35–65% of human genes undergoing alternative splicing (27,29). Differential splicing is a tissue-, developmental- and cancer-speciﬁc process (30). L1 elements have generally been considered to produce unspliced mRNA. However, studies on L1 RNA have been confounded by low expression levels and the detection of numerous low-molecular weight, L1-related transcripts that were presumed to be created from the many truncated genomic copies incorporated into other transcripts (31). Here we report that L1 contains multiple predicted SD and SA sites in both sense and antisense strands of its genome. Some of these sites are functional and their usage leads to a widespread, complex splicing pattern for most L1 transcripts. This processing results in weakening of full-length L1 expression and, like Alu, exoni- zation (32), leads to aberrant splicing of genes (5,33,34). MATERIALS AND METHODS Cell culture and transfections NIH 3T3 (ATCC CRL-1658), Ntera2 (ATCC #CRL-1973) and HeLa (ATCC CCL2) cells were maintained as described elsewhere (17). MCF7 cells (ATCC #HTB-22) were main- tained in MEM (Gibco) supplemented with 10% bovine serum (Gibco), sodium pyruvate, essential and nonessential amino acids and L-glutamine. Sk-Br-3 cells (ATCC HTB-30) were maintained in RPMI medium1640 supplemented with 15% fetal bovine serum (Gibco). Human mammary epithelial (HME) cells (CRL-4010) were maintained in MEBM (Clonetics) supplemented with MEGM SingleQuots (Clonetics). Transfections of all cell lines were performed by Lipofectamine with Plus reagent (Invitrogen) as reported pre- viously (17). Brieﬂy, two T75 ﬂasks with 4–5 · 106 cells were seeded and transfected with 6 mg of CsCl puriﬁed DNA 18– 20 h later. Total RNA was isolated by TRiZol reagent 24 h post transfection (Invitrogen) followed by chloroform extraction and isopropanol precipitation. Total RNA was poly(A) selec- ted with poly(A) selection kit (Promega) according to the manufacturer’s protocol. Poly(A)-selected RNAs were preci- pitated overnight in isopropanol. Northern blot analysis was performed as described elsewhere (17). The results of the northern blot assays were quantiﬁed on a Fuji Phosphorim- ager. DNA template for the probe was produced by PCR with the primers that ampliﬁed either LINE-1.3 50-untranslated region (50-UTR), the second exon of the neoR cassette, the intron of the neoR cassette [as described in (17)], the ﬁrst 100 bp (50UTR100 probe) (50-GGAGCCAAGATGGCCG- AATAGGAACAGCT-30 and 50-ACCTCAGATGGAAA- TGCAG-30) or 583–698 bp region (50UTR600 probe) (50-GCAGTAACCTCTGCAGAC-30 and 50-CCACTTGAG- GAGGCAG-30) of the 50-UTR. The T7 promoter sequence was included in the reverse primer of each pair. Site-directed mutagenesis The QuikChange Site-Directed Mutagenesis kit (STRATAGENE) was used to change the position 97 splice site sequence from T to C at position 99 of L1.3 as described elsewhere (17). The 1M mutation in the L1neo and L1notag vectors was the same as published previously (17). Nucleic Acids Research, 2006, Vol. 34, No. 5 1513 RT–PCR Total RNA from HeLa or NIH 3T3 cells transfected with L1notag vector was extracted and poly(A) selected as described elsewhere (17). First-strand synthesis was per- formed with 30-UTR( ) (50-GGTTAGTTACATATGTA- TAC-30 and ORF2( ) (50-CTGTGTCTTTTAATTGCAGA ATTTAGTCC-30) primers with an RT–PCR kit (Promega) according to the manufacturer’s protocol followed by PCR with 48(+) primer 50-GGAGCCAAGATGGCCGAATAG- GAACAGCT-30. The 30 end of the ORF2( ) primer is complementary to the position 2038 and 1359 of L1.3. PCR products were fractionated on a 1% low-melting agarose gel. The isolated DNA fragments were sequenced (TGEN, AZ). Human EST database search To identify examples of endogenous L1 expressed sequence tags (ESTs) that participated in splicing events, NCBI dbEST was searched via BLAST (blastn, E ¼ 1) (35) with the ﬁrst 210 bp of L1.3 consensus sequence, which encompassed the position 97 SD site. Matches where the similarity with the L1 consensus discontinued within 3 bp of the 97 SD position were retained for additional analysis. Candidate splices were sub- sequently located in the genome using BLAT (36) and examined for the position and orientation of L1 relative to the gene or other sequences participating in the splice event. In addition, sequences were manually examined for the usage of the 97 bp L1 SD and associated SA site. Finally, in order to exclude the possibility that the putative L1 splice event was the result of transcription from a genomic sequence that mirrored the splice form (either due to spurious deletions or previously retrotransposed spliced RNA), all candidate splices were checked via BLAST and BLAT for identical contiguous matches to genomic DNA. RESULTS LINE-1 elements contain functional splice sites The BDGP program (http://www.fruitﬂy.org/seq_tools/splice. html) predicted numerous 50 and 30 splice sites distributed throughout the sense strand of both the human L1.3 (L19088) and mouse L1spa (AF016099) elements (Figure 1A). The same program also predicted multiple SD and SA sites in the antisense sequence of both elements (data not shown). To characterize some of the mRNAs produced by the L1.3 element tagged with the neomycin-resistance (NeoR) cassette (L1.3Neo) (20,37) (Figure 1B), we used a strand-speciﬁc probe to the second exon and the intron of the NeoR gene (Figure 1B and C, lane NeoEx and NeoIN) to detect the L1 sense strand transcripts. Full-length mRNAs were detected with, and without, the intron interrupting the NeoR cassette (Figure 1C, bands FL1.3NeoIN and FL1.3Neo). Highly abund- ant, faster-migrating bands were also detected with both probes. These bands contained NeoR gene sequences, but were too small to include much L1.3 sequence. One transcript did not contain the intron of the NeoR cassette as detected by the intron-speciﬁc probe for the Neo resistance gene (Figure 1B and C, SpX) while the slower band contained