EMBO/soda-nel huggingface dataset:
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 1

Figure 1. Nup358 positive AL structures associate with P bodies and SGs, and Nup358 depletion leads to impairment of P body assembly

A. Confocal microscopic image showing HeLa cells immunostained for Nup358 (green, upper panel) or Nup214 (green, lower panel) and RanGAP1 (red) using specific antibodies. DNA was stained with Hoechst 33342 (blue). Scale bars, 10 μm.

B. COS-7 cells transfected with GFP-Nup358 and one of the cells was subjected for live imaging using confocal microscopy and the frames at indicated time points have been provided. Arrows denoted by different colors indicate individual AL structures. Note that one of the AL structures (marked by yellow arrow) originated from the NE (dotted line) and fused with another AL structure (marked by red arrow), which later fused with a different AL structure (marked by blue arrow). N, nucleus; C, cytoplasm. Scale bar, 5 μm.

C. Maximum intensity projection confocal image of a sodium arsenite treated HeLa cell immunostained for endogenous Nup358 (green), P bodies (red, Dcp1a as a marker) and SGs (blue, eIF3 as a marker) using specific antibodies. Scale bar, 10 μm. The histograms represent fluorescence intensity profile along the dotted arrows. Adjacent graph represents quantitative data showing percentage of P bodies (top) or SGs (bottom) associated with nuclear envelope (NE), Nup358 positive AL, with each other or unassociated with any of the other mentioned structures (others). Data are presented as mean SD (n = 3).

D. HeLa cells were transfected with Nup358 siRNA (siNup358) or Nup214 siRNA (siNup214). Cells were fixed and stained for endogenous Nup358 (green, upper panel) or Nup214 (green, lower panel) and endogenous P body marker (Dcp1a, red). Graph represents the quantitative data as mean ± SD. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact microscopically distinct P bodies and expressed as percentage. Western blot (WB) indicates the extent of Nup358 depletion and the level of Dcp1a.
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 2

Figure 2. Nup358 is required for miRNA function

A. Western analysis of HeLa cells, treated with control (siControl), Dicer (siDicer), Nup214 (siNup214) or Nup358 (siNup358) siRNA, for assessing the extent of protein depletion using indicated antibodies. Vinculin was used as loading control.

B. HeLa cells were initially transfected with indicated siRNAs, followed by renilla luciferase (RL) reporter constructs; RL-control (no let-7a binding site in the 3’UTR) or RL-3xBulge (3 imperfect let-7a binding sites in the 3’-UTR). Firefly luciferase (FL) was co-transfected to serve as internal control. RL/FL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.

C. HEK293T cells were transfected with indicated siRNAs, followed by pCMV-FL-miR30 (P) reporter with either pSUPER-control or pSUPER-miR30 constructs. RL was co-transfected as internal control. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.

D. HeLa cells were initially transfected with control (siControl), Dicer (siDicer) or Nup358 (siNup358) specific siRNAs, followed by FL constructs containing wild type (HMGA2-wt) or mutant (HMGA2-mut) HMGA2 3’-UTR, along with RL as internal control. Graph was plotted using data from three independent experiments. FL/RL luminescence ratio was calculated. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test.
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 4

Figure 4. AGO proteins interact with IR region of Nup358

A. Schematic diagram representing the domains and constructs of human (h) and zebrafish (z) Nup358 used in this study. TPR, tetratricopeptide repeat; LRR, leucine rich region; R1-R4, RanGTP binding domain; ZnF, zinc finger domains; IR, internal repeats; CHD, cyclophilin-homology domain. Dotted line indicates the deleted region in the indicated construct. FG and FxFG sequence positions are represented as short and long black lines, respectively. Amino acid positions are indicated in numbers. FL, full-length; N, amino-terminal region; M, middle region; C, carboxy-terminal region.

B. HEK293T cells were co-transfected with GFP-maltose binding protein (MBP)-control or GFP-tagged version of indicated Nup358 fragments along with HA-AGO2. Immunoprecipitation (IP) was performed using GFP antibodies and the presence of AGO2 was detected by western blotting (WB) using HA antibodies.

