Molecular insights into mRNA export regulation by the human TREX-2 complex

Domain: chemistry
Published: 2026-02-26
Editorial outcome: major_revision

Abstract
The nuclear export of mRNA represents a critical regulatory node in eukaryotic gene expression. This process is orchestrated by two conserved multi-subunit assemblies: the transcription-and-export complex (TREX) and TREX-2. While TREX facilitates mRNP packaging through multivalent RNA-protein interactions, the precise mechanism by which TREX-2 contributes to mRNA export has remained elusive. Here, we report a functional interaction between UAP56 and TREX-2 and resolve the structures of TREX-2 in both apo and UAP56-bound states. UAP56 engages TREX-2 via its N-terminal region, positioning its RecA domains on the V-shaped surface of the complex. A conserved loop from NTREX-2 inserts between the RecA domains of UAP56, stabilizing an open conformation. Biochemical assays demonstrate that TREX-2 significantly stimulates the ATPase activity of UAP56, thereby promoting RNA release. These findings provide structural and mechanistic insights into TREX-2-mediated regulation of mRNA export through UAP56 remodeling.

Introduction
In eukaryotes, gene expression involves a series of tightly coordinated nuclear events, such as transcription, 5'-capping, splicing, and 3'-end processing1-6. During these processes, messenger RNA (mRNA) becomes associated with multiple RNA-binding proteins, forming a messenger ribonucleoprotein (mRNP) complex. The proper assembly of mRNP is essential for the efficient export of mRNA through nuclear pore complex (NPC)7-13.

Two evolutionarily conserved transcription-export complexes, TREX and TREX-2, play essential roles in mRNA nuclear export14-19. TREX is assembled via association of THO complex with ATPase/helicase UAP56 (also known as DDX39B) and the mRNA-binding protein ALYREF10,12,16,20. It forms multivalent interactions with exon-junction complex (EJC) and mRNA, compacting them into export-competent globules11,21,22. In contrast, TREX-2 consists of GANP, PCID2, DSS1, ENY2, and CETN2/CETN3, and is stably anchored to the NPC15,23-28. Both TREX and TREX-2 deliver their mRNA cargo to the principal export receptor NXF1-NXT1 heterodimer, which directly interacts with FG-nucleoporins lining the NPC channel to mediate mRNA translocation29-33. Beyond their mRNA export functions, TREX and TREX-2 are also involved in transcriptional regulation and maintenance of genome integrity20,34,35.

The structure and function of TREX in mRNA export have been extensively characterized9,21,22,36-42; however, the mechanism governing TREX-2-mediated export remain insufficiently understood28,43-53. Two recent studies revealed a crucial functional link between mRNP-engaged UAP56 and TREX-254,55. Key questions persist regarding the nature of mRNP-TREX-2 interactions and the mechanistic details of mRNA handover from TREX-2 to NXF1-NXT1 (Fig. 1a). Moreover, it is still unclear whether TREX and TREX-2 mediate mRNA export through independent pathways or function synergistically to enable selective mRNA export2,56.

In this study, we explored the potential functional crosstalk between TREX and TREX-2, revealing UAP56 as a co-factor of the TREX-2 complex. Through an integrated approach combining biochemical analyses, cryo-EM studies, and functional assays, we provide key structural and functional insights into the mechanisms underlying TREX-2-mediated mRNA export.

Results
UAP56 association with TREX-2
To investigate the potential interactions between TREX-2 and mRNP, we established two Flp-In T-REx 293 cell lines that stably expressing either Strep-tagged GANP or Strep-tagged PCID2 – the scaffold components of human TREX-2, whose depletion causes growth impairment and mRNA export defects56. Immunopurification followed by mass spectrometry revealed enrichment of TREX-2 components and the export receptor NXF1 in Strep-GANP pull-downs (Supplementary Figure 1a, b). Notably, UAP56 peptides were abundantly detected, whereas other components of TREX complex (e.g., THO subunits) showed minimal association. This selectivity was corroborated in Strep-PCID2 immunoprecipitations, where UAP56 – but not other TREX complex factors – exhibited significant enrichment (Fig. 1b). These mass spectrometry analyses suggest a direct physical interaction between UAP56 and TREX-2.

To validate this interaction, we initially purified the recombinant five-subunits TREX-2 complex (containing GANP580-1264, PCID2, DSS1, ENY2, and CETN2/3). However, its poor biochemical behavior impeded further structural and functional analysis (Supplementary Figure 1c). Consequently, we focused on the core module of TREX-2 (hereafter referred to as TREX-2M), comprising the Sac3 domain of GANP (residues 580-1000), PCID2, and DSS148 (Fig. 1c; Supplementary Figure 1d). THO complex (THOC1, -2, -3, -5, -6, -7), UAP56 and ALYREF were also expressed and purified to homogeneity (Supplementary Figure 1e, f). Pull-down assays demonstrated weak but specific binding between TREX-2M and UAP56, with no detectable specific interaction with THO or ALYREF (Fig. 1d; Supplementary Figure 1g). Strikingly, stoichiometric UAP56-TREX-2M complexes formed exclusively in the presence of ADP, ATP or its non-hydrolysable analog AMPPNP, suggesting that the nucleotide-bound state of UAP56 governs its association with TREX-2 (Supplementary Figure 1h). Notably, DDX39A (also known as URH4957), a paralog of UAP56, was also enriched in PCID2 immunoprecipitates (Fig. 1b). However, pulldown assays revealed substantially weaker binding of DDX39A to TREX-2 compared to UAP56 (Supplementary Figure 1i). These findings suggest a model wherein UAP56 acts as a functional adaptor bridging TREX-2 and export-competent mRNP.

Structure of the human UAP56-TREX-2M complex
To gain structural insights, we first reconstituted UAP56-RNA complex by incubating UAP56 with poly-U RNA and AMPPNP. Subsequent addition of TREX-2M enabled assembly of UAP56-TREX-2M complex. Single particle cryo-EM analysis yielded structures of TREX-2M in the apo form and UAP56-bound state at resolutions of 3.1 and 3.3 Å, respectively (Supplementary Figures 2 & 3). Both reconstructions exhibited well-defined density for key structural motifs, enabling unambiguous modeling of side chains (Fig. 2; Supplementary Figures 4-6; Supplementary Table 1).

The human TREX-2M complex adopts a V-shaped architecture formed by GANP and PCID2, mirroring the structural organization of its yeast homolog47-53 (Fig. 2a; Supplementary Figure 5a). DSS1, while predominantly unstructured, stably associates with the PCID2 (Fig. 2a). In the UAP56-TREX-2M structure, conformations of PCID2-DSS1 remain largely unchanged, but the N-terminal half of GANP Sac3 domain rotates slightly towards PCID2 due to the binding of UAP56 (Fig. 2b-d; Supplementary Figure 5b). Intriguingly, despite reconstituting the complex with RNA-bound UAP56, the cryo-EM map revealed UAP56 in an open conformation with no observable RNA density. The RecA1 of UAP56, positioned within the cleft of the V-shaped GANP-PCID2 scaffold, was resolved with high confidence (Fig. 2b), whereas the RecA2 domain exhibited lower resolution, permitting only partial docking into the cryo-EM map (Supplementary Figure 4f). Crucially, two N-terminal UAP56 loops anchor at the GANP-PCID2 interface, forming key interaction nodes (Fig. 2c; Supplementary Figure 4a-c). These structural observations suggest that TREX-2 not only interacts with UAP56 but also remodels its RNA-binding conformation, potentially priming the helicase for ATP hydrolysis and subsequent mRNA release.

The N-terminal region of UAP56 is crucial for TREX-2 binding
The N-terminal region (NTR) of UAP56 was previously shown to be essential for mRNA export58, and a recent study reveals structural differences at the NTR between UAP56 and DDX39A shape selective mRNA export pathways57. Despite these tantalizing clues, the exact role of the NTR remains enigmatic. Our structure suggests the NTR of UAP56 may be responsible for TREX-2 binding (Fig. 2c). Two fragments of the NTR were structurally resolved in our UAP56-TREX-2M reconstitution: Loop-1 (residues 8-16) and Loop-2 (residues 36-45) (Fig. 3a, b; Supplementary Figure 4a, b).

Loop-1 (residues 8-16) is enriched with negatively-charged residues and engages a positively-charged cleft at the PCID2-GANP interface, a region previously assigned as a putative mRNA binding site of TREX-248 (Fig. 3a; Supplementary Figures 5c, d & 6a). This interface features several hydrogen bonds: side chains of Glu9, Asp12, and Asp15 from UAP56 forms H-bonds with Gln337, Lys388 and Gln389 of PCID2, respectively. The conserved Tyr13 from UAP56 makes Van der Waals contacts with Tyr376 and Val385 from PCID2 and V891 from GANP (Fig. 3a; Supplementary Figure 4a). In addition, Loop-2 (residues 36-45) further stabilizes the interaction via H-bonds and a cation-π interaction between Lys817 of GANP and His43 of UAP56 (Fig. 3b; Supplementary Figure 4b). Two auxiliary interfaces likely reinforce UAP56 anchoring on TREX-2M (Fig. 3c; Supplementary Figure 4c).

To validate our structural findings, we conducted in vitro pull-down assays using MBP-tagged TREX-2M and UAP56 variants. Wild-type (WT) UAP56 robustly bound TREX-2M, with input-normalized complex formation set as 100% (Fig. 3d, lane 1; Supplementary Figure 7). Mutations in Loop-1 significantly impaired binding: L10A and Y13A reduced relative binding to nearly 20% (Fig. 3d, lanes 3 & 4; Fig. 3e); triple alanine substitution E14A/D15A/D16A (AAA) and entire Loop-1 deletion (∆16) also abolished TREX-2M interaction (Fig. 3d, lanes 5 & 6; Fig. 3e). Similarly, the H43A/H73A double mutation, which disrupts the interface between Loop-2 and TREX-2 (Fig. 3b), significantly reduced UAP56-TREX-2 binding (Fig. 3d, lane 7 & 8; Fig. 3e). These results, combined with our structural data, establish the NTR of UAP56 as essential for TREX-2 recognition, with both Loop-1 and Loop-2 acting as critical interaction motifs.

TREX-2 stimulates the ATPase activity of UAP56
We next asked what is the biological relevance of the UAP56-TREX-2 interaction. Previous studies in yeast suggested that TREX-2 facilitates the exchange of Sub2 (UAP56 homolog) for Mex67 (NXF1 homolog) on mRNP, yet the molecular basis remained elusive59. Our structure of UAP56-TREX-2M complex reveals a potential regulatory mechanism: despite being reconstituted with RNA-bound UAP56 and AMPPNP, the structure exhibits UAP56 in an open conformation unclamped from RNA (Fig. 2c; Supplementary Figure 4f). Structural overlay with RNA-bound UAP5660 reveals that a conserved loop from GANP (residues 672-688, hereafter referred to as UAP56-activating loop (UAL)) inserts between the RecA1 and RecA2 domains of UAP56, displacing the RecA2 lobe from its RNA-bound position (Fig. 4a, b). Thus, UAL likely plays a role during the close-to-open conformational change of UAP56. Importantly, AMPPNP binds at the UAP56 ATP-binding pocket and is jointly coordinated by residues from UAP56 and UAL of GANP (Fig. 4c; Supplementary Figure 4d, e). Specifically, the adenine ring of AMPPNP is recognized by Phe65 from UAP56 and Arg678 from UAL, with Arg678 forming a potential H-bond with the adenine moiety (Fig. 4c). Arg678 shares overlapping position with Phe381 of UAP56 – a residue critical for recognizing ATP in RNA-clamped UAP5660. Notably, sequence conservation analysis identifies Arg678 as highly conserved from yeast to human (Supplementary Figure 6b). This repurposing of nucleotide-binding interactions suggests that GANP UAL acts as a structural cofactor, reconfiguring the conformation of UAP56 to prime RNA release.

