Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export - PubMed (original) (raw)

Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

Lauren Duan et al. Cell Rep. 2022.

Abstract

Nuclear clearance of the RNA-binding protein TDP-43 is a hallmark of neurodegeneration and an important therapeutic target. Our current understanding of TDP-43 nucleocytoplasmic transport does not fully explain its predominantly nuclear localization or mislocalization in disease. Here, we show that TDP-43 exits nuclei by passive diffusion, independent of facilitated mRNA export. RNA polymerase II blockade and RNase treatment induce TDP-43 nuclear efflux, suggesting that nuclear RNAs sequester TDP-43 in nuclei and limit its availability for passive export. Induction of TDP-43 nuclear efflux by short, GU-rich oligomers (presumably by outcompeting TDP-43 binding to endogenous nuclear RNAs), and nuclear retention conferred by splicing inhibition, demonstrate that nuclear TDP-43 localization depends on binding to GU-rich nuclear RNAs. Indeed, RNA-binding domain mutations markedly reduce TDP-43 nuclear localization and abolish transcription blockade-induced nuclear efflux. Thus, the nuclear abundance of GU-RNAs, dictated by the balance of transcription, pre-mRNA processing, and RNA export, regulates TDP-43 nuclear localization.

Keywords: CP: Molecular biology; RNA; TDP-43; amyotrophic lateral sclerosis; frontotemporal dementia; neurodegeneration; nuclear transport; splicing; transcription.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.. NVP2-induced RNA pol II inhibition promotes TDP-43 nuclear export

(A) Nascent RNA labeled with AF488-picolyl azide via 5-EU/click chemistry (top) and TDP-43 IF (bottom) in HeLa cells treated with NVP2 for 1 h. Arrows indicate rRNA puncta unaffected by NVP2. Scale bar, 25 μm. (B and C) Nascent RNA (AF488) mean nuclear intensity (B) and TDP-43 nuclear/cytoplasmic ratio (N/C) (C) expressed as percentage of DMSO control. Mean ± SD is shown for >2,000 cells/group in each of three biological replicates. IC50 at 1 h was calculated by non-linear regression. See also Figure S1. (D) Steady-state TDP-43 N/C in HeLa cells, mouse primary cortical neurons, human HFF1 fibroblasts, and human retinal pigment epithelial (RPE1) cells fixed and immunostained with mouse monoclonal anti-TDP-43. Mean ± SD is shown for 6–7 biological replicates. See also Figure S2. (E) NVP2-induced shift in TDP-43 N/C, shown as percentage of DMSO control, following 3 h with 250 nM NVP2. Mean ± SD is shown for 3–5 biological replicates. (F) RBP N/C in HeLa cells treated with 250 nM NVP2 for 30 min to 6 h. Curves were fit by non-linear regression using the mean of 3–4 biological replicates per RBP. See also Figure S3. In (D), (E), and (F), >2,000 cells/replicate for cell lines and 60–80 cells/replicate for neurons. ns, not significant; *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey’s test.

Figure 2.. TDP-43 exits the nucleus by passive diffusion in permeabilized cells

(A) Permeabilized cell TDP-43 passive export assay. See also Figure S4. (B) TDP-43 IF (top) and Hoechst (bottom) in HeLa cells fixed immediately (time 0) or 60 min post permeabilization, following incubation at 4°C or 37°C. The intensity histogram for TDP-43 images was normalized to time 0, prior to pseudo-color look-up table (LUT) (middle). Scale bar, 50 μm. (C) TDP-43 integrated nuclear intensity in permeabilized HeLa cells normalized to time 0. Mean ± SD is shown for >2,000 cells/well in five biological replicates. (D) TDP-43 IF (top) and Hoechst (bottom) in permeabilized HeLa cells incubated for 30 min at 4°C versus 37°C with 1,6-HD. The intensity histogram for TDP-43 images was normalized to untreated cells at 4°C. Scale bar, 50 μm. (E) TDP-43 integrated nuclear intensity in permeabilized HeLa cells incubated for 30 min at 4°C versus 37°C with 1,6-HD, normalized to untreated cells at 4°C. Mean ± SD is shown for >2,000 cells/well in three biological replicates. In (C) and (E), ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey’s test.

Figure 3.. Acute NXF1 ablation does not alter NVP2-induced TDP-43 nuclear export

(A) NXF1 immunoblot in DLD1-wild-type versus NXF1-AID cells treated with 0.5 mM auxin for 0–4 h. Note the increased molecular weight for AID-tagged (*)versus endogenous NXF1 (>). (B) PolyA-FISH (Cy3-OligodT(45)) in DLD1-wild-type versus NXF1-AID cells treated with 0.5 mM auxin. Scale bar, 25 μm. (C) PolyA-RNA N/C shown as percentage of wild-type untreated cells. (D) TDP-43 N/C in DLD1-wild-type cells (left) and NXF1-AID cells (right) pretreated with 0.5 mM auxin for 0–8 h, followed by 2 h auxin ± 250 nM NVP2. Data in both panels are normalized to untreated DLD1-wild-type cells (dotted line). (E) TDP-43 N/C in NVP2 treated versus untreated cells in DLD1-wild-type (black) versus NXF1-AID (red) cells. These are the same data as (D), adjusted for differences in the steady-state TDP-43 N/C ratio to permit comparison of NVP2-induced TDP-43 nuclear exit. In (C), (D), and (E), mean ± SD is shown for >3,000 cells/well in four biological replicates. ns, not significant; ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Sidak’s test.