C. Cells were transfected with indicated constructs and IP and WB were performed as described for B.

D. HEK293T cells transfected with GFP-MBP (control), GFP-Nup358-C or GFP-Nup358-CΔIR (a mutant devoid of IR region) and HA-AGO2. IP and WB analyses were performed as indicated.

E. Cells were transfected with HA-tagged version of indicated AGO subfamily member and GFP-Nup358-IR. IP and WB analyses were performed using indicated antibodies.

F. Lysates from cells expressing GFP-control, GFP-human (h) Nup358-IR or GFP-zebrafish (z) Nup358-IR and HA-AGO2 were immunoprecipitated using GFP antibodies and the immunoprecipitates were analyzed for the presence of HA-AGO2 by western blotting.
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 5

Figure 5. SIM is the minimum region in Nup358-IR required for binding to AGO2

A. Schematic representation of human Nup358-IR region and the constructs used in this study. IR, internal repeats; SIM, SUMO-interacting motif; 1, 2, SIM1 and SIM2. Amino acids substituted in Ubc9 mutant are indicated with red asterisks.

B. HEK293T cells were transfected with indicated constructs and immunoprecipitation (IP) was performed using GFP specific antibodies and probed for HA-AGO2 by western blotting (WB) using HA antibodies. Endogenous RanGAP and Ubc9 were probed with specific antibodies.

C. Lysates prepared from cells expressing the indicated constructs were subjected to IP and WB using indicated antibodies. Presence of endogenous RanGAP and Ubc9 was determined by WB.

D. GFP-control, GFP-IR1+2 wild type or mutants were co-expressed with HA-AGO and IP and WB analysis were performed to detect the interaction using indicated antibodies.

E. Lysates prepared from cells expressing the indicated proteins were immunoprecipitated with GFP specific antibodies and the presence of specific proteins in the immunoprecipitates was detected by WB with indicated antibodies.
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 6

Figure 6. SIM provides a conserved platform for interaction with AGO family of proteins

A. Top panel; depicts the amino acid sequence corresponding to the Nup358-SIM1 region and substitutions introduced in the SIM1 mutant. Bottom panel: HEK293T cells were co-transfected with GFP-control, GFP-SIM1 or GFP-SIM1-mut along with HA-AGO2 and immunoprecipitation (IP) was performed with GFP specific antibodies and western analysis (WB) of the input lysate and immunoprecipitates were carried out using indicated antibodies.

B. Top panel; depiction of amino acid sequence corresponding to the SIMs in the indicated proteins used in this study. Hydrophobic residues involved in SUMO binding are indicated in red. Bottom panel; constructs expressing GFP-control or GFP-fused SIMs of indicated proteins were co-transfected with HA-AGO2 in HEK293T cells and IP and WB were performed to determine the extent of interaction.

C. HA-MBP-control, HA-AGO1 or HA-AGO2 was co-expressed with GFP-SIM1 and the cell lysates were subjected to IP with HA antibodies and WB analysis with indicated antibodies to detect the presence of proteins in the immunoprecipitates.

D. SIM1 directly interacts with AGO2. Bacterial lysates expressing MBP control or MBP-AGO2 and GST or GST-SIM1 were mixed and GST pull down assay was performed. The presence of proteins in the pull down samples was analyzed by WB using specific antibodies. The extent of GST pull down was monitored by coomassie staining of the membrane (coomassie).

E. SIM1 interacts with PIWI subfamily of proteins. HEK293T cells were co-transfected with indicated constructs expressing HA-tagged version of PIWI proteins and GFP-SIM1. IP was performed using HA antibodies. The immunoprecipitates were probed for the presence of GFP-SIM1 using GFP specific antibodies by WB.

