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. 2013 Apr 29;8(4):e62125. doi: 10.1371/journal.pone.0062125

Figure 3. Stable expression of the mutant U5-200kD protein affected alpha-tubulin pre-mRNA splicing.

Figure 3

A) Detection of alpha-tubulin pre-mRNA. Lanes 3, 5, 7 and 9 are RT-PCR reactions for detection of the alpha-tubulin pre-mRNA. Absent PCR product in lanes 3 and 5 demonstrate complete splicing of the endogenous alpha-tubulin pre-mRNA. The band in lanes 7 and 9 is the alpha-tubulin pre-mRNA that accumulated in the splicing-defective SMC10 cell lines. The stronger band for PCR products (lane 9) indicates increased accumulation of the pre-mRNA due to induced expression of the mutant U5-200kD protein. Lanes 1 and 10 are the 100 bp ladders. RT-PCR products were resolved in a 1.8% agarose gel and stained with ethidium bromide. B) RT-PCR analysis of the alpha-tubulin mRNA. The upper band in lanes 3, 5, 7 and 9 is the histone product amplified from the uninduced and induced 293/EcR and SMC10 RNAs, respectively. The lower band in those lanes is the alpha-tubulin amplified product. Lanes 2, 4, 6 and 8 are the minus reverse transcriptase reactions for each sample. Lanes 1 and 10 are the 100 bp ladders. Samples were resolved in a 1.8% agarose gel and stained with ethidium bromide. C) Semi-quantitative analysis of the alpha-tubulin mRNA expression. Compared to 293/EcR cells, the alpha-tubulin mRNA expression in the SMC10 cells has decreased approximately thirty-five percent. Induction of the mutant protein had little detectable effect on alpha-tubulin mRNA levels in the SMC10 clone.