FIGURE 5.

Loss of pseudouridylation decreases EBER2 stability. (A) Total RNA isolated from various PUS-knockdown cells was subjected to northern blot analysis. EBER2 levels were reduced only in cells depleted for DKC1. Northern blot analysis for EBER1 served as a loading control. Quantification of three independent experiments is shown in the graph (asterisk indicates P < 0.001). (B) Two independent DKC1-knockdown cell lines were examined for decreased EBER2 levels. Abundance was determined by qRT-PCR and using EBER1 levels for normalization. Results are the average of three independent experiments; error bars represent standard deviation. (C) Lower EBER2 levels are not caused by a decreased transcription rate. DKC1-knockdown cells grown 5 d in the presence of Dox were cultured for 1 h with tritiated uridine followed by ASO-mediated selection of EBER1 and EBER2. The level of nascent labeled EBER2, as shown in the representative autoradiograph, remained unchanged after DKC1 knockdown, indicating the same rate of transcription in both wild-type and knockdown cells.