F. Putative SIM-binding regions in AGO2. Three distinct superimpositions of AGO2 (grey) and SUMO (blue), in complex with Nup358-SIM1(red), are shown in cartoon representation rendered with Chimera [90]. The residue numbers on the AGO2 that could be the putative SIM-binding regions are labelled. To identify possible Nup358 binding sites on AGO2, the 3D structure of AGO2 (PDB ID: 4W5O) [89] was compared with that of SUMO (PDB ID: 1Z5S) [47].
Nup358 binds to AGO proteins through its SUMOinteracting motifs and promotes the association of target mRNA with miRISC (2016)
Figure 7

Figure 7. Functional relevance of the AGO-interacting motif in miRNA pathway

A. Left panel; HeLa cells were co-transfected with GFP-MBP (control), GFP-SIM1 or GFP-SIM1-mut (AGO interaction defective) along with RL-control or RL-3xbulge. Firefly luciferase was used as transfection control. Dual luciferase assay was performed. RL/FL luminescence ration was plotted after normalization with GFP-control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test. Right panel; Western blots showing the expression of transfected constructs using indicated antibodies. -tubulin was used as loading control.

B. Top panel; Reporter construct used in this study [61]. Bottom left panel; HEK293T cells were co-transfected with indicated expression constructs and the RL reporter. Firefly luciferase (FL) was used as transfection control. Dual luciferase assay was performed. The ratio of RL/FL luminescence was plotted after normalization with N-HA-MBP control. Data are presented as mean SD (n = 3), P‐value was calculated using Student's t‐test. Bottom right panel; Western blot for monitoring the expression levels of indicated proteins using antibodies. -tubulin was used as loading control.

C. Ectopic expression of AGO2 restores P body formation, but fails to rescue the impairment in miRNA function in Nup358 depleted cells. HeLa cells were transfected with control (siContriol) or Nup358 (siNup358) specific siRNA, followed by indicated constructs. Left panel: Quantitative representation showing the number of cells showing microscopically distinct P bodies in different indicated conditions. The data was obtained from three independent experiments, and in each experiment, 100 cells were counted from different fields for the presence of intact P bodies and expressed as percentage. Middle panel: HeLa cells were transfected with indicated siRNAs, followed by expression constructs and miRNA RL reporters. FL was used as transfection control. The ratio of RL/FL luminescence was calculated. Data are presented as mean SD (n = 3), P‐value was obtained using Student's t‐test. Right panel: Western blot analysis for the relative expression of indicated constructs in HEK293T cells. -tubulin is used as loading control.

D. HeLa cells were transfected with siControl or siNup358 and subjected to membrane flotation assay. Fractions were collected and analyzed for the presence of indicated proteins by western blotting.

E. Model for the role of Nup58 in miRNA-mediated translational suppression. Exported mRNAs could be sorted for miNRA-mediated suppression at two potential sites in the cytoplasm; Site 1 - the nuclear pore complex (NPC) as a part of nuclear envelope (NE) and / or Site 2 - annulate lamellae (AL) as a part of endoplasmic reticulum (ER). Nup358, a component of the NPC and AL, plays an important role in the coupling of target mRNA with miRISC.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 1

Figure 1. Repression of telomerase activity in ALT+ hybrids and progressive silencing of hTR gene in SI24/ALT+ cells.

A. Left: Telomerase activity measured using ddTRAP assay. The horizontal pink lines represent the threshold used in these samples for background correction. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for fluorescent signal. Right: Quantification of TRAP activity after normalization to SW39/TEL+ cells (n=3).

B. hTR and hTERT expression, normalized to ACTB mRNA and to SW39/TEL+.

C. RNA Pol II binding at hTR promoter in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines, normalized to H3 ChIP. Control loci with, respectively, high or low transcriptional activity: c-fos promoter and subtelomeric 4qHox locus (n=3). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.

D. H3K9me3 and H3K27me3 (left) or H3K4me3 and H3Ac (right) densities at hTR promoter in SI14/TEL+ and SI24/ALT+, normalized to H3 (n=3). Control loci as in C.

