Fig 2: Spliced reporter mRNAs are less dependent on TPR for their nuclear export. (A) Schematic of the ftz reporter with and without an intron (±i). (B, C) Control- or TPR-depleted cells were transfected with various ftz reporter plasmids. 18–24 h later the cells were fixed and the mRNA was visualized by FISH. Representative images are shown in (B), quantification is shown in (C). Scale bar = 10 μm. Each bar is the average and standard error of three independent experiments, each experiment consisting of at least 60 cells. Note that TPR-depletion caused nuclear accumulation of the ftz-Δi mRNAs, but not the spliced ftz-i mRNAs. (D) Schematic of βG reporters with the ftz intron inserted in place of the endogenous βG introns. (E, F) Control- or TPR-depleted cells were transfected with various βG reporter plasmids. 18–24 h later the cells were fixed and the mRNA was visualized by FISH. Representative images are shown in (E), and the nuclear and cytoplasmic ratios were quantified in (F). Scale bar = 10 μm. Each bar is the average and standard error of three independent experiments, each experiment consisting of at least 60 cells. Note that TPR-depletion caused nuclear accumulation of βG mRNAs with the ftz introns, βG-ftz-2i, but not the βG reporter with endogenous introns. Student t-test was performed in C and F, * P < P < 0.05, ** P < 0.01, *** P < P < 0.001.

Fig 3: Aberrant Tpr expression correlates with altered NPC counts in hippocampal NSPCs in a mouse model of AD. (A) Schematic diagram illustrating the workflow of the automated NPC counting in NSPCs in hippocampal tissue sections. Scale bar: 2 µm (B) Immunolabeling for FG-Nups (red) in Tpr-depleted SVZ-derived NSPCs and control cells in vitro. Quantification of NPC counts in Tpr-depleted SVZ-derived NSPCs compared with controls (n = 3 mice, Ctrl siRNA = 30 total Nuclei and Tpr siRNA = 33 total Nuclei were analyzed). Scale bar: 2 µm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, * p < 0.05). (C) Immunolabeling for Tpr (red) in the SGZ of the hippocampus of 5xFAD and WT mice sacrificed at 6 weeks of age. Quantification of NPC counts in the hippocampal SGZ of 5xFAD and WT mice sacrificed at 6 weeks of age (n = 3 mice per condition, WT = 32 total Nuclei and 5xFAD = 29 total Nuclei were analyzed). Scale bar: 2 µm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, ** p < 0.01). (D) Immunolabeling for Tpr (red) in the SGZ of the human hippocampus of AD individuals compared with healthy age-matched controls. Quantification of Tpr expression in single NSPCs of the human hippocampal SGZ in AD individuals and in healthy age-matched controls (AD (AD 1–5): n = 5; healthy controls (Ctrl 1–5): n = 5, AD = 53 total single cells and healthy controls = 44 single cells were analyzed). Scale bar: 2 µm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, ** p < 0.01).

