Figure 6

Polyadenylation at the 3′LTR is splice-donor-independent, and 13 nucleotides from U3 are sufficient to promote polyadenylation. (A) Schematic representation of the U3 deletion reporter plasmids used for analysis of polyadenylation at the 3′LTR and the position of the Northern blotting probe. (B) Northern blotting analysis of RNA from HEK 293T cells transfected with pHSRV13 and SDm2 or SDm4 derivatives. The RNAs were preferentially polyadenylated at the 3’LTR polyadenylation site. Neither SDm2 nor SDm4 influenced the poly(A) site selection. To analyse determinants of the poly(A) selection, the U3 region (777 nucleotides) was shortened to encompass nucleotides from −350, -200, -100, or −13 to +1. In addition, a plasmid encoding the RU5 region with a complete deletion of U3 was analysed. rRNA amounts are shown as loading control, since the RU5 and −13 signals migrate at the same height as GAPDH RNA. (C) Luciferase assay of cells transfected with CMV-RU5, CMVRU5-SDm2, and CMV -13RU5 showing that introns, exons, and coding sequences are not required for efficient polyadenylation. Assays were performed in triplicate, and error bars represent the standard deviation from the mean. The significances of the reductions by the MSD mutations were calculated by paired two-sample t-test. p-values are indicated.