Journal: Scientific Reports

Article Title: Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

doi: 10.1038/s41598-021-92806-8

Figure Lengend Snippet:

Article Snippet: NELFE , 1 μg/mL , Rabbit , Monoclonal , A301-913A , Bethyl laboratories.

Techniques: Combined Bisulfite Restriction Analysis Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Rs79208225 SNP in HCC patients is associated with improved recurrence-free survival and decreased NELFE expression (A) Snapshot of Sanger sequencing from a wild type (WT) and a heterozygous NM_002904.6 :c.943-110C>T (rs79208225) variant carrying patient. (B) Kaplan-Meier 5 year recurrence-free survival curve for WT and rs79208225 SNP patients (WT n = 73, SNP n = 8, log rank test). (C) TaqMan RT-qPCR to evaluate NELFE mRNA expression in WT and rs79208225 SNP patient tumor (WT n = 32, SNP n = 8) and non-tumor (WT n = 33, SNP n = 6) tissue. Data are presented as mean ± SD. Student’s t test, ∗ p <0.05. (D) Representative images of hematoxylin & eosin and corresponding NELFE immunohistochemistry on WT or rs79208225 SNP patient tumors. Scale bars, 50 μm (WT n = 6, SNP n = 6). Data presented as mean ± SEM. p value from Welch’s test.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Expressing, DNA Sequencing, Variant Assay, Quantitative RT-PCR, Tissue, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Rs79208225 SNP promotes alternative splicing of NELFE exon 10 producing an uncharacterized NELFE mRNA isoform (A) Top, schematic of the minigene reporter. SNP minigenes have the rs79208225 C>T variant at the red line. WT minigenes retain the C allele. Arrows indicate primers for RT-PCR. Dotted line represents exon 10 skipping. Bottom, RT-PCR agarose gel of WT or rs79208225 SNP minigene transfected HCC cells. Rs79208225 SNP minigene cells contain a smaller band. CMV, cytomegalovirus; ex, exon; GFP, green fluorescent protein. (B) Top, schematic of the psiCheck dual luciferase firefly (fluc) and Renilla (Rluc) reporter for WT or rs79208225 SNP minigenes. Bottom, cells were transfected with WT or SNP reporter to quantify alternative splicing. Data presented as mean ± SD ( n = 3). Student’s t test, ∗ p <0.05; ∗∗ p <0.01. EV, empty vector control. (C) Digital PCR graph of d.ex10 mRNA isoform in HCC tumors from patients that harbor the rs79208225 SNP. Data presented as concentration (copies/μL) ± 95% confidence intervals. (D) RPIseq scores of the interaction probability of different splicing related proteins and the 41 nt sequence around the rs79208225 locus (20 nt on each flank) on the minus strand. Probability > 0.5 indicates positive interaction. SVM, support vector machine,; RF, random forest classifier. (E) Top, schematic of minigene reporters with RT-qPCR primers. Bottom, U2AF2 RNA immunoprecipitation PCR analyses of WT or SNP minigene transcripts. Data are presented as mean ± SD ( n = 3). Student’s t test, ∗∗ p <0.01 ; ∗∗∗ p <0.001. (F) RT-PCR agarose gel of FL NELFE and isoform at 48 h of MO treatment in HCC cell lines. (G) RT-qPCR detection of total NELFE mRNA and (H) d.ex10 isoform after 48 h of MO treatment. Total NELFE mRNA was evaluated with primers in exon 8. D.ex10 isoform was evaluated with a TaqMan probe for the exon9-exon11 junction. Data are presented as mean ± SD ( n = 4). Student’s t test, ∗∗ p <0.01, ∗∗∗ p <0.001. (I) Top, RT-PCR agarose gel of exon 10 deletion CRISPR model (mut) in Huh1 cells. Bottom, RT-qPCR analysis of total NELFE mRNA (left) and d.ex10 isoform (right) ( n = 4). Student’s t test, ∗∗ p <0.01; ∗∗∗∗ p <0.0001.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Alternative Splicing, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Luciferase, Plasmid Preparation, Control, Digital PCR, Concentration Assay, Sequencing, Quantitative RT-PCR, RNA Immunoprecipitation, CRISPR, Analysis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: d.ex10 mRNA isoform has a similar metabolism as FL mRNA transcripts and produces an uncharacterized NELFE protein isoform (A) Schematic of the effect of exon 10 skipping on NELFE stop codon location and 3′ UTR length. Depending on which poly(A) site is utilized, d.ex10 may have a 3′ UTR length of either 11 or 153 bp. Ex, exon; FL, full length; UTR, untranslated region. (B) mRNA stability evaluation of FL and d.ex10 mRNA transcripts following 5 μg/mL actinomycin D (Act. D) treatment in MO transfected cells or (C) 60 μM 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) treatment in exon 10 CRISPR deletion model. Exon 10 included transcripts were detected with a TaqMan probe at the exon10-exon11 junction. D.ex10 transcripts were detected with a TaqMan probe at the exon9-exon11 junction. Dotted lines are at 50%. Data are presented as mean ± SD with one-phase decay curves ( n = 3 in B). (D) Top, schematics of the 3′ UTR psiCHECK-2 dual luciferase reporters. NELFE 3′ UTR sequences were cloned immediately downstream of Renilla’s (RLuc) stop codon. Bottom, HCC cells were transfected with dual luciferase constructs and relative luciferase expression was measured by normalizing Renilla luciferase signal to firefly luciferase signal. Data are presented as mean ± SEM ( n = 4). Student’s t test, ∗∗∗∗ p <0.0001. FLuc, firefly luciferase. (E) Representative immunoblots of NELF complex subunits after 72 h of MO treatment. (F) Representative immunoblots of NELF complex subunits in Huh1 WT and d.ex10 mutant (mut) cell lines. (G) Representative immunoblots of NELFE after 72 h of knockdown with either an siRNA targeting exon 4 (e4) or exon 10 (e10) to validate the lower band is a NELFE isoform that lacks exon 10 (d.ex10). (H) Left, representative immunoblot of FL or d.ex10 protein rescues tagged with GFP in stable shNELFE knockdown in Hep3B cells. Right, representative cell proliferation of Hep3B cells to assess effect of d.ex10 on cell phenotypes. Data are presented as mean ± SD ( n = 3). Student’s t test performed at 96 h. ∗∗ p <0.01, ∗∗∗ p <0.001, ∗∗∗∗ p <0.0001.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Stability, Transfection, CRISPR, Luciferase, Clone Assay, Construct, Expressing, Western Blot, Mutagenesis, Knockdown