Fig 4: Translocated promoter region (Tpr) phosphorylation determines subcellular localization in neural stem/precursor cells (NSPCs). (A) Scheme illustrating the localization of Tpr and phenylalanine-glycine repeat nucleoporins (FG-Nups) in nuclear pore complexes (NPCs). (B) Comparative imaging of NPCs in subventricular zone- (SVZ-) derived NSPCs immunolabeled with Tpr (red) and FG-Nups (green) using confocal laser scanning microscopy (CLSM) (left) and SR Airyscan microscopy (right). Enlargements at the right of each panel indicate representative resolutions of the Tpr and FG-Nup signals, and white arrowheads indicate the distinct localizations of TPR and FG-Nups resolved with SR Airyscan microscopy. Nuclei are stained with DAPI (blue). Scale bars: 2 µm, 500 nm (enlargement). Representative images from three independent experiments (10 nuclei were analyzed in total). (C) Scheme illustrating Tpr localization relative to FG-Nups in an NPC after SR Airyscan microscopy and Imaris 3-D processing. (D) Representative SR Airyscan microscopy and Imaris-processed image of an SVZ-derived NSPC nucleus immunolabeled for Tpr (red) and FG-Nups (green). Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 2 µm. (E) 3-D visualization revealing distinct localizations for Tpr (red) and FG-Nups (green) in the NPC of SVZ-derived NSPCs. Representative image from three independent experiments (10 nuclei were analyzed in total). Scale bar: 1 µm. (F) 3-D visualization of confocal images revealing nuclear phospho-Tpr (P-Tpr, red) localization in NSPCs in vitro. Representative images from three independent experiments (10 nuclei were analyzed in total). Scale bars: 2 µm, 500 nm (enlargement). (G) Immunolabeling for P-Tpr (red) in combination with doublecortin (DCX) (green, marker for neuroblasts) in the subgranular zone (SGZ) of the hippocampus in adult wild-type (WT) mice. Enlargements indicate a DCX+ cell with nuclear P-Tpr and a DCX−cell with no nuclear P-Tpr. Quantification of nuclear or nuclear envelope P-Tpr localizations in Ki-67+, DCX+, and NeuN+ cells in the SGZ of the hippocampus in adult WT mice (n = 4 mice, 79–98 single cells were analyzed per Ki-67, DCX, or NeuN condition). Scale bars: 3 µm, 5 µm (enlargement). Values are the mean ± SEM (p-values calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, * p < 0.05, ** p < 0.01) (H) Electron microscopy determining the localization of Tpr and P-Tpr in comparison to FG-Nups in NSPCs of the hippocampal SGZ in adult WT mice. Quantification of nuclear or nuclear envelope Tpr- and P-Tpr-IR in NSPCs of the hippocampal SGZ in adult WT mice (n = 3 mice, 27–30 single cells were analyzed per Tpr or P-Tpr localization). Scale bar: 100 nm. Values are the mean ± SEM (p-values calculated by unpaired Student’s t-test, * p < 0.05, ** p < 0.01).

Fig 5: mRNAs that are retained in TPR-depleted cells accumulate in nuclear speckles and are bound to Nxf1. (A–C) Control- or TPR-depleted cells were microinjected with plasmid ftz-Δi reporter plasmids. After the indicated times, cells were fixed and stained for ftz mRNA by FISH and for the speckle marker SC35 by immunofluorescence. (A) An example of cells fixed 2 h post-injection with each row represents a single field of view with blue arrow pointing to examples of ftz/SC35 co-localization. Scale bar = 10 μM. (B) The degree of ftz/SC35 co-localization by Pearson correlation coefficient analysis was quantified as described in (24). As a control, the co-localization of microinjected 70 kD dextran conjugated to Oregon Green (‘OG’) with SC35 speckles was also tabulated. Each bar is the average and standard error of three independent experiments, each consisting of 150–200 nuclear speckles (see Materials and Methods for more details). (C) The amount of ftz-Δi mRNA present in nuclear speckles (defined by the brightest 10% pixels in the nucleus, using SC35 immunofluorescence - described in (24)) as a percentage of either the total nuclear (‘spec/nuc’) or total cellular (‘spec/total’) mRNA level in cells 1 h post-microinjection. Each data point represents the average and standard error of the mean of 10–20 cells. (D) Western blot showing that FLAG-TPR was successfully expressed following doxycycline treatment for 4 h. (E) Flp-In FLAG-TPR U2OS cells were treated with doxycycline for 4 h, then lysed and immunoprecipitated with anti-FLAG M2 or protein A beads. 2% of the input lysate, Immunoprecipitates (IP), or 10% of the unbound fraction were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Note the presence of IgG heavy band (*) in the anti-tubulin immunoblot. (F) Same as (E), except following doxycycline treatment, the Flp-In FLAG-TPR U2OS cells were transfected with either βG-i or βG-ftz-2i reporter plasmids for 18 to 24 h. These cells were then lysed and immunoprecipitated as in (E). The amount of βG mRNA and the 7SL ncRNA in the immunoprecipitates were quantified by RT-qPCR. Each bar is the average and standard error of at least three independent experiments. Student t-test was performed, * P < 0.05. (G) Control- or TPR-depleted cells were co-transfected with FLAG-Nxf1 and either the βG-i or βG-ftz-2i reporter plasmids for 18–24 h, then lysed and immunoprecipitated with anti-FLAG M2 beads or protein A beads. The amount of βG and the 7SL ncRNA in the immunoprecipitates were quantified by RT-qPCR. Each bar is the average and standard error of at least four independent experiments.