To test this hypothesis, we first investigated whether TREX-2 regulates the ATPase activity of UAP56. In vitro ATPase assays revealed that WT TREX-2M robustly stimulates the ATPase activity of UAP56 by ~10-fold in the presence of RNA (Fig. 5a, lanes 1, 3, 4). Michaelis–Menten equation fitting showed TREX-2M binding increased the maximum ATPase/Pi release velocity (Vmax) of UAP56 from 1.43 ± 0.48 nmol-1min-1mg-1 to 12.36 ± 0.46 nmol-1min-1mg-1 protein. (Fig. 5b; Supplementary Table. 2). This activation depends critically on the UAP56-TREX-2 interaction: the GANP R678A mutant – which disrupts AMPPNP coordination at the UAL interface (Fig. 4c) – reduced the stimulatory effect (Fig. 5a, lanes 2, 3, 5). Similarly, the UAP56 Δ16 truncation, which impairs TREX-2 binding (Fig. 3d, e), exhibited diminished ATPase activity when combined with WT or R678A mutant of TREX-2M (Fig. 5a, lanes 6-8). Together, these results demonstrate that TREX-2 directly activates the ATPase activity of UAP56 through a physical interaction mediated by UAL.

TREX-2 drives ATPase-dependent RNA release from UAP56
Integrating structural and biochemical evidence, we propose that TREX-2 facilitates mRNP handover from UAP56 to NXF1/NXT1 by activating the ATPase activity of UAP56. To test this model, we developed an in vitro RNA loading-release assay: poly-U RNA was first loaded onto UAP56 in the presence of ATPγS (an ATP analog with slow hydrolysis rate), forming stable UAP56-RNA complexes (Fig. 5c, lane 1-2). Subsequent addition of TREX-2M triggered progressive RNA release, quantified by native gel electrophoresis (Fig. 5c; Supplementary Figure 8). WT TREX-2M dose-dependently displaced RNA from UAP56 (Fig. 5c, lanes 3-5). In contrast, the R678A mutant of UAL, which is defective in nucleotide coordination and ATPase activation, shows compromised activity (Fig. 5c, lanes 6-8). The ∆16 mutant of UAP56 retains RNA-binding capacity but fails to respond to TREX-2M (Fig. 5c, lanes 9-16), aligning with its impaired binding to TREX-2 (Fig. 3d, e).

Together, these results establish a direct causal link between UAP56-TREX-2 interaction, ATPase activation, and RNA release. By coupling structural remodeling of UAP56 with catalytic activation, TREX-2 liberates mRNP from UAP56 to allow NXF1-NXT1 recruitment. Our study show the mechanistic basis of export factor exchange conserved from yeast to humans33.

Methods
GANP and PCID2 immunoprecipitation
The open reading frames of full-length GANP (Uniport ID: O60318-1) and PCID2 (Uniprot ID: Q5JVF3-1) were individually cloned into the pcDNA5-FRT-TO vector, generating N-terminal GFP and C-terminal Strep-tag fusions. The resulting plasmids were transfected into HEK293 Flp-In T-REx cells (Thermo Fisher Scientific) to establish stable cell lines. Protein expression was induced with 0.5 μg/mL tetracycline. Cells were lysed in lysis buffer (25 mM HEPES-KOH pH 7.9, 150 mM NaCl, 3 mM MgCl2, 0.05% NP40, 1 mM DTT) supplemented with 2 U/mL Benzonase (Beyotime) and 1× protease inhibitor cocktail (Yeasen). After centrifugation at 11,000g for 30 min at 4°C, supernatants were incubated with Strep-Tactin beads (IBA) for 2 h at 4°C on a head-over-tail rotor. After six washes with lysis buffer, proteins were eluted with lysis buffer containing 50 mM biotin (Yeasen) and analyzed on SDS-PAGE gels.

Mass spectrometry analysis
Immunoprecipitates of Strep-GANP, Strep-PCID2 and Strep controls were performed label-free and in triplicates. Proteins were precipitated with 15% trichloroacetic acid (TCA) and vacuum-dried. The resulting pellet was dissolved in 8 M urea, 100 mM Tris 8.5, followed by TCEP reduction, IAA alkylation, and trypsin digestion. Tryptic peptides were resuspended in 0.1% formic acid (w/w), desalted using Pierce C18 spin column (Thermo Fisher), and separated on an EASY-nLC1200 liquid chromatography (LC) system by applying a gradient of 0-80% acetonitrile (ACN) in 0.1% formic acid over 120 minutes. Eluates were analyzed by LC-MS using an Easy-nLC system directly coupled to a Thermo Scientific Orbitrap Exploris 480 mass spectrometer. Mass spectrometry data were acquired in Data-Dependent Acquisition (DDA) mode and searched against the UniProt protein sequence database using MaxQuant, with “match between runs” and “Label-Free Quantification (LFQ)” enabled. The resulting MaxQuant protein group output was further analyzed using the DEP package for downstream processing, including normalization, imputation, and differential analysis. Log fold LFQ changes of interactor signals in the individual immunoprecipitates over cell line controls were plotted against -log10 of Student’s t-test p-values calculated over triplicate data sets.

Expression and purification of TREX-2M
The cDNA constructs of GANP580-1000, PCID2, DSS1 were individually cloned into a modified pCAG mammalian expression vector67. To facilitate protein purification, GANP580-1000 and DSS1 were each fused to an N-terminal Flag tag; PCID2 was fused to an N-terminal Twin-Strep tag. HEK293F cells (Invitrogen) were cultured in SMM 293T-II medium (Sino Biological Inc.) under 6% CO2 at 37 °C with constant shaking. Equal amounts of the plasmids for the five subunits of TREX-2M were co-transfected into cells using 40-kD linear polyethylenimines (Yeasen). Transfected cells were incubated for 72 hours and harvested by centrifugation at 4,000×g for 10 minutes. Cell pellets were resuspended in lysis buffer (50 mM HEPES-KOH pH 7.9, 150 mM NaCl, 3 mM MgCl2, 0.05% NP40, 1 mM DTT) and disrupted by sonication. Clarified cell lysates were loaded into columns containing anti-Flag M2 affinity gel (Sigma) with gravity flow. After extensive washing using lysis buffer, proteins were eluted with lysis buffer containing 0.3 µg/mL Flag peptide. The eluent was loaded to a column containing 2 mL of Strep-Tactin beads (IBA). After five washes with lysis buffer, target proteins were eluted with 50 mM biotin and applied to size exclusion chromatography (SEC, Superdex-200 Increase, Cytiva) with GF75 running buffer (25 mM HEPES-KOH, pH 7.9, 75 mM NaCl, 1.5 mM MgCl2). Peak fractions were analyzed on SDS-PAGE gels followed by Coomassie staining.

Expression and purification of THO
cDNA constructs of THOC1, THOC21-1195, THOC3, THOC5, THOC6 and THOC7 were individually cloned into a modified pCAG expression vector. THOC21-1195, THOC5 and THOC6 were each fused to an N-terminal Flag tag; THOC1 was fused to an N-terminal MBP tag. Plasmids for the six subunits of THO were co-transfected into HEK293F cells and cultured for 72 hours for protein production. Cells were harvested by centrifugation, resuspended in lysis buffer and lysed by sonication. Protein complexes were sequentially purified through affinity selection using anti-Flag M2 affinity gel (Sigma) and Dextrin beads (Smart-LifeSciences). The eluent was further purified using SEC (Superose-6 Increase, Cytiva) with GF75 running buffer (25 mM HEPES-KOH, pH 7.9, 75 mM NaCl, 1.5 mM MgCl2). Peak fractions were analyzed on SDS-PAGE gels followed by Coomassie staining.

Preparation of UAP56 and ALEREF
UAP56 was fused with an N-terminal 8×His tag and sub-cloned into pET-21b expression vector. Protein was expressed in E. coli Rosetta (DE3) cells grown in SLB medium. Protein expression was induced at OD600 of 0.8 by adding 0.25 mM IPTG and incubated at 16℃ for 16 hours. Cells were collected in the H300 buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1.5 mM MgCl2, 25 mM imidazole, 0.5 mM PMSF) and lysed by sonication. After centrifugation, the supernatant was loaded to columns containing Ni-NTA agarose gel (Smart-Lifesciences) and allowed to flow by gravity. After six washes with H300 buffer, protein was eluted with GF75 buffer (25 mM HEPES-KOH, pH 7.9, 75 mM NaCl, 1.5 mM MgCl2) containing 300 mM imidazole. The eluent was further purified through ion-exchange chromatography using a heparin column (HiTrap Heparin 5mL, Cytiva). Peak fractions of target protein were pooled, concentrated with 30 kDa Amicon Filter Unit (Millipore) and applied to SEC (Superdex-200 Increase, Cytiva) with GF75 running buffer. Purified protein was concentrated, flash frozen and stored at -80℃. Mutants and truncations of UAP56 were generated by Quick-change mutagenesis (Vazyme) and purified identically.

8×His-MBP tagged ALYREF was expressed and purified similarly with following exceptions: first, the NaCl concentration of the running buffer for SEC was adjusted to 150 mM; second, proteins were used for pull-down or cryo-EM sample preparation immediately after purification, without frozen and thaw.

TREX-2M pull-down assay
To assess the potential interactions between TREX-2 and TREX components, 1 µM purified TREX-2M were individually mixed with 1.5 µM of THO, 2 µM of UAP56 and 2 µM of ALYREF in GF75 buffer (25 mM HEPES-KOH, pH 7.9, 75 mM NaCl, 1.5 mM MgCl2). The solutions were incubated at 4°C for 2 hours to allow complex formation. Proteins were captured relying on the Strep-tag at the N-termini of PCID2. After extensive washes with GF75 buffer, proteins were eluted with 50 mM biotin and analyzed on SDS-PAGE gels.

Cryo-EM sample preparation and data collection
Purified TREX-2M were concentrated to ~1.2 mg/mL and directly used for cryo-EM specimen preparation. The UAP56-TREX-2M complex were reconstituted in the presence 2 mM AMP-PNP and 10 µM of 20 nt poly-U RNA. Samples were applied to glow-discharged amorphous NiTi foil grids (Au300-1.2/1.3, Nanodim Tech.), blotted for 3 seconds and rapidly plunged into liquid ethane using Vitrobot Mark IV operating at 8°C and 100% humidity. Cryo-EM data was collected with EPU on a 300-kV Titan Krios electron microscope (Thermo Fisher Scientific) using a normal magnification of 105,000×. Movies were recorded using a Gatan K3 detector equipped with a GIF Quantum energy filter (slit width 20 eV) at pixel size of 0.82 Å. The defocus value varied from -1.2 to -1.8 μm. 3,651 and 9,848 micrographs were collected for TREX-2M and UAP56-TREX-2M, respectively.