E. NFYB binding at hTR promoter in SI14/TEL+, SI24/ALT+, SW39/TEL+ and IMRB/ALT+ cells normalized to H3. Control loci with, respectively, high and low NFYB binding: hnRNPA1 promoter and subtelomeric 4qHox (n=3).

Data information: Data are presented as mean ± SD. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution unless specified otherwise. PTM, post-translational modification.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 2

Figure 2. NHP2 protein levels are downregulated in 8G12/ALT+ cells and ectopic NHP2 overexpression silences hTR.

A. Western blot for hTR chaperone proteins in TEL+ and ALT+ hybrids. β-Actin is used as loading control. Quantification of western blots is shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=2-4). Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.

B. NHP2 mRNA expression levels, normalized to ACTB mRNA and to SI14/TEL+ cells (n=3).

C. Western blot for NHP2 in SI14/TEL+, SI24/ALT+ and 8G12/ALT+ cell lines treated with DMSO or 10 μM MG132 for 6 h. β-Actin served as loading control. Quantifications are shown on the right, normalized to β-Actin and to SI14/TEL+ cells (n=5). Statistical analyses were performed with Student's two-tailed unpaired t-test.

D. hTR RNA and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+.

E. Telomerase activity measured with ddTRAP assay in NHP2-overexpressing 8G12/ALT+cells, normalized to NHP2-overexpressing SI14/TEL+.

F. (Top) Western blot for NHP2 overexpression in 8G12/ALT+ clonal cell lines. β-Actin served as loading control. SI14/TEL+ cells are shown as reference for NHP2 expression in hybrids. (Bottom) Western blot for NHP2 overexpression in SI14/TEL+ and 8G12/ALT+ cells compared to empty vector control. β-Actin served as loading control.

G. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to SI14/TEL+ (n=3).

H. RNA Pol II binding at hTR promoter, normalized to H3 ChIP (n=3). See Fig. 1C legend for control loci. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.

Data information: Data are presented as mean ± SD.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 3

Figure 3. hTR-expressing ALT+ cell lines display reduced NHP2 protein levels.

A. hTR RNA, hTERT and NHP2 mRNA expression, normalized to ACTB mRNA and to 143B/TEL+ cells, in a series of TEL+ and ALT+ human cell lines as indicated (n=3).

B. Western blot for NHP2 expression. β-Actin served as loading control. Quantification is shown on the right after normalization to β-Actin and to 143B/TEL+ cells (n=3).

Data information: Data are presented as mean ± SD. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 4

Figure 4. Ectopic expression of hTR in ALT+/hTR-/hTERT- cells decreases p-RPA32S33 abundance at telomeres.

A. hTR RNA expression, normalized to ACTB mRNA and to U2OS cells (n=3).

B. Western blot for NHP2 expression in the early passages of hTR-expressing IMRB or U2OS cells, with β-Actin as loading control.

C. Quantification of doubling time for U2OS, U2OS-hTR, IMRB and IMRB-hTR cell lines (n=3). Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution

D. Western blot in U2OS and U2OS-hTR cells treated with or without Hydroxyurea (2 mM for 24 h), for the indicated proteins. β-Actin and Vinculin served as loading controls.

E. Representative images for co-localization between p-RPA32S33 (green) and telomeres (red FISH probe). White arrows indicate co-localization events. Scale bars: 5 μm. Quantification is shown on the right. At least 50 nuclei were scored in each of the three independent experiments performed. Each dot represents the number of p-RPA32S33-telomere co-localizations in one nucleus. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.

F. Quantification of live cell numbers with trypan blue staining and manual counting, 72 h post-transfection with siLuc, sihTR#1 or sihTR#2, measured as the percentage of siLuc-treated cells (n=3). On the right, representative images of U2OS cells treated with sihTR#1 for 72 h. Statistical analyses were performed using one-way ANOVA with post-hoc Tukey's test for multiple comparisons.

G. Western blot analyses in the indicated cell lines cells transfected for 48 h with siLuc or sihTR, with the indicated antibodies. U2OS cells treated with Hydroxyurea (2 mM for 24 h) were used as positive controls.

