Journal: Human Molecular Genetics

Article Title: Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS

doi: 10.1093/hmg/ddaa159

Figure Lengend Snippet: FUS is acetylated. (A) Endogenous FUS-IP from N2A cells treated with DACi cocktail (nicotinamide (30 mM), sodium butyrate (50 mM) and TSA (3 μM)) for 6 h. Immunoblotting was performed using the indicated antibodies. (B) Mass spectrometric identification of the acetylated FUS peptide RGGRGGGDRGGFGPGK510MDSRGEHRQDRRERPY. (C) 3× FLAG-tagged WT, K315Q/K316Q, K510Q, K315Q/K316Q/K510Q or FLAG vector control were transfected into HEK293T cells. After 24 h, cells were treated with DACi cocktail for 6 h, followed by FLAG-IP and immunoblotting with the indicated antibodies. (D) Quantification of FUS acetylation from three independent experiments ±standard deviation (SD). Student’s t-test was performed for individual comparisons against WT (*P ≤ 0.05). (E) The domain structure of FUS showing the RRM domain sequence and acetylation sites. (F) NMR solution structure of FUS RRM domain (pink) showing K315 and K316 in the KK-loop bound to stem–loop RNA (protein data bank entry 6GBM). (G) Crystal structure of TNPO1/FUS-NLS (protein data bank entry 4FQ3) illustrating the FUS-NLS domain (pink) K510 adjacent to TNPO1 (purple) D693. Molecular graphics of FUS RRM and NLS domains visualized using UCSF ChimeraX (79).

Article Snippet: The rabbit anti-acetylated-K510 FUS antibody was raised against the peptide antigen N-G 504 GFGPG (K 510 Ac) MDSRG 515 -C. The peptide was synthesized with C-terminal amidation and conjugated to Keyhole Limpet Hemocyanin (KLH) for immunization (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Western Blot, Plasmid Preparation, Control, Transfection, Standard Deviation, Sequencing

Journal: Human Molecular Genetics

Article Title: Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS

doi: 10.1093/hmg/ddaa159

Figure Lengend Snippet: Acetylation of FUS impairs RNA binding and the FUS-NLS-TNPO1 interaction. (A) 3× FLAG-tagged FUS constructs were transfected into N2A cells. After 48 h, cells were lysed and FLAG-IP followed by reverse transcription and qPCR against FUS pre-mRNA surrounding exon 7 was performed. FUS mRNA was normalized with the FLAG levels in the FLAG-FUS-IPs and with the Rpl13a mRNA levels in the total extracts. Averages of three independent experiments are shown, ±SD. Student’s t-test was performed for individual comparisons against WT. (B) The indicated 3× FLAG-tagged FUS constructs were transfected into N2A cells and treated with DACi cocktail where indicated. FLAG-IP was performed 48 h after transfection with the inclusion of RNase cocktail as indicated, followed by immunoblotting with the indicated antibodies. (C) Quantification of (B) from three independent experiments. Student’s t-test was performed for individual comparisons against WT. (D) In vitro TNPO1 pulldown with Sulfolink-immobilized acetylated or non-acetylated FUS-NLS peptides. Different amounts of GST–TNPO1 were incubated with FUS-NLS peptide immobilized on the beads at 4°C for 3 h. The amount of TNPO1 pulled down with FUS-NLS peptides were evaluated by western blot. (E) Quantification of (D) from three independent experiments, ±SD. Student’s t-test was performed comparing the band intensities of non-acetylated and acetylated peptide pulldowns at the same concentration (*P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001; NS, not significant).

Article Snippet: The rabbit anti-acetylated-K510 FUS antibody was raised against the peptide antigen N-G 504 GFGPG (K 510 Ac) MDSRG 515 -C. The peptide was synthesized with C-terminal amidation and conjugated to Keyhole Limpet Hemocyanin (KLH) for immunization (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: RNA Binding Assay, Construct, Transfection, Reverse Transcription, Western Blot, In Vitro, Incubation, Concentration Assay

Journal: Human Molecular Genetics

Article Title: Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS

doi: 10.1093/hmg/ddaa159

Figure Lengend Snippet: K510 acetylation is increased in familiar FUS ALS. (A) FUS-K510 acetylation levels in ALS patients with R521G or P525R FUS mutations versus control subjects. Immunoblotting was performed using the indicated antibodies. (B) Quantification of FUS-K510 acetylation normalized against total FUS levels. Error bars represent SD between individuals. One-way ANOVA was performed to determine statistical significance (*P ≤ 0.05).