Cryo-EM data processing
For TREX-2M, 3,651 micrographs were collected and imported into Cryo-SPARC v4.2 for further processing. After template picking, a total of 4,635,704 particles were picked, extracted and split into 10 subsets. Two rounds of two-dimensional (2D) classification were performed for each subset of the particles, resulting a total of 2,442,528 good particles. This data set of particles was then used for generation of initial models using Ab-initio reconstitution. Through several rounds of hetero-refinement, non-uniform refinement and 3D classification, a final reconstruction at 3.1 Å for TREX-2M was obtained from 108,904 particles.

For UAP56-TREX-2M complex, 9,848 micrographs were collected, from which 7,994,980 particles were picked using Topaz picking68. After particle extraction, two rounds of 2D classification were performed, generating 3,073,993 good particles. Six classes of initial model were generated using Ab-initio reconstitution, of which class 1 and class 4 were selected for further processing. After three rounds of hetero-refinement, NU-refinement and 3D classification, a final reconstruction at 3.3 Å for UAP56-TREX-2M was obtained from 147,130 particles. Reported resolution limits were calculated on the basis of the Fourier shell correlation (FSC) 0.143 criterion69. Local resolution variations were estimated using CryoSPARC70.

Model building
The AlphaFold2 predicted structures for GANP580-1000, PCID2, DSS1 were fitted into the cryo-EM map of TREX-2M in ChimeraX to generate an initial model. The model was then manually adjusted in Coot71 based on the fine features of EM map. The final models of TREX-2M were refined against the EM maps using Phenix.real_space_refine72, with secondary structure restraints. For model building of UAP56-TREX-2M, the model of TREX-2M (this study) and UAP56 (PDB: 8ENK60) were fitted to the 3.3 Å EM map for UAP56-TREX-2M complex. The atomic models were validated through examination of the Molprobity scores and statistics of the Ramachandran plots73.

Binding assay for TREX-2M and UAP56
1 µM of purified TREX-2M were mixed with 2 µM of wildtype or mutant of UAP56 in GF75 buffer (25 mM HEPES-KOH, pH 7.9, 75 mM NaCl, 1.5 mM MgCl2). The solutions were incubated at 4°C for 2 hours to allow complex formation. Proteins were captured relying on the MBP-tag at the N-termini of GANP580-1000. After six washes with GF75 buffer, proteins were eluted with 20 mM maltose and analyzed on SDS-PAGE gels. Each group were repeated for three times.

ATPase activity assay
ATPase activities of WT and truncated (∆16) UAP56 in the presence of RNA were measured by the ATPase Colorimetric Assay Kit (Innova Biosciences). 5 μM of purified protein was added to the reaction buffer (25 mM HEPES-KOH, pH 7.9, 150 mM NaCl, 5 mM MgCl2 and 10 μM 20 nt poly-U RNA) and pre-incubated on ice for 20 min. Hydrolysis was initiated by adding 3 mM ATP and incubating at 37°C for 10 min. Released inorganic phosphate was measured quantitatively with the ATPase Colorimetric Assay Kit by determining the light absorbance at 650 nm with the BioTek Synergy H1 Multimode Reader. To assess the effect of TREX-2M on the ATPase activity of UAP56, the assay was parallelly performed in the presence or absence of 5 μM WT or mutant (R678A) TREX-2M. Each of the reaction group were repeated for three times. Statistical analysis was done using GraphPad Prism 8.

Electrophoretic mobility shift assay
First, 40 nM of 5’-FAM labeled poly-U RNA were incubated with 4 μM of WT or ∆16 mutant of UAP56 in buffer containing 25 mM HEPES-KOH (pH 7.9), 75 mM NaCl, 1.5 mM MgCl2, 0.5 U/μL RNase inhibitor (Yeasen) and 2 mM ATPγS (Sigma). The mixtures were incubated at room temperature for 30 min to allow RNA binding. Then, increasing concentrations of WT or R678A mutant TREX-2M were added to the pre-formed UAP56/RNA complexes and incubated for another 30 min at room temperature. Samples were resolved on 6% native polyacrylamide gels by electrophoresis in 0.5× Tri Borate EDTA (TBE) running buffer at room temperature. Gels were imaged using Amersham Imager 680 (GE Healthcare).