Data information: Data are shown as mean ± SD, with the red line marking the mean value.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 5

Figure 5. Ectopic expression of hTR in ALT+/hTR-/hTERT- cells exacerbates telomeric damages and C-circle formation.

A. Representative images for fragile telomeres using the CO-FISH assay. Lagging strands (C-probe) and leading strands (G-probe) were marked with green PNA probes and red LNA probes, respectively. White circles and arrows indicate "smeary" or "doublet" telomeric signals, respectively. Quantification of fragile telomeres is shown on the right as the percentage of total telomeres counted (n=932-1073). Comparison of fragile to non-fragile telomere percentage was performed using Χ2-test.

B. Representative images for co-localization between RAD51 (green) and telomeres (red FISH probe). White arrows indicate representative RAD51-telomere co-localization events. Quantifications are shown on the right. A total of 120 nuclei were scored from two independent experiments.

C. Representative images for TIF quantification: co-localization between 53BP1 (green) and telomeres (red FISH probe) in U2OS or IMRB cells with or without ectopic hTR overexpression. White arrows indicate TIF. Quantification of TIF is on the right. A total of 110-170 nuclei were scored from three independent experiments. Each dot represents the number of TIF per nucleus.

D. Representative images for TIF quantification, as in (A), in U2OS or U2OS-hTR cells, treated as indicated with DMSO or 5 μM ATRi or 5 μM ATMi for 20 h. White arrows indicate TIF.

E. Quantification of TIF from (D). At least 65 nuclei were scored in each of the two independent experiments performed. Each dot represents the number of TIF per nucleus. Statistical analyses were performed using one-way ANOVA with post-hoc Games-Howell's test for multiple comparisons.

F. Representative images for co-localization between POLD3 (red) and RAP1 (green). White arrows indicate co-localization events. Quantification of co-localization events between POLD3 and RAP1 is shown on the right. A total of 190-230 nuclei were scored from three independent experiments.

G. C-circle analysis using slot-blot assay hybridized with radioactive C-rich telomeric probe. Quantification is shown below (n=7). Statistical analyses were performed using the non-parametrical Mann Whitney test.

H,I. C-circle analyses in the indicated cell lines 72 h after treatment with siPOLD3 or siLuc used as control for transfection. Quantifications are shown below (n=3). siSMARCAL1 was used as positive control for increased C-circle production in cells with defective fork reversal activity (Zhang et al, 2019b).

Data information: All scale bars: 5 μm. Data are shown as mean ± SD. B, C, E and F: the red line marks the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-tests with Welch's correction for normality of distribution, unless specified otherwise.
NHP2 downregulation counteracts hTR-mediated activation of the DNA damage response at ALT telomeres (2021)
Figure 6

Figure 6. More APBs are actively engaged in G2/M telomeric DNA synthesis in ALT+ cells ectopically expressing hTR.

A. Representative images for co-localization between PML (green) and telomeres (red FISH probe). White arrows indicate co-localization events, marking APBs. Quantification of images is shown on the right. A total of 85-300 nuclei were scored from four independent experiments. Each dot represents the number of events per nucleus: PML foci or APBs.

B. Experimental scheme for ATSA (ALT telomere DNA synthesis in APBs), used to measure G2/M telomeric DNA synthesis, within PML bodies.

C. Representative images for co-localization between EdU (green) and telomeres (red FISH probe) within PML bodies (cyan) in U2OS and U2OS-hTR cells. White arrows indicate co-localization events.

D, E. Quantification of images shown in (C). Co-localization events between EdU foci and APBs were analyzed (D) and the percentage of APBs that are actively engaged in G2/M telomeric synthesis was calculated (E). A total of at least 50 nuclei were scored from three independent experiments.

Data information: All scale bars: 5 μm. Data are shown as mean ± SD, with the red line marking the mean value. Statistical analyses were performed with Student's two-tailed unpaired t-test with Welch's correction for normality of distribution.