP56 and URH49 determines the export of a specific subset of mRNAs.
This study suggests that the formation of the two distinct mRNA export complexes depends on the distinct structural differences of homologous key helicases (UAP56 and URH49) in the complexes.
This reviewer appreciates that the study presents mechanistic insights into the gene regulation by the distinct mRNA export complexes formation/composition (TREX and AREX).With that said, the rational explanation and discussion of the different structures of the apo UAP56 and URH49 are not provided.The structural differences between UAP56 and URH49 in their apo-forms are keys to the different mRNA exports.The structural comparison presented in the manuscript does not provide mechanistic insights into the distinct mRNA export complex formation (TREX or AREX).
The following points could be clarified before further consideration.
Major points 1) To enhance the accessibility of this study, the summary could be rewritten and reorganized.This reviewer thinks that the wording apo-complex in the abstract is confusing and that the potential readers would feel it difficult to follow the content because the description is too specific.
We appreciate your comment.
We rewrote the entire Summary section.
2) It is not clear why the spatial positions of the N-and C-domains of UAP56 and URH49 are different, even though the authors compare their structures.A single amino acid substitution in the N-terminal domains of UAP56 and URH49 can alter the composition of the TREX and AREX complex, and export a subset of mRNAs.This finding is one of the main ones in this manuscript.Thus, a rational explanation or pieces of evidence should be provided by the authors.
We are grateful for your comment.
As mentioned, we created a model based on homology modeling using Modeller (https://salilab.org/modeller/)(For Reviewer's Figure (RFig).5).To estimate the plausibility of the structural model obtained by the analysis, a structural model of URH49 was created based on the UAP56 crystal structure (1XTI) (upper panel, URH49Δ41 model).The resulting URH49 structure model is almost identical to the UAP56 crystal structure (1XTI) (RMSD = 0.22 Å), unlike the structure model (Fr48) derived from the actual URH49 crystal structure.This result suggests that in UAP56 and URH49, where the total amino acid homology is high, is unable to build a structural model from the template by molecular modeling; even if the URH49Δ41 C223V model is created from the URH49 structural model (Fr48), the structure of the URH49Δ41 C223V model was similar to that of the URH49 structural model (Fr48) (lower panel).Therefore, molecular modeling could not validate whether UAP56 V224 and URH49 C223 were key to the structural differences between the two.Therefore, to identify the residues that could potentially cause global structural differences between UAP56 and URH49, we exhaustively calculated the alteration of all backbone dihedral angles between the two molecules and detected the critical residues whose local conformations differed significantly (dihedral angles greater than 120°, as shown in magenta in Fig. 5C, see also Table .

S3).
Some residues were located in the linker region of UAP56 and URH49 (in the linker region: UAP56-254E, -257 L, URH49-253E, and -256 L).This suggests that the differences in the arrangement of these residues may be the cause of the structural differences between UAP56 and URH49 in linker region.Among these amino acid residues, the amino acids closest to UAP56 V224 and URH49 C223 were UAP56 D245 and URH49 D244, located at the UAP56 243-245 linker and URH49 242-244 linker, respectively (Fig. 5C).In addition, linker sites are generally susceptible to structural changes.We examined the amino acid residues that interact with UAP56 V224 and URH49 C223 on each linker and found UAP56 M243 and URH49 M242 residues (Fig. 5D).We observed that UAP56 M243 and URH49 M242 directly interacted with UAP56 V224 and URH49 C223, respectively, altering the spatial arrangement of UAP56 M243 and URH49 M242, which in turn altered the arrangement of UAP56 D245 and URH49 D244 (Fig. 5D).These spatial arrangements influence subsequent linkers (UAP56 243-245 linker and URH49 242-244 linker) and interdomain linkers and are expected to eventually contribute to the C-domain arrangement.Thus, URH49 C223 and UAP56 V224 may be the key amino acids responsible for their structural differences.This point was stated in the text (Result line 344).
3) For the limited proteolysis of proteins (UAP56 and URH49) in the presence or absence of ADP, the authors assume that the ADP-bound and ATP-bound forms of UAP56 and URH49 are similar based on the data in Extended Data Fig. 1 (page 9 lines 247-250).It seems that there are differences in composition for AREX-complex formation in the presence of +ADP and +AMP-PNP.Need to explain.
We appreciate your comment.
UAP56 binds AMP-PNP with affinities at least 10 times lower than that of ATP and binds ADP with similar affinities as ATP (doi:10.1128/MCB.01341-07).Therefore, in the presence of AMP-PNP, it is possible that a part of apo-URH49 molecules do not bind to AMP-PNP.Consequently, we hypothesized that with the addition of AMP-PNP, both the ATP-TREX (URH49) complex and partially the apo-AREX complex may coexist in the immunoprecipitates of URH49, making it appear different from the ATPbound form of URH49.To prove this experimentally, we performed immunoprecipitation of URH49 in the presence of excess AMP-PNP.Under conditions of excess AMP-PNP, immunoprecipitation of Fig. 1B).This point has been stated in the text (Result line 121).4) Related to 3).Did the authors do the limited proteolysis of proteins (UAP56 and URH49) in the presence of ATP?The same results could be obtained in the presence of ADP.(Is the remodeling ATP-hydrolysis dependent?) We appreciate your comment.
We have added additional information regarding the limited proteolysis assay in the presence of ATP.
The limited proteolysis of the proteins (UAP56 and URH49) in the presence of ATP closely resembled those obtained when ADP was added (Extended Data Fig. 9A).This suggests that ATP-dependent structural changes in URH49 are likely to be ATP hydrolysis-independent.These findings are consistent with the notion that the complex remodeling of URH49 is induced by the addition of ADP (Extended Data Fig. 1A).This information has been added to the revised text (line 280).We appreciate your comment.
We did not crystallize UAP56, but analyzed the crystal structure of UAP56 (1XTI) reported in the PDB database.The validity values of this crystal structure model are as follows and are considered sufficiently certain.Resolution: 1.95 Å, R-Value Free:0.257,R-Value Work:0.218(1XT1 was obtained from the PDB database, https://www.rcsb.org/structure/1xti).The amino acid sequence of the linker region between the N and C domains is conserved in UAP56 and URH49.In the original submission, we could not effectively highlight the linker region.Therefore, we have made a change to address this issue.
The crystal structure generally reflects one of the possible structures in solution.We found that the RecA1 and RecA2 domains of UAP56 and URH49 differ by 34.82 °in their crystal structures (Fig.