Statistical analysis
Data were analyzed with GraphPad Prism 8. Data were considered significant at P < 0.05 by paired two-tailed Student’s t test. The sample sizes were chosen to ensure the reproducibility of the experiments and to obtain meaningful results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "My expertise in the biochemistry structural biology of protein/RNA complexes, RNA metabolism and regulation of gene expression.\n\nMajor comments\n1) As entry points, the authors use mass spectrometry approaches to determine the interactome of GANP and PCID2. For GANP, a volcano plot is shown, and the number of replicates is indicated. However, for PCID2, only the number of unique peptides for various proteins is displayed. Additionally, the control experiment is missing here, which would show how many of these peptides are bound to the beads only. It is impossible to compare the two experiments because of the different data representations. For GANP, there are many more hits that are more enriched than the selectively labeled proteins of interest to the authors. To compare both experiments effectively, they should be presented in the same way. This might also allow to plot the enrichment of GANP/PCID2 to see the overlapping proteome.\n2) After the mass spec, the authors switch to the TREX-2 core complex, called TREX-2M. However, they do not explain how they identified the region in GANP as sufficient to interact with DSS1 and PCID2. Please clarify your reasoning or cite the relevant literature.\n3) The pulldown experiments suggest that TREX-2M weakly binds UAP56 and ALYREF. Unfortunately, the key controls for the pulldown are in a different figure (different color; grey vs. blue), which makes comparison difficult. I would argue that in Extended Data Figure 1f, ALYREF binds nonspecifically to the beads, and the band has a similar intensity as in Figure 1d. Since the pulldown and control are not on the same gel, drawing conclusions is challenging. Information about the number of experiment replicates is not provided here. I suggest the authors repeat the experiment and show the control on the same gel.\nAdditionally, Figure 1e lacks a control for nonspecific binding. The authors should demonstrate that ADP alone cannot trigger the formation of a stoichiometric complex.\nOverall, the first section contains interesting data but needs consolidation. It is crucial to know if the binding of UAP56 to TREX-2 is nucleotide-dependent and which nucleotides support interaction (ADP, ATP, AMPPNP or ADP:AlFx)\nStructure of TREX-2M\n4) The structures described here are interesting but very similar to those of yeast TREX-2M (Ren lab) and human TREX-2 (Plaschka lab). I could not find the citation for the paper from the Plaschka lab, which has been on BioRxiv for 1.5 years. This study must be cited and used for comparison. Additionally, the comparison of Sub2 binding to yeast TREX-2 should be expanded.\nThe interaction of UAP56 loop1 appears clear. However, in loop2, the authors modeled a metal called m1, which is coordinated by three histidines. The manuscript does not specify what the metal is, but inspection of the PDB reveals that the authors likely chose to model a Mg2+ ion. Looking at the model, this seems most likely to be incorrect for several reasons:\ni) Mg2+ has six coordination sites, but only three are filled here.\nii) Mg2+ prefers hard atoms like oxygen and is rarely coordinated by soft atoms like nitrogen.\niii) The distances are too short; Mg2+ N (imidazole) bonds typically range from approximately 2.1 to 2.3 Å. In this model, the distances are 1.9 Å, and one carbon from the imidazole ring is the closest atom to Mg2+ at 1.64 Å. Also, His143 and His186 are unrealistically close. The density does not support the presence of Mg2+, and positioning the three His side chains is problematic. This modeling is implausible and needs correction.\nFigure3d: Pulldown assays are quantified but again lack the negative control! Was this done in replicates?\nTREX-2 stimulates ATPase activity.\nThe authors claim that R678 forms a cation/pi interaction with the adenine ring of ATP. I have examined the model and the map. While the modeling of AMPPNP is reasonable, Arg678 in the authors' model does not form cation/pi interactions with the adenine ring. In their model, the arginine is tilted away, and the close contact is between the Arg-NE atom and N3 of adenine - possibly a hydrogen bond. please see attached screenshot. I tried to correct the Arg rotamer to make it more extended. Even then, the Arg is not positioned over the adenine ring for cation/pi interactions. In short, the map does not justify this modeling or the conclusion. This needs to be corrected.\nA mechanistic model is missing here. When does hydrolysis happen, before or after TREX-2 binding? So, there are too many unknowns here.\nOverall, the paper touches an important topic. However, in my opinion the paper seems in a premature state. There are problems with the modeling that are crucial to fix; at the moment the model is not fully supported by the data. The pulldown experiments lack controls. This manuscript requires major revisions.\nAlso it lacks a detailed comparison to the data and models from the Ren and Plaschka labs. In the current state, the paper does not fit to the high quality and novelty required to publish in nature commincations.\nMinor comments\nAt various points, ‘the’ is missing in the text. Also, for some buffers, it is not indicated how the pH was adjusted.\nThe labels for the SDS PAGE gel in figures 1d, e, and f might be too small to read. It was difficult when printed on A4 full page. Please check the font size.\nExtended Figures 1c and d: SEC fractions are indicated above the gels but are missing in the chromatogram. For 1c, there is only a peak, but in the case of 1d, it is unclear which peak corresponds to the THO complex.", "reviewer_2": "This manuscript provides a comprehensive description of the mechanism by which the human TREX-2 complex facilitates the removal of the DEAD-box ATPase UAP56 from mRNPs as a preliminary to generating export-competent complexes and will be of considerable general interest. Although the claim that this work provides a “molecular basis for mRNA export regulation” is exaggerated, this manuscript still provides important structural and biochemical information about this process. The authors provide a cryo-EM structure of UAP56 bound to the TREX-2M complex and identify a key loop in the TREX-2 GANP component that has the potential to stimulate the UAP56 ATPase and provide biochemical evidence to support this hypothesis. The quality of the work is high, and the interpretations are robust. Overall, the work confirms two recent manuscripts (Nature comms 16:5471 and BioRxiv doi:10.1101/2024.03.24.586400) that have reached similar conclusions.\nComments:\n1. Reference should be made to the two recent papers that have reached similar conclusions about the role of the TREX-2 complex in mRNA export (Nature Communications 2005, 16:5471 and BioRxiv 2004 doi:10.1101/2024.03.24.586400) and differences and similarities betwen these manuscripts and the present work discussed in detail.\n2. The claim that that this work provides a “molecular basis for mRNA export regulation” should be toned down since no direct evidence is provided regarding how the release of UAP56 actually facilitates binding of the NXF1-NXT1 export factor (I do not know of any evidence that UAP56 and NXF1-NXT1 share a binding site on mRNA, for example). Instead, it seems to me that this work is demonstrating how TREX-2 functions to remove the last component of the TREX complex from the mRNP as a preliminary to making it export-competent.", "reviewer_3": "This manuscript presents cryo‑electron‑microscopy snapshots of a minimal human TREX‑2 core (GANP Sac3 560-1000 bound to PCID2 and DSS1) captured both alone at 3.1 Å resolution and in complex with the DEAD‑box helicase UAP56 at 3.3 Å. Together with a carefully designed series of biochemical assays, these structures reveal how TREX‑2 stimulates UAP56 ATPase activity, and the authors propose how this may orchestrate the release of mRNPs at the nuclear pore.\nThe quality of the structural work is sound. Side‑chain density is well resolved throughout the TREX‑2 scaffold and at the interfaces with UAP56, allowing the authors to identify two acidic N‑terminal loops in UAP56 (residues 8-16 and 36-45) that dock into a basic channel formed by GANP and PCID2. The authors further pinpoint a conserved “UAP56‑activating loop” in GANP (residues 672-688); its Arg678 side chain slots into the adenine‑stacking pocket of UAP56, completing the catalytic site and offering a direct structural explanation for the ten‑fold jump in ATPase activity they measure in vitro. Mutational analysis is rigorous: alanine substitution or deletion of Loop‑1 abolishes binding, histidine replacements in Loop‑2 markedly weaken the interaction, and an R678A substitution in GANP abrogates ATPase stimulation and RNA release. The biochemical section is consequently both compelling and internally consistent with the structural data, altogether presenting a plausible and coherent model in which TREX‑2 remodels export‑competent mRNPs by activating UAP56 and dislodging bound RNA.\nAlthough the study is convincing, several issues deserve attention before the work can be regarded as complete. First, only partial density is visible for the RecA2 lobe of UAP56 and no RNA is resolved, despite assembly with poly‑uridylate. The authors should clarify whether this reflects particle heterogeneity, preferred orientation during data collection, or a biologically relevant pre‑release intermediate. A brief account of two‑dimensional or focused classifications on the RecA2 region would help the reader judge the state of the helicase. Second, the intriguing metal ion modelled at the Loop‑2 interface remains speculative; a targeted mutation of the histidines that coordinate the ion (for example, PCID2 H186A) would verify its presence and functional relevance. Third, the kinetics of ATPase stimulation are reported as end‑point values; Michaelis-Menten analysis would provide quantitative k_cat and K_M parameters and offer a rigorous measure of the ten‑fold stimulation the authors purport. Fourth, because human cells express a paralogous helicase, URH49, it would be very interesting to demonstrate that TREX‑2 fails to stimulate URH49, thereby confirming that the acidic loop determinants drive isoform specificity. In general, how specific is TREX-2 for its helicase?\nIt would be helpful to discuss why the complex was captured in an RNA‑free state.\nWhere the manuscript falls short is not in experimental choice but in the depth of reporting and statistical treatment. Very few of the quantitative data are accompanied by information on biological versus technical replicate number, variance, or the statistical tests employed. Pull‑down efficiencies, ATPase rates and RNA‑release percentages are each presented as single bars or end‑point values, with a terse statement that “three independent repeats” were performed; yet the reader never sees the underlying replicate points, the mean +/- SD (or SEM) error, or a P‑value that would allow judgement of significance. A concise Statistics subsection should be added that specifies, experiment by experiment, how n is defined, how normality was assessed (or assumed), which tests were applied (e.g., two‑tailed unpaired t‑tests for pairwise comparisons or one‑way ANOVA with post‑hoc corrections when multiple mutants are compared), and what software was used for statistical calculations.\nThe biochemical assays would benefit from modest additional rigor. Michaelis-Menten analyses should replace or at least complement end‑point ATPase measurements so that catalytic efficiencies (k_cat and K_M) can be compared quantitatively across mutants. Microscale thermophoresis or isothermal‑titration calorimetry, performed on wild‑type and loop‑mutant proteins, would give a quantitative affinity that corroborates the qualitative pull‑down impressions.\nFinally, the manuscript should make clear whether key experiments were repeated on separate protein preparations (true biological replicates) and whether any randomisation or blinding was relevant (and, if not, explicitly state this).\nA couple of minor textual points:\nLine 141 “Loop‑1 … previously assigned as a putative mRNA binding site” - cite the specific structural study.\nLines 228-238: the “unified model” would benefit from a statement that the current study addresses only the final hand‑off step and does not yet capture THO disengagement.\nTaken as a whole, the manuscript offers an elegant and highly informative look at how TREX‑2 acts as a molecular switch to catalyse UAP56 activity and drive mRNA export. The high‑resolution structures, combined with mutagenesis and functional assays, advance the field significantly and answer a long‑standing question about the mechanics of export‑complex remodelling at the nuclear pore. Once the critiques I have put forward are addressed, this manuscript will be a strong candidate for publication."}, "author_response": "Point-by-Point response to reviewers’ comments\nWe sincerely appreciate the reviewers' constructive comments. All questions\nhave been addressed in detail, and the manuscript has been revised\naccordingly.\nReviewer #1:\nMy expertise in the biochemistry structural biology of protein/RNA complexes,\nRNA metabolism and regulation of gene expression.