Table 1. dbEST examples of L1 SD (97 bp) participating in splicing events with adjacent genic sequence Gene ID BNI3PL C14ORF161 CYSLTR2 DOCK3 DST FLJ10986 FLJ22028 FLJ30851 FLJ39873 GABRR1 GFM1 GUCY1B2 IGSF11 KRTAP4-10 LOC196394 MS4A5 Myosin 1D PDGFRA RGL1 STIM1 TAF9L 2 1 3 1 1 1 1 1 2 3 1 2 6 1 6 1 2 1 1 1 1 L1 insert location intron 3 intron 15 50 of gene intron 5 intron 1 intron 3 50 of gene 50 of gene 50 of gene 50 of gene 50 of gene 50 of gene introns 2,3d 50 of gene intron 1 intron 4 intron 17 intron 1 intron 1 intron 2 50 of gene Gene SA exon 4 exon 15 exon 1c exon 6 exon 2 exon 4 exon 2 exon 2 exon 2 exon 3 50 UTRc exon 14 exon 3 50-UTRc intron 1c 30-UTRc 17 2 intron 1c 3 1 Number of exonsb 6 26 1 53 22 16 12 13 4 10 17 16 (7,9)e 2 7 5 21 20 18 12 7 Abbreviations: ID—GenBank gene identifier; SA—splice acceptor. aL1 subfamily assignment was performed with RepeatMasker (www. repeatmasker.org). bThe numbers in the last column of the table correspond to the total number of the exons identified in the gene. cIndicates that L1 SD spliced to a cryptic SA within the gene region indicated. dDenotes the presence of an L1 that spans two different introns due to a portion of its sequence being incorporated into an exon. eIndicates alternative exons numbering based on different splice products. L1. Because of the observed cis preference of L1 RNA for its translation products (47), RNAs that do not encode both ORFs would not retrotranspose well and therefore almost all of the L1 splicing events will result in reduction of retrotransposi- tion. The potential exceptions are the splices that primarily remove the 50-UTR sequences (e.g. splices ‘a’ and ‘b’ in Figure 2A). These splice variants could express both ORFs and therefore be retrotranspositionally competent. Finding a number of full-length L1 elements that have inserted in the genome precisely missing those ‘intronic’ sequences demon- strates that these spliced mRNAs have undergone retrotrans- position. Because the splicing events remove most of the promoter (19), any copies inserted by this mechanism would be less capable of further retrotransposition. The products of splicing appear to be similar in quantity to the abundant premature polyadenylation transcripts. However, we cannot be sure that all spliced RNAs would have similar stabilities to the full-length RNAs. In particular, some would have very poor translational potential and, therefore, they might be subject to degradation by pathways such as nonsense-mediated decay (48,49). Thus, our observations represent a minimum estimate of L1 silencing by splicing. Whether splicing has any major inﬂuence on L1 retrotrans- position other than lessening expression is not clear. Between premature polyadenylation and splicing, we would expect pro- duction of mRNAs that could translate either ORF1 or ORF2 alone, as well as various truncated versions of these proteins. Production of the ORF2 protein via splicing is most likely not required for L1 retrotransposition because of the cis preference Number of ESTs Subfamilya L1PA2 L1PA2 L1PA2 L1PA3 L1PA3 L1PA3 L1PA2 L1PA6 L1PA3 L1PA2 L1PA2 L1PA6 L1PA3 L1PA2 L1P-AN L1PA2 L1PA2 L1PA3 L1PA3 L1PA3 L1PA4 Nucleic Acids Research, 2006, Vol. 34, No. 5 1519 of L1 for its translation products (50). However, it would be expected to be sufﬁcient to drive Alu retrotransposition (51). It is also possible that some of the other translation products may serve to either assist, or hinder, the L1 retrotransposition process. Although we commonly think of splicing in terms of mRNA maturation, it is worth considering that L1 must return to the nucleus in order to be inserted and may be re-exposed to parts of the splicing apparatus. One observation that supports this association is that L1 elements commonly fuse during integ- ration to spliceosome-associated U6 snRNA (52). Such chi- meras can arise by a template switching mechanism, possibly facilitated by U6 snRNA being bound to the L1 mRNA molecule undergoing retrotransposition (52,53). The genomic impact of L1 splicing A number of studies have demonstrated that extant Alu ele- ments contribute to extensive alternative splicing of genes through a process termed Alu exonization (54). Splice sites donated by Alu arise from mutations in the sequence of these elements that create consensus splice sites. In contrast, L1 elements already contain functional splice sites in their sequences prior to integration. Our ﬁnding of multiple examples of splicing events between L1 elements and human genes in the human EST database is consistent with several previous reports of genetic defect-causing hybrid spli- cing between L1 elements in either orientation and nearby genes in both human and mouse (5,33,34). We believe that our study is biased against the hybrid splicing events that severely compromise normal gene expression and splicing events that result in unstable transcripts. Plausible scenarios for L1 interference with gene expression include exon skip- ping via splicing between intronic L1s or an L1 and a SA site of a gene. These events would result in frame shift/nonsense mutations or in production of a protein with potential dominant mutant function. For example, previously reported splicing between L1 sequence and estrogen receptor (ER) gene pro- duces a tumor-speciﬁc transcript encoding a protein that lacks hormone-binding domain of the normal ER (55). At least one of the genes in Table 1, GFM1, was reported as utilizing an alternative promoter to generate an alternative exon 1. This alternative exon is derived from the L1 promoter region. Because L1 elements contain splice sites in both the sense and antisense strands, we would speculate that altered splicing of genes due to L1 elements inserted in introns could be one of their major negative impacts. The most commonly occurring 50 and 30 ESE is G/AAAG/AAA (26), suggesting that the A-rich sense strand of L1 elements may have a potential to support more efﬁcient splicing. An ESE analysis program that predicts ESE hexamers (http://genes.mit.edu/burgelab/rescue- ese/) (26,56) identiﬁed four times as many ESEs in the sense strand of L1.3 as in the antisense. This suggests that there might be a difference in the strength of the splice sites of L1 strands which is consistent with the general ﬁnding that the limited L1 sequences found in introns are preferentially located in the antisense orientation (3,4). Predicted ESEs in the A-rich L1 sequence have a potential to inﬂuence the strength of the SA and SD sites of genes they have inserted. The presence of functional splice sites in the L1 genome may also contribute to the previously demonstrated decrease of transcripts containing L1 fragments (18).