ZFC3H1 and U1-70K function in the same pathway for the nuclear retention of 5′SS motif containing mRNAs. (A) U2OS cells were treated with lentivirus shRNA against either U1-70K, ZFC3H1, or a mixture of the two. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for U1-70K, ZFC3H1, and mAb414. Note that to effectively deplete U1-70K, cells were treated with lentivirus containing four shRNA plasmids. Also note that the asterisk (*) denotes a non-specific band. (B,C) Control-, U1-70K-, ZFC3H1-, or codepleted cells were transfected with the intronless ftz reporter ±5′SS as described in Figure 2. Note that the cytoplasmic/nuclear distribution of ftz-Δi-5′SS mRNA in cells codepleted of U1-70K and ZFC3H1 resembles the distribution in single depletion cells, suggesting that both proteins function in the same pathway. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D. Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t-test was performed for C. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. (D) HEK cells expressing carboxy-terminally tagged ZFC3H1 (ZFC3H1-FLAG) were lysed and subjected to immunoprecipitation reactions with FLAG M2 beads or mouse IgG (“Control IP”). Immunoprecipitates were separated by SDS-PAGE and immunoprobed for FLAG and U1-70K. For comparison, 1% of the input lysate was also analyzed. The full-length ZFC3H1-FLAG protein is denoted by the asterisk (*) and a shorter band, likely a degradation product, is denoted by the pound (#) sign.