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Loss of NELFE exon 10 is associated with reduced cancer cell phenotypes (A) Cell proliferation assay in MO treated HCC cell lines. Cells were plated for xCELLigence 48 h after MO transfection and measured every 12 for 72 h. Data are presented as mean ± SD ( n = 3–4). Statistical analysis was conducted at 72 h using a Student’s t test. ∗∗∗∗ p <0.0001. (B) Cell colony formation assay in MO treated HCC cell lines. Cells were plated in a 6-well plate 48 h after MO transfection. Left, quantification of colony formation after 10–14 days. Right, representative crystal violet stains of colonies. Data is presented as mean ± SD ( n = 3), Student’s t -test. ∗∗ p <0.01 and ∗∗∗∗ p <0.0001. (C) Spheroid formation assay in MO treated HCC cell lines. Cells were plated in a 6-well AlgiMatrix 48 h after MO transfection. Left, quantification of spheroid formation after 14 days. Right, representative spheroids imaged at 20×. Scale bars, 200 μm. Data are presented as mean ± SD ( n = 3). Statistical analysis was conducted by a Student’s t test. (D) Cell proliferation assay in Huh1 WT and Huh1 d.ex10 mut cell lines. Data are presented as mean ± SD ( n = 3). Student’s t test performed at 72 h. (E) Spheroid formation assay in Huh1 WT and Huh1 d.ex10 mut cell lines. Left, quantification of spheroid formation after 14 days. ∗∗∗∗ p <0.0001. Right, representative spheroids imaged at 20×. Scale bars, 200 μm. (F) MHCC97H tumor growth curve in athymic nude mice treated with intraperitoneal injections of 80 μM vivo-MOs. Gray arrows indicate treatment. Data presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 5). Two-way ANOVA with Dunnett’s multiple comparisons, ∗ p <0.05. (G) Graph representing tumor weights for indicated groups at the conclusion of the study. Data presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 5). Statistical analysis was conducted using a Student’s t test. (H) Left, RT-qPCR using a TaqMan probe spanning the exon 2-exon 3 junction to detect total NELFE mRNA from a less than 50 mg section of tumor tissue. Right, RT-qPCR using a TaqMan probe spanning the exon 9-exon 11 junction to detect d.ex10 mRNA transcripts. Data are presented as mean ± SEM (vivo-Ctrl n = 5, vivo-i9 n = 6, vivo-e10 n = 4). Student’s t test with Welch’s correction, ∗ p <0.05 (I) Representative hematoxylin and eosin (H&E) stain and NELFE immunohistochemistry of MHCC97H tumors with different vivo-MO treatments. Scale bars, 100 μm.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: Proliferation Assay, Transfection, Analysis, Colony Assay, Tube Formation Assay, Spheroids, Quantitative RT-PCR, Tissue, Staining, Immunohistochemistry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Morpholino-mediated splice switching of NELFE suppresses hepatocellular carcinoma cell phenotypes in vitro and in vivo

