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. 2019 Jul 10;8:e44752. doi: 10.7554/eLife.44752

Figure 3. Validation of single-molecule translation reporter assay.

(A) Schematic of translation reporter constructs for labeling of RNA and nascent protein chains. (B) Live cell imaging snapshot of a cell expressing the control translation reporter. The mCherry channel detects the 3x-mCherry-PCP protein. Bright spots correspond to RNA molecules. Diffuse signal results from free 3x-mCherry-PCP. The GFP channel detects the scFv-GFP antibody. Bright spots overlap with RNA spots (merge image) and correspond to nascent protein at translation sites. Diffuse signal results from free scFv-GFP or scFv-GFP bound to the reporter protein released after translation. (C) Cells expressing the control translation reporter, containing PP7 repeats (+PP7), or a reporter without PP7 repeats (-PP7), were imaged live. mCherry intensity overlapping with translation sites (GFP spots) was measured and normalized to the intensity observed in nearby cytoplasmic regions with diffuse signal. Value of 1 indicates that there is no mCherry concentration at translation sites. (D) The same cytoplasmic areas, of cells expressing the control translation reporter, were imaged before and after puromycin addition. GFP/mCherry intensity of individual spots was calculated as a measure of translational efficiency. n > 100 (C) and n > 500 (D) spots from multiple cells observed in three independent experiments; error bars: standard error; ****: p-value<0.0001 by Student’s t-test. Scale bars: 5 μm.

Figure 3.

Figure 3—figure supplement 1. Translation signal of localized reporters reflects active translation.

Figure 3—figure supplement 1.

Cells expressing the indicated translation reporters were imaged just prior to (t = 0 min), or 15 min (t = 15 min) after, addition of the translation inhibitors cycloheximide, harringtonine or lactimidomycin. Graphs indicate the relative change in number of detectable translation sites (one indicating no change). n = 13–17 cells per condition. error bars: standard error; ****: p-value<0.0001 by one way ANOVA with Tukey’s multiple comparisons test. Scale bars: 10 μm.