\nMajor comments\n1) As entry points, the authors use mass spectrometry approaches to\ndetermine the interactome of GANP and PCID2. For GANP, a volcano plot is\nshown, and the number of replicates is indicated. However, for PCID2, only\nthe number of unique peptides for various proteins is displayed. Additionally,\nthe control experiment is missing here, which would show how many of these\npeptides are bound to the beads only. It is impossible to compare the two\nexperiments because of the different data representations. For GANP, there\nare many more hits that are more enriched than the selectively labeled\nproteins of interest to the authors. To compare both experiments effectively,\nthey should be presented in the same way. This might also allow to plot the\nenrichment of GANP/PCID2 to see the overlapping proteome.\nResponse: We appreciate the reviewer's insightful suggestion. In the revised\nmanuscript, we have now analyzed the proteomic data for PCID2\nimmunoprecipitates and included a volcano plot of enriched proteins (revised\nFig. 1b), consistent with the presentation for GANP. While many hits showed\ngreater enrichment than our selectively labeled proteins of interest, many of\nthese highly enriched proteins (e.g., chaperones, transcription factors,\ntubulins) are poorly characterized in the context of mRNA binding or nuclear\nexport. Therefore, only known mRNA export factors are labeled in the volcano\nplots. In both datasets, DDX39B (UAP56) - but not THO complex components\n- was significantly enriched relative to controls. UAP56 is particularly notable\ngiven its established roles in mRNA packaging by TREX and as an mRNP\nmarker, potentially bridging TREX-2 and mRNA. This is further supported by\nour structural and biochemical evidence demonstrating TREX-2-mediated\nremodeling of UAP56 for mRNA release. Notably, DDX39A (also known as\nURH49), a paralog of UAP56, was also enriched in PCID2\nimmunoprecipitates (revised Fig. 1b). However, pulldown assays performed\nduring revision revealed substantially weaker binding of DDX39A to TREX-2\ncompared to UAP56 (revised Extended Data Fig. 1i). We concur that other\nenriched factors merit further investigation, such as LENG8 (recently identified\nas a PCID2-interacting factor in the TREX-2.1 complex (Clarke, B.P. et al. Nat\nCommun 2025)) and SAC3D1, which forms a complex with PCID2-DSS1\n(Extended Data Fig. 10e), but requires functional characterization. We have\nadded a Discussion section addressing these two GANP-like factors.\n2) After the mass spec, the authors switch to the TREX-2 core complex, called\nTREX-2M. However, they do not explain how they identified the region in\nGANP as sufficient to interact with DSS1 and PCID2. Please clarify your\nreasoning or cite the relevant literature.\nResponse: We thank the reviewer for raising this important point. The TREX-\n2M was previously established as the minimal unit sufficient for mediating\nDSS1-PCID2 interactions in both yeast and human, as documented in prior\nliterature (Ellisdon, A. et al., Nat Struct Mol Biol 2012; James M. B. et al.,\nNucleic Acids Research, 2017). We have now explicitly cited these\nfoundational works in our revised manuscript. Regarding complex selection,\nwhile we initially purified the full TREX-2 complex (GANP-PCID2-DSS1-\nCETN3-ENY2), its poor behavior during gel filtration chromatography\nrendered it unsuitable for structural and biochemical studies (see below). In\naddition, AlphaFold prediction suggests TREX-2M is responsible for UAP56\nbinding, and the ENY2/CETN2 region is only connected by flexible linkers of\nGANP.\n3) The pulldown experiments suggest that TREX-2M weakly binds UAP56 and\nALYREF. Unfortunately, the key controls for the pulldown are in a different\nfigure (different color; grey vs. blue), which makes comparison difficult. I would\nargue that in Extended Data Figure 1f, ALYREF binds nonspecifically to the\nbeads, and the band has a similar intensity as in Figure 1d. Since the\npulldown and control are not on the same gel, drawing conclusions is\nchallenging. Information about the number of experiment replicates is not\nprovided here. I suggest the authors repeat the experiment and show the\ncontrol on the same gel. Additionally, Figure 1e lacks a control for nonspecific\nbinding. The authors should demonstrate that ADP alone cannot trigger the\nformation of a stoichiometric complex.\nOverall, the first section contains interesting data but needs consolidation. It is\ncrucial to know if the binding of UAP56 to TREX-2 is nucleotide-dependent\nand which nucleotides support interaction (ADP, ATP, AMPPNP or ADP:AlFx).\nResponse: We appreciate the reviewer's meticulous assessment of these\nexperiments. To clarify: our original conclusions explicitly excluded specific\nbinding between TREX-2M and ALYREF due to the observed nonspecific\nbinding to beads, as stated: \"Pull-down assays demonstrated weak but\nspecific binding between TREX-2M and UAP56, with no detectable specific\ninteraction with THO or ALYREF\" (Fig. 1d; Extended Data Fig. 1g). This\nnonspecific binding persisted across bead types (we tried Ni-NTA, Anti-Flag-\nMS2 and Strep-Tactin beads), likely reflecting the poor solubility nature of\nALYREF.\nWe have rectified the suboptimal figure arrangement that complicated\ncomparison of pulldowns and controls. The revised manuscript now displays\nthese controls in unified panels with consistent color coding, and specifies\nexperimental replicates in figure legends.\nRegarding nucleotide dependence, our additional experiments during\nrevision demonstrate that not only ATP and AMPPNP, but surprisingly also\nADP enhances UAP56-TREX-2 binding (Extended Data Fig. 1h). This\nsuggests the adenine moiety, rather than the phosphate groups, contributes to\nthe interaction. Consistent with this observation, the Arg678 of GANP\nengages the adenine ring of bound AMPPNP in our structure with a hydrogen\nbond. This interpretation has been revised in our manuscript.\nStructure of TREX-2M\n4) The structures described here are interesting but very similar to those of\nyeast TREX-2M (Ren lab) and human TREX-2 (Plaschka lab). I could not find\nthe citation for the paper from the Plaschka lab, which has been on BioRxiv\nfor 1.5 years. This study must be cited and used for comparison. Additionally,\nthe comparison of Sub2 binding to yeast TREX-2 should be expanded.\nResponse: Point accepted. The revised manuscript now appropriately cites\nboth the Ren Lab's yeast TREX-2M structure and the Plaschka Lab's human\nTREX-2 structure. We have prepared a new comparative figure (Extended\nData Fig. 10) and expended our discussion on the conserved and divergent\nfeatures across these complexes.\nThe interaction of UAP56 loop1 appears clear. However, in loop2, the authors\nmodeled a metal called m1, which is coordinated by three histidines. The\nmanuscript does not specify what the metal is, but inspection of the PDB\nreveals that the authors likely chose to model a Mg2+ ion. Looking at the\nmodel, this seems most likely to be incorrect for several reasons:\ni) Mg2+ has six coordination sites, but only three are filled here.\nii) Mg2+ prefers hard atoms like oxygen and is rarely coordinated by soft\natoms like nitrogen.\niii) The distances are too short; Mg2+ N (imidazole) bonds typically range from\napproximately 2.1 to 2.3 Å. In this model, the distances are 1.9 Å, and one\ncarbon from the imidazole ring is the closest atom to Mg2+ at 1.64 Å. Also,\nHis143 and His186 are unrealistically close. The density does not support the\npresence of Mg2+, and positioning the three His side chains is problematic.\nThis modeling is implausible and needs correction.\nResponse: We thank the reviewer for his/her expert structural assessment.\nFollowing consultation with bioinorganic specialists and re-examination of the\ndensity maps, we agree that the initial Mg2+ assignment was likely inaccurate.\nWe have therefore modeled this density as a Zn2+ ion in the revised structure,\nwith a refined Zn²⁺-N(imidazole) bond length of 2.3 Å (see the image below).\nThis coordination is similar to a previously report structure (PDB: 1MOO). We\nacknowledge that at the current resolution, this assignment remains tentative.\nThus, we would be pleased to remove the assignment should the reviewer\nconsider this the most conservative interpretation of the data. Importantly, the\npresence or identity of this metal ion does not impact our core mechanistic\nconclusions.\nFigure3d: Pulldown assays are quantified but again lack the negative control!\nWas this done in replicates?\nResponse: We thank the reviewer for this important methodological critique.\nWhile we initially considered wild-type protein comparisons sufficient for\nassessing mutational effects, we fully acknowledge that dedicated negative\ncontrols are essential for unambiguous interpretation. In response, we have\nre-performed pull-down assays including appropriate negative control and\nincorporated these data into Extended Data Fig. 5. Both WT and mutants of\nUAP56 exhibits no non-specific binding to the beads. Additionally, two\nindependent biological replicates demonstrating reproducibility are presented\nas requested.\nTREX-2 stimulates ATPase activity.\nThe authors claim that R678 forms a cation/pi interaction with the adenine ring\nof ATP. I have examined the model and the map. While the modeling of\nAMPPNP is reasonable, Arg678 in the authors' model does not form cation/pi\ninteractions with the adenine ring. In their model, the arginine is tilted away,\nand the close contact is between the Arg-NE atom and N3 of adenine -\npossibly a hydrogen bond. please see attached screenshot. I tried to correct\nthe Arg rotamer to make it more extended. Even then, the Arg is not\npositioned over the adenine ring for cation/pi interactions. In short, the map\ndoes not justify this modeling or the conclusion. This needs to be corrected.\nA mechanistic model is missing here. When does hydrolysis happen, before\nor after TREX-2 binding? So, there are too many unknowns here.\nResponse: We appreciate the reviewer's rigorous assessment of the\nstructural model. Regarding the Arg678-adenine interaction, we acknowledge\nthat our initial description of adenine “sandwiching” was inaccurate. As the\nreviewer pointed out, the arginine is tilted away and forms a hydrogen bond\n(rather than cation/pi interaction) with the Adenine ring of AMPPNP. We have\nrevised the text to clarify this point.\nOur structural and biochemical data suggests ATP hydrolysis occurs\nafter TREX-2 binding. This temporal sequence is evidenced by: first, UAP56\nadopts a closed conformation when bound to ATP and RNA; second, TREX-2\nbinding stimulates the ATPase activity of UAP56 as evidenced by a 10-fold\nincrease of the Vmax (Fig. 5b); third, this TREX-2-mediated activation of\nhydrolysis facilitates RNA release through conformational changes (Fig. 4;\nFig. 5c).\nOverall, the paper touches an important topic. However, in my opinion the\npaper seems in a premature state. There are problems with the modeling that\nare crucial to fix; at the moment the model is not fully supported by the data.\nThe pulldown experiments lack controls. This manuscript requires major\nrevisions.\nAlso it lacks a detailed comparison to the data and models from the Ren and\nPlaschka labs. In the current state, the paper does not fit to the high quality\nand novelty required to publish in nature communications.\nResponse: We sincerely appreciate the reviewer's recognition of the\nsignificance our study and his/her thorough evaluation. Throughout the\nrevision process, we have: 1) Performed all requested additional experiments\nto include essential controls; 2) Comprehensively reorganized figures for\nenhanced clarity; 3) Rigorously refined the structural model according to the\nreviewer's specific guidance; 4) Implemented detailed structural comparisons\nwith Ren and Plaschka lab models. These substantive revisions have\nstrengthened both the scientific rigor and conceptual clarity of our work. We\nbelieve the revised manuscript now meets the standards of the journal.\nMinor comments\nAt various points, ‘the’ is missing in the text. Also, for some buffers, it is not\nindicated how the pH was adjusted.\nResponse: We have carefully reviewed the entire manuscript to avoid\ngrammatical omissions and specified pH adjustment methods for every\nbuffers.\nThe labels for the SDS PAGE gel in figures 1d, e, and f might be too small to\nread. It was difficult when printed on A4 full page. Please check the font size.\nResponse: We have increased label font sizes by 20% in Fig. 1.\nExtended Figures 1c and d: SEC fractions are indicated above the gels but\nare missing in the chromatogram. For 1c, there is only a peak, but in the case\nof 1d, it is unclear which peak corresponds to the THO complex.\nResponse: We have added the labels of fractions onto the chromatogram.\nThe peak of full THO complex has now been indicated.\nWe thank this reviewer for his/her constructive comments.\nReviewer #2 (Remarks to the Author):\nThis manuscript provides a comprehensive description of the mechanism by\nwhich the human TREX-2 complex facilitates the removal of the DEAD-box\nATPase UAP56 from mRNPs as a preliminary to generating export-competent\ncomplexes and will be of considerable general interest. Although the claim\nthat this work provides a “molecular basis for mRNA export regulation” is\nexaggerated, this manuscript still provides important structural and\nbiochemical information about this process. The authors provide a cryo-EM\nstructure of UAP56 bound to the TREX-2M complex and identify a key loop in\nthe TREX-2 GANP component that has the potential to stimulate the UAP56\nATPase and provide biochemical evidence to support this hypothesis. The\nquality of the work is high, and the interpretations are robust. Overall, the work\nconfirms two recent manuscripts (Nature comms 16:5471 and BioRxiv\ndoi:10.1101/2024.03.24.586400) that have reached similar conclusions.