doi: 10.1016/j.omtn.2026.103124

Figure Lengend Snippet: Targeted destruction of NELFE MO-loaded MBs extends survival in vivo (A) Schematic of locally released MOs from UTMD of MO loaded MBs. MO, morpholino; MB, microbubble; RO, retroorbital injection. (B) Microscopy images of Definity MBs loaded with fluoresceinated control vivo-MO. Images taken at 20×. Scale bars, 10 μm. (C) Ultrasound imaging of MB destruction and replenishment sequence. White circle outlines the tumor. Yellow line indicates the liver. (D) Immunoblot of Huh1 spheroids treated with two different concentrations of vivo-MO alone, loaded on MBs without UTMD, or loaded on MBs that subsequently underwent UTMD. NELFE band intensity was normalized to respective GAPDH then to WT. (E and F) Top, schematics of the experimental approach assessing MB + MO effect on Huh1.luc (E) and Huh7.luc (F) orthotopic tumors in NOD/SCID mice. Blue arrows indicate IVIS pre- and post-treatment imaging days. Black arrows indicate treatment days of UTMD of 50 μL MB + 80 μM MO administered through retro-orbital injections. Left, representative bioluminescence images of Huh1.luc (E) and Huh7.luc (F) mice pre-MB+MO treatment and 1-week post- final MB + MO treatment. Scale is in radiance (p/sec/cm/sr). Right, Kaplan-Meier survival curves of Huh1.luc (E) and Huh7.luc (F) vivo-MO treated mice (Huh1.luc experiment: MB + Ctrl n = 6, MB + i9 n = 8, MB + e10 n = 6; Huh7.luc experiment: MB + Ctrl n = 7, MB + i9 n = 5, MB + e10 n = 6). Survival analyses were conducted by a log-rank test.

Article Snippet: Following knockdown validation, 1 × 10 cells were replated in 6-well plates and transduced with lentiviral vectors expressing full-length NELFE (pLenti-NELFE FL; OriGene, Cat. No. RC208658L2), NELFE lacking exon 10 (pLenti-NELFE Δexon10; generated by In-Fusion cloning using the In-Fusion HD Cloning Kit, Takara, Cat. No. 638948), or the corresponding empty vector (pLenti-EV).

Techniques: In Vivo, Injection, Microscopy, Control, Imaging, Sequencing, Western Blot, Spheroids

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3

Article Snippet: Biotinylated NELFE peptide (QPFQRSIpSADDLQE) was synthesized (GenScript) and bound to NeutrAvidin agarose.