Article Snippet: The rabbit anti-acetylated-K510 FUS antibody was raised against the peptide antigen N-G 504 GFGPG (K 510 Ac) MDSRG 515 -C. The peptide was synthesized with C-terminal amidation and conjugated to Keyhole Limpet Hemocyanin (KLH) for immunization (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Control, Western Blot

Journal: Human Molecular Genetics

Article Title: Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS

doi: 10.1093/hmg/ddaa159

Figure Lengend Snippet: The regulators of FUS acetylation. (A) 3× FLAG-FUS or 3× FLAG-vector was co-transfected with HA-CBP or HA-vector into N2A cells. FLAG-IP was performed, followed by immunoblotting using a pan-acetylated lysine antibody and other indicated antibodies. (B) N2A cells were treated with different concentrations of the CBP/p300 inhibitor A-485. Immunoblotting was performed using the FUS K510 acetylation antibody and other antibodies as indicated. (C) Quantification of (B) from three independent experiments ±SD. Student’s t-test was performed for individual comparisons against no treatment (*P ≤ 0.05). (D) N2A cells were transfected with 3× FLAG-vector or 3× FLAG-FUS and treated with 8 μM CBP/p300 inhibitor A-485 for 16 h in the presence of DACi cocktail. FLAG-IP was performed followed by immunoblotting with a pan-acetylated lysine antibody and a FLAG antibody. The cell lysate was examined using the FUS K510 acetylation antibody and other indicated antibodies. (E). N2A cells were treated with 30 mM nicotinamide and/or 3 μM TSA for 6 h. FLAG-IP was performed followed by immunoblotting with a pan-acetylated lysine antibody and a FLAG antibody. (F) Quantification of (E) from three independent experiments, ±SD. One-way ANOVA was performed to determine statistical significance (*P ≤ 0.05; **P ≤ 0.005). (G). N2A cells were treated with 30 mM nicotinamide and/or 3 μM TSA for 6 h. Cells were harvested and lysed after treatment and immunoblotting was performed using the FUS K510 acetylation antibody and other indicated antibodies. (H) Quantification of (G) from three independent experiments ±SD. One-way ANOVA was performed to determine statistical significance (*P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.001). (I) HEK293T cells were transfected with 3× FLAG-empty vector, 3× FLAG-SIRTs 6–7, 3× FLAG-HDAC3 and 3× FLAG-ROA1 (hnRNPA1) used as a positive control. After 48 h, endogenous FUS-IP was performed, followed by immunoblotting using the indicated antibodies.

Article Snippet: The rabbit anti-acetylated-K510 FUS antibody was raised against the peptide antigen N-G 504 GFGPG (K 510 Ac) MDSRG 515 -C. The peptide was synthesized with C-terminal amidation and conjugated to Keyhole Limpet Hemocyanin (KLH) for immunization (Pocono Rabbit Farm and Laboratory, Canadensis, PA).

Techniques: Plasmid Preparation, Transfection, Western Blot, Positive Control

Journal: Journal of Cell Science

Article Title: The lncRNA hsrω regulates arginine dimethylation of human FUS to cause its proteasomal degradation in Drosophila