\nResponse: We sincerely thank Reviewer #2 for the thorough evaluation and\npositive assessment of the scientific quality and significance of our work. We\nare encouraged by your recognition that our findings provide “important\nstructural and biochemical information” about the TREX-2–UAP56 regulatory\nmechanism and will be of “considerable general interest”. In response to\nreviewer’s concern on the title, we have modified it as “Molecular insights into\nmRNA export regulation by human TREX-2 complex”.\nComments:\n1. Reference should be made to the two recent papers that have reached\nsimilar conclusions about the role of the TREX-2 complex in mRNA export\n(Nature Communications 2005, 16:5471 and BioRxiv 2004\ndoi:10.1101/2024.03.24.586400) and differences and similarities between\nthese manuscripts and the present work discussed in detail.\nResponse: We appreciate this point. In our revised manuscript, we have\nformally cited both the Nature Communications study (2024, 16:5471) and the\nBioRxiv preprint. Additionally, a dedicated section in the Discussion now\ndetails our comparative analysis of these parallel studies alongside our work.\n2. The claim that that this work provides a “molecular basis for mRNA export\nregulation” should be toned down since no direct evidence is provided\nregarding how the release of UAP56 actually facilitates binding of the NXF1-\nNXT1 export factor (I do not know of any evidence that UAP56 and NXF1-\nNXT1 share a binding site on mRNA, for example). Instead, it seems to me\nthat this work is demonstrating how TREX-2 functions to remove the last\ncomponent of the TREX complex from the mRNP as a preliminary to making it\nexport-competent.\nResponse: Point accepted. We agree with the reviewer that the original title\nclaiming a \"molecular basis for mRNA export regulation\" was too strong, as\nour study doesn't provide direct evidence for how UAP56 release enables\nNXF1-NXT1 binding. Our work instead demonstrates the crucial UAP56-\nTREX-2 interaction and how TREX-2 regulates the ATPase activity of UAP56\nto release mRNP, thereby positioning it for export competency. Since the\nmechanism of NXF1-NXT1 binding remains a fundamental open question in\nthe field, we have revised the title to “Molecular insights into mRNA export\nregulation by human TREX-2 complex” and updated the manuscript text\nthroughout to better align with these findings.\nWe thank this reviewer for his/her constructive comments.\nReviewer #3 (Remarks to the Author):\nThis manuscript presents cryo‑electron‑microscopy snapshots of a minimal\nhuman TREX‑2 core (GANP Sac3 580-1000 bound to PCID2 and DSS1)\ncaptured both alone at 3.1 Å resolution and in complex with the DEAD‑box\nhelicase UAP56 at 3.3 Å. Together with a carefully designed series of\nbiochemical assays, these structures reveal how TREX‑2 stimulates UAP56\nATPase activity, and the authors propose how this may orchestrate the\nrelease of mRNPs at the nuclear pore.\nThe quality of the structural work is sound. Side‑chain density is well resolved\nthroughout the TREX‑2 scaffold and at the interfaces with UAP56, allowing\nthe authors to identify two acidic N‑terminal loops in UAP56 (residues 8-16\nand 36-45) that dock into a basic channel formed by GANP and PCID2. The\nauthors further pinpoint a conserved “UAP56‑activating loop” in GANP\n(residues 672-688); its Arg678 side chain slots into the adenine‑stacking\npocket of UAP56, completing the catalytic site and offering a direct structural\nexplanation for the ten‑fold jump in ATPase activity they measure in vitro.\nMutational analysis is rigorous: alanine substitution or deletion of Loop‑1\nabolishes binding, histidine replacements in Loop‑2 markedly weaken the\ninteraction, and an R678A substitution in GANP abrogates ATPase stimulation\nand RNA release. The biochemical section is consequently both compelling\nand internally consistent with the structural data, altogether presenting a\nplausible and coherent model in which TREX‑2 remodels export‑competent\nmRNPs by activating UAP56 and dislodging bound RNA.\nResponse: We sincerely thank Reviewer #3 for the thorough evaluation and\npositive assessment of our structural and biochemical work. We're deeply\nencouraged by his/her recognition of the quality and significance of our\nfindings. These insightful feedbacks significantly strengthened the scientific\nrigor and clarity of our manuscript.\nAlthough the study is convincing, several issues deserve attention before the\nwork can be regarded as complete. First, only partial density is visible for the\nRecA2 lobe of UAP56 and no RNA is resolved, despite assembly with poly‑\nuridylate. The authors should clarify whether this reflects particle\nheterogeneity, preferred orientation during data collection, or a biologically\nrelevant pre‑release intermediate. A brief account of two‑dimensional or\nfocused classifications on the RecA2 region would help the reader judge the\nstate of the helicase.\nResponse: We agree that RecA2 is poorly resolved in our cryo-EM map,\nwhich is likely due to its inherent flexibility. This mobility arises from RecA1\nbeing anchored within the GANP/PCID2 cleft, whereas RecA2 lacks direct\ncontacts with TREX-2. Our structural data supports a mechanism whereby\nTREX-2 binding splits the two RecA domains of UAP56—via insertion of the\nconserved UAP56-activating loop—to promote ATP hydrolysis and\nsubsequent RNA release. During revision, focused 3D classification on RecA2\nconfirmed its dynamic displacement but did not yield improved local\nresolution. Notably, the mobile nature of RecA2 aligns with two parallel\nstudies from Ren lab and Plaschka lab (Nature Communications 2025,\n16:5471 and BioRxiv doi:10.1101/2024.03.24.586400).\nSecond, the intriguing metal ion modelled at the Loop‑2 interface remains\nspeculative; a targeted mutation of the histidines that coordinate the ion (for\nexample, PCID2 H186A) would verify its presence and functional relevance.\nResponse: We appreciate this suggestion. To address the functional\nrelevance of the metal ion at the Loop-2 interface, we generated the\nH43A/H73A double mutation targeting its histidine coordination sites. As\nshown in Fig. 3d and Extended Data Fig.5, this mutation specifically\nattenuates the interaction, supporting both the presence and functional\nsignificance of the metal ion.\nThird, the kinetics of ATPase stimulation are reported as end‑point values;\nMichaelis-Menten analysis would provide quantitative k_cat and K_M\nparameters and offer a rigorous measure of the ten‑fold stimulation the\nauthors purport.\nResponse: We thank the reviewer for this thoughtful suggestion. We have\nnow performed full Michaelis-Menten analyses to quantify the ATPase activity\nof UAP56 both alone and in complex with TREX-2M. The results are presented\nin Fig. 5b of the revised manuscript. Our analysis reveals that TREX-2M\nstimulates the ATPase activity of UAP56 by increasing V approximately 10-\nmax\nfold (Fig. 5b). This provides direct kinetic validation of the 10-fold stimulation\nreported in our original end-point assays.\nFourth, because human cells express a paralogous helicase, URH49, it would\nbe very interesting to demonstrate that TREX‑2 fails to stimulate URH49,\nthereby confirming that the acidic loop determinants drive isoform specificity.\nIn general, how specific is TREX-2 for its helicase?\nResponse: We appreciate this insightful suggestion regarding helicase\nspecificity. To address whether TREX-2 discriminates between UAP56 and its\nparalog URH49 (DDX39A), we performed new co-immunoprecipitation assays\nwhich revealed significantly weaker binding between TREX-2 and URH49\ncompared to UAP56 (revised Extended Data Fig. 1i). As the reviewer pointed\nout, this selectivity may arise from the unique N-terminal motifs of UAP56,\nwhich we show mediate the specific binding between TREX-2 (as\ndemonstrated in Fig. 3d). Together, these results provide mechanistic insight\ninto how TREX-2 achieves helicase isoform specificity, as previously\nsuggested in studies of mRNA export pathways (Fujita et al., Nature\nCommunications, 2023).\nIt would be helpful to discuss why the complex was captured in an RNA‑free\nstate.\nResponse: Our structural data reveal that TREX-2 binding actively remodels\nthe conformation of UAP56: while UAP56 typically forms a closed ATP/RNA-\nbound state with its RecA domains clamped on RNA, TREX-2 inserts a\nconserved loop between these domains, forcing them into an open\nconformation incompatible with RNA binding. This mechanistically explains\nthe observed RNA-free complex, likely representing a transitional state during\nTREX-mediated mRNA release prior to export complex assembly.\nWhere the manuscript falls short is not in experimental choice but in the depth\nof reporting and statistical treatment. Very few of the quantitative data are\naccompanied by information on biological versus technical replicate number,\nvariance, or the statistical tests employed. Pull‑down efficiencies, ATPase\nrates and RNA‑release percentages are each presented as single bars or end\n‑point values, with a terse statement that “three independent repeats” were\nperformed; yet the reader never sees the underlying replicate points, the\nmean +/- SD (or SEM) error, or a P‑value that would allow judgement of\nsignificance. A concise Statistics subsection should be added that specifies,\nexperiment by experiment, how n is defined, how normality was assessed (or\nassumed), which tests were applied (e.g., two‑tailed unpaired t‑tests for\npairwise comparisons or one‑way ANOVA with post‑hoc corrections when\nmultiple mutants are compared), and what software was used for statistical\ncalculations.\nThe biochemical assays would benefit from modest additional rigor. Michaelis-\nMenten analyses should replace or at least complement end‑point ATPase\nmeasurements so that catalytic efficiencies (k_cat and K_M) can be\ncompared quantitatively across mutants. Microscale thermophoresis or\nisothermal‑titration calorimetry, performed on wild‑type and loop‑mutant\nproteins, would give a quantitative affinity that corroborates the qualitative pull\n‑down impressions.\nFinally, the manuscript should make clear whether key experiments were\nrepeated on separate protein preparations (true biological replicates) and\nwhether any randomization or blinding was relevant (and, if not, explicitly state\nthis).\nResponse: We sincerely thank the reviewer for these essential\nrecommendations to strengthen our statistical and biochemical rigor. We have\nthoroughly addressed these concerns by implementing comprehensive\nstatistical reporting throughout the manuscript. All quantitative data now\nexplicitly indicate biological versus technical replication strategies, display\nindividual replicates with error measurements, and specify the statistical tests\napplied. In addition, we further quantified the catalytic efficiency of UAP56\nthrough Michaelis-Menten analyses, confirming the binding of TREX-2M\ninduces a 10-fold increase of the V . Although microscale thermophoresis\nmax\n(MST) was employed to measure binding affinities, recurrent technical\nvariability prevented consistent quantification from replicate samples.\nCrucially, all key experiments incorporated multiple biological replicates, now\ncompiled in the Extended Data Figs. 7 & 8.\nA couple of minor textual points:\nLine 141 “Loop‑1 … previously assigned as a putative mRNA binding site” -\ncite the specific structural study.\nResponse: Cited.\nLines 228-238: the “unified model” would benefit from a statement that the\ncurrent study addresses only the final hand‑off step and does not yet capture\nTHO disengagement.\nResponse: Point accepted. We agree with this observation and have revised\nthe manuscript to explicitly state that our study focuses specifically on the final\nhand-off step mediated by TREX-2, while the preceding THO complex\ndisengagement process remains outside the scope of this study. The\nmodification maintains the integrity of our structural and biochemical evidence\nwhile acknowledging this important boundary for future investigations.\nTaken as a whole, the manuscript offers an elegant and highly informative\nlook at how TREX‑2 acts as a molecular switch to catalyse UAP56 activity\nand drive mRNA export. The high‑resolution structures, combined with\nmutagenesis and functional assays, advance the field significantly and answer\na long‑standing question about the mechanics of export‑complex remodeling\nat the nuclear pore. Once the critiques I have put forward are addressed, this\nmanuscript will be a strong candidate for publication.\nResponse: We sincerely thank Reviewer #3 for his/her comprehensive and\ninsightful critiques, which have significantly enhanced both the clarity and\nscientific rigor of our manuscript. We're particularly grateful for the recognition\nof our work's contribution to resolving longstanding mechanistic questions\nabout the role of TREX-2 in mRNA export, and for guiding us toward making\nthis study publication-ready.\nWe thank this reviewer for his/her constructive comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Structural Modeling\nZn²⁺ Coordination\nThe proposed coordination of Zn²⁺ in the structural model appears insufficiently supported by the current evidence. In most proteins, Zn²⁺ adopts tetrahedral coordination geometry with four ligands; catalytic zinc centers, as observed for example in carbonic anhydrase, may involve a water or hydroxide ligand in place of a canonical side-chain donor. Such cases represent enzymatic rather than purely structural zinc sites and therefore may not serve as an appropriate analogy in the present context.