Journal: Nature
Article Title: mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis
doi: 10.1038/s41586-018-0538-8
Figure Lengend Snippet: a, EM images of polyribosome with METTL3-gold particle labeling. Red arrows indicate METTL3 with immuno-gold particle (6 nm). Three independently performed experiments show similar results. b, Counting of METTL3 with gold particle labeling in each polyribosome. c, EM images of polyribosome with METTL3 and eIF4E. Red arrows indicate METTL3 with immuno-gold particle (6 nm) and yellow arrows indicate eIF4E with immuno-gold particle (10 nm). Four independently performed experiments show similar results. d, Average distance between immuno-gold particles was measured. n = 6 biologically independent samples from at least three independent experiments. Error bars represent mean ± SD. e, Colloidal Coomassie blue staining of recombinant protein His-METTL3 or His-METTL3 1-200 amino acid fragments (1-200). Two independently performed experiments show similar results. f, Colloidal Coomassie blue staining of recombinant GST-tagged protein eIF3g, eIF3h, eIF3i, eIF3j or eIF3m. Two independently performed experiments show similar results. g, GST-eIF3h was co-purified with His-METTL3 in the presence of either rabbit IgG (rIgG) or α-METTL3 antibody. Levels of co-purified His-METTL3 were analyzed by Western blotting. Two independently performed experiments show similar results. h, Schematic diagram of human eIF3h deletion mutants. i, Colloidal Coomassie blue staining of recombinant GST-eIF3h, -eIF3h (1-222) or -eIF3h (29-222). n = 1 independent experiments. j, GST pull-down of indicated eIF3h deletion mutants. Co-purified His-METTL3 was analyzed by Western blotting. n = 1 independent experiments. k, Western blotting demonstrates efficient knockdown of eIF3h protein. Three independently performed experiments show similar results. l, qRT-PCR analysis demonstrates efficient down regulation of eIF3h mRNA. Error bars represent mean ± SD; n = 3 biologically independent samples; two-sided t-test. m, qRT-PCR analysis of reporter mRNAs. FLuc-MS2bs reporter mRNAs were normalized to RLuc mRNAs. The FLuc:RLuc ratio obtained in FLAG-MS2 was set to 1. Error bars represent mean ± SD; n = 3 biologically independent samples.
Article Snippet: Where indicated, during the elution, α-METTL3 antibody (Proteintech, 15073-1-AP) and gold nanoparticle (6nm) conjugated α-rabbit IgG were added with/without either α-CBP80 antibody or α-eIF4E antibody that was gold nanoparticle (10nm) conjugated using GOLD conjugation kit (Abcam, ab201808) according to the manufacturer’s instructions.
Techniques: Labeling, Staining, Recombinant, Purification, Western Blot, Quantitative RT-PCR
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