Techniques: Phospho-proteomics, Binding Assay, Multiplex sample analysis, Labeling, Expressing, Irradiation, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Knockdown, Transfection, Recombinant, Control, Inhibition, Lysis

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: NELFE phosphorylation on S115 is required for the interaction with 14-3-3. a Schematic representation of NELFE domain organization and phosphorylation sites that were identified by phosphoproteomics. The SILAC ratios quantified for phosphorylation sites on NELFE after UV light and p38 inhibition are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. b The table shows all phosphorylation sites identified on NELFE by phosphoproteomics. The position, SILAC ratios, 14-3-3 binding prediction and sequence window are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. c Serine 115 phosphorylation is required for the interaction of NELFE and 14-3-3. U2OS cells expressing GFP-tagged wild-type NELFE or NELFE serine-to-alanine mutants were irradiated with UV light. Protein extracts were incubated with GST-14-3-3 and enriched proteins were resolved on SDS-PAGE. d Mass spectrometric parent ion scan of the peptide SISADDDLQESSR corresponding to S115 in NELFE. The SILAC triplet shows the relative abundance and mass to charge (m/z) of the phosphorylated peptide in mock-treated cells and cells irradiated with UV light without or with pretreatment with the p38 inhibitor. e Absolute occupancy of serine 49, 51, 115, and 251 phosphorylation in NELFE in undamaged cells and after UV light was determined by MS. f NELFE S115A mutant does not bind to 14-3-3. SILAC-labeled cells overexpressing GFP-tagged wild-type NELFE or NELFE S115A mutant were irradiated with UV light. UV-light-irradiated U2OS cells overexpressing GFP alone were used as control. Cells were lysed and protein extracts were incubated with GFP Trap agarose. The scatter plot shows the logarithmized SILAC ratios of quantified proteins. The color-coding indicates the density. g Recombinant 14-3-3 binds to phosphorylated NELFE peptide. Biotinylated phosphorylated NELFE peptide corresponding to serine 115 was bound to NeutrAvidin agarose. Phosphorylated and dephosphorylated peptide were incubated with purified 14-3-3. h Structure of 14-3-3 epsilon in complex with NELFE phosphorylated peptide QPFQRSI(p)SADDDLQE. Structure of the 14-3-3 epsilon in cartoon representation (Yellow and Cyan) and NELFE phosphorylated peptide in ball and stick model (Green). The inset on the right shows the 14-3-3 epsilon–NELFE phosphorylated peptide interaction

Article Snippet: Biotinylated NELFE peptide (QPFQRSIpSADDLQE) was synthesized (GenScript) and bound to NeutrAvidin agarose.

Techniques: Phospho-proteomics, Multiplex sample analysis, Inhibition, Labeling, Binding Assay, Sequencing, Expressing, Irradiation, Incubation, SDS Page, Mutagenesis, Control, Recombinant, Purification

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: p38-dependent phosphorylation of NELFE promotes its dissociation from chromatin. a Chromatin-associated proteins were extracted from untreated, UV-light-treated, and p38i, UV-light-treated U2OS cells, and analyzed by SILAC-based quantitative mass spectrometry. The bar plot shows the GO-BP (biological process) terms associated with proteins specifically enriched on or removed from chromatin after UV light (40 J/m 2 , 1 h recovery). The significance of the enrichment of a specific term was determined using Fisher’s exact test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. b The bar plot shows selected proteins associated with DNA repair and cell cycle that are significantly recruited or removed from chromatin after UV light. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. c The NELF complex subunits are removed from chromatin in a UV light and p38-dependent manner. The bar plot shows selected proteins whose removal from chromatin after UV light is dependent on p38. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. d NELFE dissociates from chromatin after UV light. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies (left). The levels of NELFE on chromatin were quantified from three replicate experiments and normalized to MCM7 levels (right). The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01). e Dynamics of NELFE removal from chromatin. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (** p -value < 0.01). f Knockdown of NELFE reduced the ability of U2OS cells to form colonies after UV light. The error bars show the mean and SD of results obtained in three replicate experiments performed in three technical replicates. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01)

Article Snippet: Biotinylated NELFE peptide (QPFQRSIpSADDLQE) was synthesized (GenScript) and bound to NeutrAvidin agarose.