\nTwo histidine residues display orientations that are, in principle, compatible with metal coordination, although the observed metal–ligand distances of approximately 2.2 Å are unusually long. Furthermore, one of these histidines is not fully defined in the electron density. The third putative histidine ligand appears incorrectly modeled, with the CE atom positioned closer to the metal ion than the imidazole nitrogens, which is inconsistent with canonical coordination chemistry.\nGiven the lack of independent evidence for Zn²⁺ or other divalent ions, such as atomic absorption spectroscopy or related analytical approaches, the inclusion of a Zn²⁺ ion in the structural model does not appear experimentally justified. As the metal ion is not essential for the mechanistic interpretation presented in the manuscript, its removal from the model is recommended.\nArg678\nThe conformation of Arg678 could be correctly modeled. But the modeling is not ideal: The adenine base engages in π–π interactions with Phe65; however, the two aromatic systems adopt an orientation of approximately 45°, rather than a stacked (0°) or T-shaped (90°) arrangement typically obversev/expected. This geometry raises concerns that the refinement parameters (weight of geometry over map) may not have adequately chosen. This is an important region and has a the central role of this contact in the mechanistic analysis.\nSimilarly, the proposed hydrogen bond between Arg678 and adenine is uncertain. Although the distance is reasonable, the angle and displacement deviate from ideal parameters, rendering the interaction ambiguous.\nBiochemical Data\nPulldown Experiments: The revised pulldown assays appear robust and support the interaction model presented.\nATPase Analysis: The ATPase measurements are generally convincing. Nevertheless, the decision to fit the data with a Hill equation introduces additional parameters without clear justification. Did a regular Michaelis Menton model not fit the data? In the extended data Km and Vmax values are reported, but the Hill coefficient (n) is not reported, but forms an integral part of the fitted model. It would be informative to report n and to indicate whether it is comparable for UAP56 and UAP56–TREX-2 complexes. Biochemically, I do not understand why a Hill Model should fit the data better. More information would be required, ideally comparison of the two fits side by side. (The fit equation is also lacking in the methods section, making it unable to assess how the data were fitted.)\nInterpretation of the structure and the biochemical data: With regard to mechanistic interpretation, the data likely reflect acceleration inorganic phosphate release rather than nucleotide hydrolysis per se, given that the employed assay quantifies free phosphate. This might be achieved by opening up the two RecA domains and what the authors obverse in the structure is the final open state.\nIn several NTPase systems, phosphate release constitutes the rate-limiting step, rather than NTP hydrolysis, as documented in studies from Alfred Wittinghofer and colleagues. This interpretation could also reconcile the observation that both ADP and ATP promote TREX-2/UAP56 binding and may account for the limited effect of the R678 mutant in the ATPase assay. This would be important to clarify also in the text because this is structural mechanistic study.\nAlthough the manuscript now refers to the studies by the Plaschka and Ren laboratories, the discussion remains very brief. Given that the Plaschka study has been available on bioRxiv for an extended period and is now published in Nature, a more substantive comparison appears warranted, and the citation should be updated accordingly. But I am not insisting here. But it would help readers to see similarities and differences if they are not experts from the field.", "reviewer_2": "This manuscript contains interesting data on the role of the TREX-2 complex in the removal of the DEAD-box ATPase UAP56 as an important step in the generation of an export-competent mRNP. The manuscript has been greatly improved on revision with considerable new data and controls added and now should be a considerable interest to the structural cell biology community. However, there are still several important changes that are needed to be made, especially to make it accessible to a wider audience:\n1. I found the summary figure (Fig 6) incomprehensible and this needs to be changed. The individual components were not identified clearly and showed little resemblance to their structure or size. What, for example, is the pair of large green ellipses? Are they the two lobes of UAP56? All components need to be identified in the figure and also in the legend. It was completely unclear what point this figure was trying to make and the authors would do well to make something similar to the figures in the two reviews (see point 5 below) or the other recent papers on this subject (point 4 below). Moreover, it is incorrect to show the mRNA as a wavey line – the mRNA almost certainly folds up into a compact structure generated by stem loops, etc and by the proteins (such as ALYREF, etc) bound to it.\n2. The first sentence of the legend to Fig 6 is incorrect. Export-competent mRNA is only generated when UAP56 leaves and NXF1-NXT1 is attached. Why not say something like:\nDuring their passage through the nucleus, mRNAs are bound by a spectrum of RNA-binding proteins including the CBC and ALYREF, to form a protein decorated mRNP\n3. The Hohmann et al preprint (current reference 59) that is referenced as BioRxiv has now been published in Nature (Hohmann, U. et al. 2025. An ATP-gated molecular switch orchestrates human messenger RNA export. Nature, https://doi.org/10.1038/s41586-025-09832-z) and in places is rather different to the preprint with considerable new data, changes in emphasis, and changes in authorship. Therefore, this reference should be changed to guide the reader to the final published work.\n4. The earlier work in this area (references 59 and 60) is currently only referenced in the Discussion and should also be included in the Introduction to illustrate the existing work on which the current authors are now building.\n5. Two recent reviews have been published in this area (Faraway R, Zenklusen D, Plaschka C. 2025. Mechanisms of Messenger RNA Packaging and Export. Annu Rev Cell Dev Biol. 2025 41:479-504. doi: 10.1146/annurev-cellbio-101123-045256 and Stewart M. 2025. From transcription to export: mRNA's winding path to the cytoplasm. Trends Biochem Sci. 50:748-765. doi: 10.1016/j.tibs.2025.06.004.)and these should also be included in both the Introduction and the Discussion. These two reviews would be especially helpful in describing the earlier stages of mRNP formation. This would enable the present authors to concentrate on the new data they have generated about the role of TREX-2 in removing UAP56 and leading to the formation of an export-competent mRNP.", "reviewer_3": "Overall, I am mostly satisfied with the authors' response, but a few fixes will make it unambiguously satisfactory. Please (1) address the URH49 point exactly as asked by adding a small URH49 ATPase ± TREX-2M assay (n≈3 with statistics) showing lack of stimulation; if this is not feasible, explicitly qualify the claim to “consistent with functional specificity” and explain why. (2) Specify the subunit and residue numbers for the metal-site mutants (e.g., “PCID2 H43/H73A”), and briefly justify why these residues report on the proposed ion site. (3) Describe the kinetic parameters directly in the rebuttal (mean ± SD/SEM, n, test, P for kcat and KM, not just Vmax), and (4) please add a clear pointer to a Statistics subsection that defines n, shows individual data points, and names the exact tests used. Once this is accomplished, this manuscript will be suitable for publication."}, "author_response": "Point-by-Point response to reviewers’ comments\nWe are grateful to all three reviewers for their constructive and insightful\ncomments. Every concern raised has been addressed in our revision, and the\nmanuscript has been updated accordingly.\nReviewer #1:\nStructural Modeling\n1) Zn²⁺ Coordination\nThe proposed coordination of Zn²⁺ in the structural model appears\ninsufficiently supported by the current evidence. In most proteins, Zn²⁺ adopts\ntetrahedral coordination geometry with four ligands; catalytic zinc centers, as\nobserved for example in carbonic anhydrase, may involve a water or\nhydroxide ligand in place of a canonical side-chain donor. Such cases\nrepresent enzymatic rather than purely structural zinc sites and therefore may\nnot serve as an appropriate analogy in the present context.\nTwo histidine residues display orientations that are, in principle, compatible\nwith metal coordination, although the observed metal–ligand distances of\napproximately 2.2 Å are unusually long. Furthermore, one of these histidines\nis not fully defined in the electron density. The third putative histidine ligand\nappears incorrectly modeled, with the CE atom positioned closer to the metal\nion than the imidazole nitrogens, which is inconsistent with canonical\ncoordination chemistry.\nGiven the lack of independent evidence for Zn²⁺ or other divalent ions, such\nas atomic absorption spectroscopy or related analytical approaches, the\ninclusion of a Zn²⁺ ion in the structural model does not appear experimentally\njustified. As the metal ion is not essential for the mechanistic interpretation\npresented in the manuscript, its removal from the model is recommended.\nResponse: We fully accept this point and have removed the Zn2+ ion from our\nstructural model. We sincerely appreciate your expert guidance during model\nrefinement, which has significantly enhanced our understanding of\nmetalloprotein structural principles.\n2) Arg678\nThe conformation of Arg678 could be correctly modeled. But the modeling is\nnot ideal: The adenine base engages in π–π interactions with Phe65; however,\nthe two aromatic systems adopt an orientation of approximately 45°, rather\nthan a stacked (0°) or T-shaped (90°) arrangement typically\nobserved/expected. This geometry raises concerns that the refinement\nparameters (weight of geometry over map) may not have adequately chosen.\nThis is an important region and has a central role of this contact in the\nmechanistic analysis.\nSimilarly, the proposed hydrogen bond between Arg678 and adenine is\nuncertain. Although the distance is reasonable, the angle and displacement\ndeviate from ideal parameters, rendering the interaction ambiguous.\nResponse: We appreciate these insightful observations regarding the Arg678\nconformation and interactions. In response, we have re-evaluated and\nadjusted the refinement weights for geometry versus the map to optimize\nmodel accuracy. This has led to a correction of the Phe65 orientation,\nresulting in a biologically plausible geometry for its aromatic interaction with\nthe adenine base. The conformation of Arg678, including its angle and\ndisplacement relative to adenine, has also been rigorously refined.\nRegarding the proposed hydrogen bond between Arg678 and adenine,\nwe concur that the initial assignment was insufficiently supported by the\nmodel’s geometry. To reflect this ambiguity, the interaction is now explicitly\ndescribed as a “potential hydrogen bond” in both the revised manuscript text\nand the associated figure legends.\nBiochemical Data\nPulldown Experiments: The revised pulldown assays appear robust and\nsupport the interaction model presented.\nATPase Analysis: The ATPase measurements are generally convincing.\nNevertheless, the decision to fit the data with a Hill equation introduces\nadditional parameters without clear justification. Did a regular Michaelis\nMenton model not fit the data? In the extended data Km and Vmax values are\nreported, but the Hill coefficient (n) is not reported, but forms an integral part\nof the fitted model. It would be informative to report n and to indicate whether\nit is comparable for UAP56 and UAP56–TREX-2 complexes. Biochemically, I\ndo not understand why a Hill Model should fit the data better. More\ninformation would be required, ideally comparison of the two fits side by side.\n(The fit equation is also lacking in the methods section, making it unable to\nassess how the data were fitted.)\nResponse: We appreciate your rigorous assessment of our kinetic modeling\napproach and fully acknowledge the valid concerns regarding the use of the\nHill equation. Following thorough re-examination of our methodology and\ndatasets, we confirm that all ATPase kinetics were exclusively analyzed using\nthe standard Michaelis-Menten model—the reference to a “Hill equation”\nresulted from an inadvertent error during the back-and-forth editing of our\noriginal manuscript. We confirm the Michaelis-Menten model provided\nexcellent fits for all datasets. The kinetic parameters for UAP56 and DDX39A\nare listed in updated Extended Data Table 2. We deeply regret this oversight\nand any resulting confusion. The manuscript text and relevant figure legends\nhave been revised accordingly.\nInterpretation of the structure and the biochemical data: With regard to\nmechanistic interpretation, the data likely reflect acceleration inorganic\nphosphate release rather than nucleotide hydrolysis per se, given that the\nemployed assay quantifies free phosphate. This might be achieved by\nopening up the two RecA domains and what the authors obverse in the\nstructure is the final open state.\nIn several NTPase systems, phosphate release constitutes the rate-limiting\nstep, rather than NTP hydrolysis, as documented in studies from Alfred\nWittinghofer and colleagues. This interpretation could also reconcile the\nobservation that both ADP and ATP promote TREX-2/UAP56 binding and may\naccount for the limited effect of the R678 mutant in the ATPase assay. This\nwould be important to clarify also in the text because this is structural\nmechanistic study.