Fig. 4: Simultaneous visualization of single mRNAs, translation, and RISC-binding.
From: Single-molecule imaging of microRNA-mediated gene silencing in cells
a Schematic of the SINAPS-IF-FISH experiment to visualize translation and RISC-binding simultaneously at single-mRNA resolution. Magenta, green, and cyan spots represent reporter mRNAs, SunTag peptides, and RISC, respectively. b The images of a RISC-negative untranslated mRNA (first row), a RISC-negative translated mRNA (second row), a RISC-positive untranslated mRNA (third row), and a RISC-positive translated mRNA (fourth row) in U2OS cells are shown. Reporter mRNAs (magenta, first column), SunTag peptides (green, second column), and AGO (cyan, third column) were labeled by SINAPS and IF-FISH. Merged images are shown on the right side. Scale bar, 1 μm. c The ratio of RISC-negative untranslated (magenta), RISC-negative translated (green), RISC-positive untranslated (cyan), and RISC-positive translated (orange) mRNAs. All mRNAs were classified into these four classes based on 3D-colocalization analysis. d A trend of negative correlation between translational efficiency and RISC-binding efficiency at the single-cell level. Images were analyzed using CellProfiler and FISH-quant. Then, translational efficiency and RISC-binding efficiency were calculated as described in Supplementary Fig. 9 (see also Methods). Each circle represents a single cell (n = 50 for each condition). e, f Translated mRNAs tend to be RISC-positive mRNAs. The fraction of RISC-positive mRNAs (e) and the intensity of AGO on mRNAs (f) are shown. In f, the means with SEM and the p-value of one-tailed Mann–Whitney test are shown. Magenta and green bars represent the values of untranslated and translated mRNAs, respectively. g, h RISC-positive mRNAs tend to be translated mRNAs. The fraction of translated mRNAs (g) and the number of ribosomes on mRNAs (h) are shown. In h, the means with SEM and the p-value of one-tailed Mann–Whitney test are shown. Magenta and cyan bars represent the values of RISC-negative and RISC-positive mRNAs, respectively. Source data are provided as a Source Data file.
