Nucleoporin TPR is an integral component of the TREX-2 mRNA export pathway

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.

Analysis of BSK-NUPs interdependence in interphase of pre-existing NPC (a) and post-mitotic NPC (b) upon loss of NUP153, TPR, or NUP50. Assembled NPC is stable, and loss of individual basket nucleoporin does not lead to loss of other nucleoporins from the NPC, except the case of NUP50, which requires NUP153 for its localization (Supplementary Data 1). NUP153 does not control localization of TPR in the pre-existing NPC, but is essential for TPR recruitment to NPC after mitotic NPC re-assembly. c, Protein regions of RANGAP1 detected by respective antibody.      Dynamics of exchange between NPC-bound and nucleoplasmic NXF1 before and after TPR loss.   Scale bar 10 µm. c, RNA localization of GANP-NXF1-specific transcripts, aen, ubl4a, and gadd45b. Corresponding transcripts were down-or upregulated upon NXF1 or GANP but not TPR loss. All RNA transcripts were enriched within the nucleus upon NXF1 or GANP loss. Note 2 h of TPR depletion affects GANP localization on the NE and can affect expression of GANP-specific transcripts at later time points. Scale bar: 10 µm.    Label-free quantitation was based on areas under peptide precursor traces (MS1) that were aligned with the Minora feature detection function in PD2.2. For TMT sets, report ion intensities of ten channels were normalized at the total peptide amount level. Then relative quantitation of individual proteins was calculated using the normalized report ion intensities of unique peptides with <1% FDR and >30% isolation purity.

Oligonucleotides PCR product length
We determined the protein-level fold changes based on the median of peptide-level fold changes from the Proteome Discoverer-produced abundances in handling both TMT and Labelfree results. Peptides that could be mapped to multiple proteins by Mascot were removed. We also discarded all keratin-related peptides based on the UniProt annotation. We separated peptides that mapped onto UniProt ID P52948 into NUP96 and NUP98, according to their mapping locations.
Peptides that are mapped to amino acids from 1 to 880 were counted for NUP96; the others were used for NUP98. To minimize the batch effect, we used the quartile normalization before calculation of fold changes in the Label-free quantification. Such fold changes were visualized after k-means clustering. In determining the optimal k parameter for clustering, we used "mclust" package to calculate Bayesian Information Criterion of the expectation-maximization model 6 . The quartile normalization, k means clustering, and data visualization were performed under R development environment (R Core Team, 2018).
Protein transport assay. AID TPR cell lines were seeded on Ibidi μ-Slide four well glass bottom slides in complete DMEM media. Cells were then changed to FluoroBrite DMEM (ThermoFisher) media and transfected with 500 ng of NLS-mCherry-LEXY plasmid (pDN122) (Addgene plasmid # 72655 7 ) using ViaFect (Promega) transfection reagent according to the manufacturer's protocol. After transfection, cells were kept in the dark for 24 h prior to imaging.
NLS-mCherry-LEXY positive cells were first excited with a 561 nm (20% of power was applied) laser line with 30 ms exposure and imaged every 30 seconds for 10 minutes. Next, the cells were exposed to 405 nm (20% of power was applied) laser line with 1 s exposure every 30 seconds to induce nuclear export of model substrate, which was monitored for 15 minutes. 405 nm laser was then shut off to induce nuclear import of model substrate, which was monitored for 20 minutes.
The cells were then exposed to 405 nm laser again to induce another round of nuclear export of model substrate for 15 minutes. During the course of the experiment, cells were imaged every 30 seconds using 561 nm laser to follow mCherry signal of the model substrate.
Image analysis was performed on Volocity (PerkinElmer) and ImageJ (National Institutes of Health) software with Time Series Analyzer V3 plugin and ROI Manager dialog box. We for 10 min at 16 0 C. SDS-PAGE and Western blotting were performed as described elsewhere 8 . In brief, the protein samples were separated on 4-20% SDS-PAGE or Bolt™ 8% Bis-Tris gels (Invitrogen) for 1 h and blotted onto PVDF membrane. The membrane was blocked in 5% non-fat milk for 1 h with the following incubation with the primary antibody overnight at 4 0 C. The membrane was rinsed and probed for 2 h with the secondary antibodies conjugated to HRP with dilution 1:15000 in 1x TN buffer (150 mM NaCl, 10 mM TrisHCl, pH 7.5, 0.1% Tween 20).