5A) but
have not yet verified the extent to which the two domains are different in solution.Further analysis is required to verify this issue, which has been added to the revised text (Discussion line 494).We have also added that unidentified factors other than the observed differences in the crystal structures of UAP56 and URH49 may have affected the differences in complex formation between UAP56 and URH49.
The amino acid sequence of the linker region was conserved, but was not effectively highlighted in the previous alignment.We corrected this in the revised manuscript (Extended Data Fig. 4).This point was stated in the text (Result line 371).We appreciate your comment.
It is important to note that quantitative analysis is required to confirm these observations.In addition, based on the experimental results described below, we believe that ongoing research will be necessary to confirm the function of URH49 C-loop in the ATP-binding of URH49.Therefore, the results of the verification of ATP-binding and helicase activities of UAP56 and URH49 (Extended Data Fig. 8G, H in the original paper) were removed from the revised manuscript, and the potential role of URH49 C-loop was described in Discussion (line 502).The removal of these data has no impact on the essence of this manuscript.Next, we describe the results of the experiments conducted to investigate the function of the URH49 C-loop.
We generated two mutants of URH49, URH49ΔC-loop mutant with a deletion of the C-loop and URH49 C-loop mutant with eleven amino acid substitutions in the C-loop, replacing them with adenine, to compare their ATP binding with the wild-type URH49.The obtained results did not meet our expectations probably because the C-loop region exists as a conserved motif (motif V in Extended Data Fig. 4) which is essential for ATP binding and ATPase activity in DEAD-box helicases to which UAP56 and URH49 belong (doi:10.1111/febs.13198).Therefore, altering the C-loop region by the deletion or mutation could lead to difficulties in proper folding of the recombinant protein produced in E. coli or a decrease in the stability of the folded protein, making it challenging to accurately evaluate ATP binding under experimental conditions.For these reasons, further investigation is required to determine whether the C-loop region of URH49 directly inhibits ATP binding.We believe that ongoing research will be necessary to confirm this.

Minor points 1) TEXT:
It is hard to follow the description.This reviewer suggests the authors rewrite and revise the text more clearly to enhance the accessibility to readers.
We rewrote the entire Summary section.
Thus, the TREX complex is remodeled from the ATP-unbound TREX complex to the ATP-bound TREX complex via ATP binding to UAP56.Page 4: line 84 ~105.There are several sentences and wording and they are confusing.rewrite and rephrase.
We rephrased two paragraphs so that these were easier for the reader to read.These paragraphs were in lines 83 ~102 of the revised manuscript.
We rephrased the followings.
We rephrased the followings.
Line 196, To identify the region(s) within UAP56 and URH49 responsible for forming different apocomplexes, we constructed plasmids expressing various mutants of UAP56 and URH49, in which different regions were swapped.We examined the formation of apo-complexes.
Line 212, To further investigate the potential contribution of amino acid differences other than UAP56-V224 and URH49-C223 to their distinct complex formation, we generated a mutant termed "UAP56 Ncore C224V."In this mutant, the N-domain of UAP56, excluding V224, was replaced with the N-domain of URH49.This mutant lost the ability to form the apo-AREX complex but retained the ability to form the apo-TREX complex.We appreciate your comment.
We are grateful for addressing the labeling errors in the previous data.The data in Extended Data Fig. 8G in the original paper were removed.The removal of this data has no impact on the essence of this manuscript.

2) Figures
Extended Data Fig. 8G, H correct the labels of figures.
We appreciate your comment.
The data in Extended Data Fig. 8G, H in the original paper were removed.The reason why these data were removed is described above.
5) Page 15: lines 414-416: "The structures of UAP56 and URH49 in apo-state showed the distinct configuration of the N-and C-domain due to different linker structures".Did the authors check the structure of UAP56 by themselves?The models in the public domain might not be well-refined, and not well-modeled.Are the amino sequences of the linkers conserved?Please show the sequence alignments in the supplementary figures.

6)
Page 15,The loop structure within the C-domain of …, and prevent its own ATP binding".Page 11: line 333.The data in Extended Data Fig.8Gdoes not support the weaker ATP binding of URH49 than UAP56.The data presented show the difference is not significant.A quantitative kinetic analysis (ATP-hydrolysis) or affinity analysis would be required.The author could substitute amino acids in the loop and test the ATPase activity or ATP-binding.
However, upon ATP binding, the N-and C-domains undergo rearrangement into similar closed structures driven by interactions with ATP.Page 9: line246-247, rephrase We rephrased the followings.Line 260, These results led us to hypothesize that the structures of UAP56 and URH49 in their apo-and ATP-bound states determine the formation of their apo-and ATP complexes.Page 10: line 295, rephrase.We rephrased the followings.Line 312, Structural differences between the apo-UAP56 and URH49.Page 11: line 333.The data in Extended Data Fig.8Gdoes not support the weaker ATP binding of URH49 than UAP56.Is this significant?See major comments 6).