Subcellular localization of non-translating (free) RNAs and translation sites. (A) Schematic maps of the mTurquoise markers used to label different subcellular sites. (B) Representative images of the NIH3T3 GFP/RFP cells co-transfected with NV-MS2 and one of each mTurquoise plasmid labeling ER, Golgi, LE, Lyso, and PM. Images were acquired on an LSM980 confocal microscope. RNA is in red and subcellular locations in hot cyan. Single z-planes are presented, and scale bars are 10 and 2 µm for zoom insets. White arrows indicate NV RNA associated with the turquoise surfaces. (C) Representative images of cells co-expressing ΔNC and one of each mTurquoise plasmid. Translating and non-translating FL RNAs associated with the subcellular surfaces (hot cyan) are indicated by yellow and white arrows, respectively. Single z-planes are presented, and scale bars are 10 and 2 µm for zoom insets. In zoom insets, the letters T, R, G, and M denote turquoise, red, green, and merge, respectively. (D) Percentage of free RNAs associated with each location determined with Imaris as the number of NV or non-translating FL RNA spots associated with each location, relative to the total number of corresponding free RNA spots in the whole cell. Black and white circles represent NV and FL untranslating RNAs, respectively. (E) Percentage of translating FL RNA spots associated with each location, relative to the total number of translation sites per cell. The graphs show the mean ± SEM. (n = 40 cells for ER, 15 cells for PM, and 10 cells for other locations). ns = nonsignificant, *P ≤ 0.05, ***P ≤ 0.001, and ****P ≤ 0.0001 (Mann–Whitney test).