Figure 6.

U1 RNA levels inversely correlate with intronic APA. (a) U1 snRNA levels decrease after UV treatment. HCT116 and RKO cells were treated with UV irradiation and allowed to recover for the indicated times, and then harvested. Nuclear RNA was isolated and cDNA was prepared using random primers. qRT–PCR reaction was performed with primers specific for U1 snRNA. qRT–PCR products of actin were used as endogenous control. The values from each sample were normalized to non-treated samples. The qRT–PCR values were calculated from three biological samples by triplicate in each determination. (b) Changes in the levels of spliceosome complex components on UV treatment. HCT116 cells were treated with UV irradiation and analyzed as in Figure 4a by qRT–PCR reaction with primers specific for U2 snRNA, U1A, U1C and U1-70K. The qRT–PCR values were calculated from three independent samples. (c) Functional depletion of U1 snRNA, but not U2 snRNA, causes an increase in intronic pA. HCT116 cells were transfected with control or anti-sense morpholino targeting U1 snRNA (U1 AMO) or U2 snRNA (U2 AMO). cDNA was prepared and used in qRT–PCR assays as in Figure 4c. The qRT–PCR values were calculated from three biological samples were analyzed by triplicate in each determination. (d) Overexpression of U1 RNA abolishes UV-induced intronic APA. HCT116 cells were transfected with two concentrations of either control or U1 snRNA expressing vectors and treated with UV irradiation. Nuclear RNA was used to prepare cDNA, which was used in qRT–PCR reactions as in Figure 2b. The qRT–PCR values were calculated from three independent samples. (e) Overexpression of U1 RNA decreases the levels of UV-induced apoptosis in HCT116 cells. The DNA fragmentation was calculated from three independent samples. APA, alternative cleavage and polyadenylation; cDNA, complementary DNA; qRT–PCR, quantitative reverse transcription–PCR; UV, ultraviolet.