Techniques: Phospho-proteomics, Multiplex sample analysis, Mass Spectrometry, SDS Page, Western Blot, Knockdown

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV light leads to an increase in transcriptional elongation. a Metagene analysis showing total RNA pol II occupancy measured by ChIP-seq in mock-treated U2OS cells and cells irradiated with UV light (40 J/m 2 , 1 h recovery). All TSSs bound by RNA pol II in untreated cells and after UV light exposure were used for the analysis. Metagene analysis shows an average of two independent replicate ChIP-seq experiments. b Exposure of U2OS cells with UV light (40 J/m 2 , 1 h recovery) promotes the release of RNA pol II into downstream regions of genes. The box plot shows the calculated RNA pol II release ratios (PRRs) in untreated cells and in cells irradiated with UV light. The average PRRs were calculated from two independent replicate experiments. The lower and upper hinges represent the first and third quartiles (25th and 75th percentiles, respectively). The line in the center of the box corresponds to the median of the data range. P -value was calculated using the Wilcoxon’s rank-sum test with continuity correction. c REACTOME terms significantly enriched among genes with UV light upregulated PRRs. The bar plot shows significantly overrepresented REACTOME terms associated with genes containing upregulated PRR compared with all RNA pol II bound genes. The significance of the enrichment of a specific term was determined using a hypergeometric test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. d UCSC Genome Browser tracks displaying the density of RNA pol II around the PLK3 and RPL11 gene in untreated cells and after exposure of cells with UV light. e Model for the NELF complex regulation by p38-MK2. Exposure of human cells to UV light leads to rapid activation of p38 and its downstream effector kinase MK2. MK2 triggers widespread phosphorylation of RNA-binding proteins, including the NELF complex subunit NELFE. Site-specific NELFE phosphorylation on S115 induces its transient interaction with 14-3-3. NELFE phosphorylation leads to dissociation of NELFE from chromatin that is accompanied by RNA pol II elongation

Article Snippet: Biotinylated NELFE peptide (QPFQRSIpSADDLQE) was synthesized (GenScript) and bound to NeutrAvidin agarose.

Techniques: ChIP-sequencing, Irradiation, Activation Assay, Phospho-proteomics, RNA Binding Assay

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β- d -ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Binding Assay, Multiplex sample analysis, Labeling, Expressing, Irradiation, Incubation, SDS Page, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Knockdown, Transfection, Recombinant, Control, Inhibition, Lysis

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: NELFE phosphorylation on S115 is required for the interaction with 14-3-3. a Schematic representation of NELFE domain organization and phosphorylation sites that were identified by phosphoproteomics. The SILAC ratios quantified for phosphorylation sites on NELFE after UV light and p38 inhibition are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. b The table shows all phosphorylation sites identified on NELFE by phosphoproteomics. The position, SILAC ratios, 14-3-3 binding prediction and sequence window are indicated. UV-light-induced, p38-dependent phosphorylation sites are labeled in red. c Serine 115 phosphorylation is required for the interaction of NELFE and 14-3-3. U2OS cells expressing GFP-tagged wild-type NELFE or NELFE serine-to-alanine mutants were irradiated with UV light. Protein extracts were incubated with GST-14-3-3 and enriched proteins were resolved on SDS-PAGE. d Mass spectrometric parent ion scan of the peptide SISADDDLQESSR corresponding to S115 in NELFE. The SILAC triplet shows the relative abundance and mass to charge (m/z) of the phosphorylated peptide in mock-treated cells and cells irradiated with UV light without or with pretreatment with the p38 inhibitor. e Absolute occupancy of serine 49, 51, 115, and 251 phosphorylation in NELFE in undamaged cells and after UV light was determined by MS. f NELFE S115A mutant does not bind to 14-3-3. SILAC-labeled cells overexpressing GFP-tagged wild-type NELFE or NELFE S115A mutant were irradiated with UV light. UV-light-irradiated U2OS cells overexpressing GFP alone were used as control. Cells were lysed and protein extracts were incubated with GFP Trap agarose. The scatter plot shows the logarithmized SILAC ratios of quantified proteins. The color-coding indicates the density. g Recombinant 14-3-3 binds to phosphorylated NELFE peptide. Biotinylated phosphorylated NELFE peptide corresponding to serine 115 was bound to NeutrAvidin agarose. Phosphorylated and dephosphorylated peptide were incubated with purified 14-3-3. h Structure of 14-3-3 epsilon in complex with NELFE phosphorylated peptide QPFQRSI(p)SADDDLQE. Structure of the 14-3-3 epsilon in cartoon representation (Yellow and Cyan) and NELFE phosphorylated peptide in ball and stick model (Green). The inset on the right shows the 14-3-3 epsilon–NELFE phosphorylated peptide interaction

