Journal: Genes & Development

Article Title: tRNA synthetase activity is required for stress granule and P-body assembly

doi: 10.1101/gad.353535.125

Figure Lengend Snippet: Inhibition of tRNA charging activates the ISR without inducing RNP granules. ( A ) U-2 OS cells were treated with 0.2% DMSO carrier, 20 µM halofuginone (HF), 250 µM sodium arsenite (As), or 1.25 µM thapsigargin (Tg) for 1 h, and Western blotting for phosphorylated and total eIF2α was done. Total protein ( n = 3 independent replicates) and molecular weights (in kilodaltons) are shown. ( B ) Translation activity was measured using bioorthogonal noncanonical amino acid tagging in cells treated as in A . Methionine or azidohomoalanine (AHA) was added 10 min prior to collection. The ratio of nascent to total protein is shown from n = 3 independent replicates. ( C ) U-2 OS cells stably expressing GFP-G3BP1 (green) were treated as in A and fixed, and nuclei were stained with Hoechst (blue). The percentage of cells with SGs was quantified from n = 3 independent experiments; ≥305 cells were counted in each condition. Scale bars, 10 µm. ( D ) Cells were treated as in C , and immunofluorescence and fluorescence in situ hybridization were done to detect UBAP2L (magenta), PABPC1 (cyan), and poly(A) RNA with oligo(dT) probes (yellow) ( n = 2 independent experiments). ( E ) Cells were treated with 0.2% DMSO, 20 µM HF plus 0.2% DMSO, 250 µM arsenite, or 1.25 µM thapsigargin for 1 h, and the percentage of cells with SGs was quantified ( n = 3 independent experiments); ≥287 cells were counted per treatment. ( F ) Cells were treated, and immunofluorescence microscopy was done as in D to detect the P-body marker DCP1A. Quantification of the percentage of cells with P-bodies (PBs) from n = 3 independent experiments is shown; ≥364 cells were counted per treatment. ( G ) Cells were treated as in C to detect XRN1 (magenta) and EDC4 (yellow) by immunofluorescence microscopy ( n = 2 independent experiments). ( H ) U-2 OS cells were treated as in E , and immunofluorescence microscopy was done to detect DCP1A. The percentage of cells with PBs from n = 3 independent experiments is shown; ≥351 cells were counted per treatment across all replicates. Representative images are shown with the average ± SEM for each experiment, and green, gray, and pink points represent the average of each replicate. Statistical significance was assessed with an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (*) P < 0.05, (**) P < 0.01, (***) P < 0.005, (****) P < 0.001.

Article Snippet: PABPC1 , Proteintech , 66809 , 1:100 (immunofluorescence).

Techniques: Inhibition, Western Blot, Activity Assay, Stable Transfection, Expressing, Staining, Immunofluorescence, Fluorescence, In Situ Hybridization, Microscopy, Marker

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: CELF1 displaces eIF4G1 from eIF4E and directly binds PABPC1. ( a ) Purified GST-tagged eIF4E or eIF4E S209D was mixed with purified DDK-eIF4G1. Complexes were pulled down with purified glutathione beads and immunoblotted with indicated antibodies. ( b ) Purified DDK-tagged eIF4G1 was mixed with purified GST-tagged eIF4E S209D (positive control) or purified 6x-His-tagged CELF1 and precipitated on glutathione or Ni 2+ resin and immunoblotted with the indicated antibodies. ( c ) Immunoblots of binding assays in which purified GST-eIF4E S209D was mixed with purified DDK-eIF4G1 in combination with increasing amounts of either purified WT (6xHis-CELF1) or mutant (6x-His-CELF1 Δ365–71 ) CELF1. Complexes were captured on glutathione resin. ( d ) Immunoblots of binding assays in which purified GST-eIF4E S209D was mixed with purified wild type ( 6xHis-CELF1 ) and increasing amounts of purified DDK-eIF4G1. Complexes were captured on Ni 2+ resin. ( e ) Immunoblots of competitive binding assays in which purified DDK-tagged PABPC1 was incubated with the indicated ratios of DDK-tagged eIF4G1 and 6xHis-tagged CELF1. Complexes were captured on Ni 2+ resin. ( f ) Purified, untagged CELF1 was mixed with purified, untagged eIF4E S209D , purified, untagged PABPC1, or both. Complexes were immunoprecipitated with IgG (negative control) or anti-CELF1 antibody and blotted with the indicated antibodies. ( g ) Immunoblots of binding assays in which the indicated concentrations of purified DDK-tagged eIF4A were mixed with increasing concentrations of purified DDK-eIF4G1 or purified 6xHis-tagged CELF1. Complexes were captured on Ni 2+ resin or via immunoprecipitation with anti-eIF4G1 antibody, as indicated. All results are representative of at least three independent experiments.

Article Snippet: The C-MYC/DDK-tagged purified recombinant human eIF4G1, eIF4A1, and PABPC1 proteins were purchased from OriGene (Rockville, MD).

Techniques: Purification, Positive Control, Western Blot, Binding Assay, Mutagenesis, Incubation, Immunoprecipitation, Negative Control

Journal: Nucleic Acids Research

Article Title: CELF1 is a non-canonical eIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

doi: 10.1093/nar/gkag123

Figure Lengend Snippet: A working model for CELF1-dependent cap-dependent translational initiation during TGF-β-mediated EMT of breast epithelial cells. During EMT of breast epithelial cells, CELF1 is stabilized and recruited to GREs within the 3′ UTRs of EMT effector mRNAs. In cis on the RNA, CELF1 then bridges eIF4E and PABPC1 to promote translation of these mRNAs. Additional mechanisms conferring specificity on this process, as well as the mechanism by which CELF1 recruits the full 43S pre-initiation complex, remain to be defined. Components are not drawn to scale.

Article Snippet: The C-MYC/DDK-tagged purified recombinant human eIF4G1, eIF4A1, and PABPC1 proteins were purchased from OriGene (Rockville, MD).

Techniques: