Figure 2. L1 but not neomycin gene splicing is delayed during the course of L1 expression.

A. A schematic representation of the neomycin tagged human L1.3 element expression cassette (L1Neo) and some of the relevant mRNA products that it makes. PRO, ORF1, ORF2, and 5’ UTR 100 probe are designated as in Figure 1. L1.3pA and SV40pA indicate the position of the respective polyadenylation signals. Stippled arrow indicates the position and orientation of the neomycin (NeoR) gene, which is interrupted by an intron (IN) in the same orientation as the L1 ORFs. Solid black lines correspond to the portions of the L1Neo expression cassette that are included into the mature mRNA product. Dashed black lines indicate sequences that are removed by splicing. Black horizontal arrow indicates relative position of the strand-specific RNA probe (5’ UTR100) used to detect both spliced and polyadenylated products. A scale bar and splice donor, acceptor, and polyadenylation sites are indicated as in 1A.B. Northern blot analysis of the transiently transfected L1Neo expression vector in NIH 3T3 cells with the 5’ UTR 100 probe at 6, 9, 24, 48 and 72 hours after the addition of the transfection cocktail. FL1NeoUnsp and FL1NeoSp indicate unspliced and spliced (intron positioned in the Neo cassette) full-length L1Neo mRNAs, respectively. pA labels prematurely polyadenylated mRNA. L1 splice products and L1/NeoRNA indicate spliced and prematurely polyadenylated mRNAs and a hybrid splice between the L1 sequence and the Neo gene as depicted in 2A (Belancio et al., 2006). C. A northern blot analysis of the transiently transfected tagged L1.3 (L1Neo) and wild-type L1.3 (L1.3) expression vectors in NIH 3T3 cells probed with the strand-specific RNA probe (5’ UTR 100). L1/Neo splice product is a hybrid mRNA formed by splicing of L1 and Neo sequences (Belancio et al., 2006). FL1.3 marks the full-length L1 mRNA produced by the wild-type L1.3 expression vector.