\nResponse: We appreciate the reviewer’s critical insight regarding the\nmechanistic interpretation of our biochemical data. Our malachite green assay\nspecifically measures inorganic phosphate (Pi) release, not direct ATP\nhydrolysis. We agree the observed TREX-2 stimulation of UAP56 likely\nreflects accelerated ADP-Pi release rather than enhanced hydrolysis per se.\nThis interpretation reconciles several key observations: 1) both ADP and ATP\npromote TREX-2/UAP56 complex formation (Extended Data Fig. 1h); 2) the\nR678A mutation shows limited impact on ATPase activity (Fig. 5a, b); and 3)\nrecent biochemical evidence demonstrates TREX-2 activates UAP56 by\npromoting ADP-Pi release (Plaschka lab, Nature 2025 Nov 6. doi:\n10.1038/s41586-025-09832-z). We have revised the manuscript to clarify this\nmechanistic point, including reference to the Plaschka lab findings.\nAlthough the manuscript now refers to the studies by the Plaschka and\nRen laboratories, the discussion remains very brief. Given that the Plaschka\nstudy has been available on bioRxiv for an extended period and is now\npublished in Nature, a more substantive comparison appears warranted, and\nthe citation should be updated accordingly. But I am not insisting here. But it\nwould help readers to see similarities and differences if they are not experts\nfrom the field.\nResponse: Point accepted. We have updated the citation and extended our\ndiscussion on these related studies (see changes in Introduction and\nDiscussion).\nWe thank this reviewer for his/her constructive comments.\nReviewer #2:\nThis manuscript contains interesting data on the role of the TREX-2 complex\nin the removal of the DEAD-box ATPase UAP56 as an important step in the\ngeneration of an export-competent mRNP. The manuscript has been greatly\nimproved on revision with considerable new data and controls added and now\nshould be a considerable interest to the structural cell biology community.\nHowever, there are still several important changes that are needed to be\nmade, especially to make it accessible to a wider audience:\nResponse: We thank the reviewer for the encouraging comments.\n1. I found the summary figure (Fig 6) incomprehensible and this needs to be\nchanged. The individual components were not identified clearly and showed\nlittle resemblance to their structure or size. What, for example, is the pair of\nlarge green ellipses? Are they the two lobes of UAP56? All components need\nto be identified in the figure and also in the legend. It was completely unclear\nwhat point this figure was trying to make and the authors would do well to\nmake something similar to the figures in the two reviews (see point 5 below)\nor the other recent papers on this subject (point 4 below). Moreover, it is\nincorrect to show the mRNA as a wavey line – the mRNA almost certainly\nfolds up into a compact structure generated by stem loops, etc and by the\nproteins (such as ALYREF, etc) bound to it.\nResponse: We thank the reviewer for these critiques on Figure 6. We agree\nthat the original schematic lacked clarity and accurate representation of the\nindividual components. In the revised version, each component is now clearly\nlabeled within the figure and thoroughly described in the legend. Specifically,\nUAP56 is shown (in salmon) with its two RecA domains and N-terminal\ndomain (NTD); in the packaged mRNP, UAP56 is clamped on the mRNA, and\nupon reaching the NPC, it is captured by TREX-2 (depicted as a pair of large\ngreen ellipses) via its NTD. The revised figure also highlights that TREX-2\nactivates the ATPase activity of UAP56, promoting mRNP release, which is\nsubsequently captured by NXF1-NXT1 (indicated in black). We apologize for\nany confusion caused by the previous version, and we hope the revised figure\nand labeling meet the reviewer's expectations.\n2. The first sentence of the legend to Fig 6 is incorrect. Export-competent\nmRNA is only generated when UAP56 leaves and NXF1-NXT1 is attached.\nWhy not say something like:\nDuring their passage through the nucleus, mRNAs are bound by a spectrum\nof RNA-binding proteins including the CBC and ALYREF, to form a protein\ndecorated mRNP.\nResponse: Thank you for highlighting this important point. We fully accept\nyour correction and have revised the wording here.\n3. The Hohmann et al preprint (current reference 59) that is referenced as\nBioRxiv has now been published in Nature (Hohmann, U. et al. 2025. An ATP-\ngated molecular switch orchestrates human messenger RNA export. Nature,\nhttps://doi.org/10.1038/s41586-025-09832-z) and in places is rather different\nto the preprint with considerable new data, changes in emphasis, and\nchanges in authorship. Therefore, this reference should be changed to guide\nthe reader to the final published work.\nResponse: Done.\n4. The earlier work in this area (references 59 and 60) is currently only\nreferenced in the Discussion and should also be included in the Introduction\nto illustrate the existing work on which the current authors are now building.\nResponse: Thank you for this thoughtful suggestion. We have now cited\nthese two studies in both the Introduction, the Main text and the Discussion.\n5. Two recent reviews have been published in this area (Faraway R,\nZenklusen D, Plaschka C. 2025. Mechanisms of Messenger RNA Packaging\nand Export. Annu Rev Cell Dev Biol. 2025 41:479-504. doi: 10.1146/annurev-\ncellbio-101123-045256 and Stewart M. 2025. From transcription to export:\nmRNA's winding path to the cytoplasm. Trends Biochem Sci. 50:748-765. doi:\n10.1016/j.tibs.2025.06.004.) and these should also be included in both the\nIntroduction and the Discussion. These two reviews would be especially\nhelpful in describing the earlier stages of mRNP formation. This would enable\nthe present authors to concentrate on the new data they have generated\nabout the role of TREX-2 in removing UAP56 and leading to the formation of\nan export-competent mRNP.\nResponse: Done. These two recent reviews (ref. 1 and 2) are now referenced\nin both the Introduction and the Discussion.\nWe thank this reviewer for his/her constructive comments.\nReviewer #3:\nOverall, I am mostly satisfied with the authors' response, but a few fixes will\nmake it unambiguously satisfactory. Please (1) address the URH49 point\nexactly as asked by adding a small URH49 ATPase ± TREX-2M assay (n≈3\nwith statistics) showing lack of stimulation; if this is not feasible, explicitly\nqualify the claim to “consistent with functional specificity” and explain why.\nResponse: Thank you for highlighting this important point regarding URH49.\nIn the first round of revision, we did not perform an ATPase assay for URH49\nbecause our in vitro pulldown assay (Extended Data Fig. 1i) demonstrated\nsubstantially weaker binding of URH49 to TREX-2M compared to UAP56.\nHowever, in response to your suggestion, we have now conducted the URH49\nATPase assay in the absence/presence of TREX-2M. Surprisingly, our new\ndata show that TREX-2 is able to stimulate the ATPase activity of URH49 to a\nsimilar extent as observed with UAP56 (Extended Data Table -2; Extended\nData Fig. 10d, e, see below)\nTo further explore the functional relevance, we tested whether ATPase\nstimulation is linked to RNA release from URH49 by TREX-2. This is indeed\nthe case: although URH49 binds poly-U RNA less efficiently, TREX-2 can\nnevertheless promote RNA release from URH49-clamped complexes\n(Extended Data Fig. 10e). These findings suggest that URH49, like UAP56,\nis regulated by TREX-2 through weak but specific (or transient) interactions.\nGiven previous reports that URH49 is responsible for exporting a subset of\nmRNA cargo, our data suggest that both URH49 and UAP56 share a common\nmechanism for mRNP release upon docking to the NPC-anchored TREX-2\ncomplex, and the cargo specificity is likely determined at earlier stages of\nmRNP assembly.\nWe have included these new results and their interpretation in the\nDiscussion section of the revised manuscript.\n(2) Specify the subunit and residue numbers for the metal-site (e.g., “PCID2\nH43/H73A”), and briefly justify why these residues report on the proposed ion\nsite.\nResponse: Thank you for your suggestion. The specific mutant examined is\nUAP56 H43A/H73A. These histidine residues were selected because they\ncoordinate the putative metal site in the structural model and are highly\nconserved in UAP56 across species. However, in response to reviewer #1’s\ncomments, we have removed the metal ion from our model, as the current\nresolution is insufficient to conclusively determine the presence or identity of a\ncoordinated metal. The text and figure legend have been updated accordingly.\n(3) Describe the kinetic parameters directly in the rebuttal (mean ± SD/SEM,\nn, test, P for kcat and KM, not just Vmax), and (4) please add a clear pointer\nto a Statistics subsection that defines n, shows individual data points, and\nnames the exact tests used. Once this is accomplished, this manuscript will\nbe suitable for publication.\nResponse: Point accepted. We have now added a Statistics subsection to\nthe Methods that defines sample size (n), presents individual data points in all\nrelevant figures, and specifies the exact statistical tests used for each\nanalysis. The kinetic parameters are as follows (mean ± SEM, n = 3):\n1) UAP56: Vmax = 1.43 ± 0.48 nmol·mg⁻¹·min⁻¹, Km = 20.81 ± 31.44 μM,\nkcat = 0.00119 s⁻¹\n2) UAP56-TREX-2M: Vmax = 12.36 ± 0.46 nmol·mg⁻¹·min⁻¹, Km = 57.06 ±\n6.14 μM, kcat = 0.0103 s⁻¹\n3) URH49: Vmax = 2.38 ± 0.10 nmol·mg⁻¹·min⁻¹, Km = 26.40 ± 4.66 μM,\nkcat = 0.00199 s⁻¹\n4) URH49-TREX-2M: Vmax = 10.78 ± 0.24 nmol·mg⁻¹·min⁻¹, Km = 105.91 ±\n5.41 μM, kcat = 0.00898 s⁻¹\nGroup comparisons were analyzed using unpaired two-tailed Student’s t-test.\nSignificant increases in kcat and Km were observed upon addition of TREX-\n2M to UAP56 (p<0.0001) and URH49 (p=0.0043).\nPoint-by-Point response to reviewers’ comments\nReviewer #1:\nThe authors have addressed all the remaining concerns I had. They also\nadapted and expanded the discussion appropriately.\nResponse: We sincerely thank the reviewer for the constructive feedback and\nsupport throughout the review process.\nReviewer #3:\nThe revision is substantially improved, and my major concerns are addressed.\nIn particular, the authors now directly test URH49/DDX39A and show TREX-2-\ndependent stimulation comparable to UAP56, and they appropriately revise\nthe model to place helicase “specificity” upstream of ATPase activation. This\nresolves the key mechanistic ambiguity raised in my initial review.\nResponse: We are pleased that the mechanistic ambiguity raised in the initial\nreview has now been addressed. We sincerely thank the reviewer for the\nconstructive feedback and support throughout the review process.\nTwo minor issues remain that I recommend the authors address for clarity and\ninternal consistency. First, the manuscript should be explicit about the\nterminology used for the malachite green assay readout. This assay reports Pi\nrelease/ATPase activity, and the Discussion appropriately notes that TREX-2\nmay accelerate nucleotide/product release rather than the chemical hydrolysis\nstep. However, parts of the Results/figure legends still describe the effect as\n“enhanced ATP hydrolysis.” I recommend standardizing phrasing throughout\nto “ATPase/Pi release rate” (or explicitly defining the readout once and using\n“ATPase activity” consistently thereafter), including in the model figure/legend.\nResponse: We thank the reviewer for this helpful comment. As suggested,\nwe have revised the relevant sections of the Results and figure legends to\navoid the phrase “ATP hydrolysis” and instead clearly define the malachite\ngreen assay readout as reflecting ATPase/Pi release rate. The term “ATPase\nactivity” is now used consistently throughout the manuscript, with an explicit\ndefinition provided upon first use.\nSecond, the kinetic/statistical reporting would benefit from one additional\ntransparency step. The authors report Km and Vmax as mean ± SEM (n=3)\nbut provide kcat values without uncertainty and state that an unpaired t-test\nwas used. I recommend reporting kcat with an associated error estimate\n(replicate-wise kcat values with mean ± SEM/SD, or clearly described error\npropagation from Vmax) and briefly clarifying whether replicates were\nnaturally paired (same preparation measured ± TREX-2). If pairing applies, a\npaired analysis (or an explicit justification for unpaired testing) would be more\nappropriate.\nWith these minor clarifications implemented, I would be satisfied that the\nrevision fully addresses my comments.\nResponse: We thank the reviewer for this thoughtful suggestion. We agree\nthat improved reporting of kinetic parameter uncertainties and replicate\nstructure enhances transparency and rigor. Accordingly, we have now\ncalculated and reported kcat values with associated uncertainty as mean ±\nSEM (n = 3) (updated Supplementary Table 2), derived from the same\nbiological replicates used for Vmax determination.\nFurthermore, we confirm that the three experimental replicates were based on\nthe same preparation, with each independent measurement performed in\nparallel in the presence or absence of TREX-2M. Given this paired\nexperimental design, we have followed the reviewer’s recommendation and\nreplaced the unpaired t-test with a paired t-test for the relevant comparisons.\nThe updated statistical analysis is now described in the Methods section, and\nappropriate changes have been made in the figure legends.\nWe appreciate the reviewer’s insightful feedback, which has helped us\nimprove the clarity and statistical rigor of our kinetic data presentation."}]

Decision letter