Figure 3.

Effects of the pA1 hexamer sequences and the downstream GU/U-rich elements on the usage of the proximal and distal pA1 signals. (A) Structure of the 3’-end-labeled probe and the expected sizes of fragments protected by RNA polyadenylated at the pA1_prox or pA1_dist cleavage site. The probe was 3’-end-labeled (asterisks) at the EagI site. (B) & (C) Polyacrylamide gel electrophoresis of the protected products derived from S1 nuclease analysis using total RNAs prepared from minigene-transfected HuH-7 cells. Structure features of the minigenes are indicated at the top of the gel where canon denotes the canonical hexamer, AAUAAA; mut denotes the AAGAAA hexamer and inv denotes an inverted sequence as indicated in Figure 1B. The U2 and U3 motifs are as shown in Figure 4. The intensity of the protected band was quantified by a phosphorimager. The pA1_dist usage was calculated as a fraction of the pA1_dist signal intensity to the combined signal intensities of pA1_dist and pA1_prox. The fragments of 409-nt along with 398-nt and those of 392-nt plus 378-nt are taken to represent pA1_dist and pA1_prox usage, respectively. In lane 6 in (B), the asterisked band was likely derived from the use of the pA1_dist signal. And in lane 2 in (C), the band with open circle was likely derived from the use of pA1_prox signal and the generation of such a band is probably due to stabilization of the DNA/RNA duplex terminus at the position of the G residue in the AAGAAA mutant. Open arrow on the left indicates non-specific band. The image of the left panel (lanes 1-8) was the result of phosphorimager and that of the right (lanes 9-12) was from autoradiography.