Journal: Disease Models & Mechanisms

Article Title: Mutation in Prkra results in cerebellar abnormality and reduced eIF2α phosphorylation in a model of DYT-PRKRA

doi: 10.1242/dmm.050929

Figure Lengend Snippet: The lear-5J protein binds dsRNA less efficiently but interacts with PKR similarly to wt PACT/RAX. (A) dsRNA-binding activity of wt PACT/RAX and lear-5J truncated protein was measured by a poly(I):poly(C)-agarose-binding assay with in vitro translated 35 S-labeled proteins. T, total input; B, proteins bound to poly(I):poly(C)-agarose. Competition lanes 5 and 6: competition with 100-fold molar excess of single-stranded RNA (ss) or dsRNA (ds). The faint band below the parent PACT/RAX band represents products of in vitro translation from an internal methionine codon in lane 1. (B) Quantification of the dsRNA-binding assay. Bands were quantified by phosphorimaging analysis, and percentage bound was calculated. Error bars: s.d. from three independent experiments. The P -value (0.003) calculated using statistical analyses indicated significant difference between percentage dsRNA-binding of wt (blue bar) and that of lear-5J mutant (red bar). (C) Co-immunoprecipitation of endogenous PKR and Flag-PACT/RAX or Flag-lear-5J overexpressed in HeLa cells. HeLa cells were transfected with Flag wt PACT/RAX or Flag-lear-5J in pCDNA3.1 expression constructs at 40% confluency and harvested 24 h post-transfection. Whole-cell extracts were immunoprecipitated at 4°C overnight using 100 ng anti-PKR antibody per immunoprecipitation. Samples were then analyzed via SDS-PAGE gel electrophoresis and western blot analysis probing for Flag-tagged wt PACT/RAX or lear-5J (co-IP panel) using monoclonal anti-Flag-M2 (Sigma-Aldrich) antibody. To ascertain that an equal amount of protein was immunoprecipitated, blots were re-probed using an anti-PKR antibody (IP panel). Input blots without immunoprecipitation demonstrate that equal amounts of each protein were present prior to immunoprecipitation. IP, immunoprecipitation; mAb, monoclonal antibody; mut, mutant; wt, wild type.

Article Snippet: They were then resuspended in lysis buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl 2 , 50 mM KCl, 400 mM NaCl, 2 mM DTT, 1% Triton X-100, 100 U/ml aprotinin, 0.2 mM PMSF, 20% glycerol) and incubated on ice for 5 min. Whole-cell lysates were centrifuged at 10,000 g for an additional 5 min. PKR was then immunoprecipitated from 100 μg of this lysate using anti-PKR monoclonal antibody (R&D Systems, MAB1980; 1:2000) in a high-salt buffer (20 mM Tris-HCl pH 7.5, 50 mM KCl, 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 100 U/ml aprotinin, 0.2 mM PMSF, 20% glycerol, 1% Triton X-100) at 4°C on a rotating wheel for 30 min. We then added 10 µl protein A-sepharose beads to each immunoprecipitate followed by an additional 1 h incubation under the same conditions.

Techniques: Binding Assay, Activity Assay, In Vitro, Labeling, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Construct, SDS Page, Nucleic Acid Electrophoresis, Western Blot, Co-Immunoprecipitation Assay

Journal: Cell reports

Article Title: NIPBL Controls RNA Biogenesis to Prevent Activation of the Stress Kinase PKR

doi: 10.1016/j.celrep.2015.12.012

Figure Lengend Snippet: (A) The heatmap shows an upregulation of immune response genes in NIPBL -MS LCLs. The average log 2 expression value is displayed. (B) There is increased p-PKR, p-eIF2α, and ATF4 in LCLs derived from patients with NIPBL -MS, -NS, and -FS mutations and HDAC8 mutations (98P and 7P), but not from LCLs with cohesin SMC1 missense ( SMC1 -MS) or SMC3 missense ( SMC3 -MS) mutations. (C) 7DG treatment can inhibit the PKR-signaling cascade, as shown by reduced levels of phosphorylation of PKR and eIF2α as well as the reduced levels of ATF4. (D) The NIPBL -MS and NIPBL -NS LCLs show poor cell proliferation, which is partially rescued by treatment with 7DG (300 nM). Error bars represent SEM. (E) The NIPBL -MS and NIPBL -NS LCLs have elevated levels of apoptosis and lower viability, both of which are rescued by treatment with 7DG. (F) NIPBL -MS and NIPBL -NS LCLs have elevated levels of ROS, which are partially reversed by 7DG treatment. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to WT; †p < 0.05 and ††p < 0.01 compared to NIPBL -MS; ◆p < 0.05 and ◆◆p < 0.01 compared to NIPBL -NS. All experiments were performed with n = 3–4. Error bars represent SEM. See also .

