Figure 7.

TTRAP SIM motif but not 5′-tyrosyl DNA phosphodiesterase activity is essential for its role in rRNA biogenesis. (a) siTTRAP-stable cells were transfected with FLAG-TTRAP wt or SIM mutant or empty vector as control and treated with 5 μM MG132 for 16 h. rRNA biogenesis was followed by qPCR relative to β-actin and GAPDH housekeeping genes. Standard deviation was calculated from three independent experiments. Significance between siTTRAP cells transfected with wt TTRAP or with empty vector was calculated with t-test (^*P<0.05). NS, nonsignificant. In the same experiment, cell lysates were prepared and analyzed by immunoblot for the presence of ectopic TTRAP protein. β-Actin was used as loading control. (b) siTTRAP-stable cells were transfected with TTRAP wt or phosphodiesterase mutants E152A or D262A or empty vector as control and processed as in (a). (c) TTRAP E152A and D262A mutants do not accumulate in the nucleolus. SiTTRAP-stable SH-SY5Y cells were transfected and treated as in (b). Double immunofluorescence was performed with anti-FLAG and anti-NCL antibodies. Images are representatives of two independent experiments Bars, 7 μm