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Multiplex sample analysis, Inhibition, Labeling, Binding Assay, Sequencing, Expressing, Irradiation, Incubation, SDS Page, Mutagenesis, Control, Recombinant, Purification

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: p38-dependent phosphorylation of NELFE promotes its dissociation from chromatin. a Chromatin-associated proteins were extracted from untreated, UV-light-treated, and p38i, UV-light-treated U2OS cells, and analyzed by SILAC-based quantitative mass spectrometry. The bar plot shows the GO-BP (biological process) terms associated with proteins specifically enriched on or removed from chromatin after UV light (40 J/m 2 , 1 h recovery). The significance of the enrichment of a specific term was determined using Fisher’s exact test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. b The bar plot shows selected proteins associated with DNA repair and cell cycle that are significantly recruited or removed from chromatin after UV light. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. c The NELF complex subunits are removed from chromatin in a UV light and p38-dependent manner. The bar plot shows selected proteins whose removal from chromatin after UV light is dependent on p38. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance. d NELFE dissociates from chromatin after UV light. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies (left). The levels of NELFE on chromatin were quantified from three replicate experiments and normalized to MCM7 levels (right). The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01). e Dynamics of NELFE removal from chromatin. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t -test was used to assess the significance (** p -value < 0.01). f Knockdown of NELFE reduced the ability of U2OS cells to form colonies after UV light. The error bars show the mean and SD of results obtained in three replicate experiments performed in three technical replicates. Two-sided Student’s t -test was used to assess the significance (*** p -value < 0.001, ** p -value < 0.01)

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: Phospho-proteomics, Multiplex sample analysis, Mass Spectrometry, SDS Page, Western Blot, Knockdown

Journal: Nature Communications

Article Title: p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage

doi: 10.1038/s41467-018-03417-3

Figure Lengend Snippet: UV light leads to an increase in transcriptional elongation. a Metagene analysis showing total RNA pol II occupancy measured by ChIP-seq in mock-treated U2OS cells and cells irradiated with UV light (40 J/m 2 , 1 h recovery). All TSSs bound by RNA pol II in untreated cells and after UV light exposure were used for the analysis. Metagene analysis shows an average of two independent replicate ChIP-seq experiments. b Exposure of U2OS cells with UV light (40 J/m 2 , 1 h recovery) promotes the release of RNA pol II into downstream regions of genes. The box plot shows the calculated RNA pol II release ratios (PRRs) in untreated cells and in cells irradiated with UV light. The average PRRs were calculated from two independent replicate experiments. The lower and upper hinges represent the first and third quartiles (25th and 75th percentiles, respectively). The line in the center of the box corresponds to the median of the data range. P -value was calculated using the Wilcoxon’s rank-sum test with continuity correction. c REACTOME terms significantly enriched among genes with UV light upregulated PRRs. The bar plot shows significantly overrepresented REACTOME terms associated with genes containing upregulated PRR compared with all RNA pol II bound genes. The significance of the enrichment of a specific term was determined using a hypergeometric test. P -values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. d UCSC Genome Browser tracks displaying the density of RNA pol II around the PLK3 and RPL11 gene in untreated cells and after exposure of cells with UV light. e Model for the NELF complex regulation by p38-MK2. Exposure of human cells to UV light leads to rapid activation of p38 and its downstream effector kinase MK2. MK2 triggers widespread phosphorylation of RNA-binding proteins, including the NELF complex subunit NELFE. Site-specific NELFE phosphorylation on S115 induces its transient interaction with 14-3-3. NELFE phosphorylation leads to dissociation of NELFE from chromatin that is accompanied by RNA pol II elongation

Article Snippet: The 14-3-3 epsilon and synthesized NELFE phospho-peptide (QPFQRSI(p)SADDDLQE, GenScript) were mixed to a molar ratio of 1:5 for crystallization.

Techniques: ChIP-sequencing, Irradiation, Activation Assay, Phospho-proteomics, RNA Binding Assay