doi: 10.1242/jcs.236836

Figure Lengend Snippet: A decline of arginine methyltransferase activity is critical for FUS toxicity in Drosophila . (A) The anti-C-terminus FUS IgG was used to immunoprecipitate FUS (IP FUS ) from total proteins extracted from the adult heads of newly eclosed flies of genotype GMR-GAL4/+; UAS -FUS/+; UAS -GFP IR/+ (FUS+Ctrl) and GMR-GAL4/+; UAS -FUS/+; UAS -hsrω IR/+ (FUS+hsrω IR), respectively, raised at 25°C. Western blots with anti-C-terminus FUS IgG antibody were performed as a control (IP:FUS). Anti-pan monomethylated (MMA) and -pan dimethylated (DMA) arginine IgG antibodies were used to screen for the final level of arginine methylation of FUS (IB:MMA and IB:DMA, respectively). Anti-Actin (IB:Actin) and anti-IgG (IB:IgG) were used as controls. The methylation status of FUS was also examined upon genetical and pharmacological manipulation of DART1 and DART5 activity. DART1 and DART5 were overexpressed in flies carrying GMR-GAL4/+; UAS- FUS/+; UAS- DART1/+ (FUS+DART1) and GMR-GAL4/+; UAS- FUS/ UAS- DART5;+ (FUS+DART5), respectively. The DART activity was impaired in FUS+hsrω IR flies fed with 25 μM of furamidine (a PRMT1 inhibitor) and GSK591 (a PRMT5 inhibitor), respectively, throughout their development as shown by the reduced intensity of the IB:DMA bands. (B) Gel plots were obtained with ImageJ32 software and used to measure the relative abundance of MMA and DMA in each fraction. (C) The figure depicts the distinct FUS domains and marks the arginine (R) to be methylated in RGG1 and those that might be similarly modified in RGG3. (H–L) Total mRNAs were extracted from the eye discs of L3 larvae at three independent times and further analyzed by qRT-PCR in triplicate. The expression of both genes encoding type I PRMTs ( DART1 , DART3 and DART4 ) (C–E) and II PRMTs ( DART5 and DART7 ) (F–G) were normalized to the level of elav . The transcript abundance is the mean of nine independent reactions for each fly line. ** P <0.01, *** P <0.001; n.s., not significant. The genotypes were as follows: GMR-GAL4/+;+; UAS -GFP IR/+ (Ctrl), GMR-GAL4/+; UAS- FUS/+; UAS -GFP IR/+ (FUS+Ctrl), GMR-GAL4/+;+; UAS- hsrω IR/ UAS- GFP IR (hsrω IR+Ctrl) and GMR-GAL4/+; UAS- FUS/+; UAS- hsrω IR/+ (FUS+hsrω IR). (M) Light and scanning electron micrographs of the external eye surface of flies of genotypes, a–a″, GMR-GAL4/+; UAS- FUS/+; UAS- lacZ/+ (FUS+Ctrl); b–b″, GMR-GAL4/+; UAS- FUS/+; UAS- DART1/+ (FUS+DART1) and c–c″, GMR-GAL4/+; UAS- FUS/ UAS- DART5;+ (FUS+DART5) raised at 25°C. Lower panels show a higher magnification. Anterior is to the left and dorsal to the top. The white dashed line highlights the area of degeneration. Scale bars: 100 μm (middle panels); 50 μm (lower panels). (N) The external eye structure of 100 newly eclosed male flies from each above fly lines were examined under a dissection microscope, and the most representative were analyzed using SEM. The area of degeneration of ∼18 individuals were measured by ImageJ software and reported as µm 2 . ** P <0.01.

Article Snippet: The membrane was blocked with PVDF blocking reagent (Toyobo) at room temperature and then incubated overnight with mouse monoclonal anti-FUS antibody (against the C-terminus, Santa Cruz Biotechnology, 4H11; 1:1000), rabbit polyclonal anti-FUS antibody (N-terminus, Bethyl A300-302; 1:5000), mouse monoclonal anti-asymmetric dimethylarginine FUS R216, R218 (Funakoshi, clone 2B12; 1:1500), rat monoclonal anti-methylated RGG3 FUS (Merck, 9G6; 1:1000), rat monoclonal anti-elav (DSHB, 7E8A10; 1:750), mouse monoclonal anti-actin antibody (clone AC-40, Sigma-Aldrich; 1:5000) or mouse monoclonal pan-ubiquitin (Ubi-1) (Abcam, ab7254; 1:1000).

Techniques: Activity Assay, Western Blot, Methylation, Software, Modification, Quantitative RT-PCR, Expressing, Dissection, Microscopy