Article Snippet: Primary antibodies for NIPBL were purchased from Bethyl (A301-799A); p-PKR (ab32036), METTL3 (ab49253), METTL14 (ab98166), and α-tubulin (ab15246) were purchased from Abcam; PKR was purchased from Santa Cruz Biotechnology (sc-6268); and p-eIF2α (3398), eIF2α (9722), and ATF4 (11815) were purchased from Cell Signaling Technology.

Techniques: Expressing, Derivative Assay, Phospho-proteomics

Journal: Cell reports

Article Title: NIPBL Controls RNA Biogenesis to Prevent Activation of the Stress Kinase PKR

doi: 10.1016/j.celrep.2015.12.012

Figure Lengend Snippet: (A) qPCR shows the lower expression of METTL3 and METTL14 RNA in NIPBL -MS cells. Ubiquitin C served as a loading control. *p < 0.05 and **p < 0.01 compared to WT. (B) The reduction of METTL3 and METTL14 protein expression is shown in western blots. (C) The scheme used to fractionate RNA is diagrammed. The mRNAs are first isolated from the total RNA, followed by ncRNAs and rRNAs (see Experimental Procedures for details). (D) m 6 A levels are significantly reduced in mRNAs, ncRNAs, and rRNAs from NIPBL -MS cells compared to WT cells. There is an especially dramatic decrease (more than 6-fold) in m 6 A levels in the ncRNAs of the NIPBL mutant LCLs. **p < 0.01 and ***p < 0.001 compared to WT. (E) Both ncRNAs and rRNAs isolated from NIPBL -MS LCLs are capable of activating recombinant PKR in vitro; 10 ng Poly I:C was used as a positive control for PKR activation. *p < 0.001 compared to untreated control; #p < 0.001 compared to WT rRNA. (F) Total RNA isolated from NIPBL -NS and NIPBL -FS, and HDAC8 (98P) and HDAC8 (7P) can induce PKR activation in vitro; 1 ng Poly I:C was used as a positive control for PKR activation. For (E) and (F), 10 ng RNA was used in each reaction.

Article Snippet: Primary antibodies for NIPBL were purchased from Bethyl (A301-799A); p-PKR (ab32036), METTL3 (ab49253), METTL14 (ab98166), and α-tubulin (ab15246) were purchased from Abcam; PKR was purchased from Santa Cruz Biotechnology (sc-6268); and p-eIF2α (3398), eIF2α (9722), and ATF4 (11815) were purchased from Cell Signaling Technology.

Techniques: Expressing, Ubiquitin Proteomics, Control, Western Blot, Isolation, Mutagenesis, Recombinant, In Vitro, Positive Control, Activation Assay

Journal: Cell reports

Article Title: NIPBL Controls RNA Biogenesis to Prevent Activation of the Stress Kinase PKR

doi: 10.1016/j.celrep.2015.12.012

Figure Lengend Snippet: NIPBL binds to the promoter/TSS of RNA-processing genes, including METTL3 and METTL14 , to promote their expression. The RNA-processing genes are essential for the RNA modifications such as m 6 A methylation (red circle), pseudouridylation, etc. In normal cells, RNAs are highly modified with m 6 A methylation and other modifications to prevent activation of PKR. However, in CdLS LCLs with loss of NIPBL function, RNA-processing genes are expressed at lower levels. RNAs are generated that contain less m 6 A modification and potentially other modifications as well. Such aberrant RNAs cause the activation of PKR that is marked by both dimerization and auto-phosphorylation.

Article Snippet: Primary antibodies for NIPBL were purchased from Bethyl (A301-799A); p-PKR (ab32036), METTL3 (ab49253), METTL14 (ab98166), and α-tubulin (ab15246) were purchased from Abcam; PKR was purchased from Santa Cruz Biotechnology (sc-6268); and p-eIF2α (3398), eIF2α (9722), and ATF4 (11815) were purchased from Cell Signaling Technology.

Techniques: Expressing, Methylation, Modification, Activation Assay, Generated, Phospho-proteomics