Figure 4.. RNase and GU-rich oligomers induce TDP-43 nuclear efflux

(A) Addition of RNase A or RNA oligomers to the permeabilized cell TDP-43 passive export assay. (B) TDP-43 IF (top) and Hoechst (bottom) in permeabilized HeLa cells incubated for 30 min at 4°C ± RNase A. Scale bar, 25 μm. (C) TDP-43 integrated nuclear intensity in permeabilized HeLa cells incubated for 30 min at 4°C versus 37°C ± RNase A. Data are normalized to untreated cells kept at 4°C. ****p < 0.0001 versus untreated by two-way ANOVA with Tukey’s test. See also Figure S5. (D) TDP-43 integrated nuclear intensity (percent untreated) in permeabilized HeLa cells incubated for 30 min at 4°C with A16, (GU)8, or “AUG12.” (E) TDP-43 integrated nuclear intensity (percent untreated) in permeabilized HeLa cells incubated at 4°C, 25°C, or 37°C for 30 min with (GU)8. (F) Integrated nuclear intensity of nuclear RBPs (percent untreated) in permeabilized HeLa cells incubated for 30 min at 4°C with (GU)8. Note that TDP-43 data are the same as in (D). (G) Integrated nuclear intensity of nuclear RBPs (percent untreated) in permeabilized HeLa cells incubated for 30 min at 4°C with the indicated oligomers. (H) Transfection of live HeLa cells with protected RNA oligomers. (I) TDP-43 N/C (percent untreated) 5 h post transfection with protected A13 or (GU)6. (J) RBP N/C (percent untreated) 5 h post transfection with protected (GU)6. Note that TDP-43 data are the same as the (GU)6 data in (I). In (F), (G), and (J), RBP in red is the most closely predicted binding partner for that motif, green is moderately similar, and gray and black denote no or low predicted binding. In (C) to (J), mean ± SD is shown for >2,000 cells/well in 3–4 biological replicates. IC50 was calculated by non-linear regression. See Table S1 for oligomer sequences.

Figure 5.. Inhibition of pre-mRNA splicing promotes TDP-43 nuclear accumulation

(A–C) qRT-PCR quantification of DNAJB1 (A), RIOK3 (B), and BRD2 (C) introns, normalized to U6 small nuclear RNA in HeLa cells treated for 4 h with IGK or PLB. Mean ± SD is shown for four biological replicates. A single outlier was removed from IGK at 100 μM (fold change 18.78) via Grubbs test (α = 0.05). **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Dunnett’s test. (D) TDP-43 IF in HeLa cells treated with IGK for 4 h, followed by 2 h ± 250 nM NVP2. Scale bar, 25 μm. (E) TDP-43 N/C (percent untreated) in IGK or IGK + NVP2-treated cells. (F) TDP-43 N/C (same data as B) shown as percentage of IGK only to permit comparison of NVP2-induced nuclear exit. IC50 Was calculated by non-linear regression. (G) TDP-43 IF in HeLa cells treated with PLB for 4 h, followed by 2 h PLB ± 250 nM NVP2. Scale bar, 25 μm. (H) TDP-43 N/C (percent untreated) in PLB or PLB + NVP2-treated cells. (I) TDP-43 N/C (same data as E) shown as percentage of PLB only, to permit comparison of NVP2-induced nuclear exit. IC50 was calculated by non-linear regression. In (D) and (G), the intensity histogram for each image was independently maximized across the full range and a pseudo-color LUT was applied. In (E), (F), (H), and (I), mean ± SD of >2,000 cells/well in four biological replicates. ns, not significant; ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey’s test. See also Figure S6.

Figure 6.. Mutation or deletion of TDP-43 RRM domains disrupts TDP-43 nuclear localization

(A) V5-tagged TDP-43 RRM mutant constructs (top) and V5 IF (bottom) in transiently transfected TDP-43 CRISPR KO HeLa cells, after 1 h with 250 nM NVP2. The intensity histogram for each image was independently maximized across the full range and a pseudo-color LUT was applied. Scale bar, 25 μm. For plasmids see Table S2. See also Figure S7. (B) V5 N/C ratio of TDP-43WT versus RRM mutants in transiently transfected TDP-43 CRISPR KO HeLa cells, following 3 h with 250 nM NVP2. Data are normalized to DMSO-treated TDP43WT cells. Mean ± SD is shown of >1,000 cells/well in six biological replicates. (C) N/C fractionation and immunoblotting of TDP-43 CRISPR KO HeLa cells, transiently transfected with V5-tagged TDP-43 constructs and treated for 3 h with 250 nM NVP2. Lamin B1, nuclear marker; GAPDH, cytoplasmic marker. (D) V5 N/C normalized to DMSO-treated TDP43WT cells. Mean ± SD is shown for three biological replicates. In (B) and (D), ns denotes not significant; ****p < 0.0001 by two-way ANOVA with Tukey’s test.

Figure 7.. Mechanisms coupling TDP-43 localization with nuclear GU-rich pre-mRNA binding and abundance

(A) Under physiological conditions, nuclear TDP-43 is highly bound to GU-rich nuclear RNAs, limiting the pool of free TDP-43 capable of exiting nuclei by free diffusion through NPCs. (B) Perturbations that deplete nuclear GU-rich RNAs or impair TDP-43 RNA binding (e.g., transcriptional blockade, RNase, RRM mutations, or putative disruption of GU-RNA homeostasis in disease) cause an initial increase in nuclear free TDP-43, accelerating its passive export until reaching steady state at a lower N/C ratio. Insets depict the cellular GU-RNA N/C gradients that dictate TDP-43 localization.

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Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export - PubMed (original) (raw)