Both the EDC and the CPA attach poly(A)site-cleaved RNA to the polymerase, and polyadenylation does not release the EDC-attached RNA. (A) Pull-down (Method 2) of EDC-attached poly(A)site-cleaved RNA that either is or is not polyadenylated. Band 1 in lanes 3 and 4 was quantitated after subtracting the background appearing at the same position in lanes 1 and 2, respectively. The averages and ranges for two independent experiments are shown. (B) Pull-down of poly(A)site-cleaved RNA (Method 3) that either has or does not have a 3′ splice site. For lanes 3 and 4 a plasmid construct with a mutant 3′ splice site (AG → ct) was used. The averages and ranges for two independent experiments are shown.
