Journal: Nature Communications

Article Title: Ribosome inhibition by C9ORF72 -ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

doi: 10.1038/s41467-022-30418-0

Figure Lengend Snippet: a Comparison of translation inhibition by PR 20 and GR 20 to that by harringtonine, cycloheximide, and GDPCP upon a 5-min preincubation with rabbit reticulocyte lysates prior to addition of nanoluciferase mRNA ( t = 0). IC50 of PR 20 and GR 20 are similar to those of cycloheximide, harringtonine, and GDPCP ( n = 3 independent experiments, mean ± SEM; RLU, relative luminescence units). b Comparison of translation inhibition by PR 20 and GR 20 to that by harringtonine, cycloheximide, and GDPCP. The inhibitors were added 300–400 s (gaps in the curves) after addition of nanoluciferase mRNA. PR 20 and GR 20 act similarly to elongation inhibitors cycloheximide and GDPCP ( n = 3 independent experiments, mean ± SEM). c Comparison of polysome profiles of rabbit reticulocyte lysate translating endogenous mRNA in the presence of translation inhibitors (Abs254, UV absorbance at 254 nm). The translation mixture was incubated for 5 min at 30 °C in the presence or absence of PR 20 , GR 20 , harringtonine, cycloheximide, and GDPCP ( n = 2 independent experiments, representative traces with concurrent uninhibited control are shown). Half-mer peaks (black arrows) are most prominent with PR 20 , GR 20 , and GDPCP. Source data are provided as a Source Data file.

Article Snippet: To measure the IC50s of the translational inhibitors cycloheximide (Akros Organics), harringtonine (AbCam), GDPCP (Jena Biosciences), PR 20 and GR 20 , we monitored the translation of nanoluciferase mRNA in nuclease-treated RRL (Promega).

Techniques: Inhibition, Incubation

Journal: Nature Communications

Article Title: Ribosome inhibition by C9ORF72 -ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

doi: 10.1038/s41467-022-30418-0

Figure Lengend Snippet: a Scheme of toeprinting experiments using two fluorescently-labeled primers, primer A and primer B. Primer extension through the open reading frame (ORF) to ribosomes stalled at the start codon is expected to yield a peak at ~115 nt for Primer A or at ~515 nt for Primer B. b Following a 5-min translation reaction at 37 °C in the absence or presence of inhibitors (no inhibitor, 4 μM PR 20 , 4 μM GR 20 , 40 μM harringtonine, 100 μg/ml cycloheximide or 100 μM GDPCP), sucrose-gradient fractionation was used to separate free mRNA from various ribosome fractions. The 80S fraction and 4–5-mer fractions (dashed boxes) were used in assays shown in panels b , c . The sucrose gradient traces are shown for no inhibitor, PR 20 and harringtonine. c The 80S fraction was subjected to toeprinting analysis with primer A via fragment analyzer (see Methods). Traces reveal peaks at the expected location of the start codon. The blue-shaded zones of the traces were quantified for panel e , indicating enrichment of ribosomes stalled at the start codon relative to a region of the ORF especially in samples treated with harringtonine and PR 20 . d As in ( c ) except that the 4-5mer polysome fraction was used for toe-printing analysis with primer B. Traces show many peaks in the ORF of the mRNA and also at the start codon. Blue and pink boxes indicate sections of the trace used in quantification in panel g . e The normalized ratio of start-codon-associated toeprints to coding-region-associated toeprints in the 80S fraction using primer A. f Independent repeat of toeprinting reaction as in e . g The normalized ratio of start-codon-associated toeprints to coding-region-associated toeprints. Ten different windows (20 bp in length each) within the coding region were quantified relative to a 20 bp window around the start codon, and the mean ± SEM is shown. h Independent repeat of toeprint reaction as in g . Source data are provided as a Source Data file.

Article Snippet: To measure the IC50s of the translational inhibitors cycloheximide (Akros Organics), harringtonine (AbCam), GDPCP (Jena Biosciences), PR 20 and GR 20 , we monitored the translation of nanoluciferase mRNA in nuclease-treated RRL (Promega).

Techniques: Labeling, Fractionation