FIGURE 4.

ZFC3H1 and U1-70K are required for nuclear retention of 5′SS motif containing mRNAs in speckles. (A,B) Control or ZFC3H1-depleted U2OS cells were microinjected with plasmids containing the βG-Δi reporter ± the 5′SS motif. After the indicated times, the cells were fixed and stained for βG mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. (A) Example images of U2OS cells fixed 2 h post-injection with βG reporter mRNA ± the 5′SS motif, with each row representing a single field of view with white arrows pointing to examples of βG mRNA/SC35 colocalization. The merged overlayed image shows βG mRNA in red, SC35 in green. (B) Quantification of the degree of βG mRNA/SC35 colocalization in cells depleted of ZFC3H1 or control shRNA treatment by Pearson correlation coefficient analysis as previously described (Akef et al. 2013). Each bar represents the average and standard error of three independent experiments, each experiment consisting of 150 to 200 nuclear speckles from 15 to 20 cells. Note that ZFC3H1 is not required for the targeting of 5′SS motif containing mRNAs to nuclear speckles. (C–E) Control, U1-70K- or ZFC3H1-depleted U2OS cells were transfected with ftz-Δi ± 5′SS motif. Eighteen to twenty-four hours post-transfection, the cells were fixed and stained for ftz mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. Representative images, with each row depicting a single field of view, is shown in C with merged overlays showing ftz mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of ftz/SC35 colocalization are indicated with arrows. (D) The degree of ftz/SC35 colocalization by Pearson correlation coefficient analysis was quantified as above, except that values <0.25 were counted as “not colocalized.” Note that U1-70K or ZFC3H1 depletion leads to decreased level of colocalization between ftz mRNA and SC35. Each bar represents the average and standard error of three independent experiments, each experiment consisting of 100–200 nuclear speckles from 10 to 20 cells. (E) The amount of ftz reporter mRNA present in nuclear speckles as a percentage of either the nuclear (“Spec/Nuc”) or total cellular (“Spec/Total”) mRNA levels in transfected cells. Each data point represents the average and standard error of the mean of at least three independent experiments, each experiment consisting of 10–20 cells. Student's t-test was performed, (**) P < 0.01, (***) P < 0.001.