FIGURE 4.

Splicing of the second intron competes with internal polyadenylation of the B19V pre-mRNA at the (pA)p site. A, C1NS1(−) is schematically illustrated, and the nucleotide substitutions at the respective splice site in each derivative mutant are shown. B–D, COS-7 cells were transfected with C1NS1(−) and its derivatives, and UT7/Epo-S1 cells were electroporated with linearized B19V N8 DNA and its derivatives as indicated. Total RNA isolated from COS-7 and mRNA isolated from UT7/Epo-S1 cells were protected by homologous probe 9 (B), homologous probe 10 (C), or probe 8 (D). E, COS-7 cells were transfected with C1NS1(−), C1B19D2KO, or C1B19D2ISE2KO (18) as indicated. Total RNA isolated was protected by probe 8. A representative experiment is shown with the identities of the protected bands indicated on the right. Quantification of the ratios of RNAs spliced (Spl) versus unspliced (Unspl) at the A2-1 site (B), the ratios of RNAs spliced versus unspliced at the A2-2 site (C), and the ratios of RNAs polyadenylated at (pA)p versus (pA)d (D) are shown as averages with the S.D., and which were calculated from the results of at least three individual experiments. Asterisk indicates likely nonspecific hybridized products (14). RT, read-through; TR, terminal repeats.