Figure 1.

Effects of poly(A) hexamer sequences and the downstream GU/U-rich elements on pA1 usage. (A) Structure of the minigene plasmid pAS-2E_Bst. The bi-exonic minigene was transcriptionally driven by the SV40 early promoter (SV). The first exon (shaded box) included the human ASS1 cDNA exons 1 through 15 where a 30-nt foreign sequence (dark box) was inserted. The 3’-portion of the minigene included the complete intron 15 (Int15) and the terminal exon 16 (Ex16) of the gene. The sequence surrounding the pA1 signal is displayed where the proximal (pA1_prox) and distal (pA1_dist) poly(A) hexamers are underlined and their respective cleavage sites are indicated by arrows. The downstream U/GU-rich elements are indicated as follows: the U-rich (U1) element is double-underlined and the GU-repeat is designated as (GU)n where n referred to repeat number. (B) S1 nuclease mapping analysis of RNAs derived from the ASS1 minigene constructs. Total RNAs were prepared from minigene-transfected HuH-7 cells and were analyzed with the depicted probe. The structure of the 3’-end-labeled probe and the expected sizes of fragments protected by the respective RNA are shown. A probe was specifically tailored for each minigene. Asterisks indicate [³²P] labels at the 3’-end of the probe. The minigene pAS-2E_Bst and its modification derivatives are listed. The region highlighted in (A) are displayed where “-“ indicates identical nucleotides to the prototype and nucleotides that are different from the prototype are shown. The intensity of the protected band was quantified by the use of a phosphorimager. The relative amount of pA1 RNA of the minigene is represented as a ratio of pA1 signal to total signals of pA1, pA2 and pA3 combined. Lane M is a 5’-end-labeled DNA marker where the fragment sizes are shown on the left. Lane Cont. was a control RNA prepared from cells transfected with the parental plasmid pSVpoly that was used in the minigene construction. The endogenous ASS1 mRNA-protected fragment (endo ASS1) is also indicated. The gel displayed is typical of two different experiments using independently prepared RNAs.