1514 Nucleic Acids Research, 2006, Vol. 34, No. 5 Figure 1. LINE-1 elements contain multiple splice sites. (A) A schematic representation of putative splice sites identified by the BDGP program in the sense strand of human L1.3 and mouse L1spa. Black (SD) and gray (SA) arrows mark splice sites using a default cutoff value of 0.4. Asterisks mark SD and SA sites that have been identified as functional based upon sequence analysis of spliced transcripts recovered by RT–PCR or as determined by sequence analysis of spliced products found re-integrated into the human and mouse genomes. PRO corresponds to the L1 promoter region, ORF1 and ORF2 identify ORFs 1 and 2. (B) A schematic representation of the L1.3 neomycin-resistance (L1.3Neo) expression cassette. The position and orientation of the NeoR gene are shown by an arrow. It is interrupted by an intron (IN) in the same orientation as the L1 ORFs. The cassette ends in L1.3 and SV40 polyadenylation signals (L1.3pA and SV40pA, respectively). The L1.3 portion that is missing in transcripts SpX and X(IN) is marked above the cassette. Sequences below the schematic of the vector demonstrate the 50 and 30 splice sites with invariant GT and AG dinucleotides shown in larger font. The resulting sequence of the splicing event is listed under the junctions. Arrows above the L1.3 expression cassette (marked 50-UTR, NeoEx and NeoIN) represent positions of the strand-specific RNA probes used for L1 RNA detection. The arrows indicate the sense of the probes. Because of the length of the 50-UTR probe (900 bp) in vitro transcription products are a mix of truncated transcripts, which are enriched for the 30 end of the L1.3 50-UTR (solid portion of the arrow). Below the sequences is a schematic representation of the splice products generated between the L1.3 and the Neo cassette [SpX(IN) and SpX] and the premature polyadenylated, unspliced L1.3 mRNAs (1–3). Solid black lines represent portions of the L1.3 and Neo cassette sequences included in the transcripts. Dotted lines denote parts that were removed by splicing. (C) Northern blot analysis of poly(A)-selected mRNAs from NIH 3T3 cells transfected with the L1.3Neo expression vector. Strand-specific probes corresponding to the L1.3 50-UTR (50UTR), second exon of the NeoR gene (NeoEx) and intron of the NeoR gene (NeoIN) were designed to detect plus-strand mRNAs. Full-length L1.3Neo mRNAs with spliced and unspliced NeoR introns are marked as FL1.3Neo and FL1.3NeoIN, respectively. Numbers 1 through 3 indicate products of premature polyadenylation (17). Potential splice species are labeled as SpX, SpX(IN), ‘a3’ and ‘b3’ [‘a’ and ‘b’ denotes splicing events (Figure 2) and a number corresponds to the poly(A) sites used to generate the 30 end of the transcripts]. Note that the most abundant band in the NeoEx lane is barely detected with the 50-UTR probe (50UTR lane) indicating that it contains a small portion of L1.3. In vitro-generated 50-UTR probe is not all full-length and therefore the detection is skewed toward the 30 end of the 50-UTR. Results of blotting with the strand- specific probe to the NeoR intron (NeoIN) indicate that SpX(IN) contains the intron while SpX does not. (D) RT–PCR analysis of the L1/NeoR splice junction. Positions of 48(+) and L1Neo( ) primers are shown in (B). RT(+) and ( ) indicate the presence or absence of RT in the respective RT–PCRs. the intron [Figure 1B and C, SpX(IN)]. The estimated size of the SpX and X(IN) products approximately corresponded to the sizes of the spliced and unspliced NeoR gene, respectively. The Sp(X) band is only weakly detected by a 50-UTR probe that is biased towards the 30 end of the 50-UTR (Figure 1B and C), suggesting that much of the 50-UTR sequence is not present in this transcript, possibly due to splicing. To conﬁrm the identity of these products, we used an upstream primer cor- responding to the beginning of the L1.3 50-UTR and the down- stream primer complementary to the beginning of the second exon of the NeoR gene to perform RT–PCR on poly(A)- selected RNAs from transfected NIH 3T3 cells (Figure 1D).