Fig. 1: NaP-TRAP measures translation through immunocapture. | Nature Communications

Fig. 1: NaP-TRAP measures translation through immunocapture.

From: NaP-TRAP reveals the regulatory grammar in 5’UTR-mediated translation regulation during zebrafish development

Fig. 1

a Schematic detailing the NaP-TRAP method. FLAG-tagged nascent chain complexes of reporter mRNAs are enriched via an anti-FLAG immunoprecipitation. Translation is measured as a ratio of reporter reads in the pulldown relative to the input. b NaP-TRAP derived translation at 6 hpf (left, N = 3 replicates; 25 embryos per replicate) and fluorescence measurements at 24 hpf (right, - MO N = 14 embryos, +MO N = 9 embryos) in the presence (+ MO) or absence (- MO) of a translation blocking morpholino (two-sided unpaired t-test: ** p < 0.005, **** p < 0.0001; NaP-TRAP + /− MO, p = 0.0036; Fluorescence +/− MO p < 10−4; error bars = SEM). c NaP-TRAP derived translation values for 3xFLAG-GFP-3xmiR-430 (blue) and 3xFLAG-GFP-3xmiR-204 (gray) at 2 hpf (N = 4 replicates; 25 embryos per replicate) and 4.3 hpf (N = 4 replicates; 25 embryos per replicate) (two-sided unpaired t-test: ** p < 0.01, *** p < 0.005; miR-204 2hpf vs 4 hpf, p = 0.0071; miR-430 2hpf vs 4 hpf = 0.0001; error bars = SD). Schematic representation of repression of translation exercised by miR-430, but not miR-204 at 4.3 hpf. d NaP-TRAP derived translation values at 2 hpf (left, N = 3 replicates; 25 embryos per replicate) and quantification of band intensity of western blot and example immunoblot (right, N = 3 replicates; 5 embryos per replicate) of GFP reporters with poly-A tails of 0, 30, 60 and 90 As in the presence of a DsRED injection control. e In HEK293T cells, NaP-TRAP derived translation values are strongly correlated with mean ribosome load (polysome profiling) for a 5’ UTR mRNA reporter library (two-sided Pearson’s R = 0.92, N = 7572 reporters). f A cumulative density plot of residuals (NaP-TRAP translation – predicted translation) for reporters containing oORFs (N = 247 reporters), uORFs (N = 3834 reporters), and no upstream start codons (N = 1993 reporters) (p-values were calculated using a two-sided Mann-Whitney U-test; oORF vs noAUG p < 10−52; oORF vs uORF p < 10−29; uORF vs noAUG p < 10−60).

Back to article page