RNA insertions into the 5′ end of βG mRNA promotes nuclear export. (A) A schematic representation of the different fusion constructs that were used in this figure. (B,C) Plasmids containing the indicated constructs were transfected into human U2OS cells. After 14–18 h, the cells were fixed, permeabilized, and stained for mRNA using a FISH probe directed against βG. The cells were imaged (B) and mRNA distribution was quantified (C). Each bar represents the average and standard error of three independent experiments, each consisting of at least 30 cells. (D–F) Plasmids containing the indicated constructs were microinjected into the nuclei of U2OS (D) or NIH 3T3 (E,F) cells. After 20 min, cells were treated with α-amanitin and the newly synthesized mRNA was allowed to be exported for an additional 2 h. Cells were fixed, permeabilized, stained for mRNA using a FISH probe directed against βG and imaged. Examples of mRNA FISH staining in NIH3T3 cells are shown in E. The mRNA distribution was quantified for injected U2OS (D) and NIH3T3 (F), with each bar representing the average and standard error of three independent experiments, with each experiment consisting of at least 30 cells. (G,H) Plasmids containing the indicated constructs were transfected into human U2OS cells. After 14–18 h, the cells were fixed and permeabilized, stained for mRNA using a FISH probe directed against βG, imaged (G) and mRNA distribution was quantified (H). Each bar represents the average and standard error of three independent experiments, each consisting of at least 30 cells. Scale bar = 20 µm.
