1518 Nucleic Acids Research, 2006, Vol. 34, No. 5 of these sequences demonstrated that one, AL807813, belongs to the Ta family (Supplementary Figure 4). Additionally, we detected at least one sequence that matches L1 splice 97–303 on chromosome #20 (HSJ581I13). Because L1 constructs in which the 50-UTR has been almost completely deleted are found to retrotranspose highly efﬁciently (20), RNAs that splice out portions of the 50-UTR would also be expected to be capable of autonomous retrotransposition. We also searched the mouse genome with sequences corresponding to some of the splicing events at the predicted splice sites in the L1spa element (Figure 1). We found 22 matches to several of the splicing events predicted to produce retrotrans- positionally competent L1spa mRNAs (SD sites at positions 27 and 239 and SA sites at 1514, 1597 and 1702 of the L1spa, Supplementary Table 1 and Supplementary Figures 5–7). L1 splicing is redundant The SD site at position 97 of the L1.3 genome appears to be the most commonly used 50 splice site. We introduced a point mutation that destroyed the conserved GU element of the splice site (97M construct). Northern blot analysis with the strand-speciﬁc NeoEx probe detected the SpY band of the size similar to the size of the SpX band, but much lower intensity, and almost complete disappearance of the SpX(IN) band (Figure 6). Detection of the SpY band is consistent with either the usage of a cryptic splice site near the mutated SD site or utilization of the SD site at position 54 of the L1 genome. Use of this SD site would result in production of a transcript of almost the same size as SpX. Additionally, another major, smaller band, SpZ, was identiﬁed (Figure 6) consistent with the usage of one of the cryptic SA sites in the exon 2 of the NeoR gene (45). Quantitative analysis detected no increase in the amount of the full-length L1.3 RNA in proportion to the truncated RNA species between the WT and the 97M splice mutant elements. The 97M splice mutant retrotransposed at Figure 6. Mutation of one of the splice sites in the L1.3 sequence results in the more efficient utilization of another splice signal. (A) An illustration of the major splicing events between either wt L1.3 [marked SpX and X(IN)] or a mutant of the 97 SD site, 97M, (marked SpY and Z) and the NeoR gene produced by the L1Neo construct. The dotted lines labeled SpY and SpZ represent predicted splices corresponding to those bands in the northern blot. (B) A northern blot analysis of the above depicted constructs with the NeoEx strand-specific probe. 60% of the efﬁciency of the wild-type element as determined by a retrotransposition assay in HeLa cells. This result was consistent with a reproducible decrease in the amount of RNA generated by the 97M splice mutant (Figure 6). The 97 splice site overlaps with a Runx3-binding site that regulates L1 pro- moter activity and the mutations we used to silence the splice site have been shown previously to silence this Runx3 site as well (13). L1.3Neo contains a CMV promoter, but the L1 promoter is also present and may explain changes in RNA levels in this mutant. Alteration in the splicing pattern of the 97M splice mutant, however, demonstrates that the removal of one splice site from the L1.3 sequence results in the more efﬁcient usage of another splice signal. This compensation of the L1 splicing process is similar to the previously reported redundancy of the premature polyadenylation (17). L1 splice sites are utilized for hybrid splicing with human genes L1 insertions into human genes can interfere with normal gene expression in numerous ways, often leading to a disease [reviewed in (46)]. Therefore, they are poorly tolerated, par- ticularly when L1s are inserted in the forward orientation. We wished to determine whether functional splice sites in the L1 sequence can be utilized in combination with the splice sites of the human genes in which they insert. We performed a BLAST search (35) of the human EST database with the 210 bp frag- ment of the beginning of the L1.3 50-UTR. Out of the total 1700 hits, 200 ESTs contained L1 sequence terminating precisely at the splice site at the position 97 of the L1.3. Of these ESTs 39 involved clear splicing events between L1 SD site at position 97 and SA sites of 21 different human genes (Table 1). Most of the other ESTs identiﬁed had sequence characteristics of authentic splices, but into sequences other than known exonic SAs. Identiﬁed splicing events between L1 elements and human genes came from libraries generated from different human tissues (bladder, brain, stomach and others) indicating that the process is not limited to any particular tissue type. We hypothesize that the number of identiﬁed ESTs of L1/gene splicing events is underrepresented due to (i) normal- ization of the majority of the libraries prior to cDNA synthesis, (ii) potential instability of the hybrid mRNAs, and (iii) most likely rapid elimination of the L1 insertion events that signi- ﬁcantly interfere with the normal gene expression (disease or potential lethality in utero). DISCUSSION Because only full-length L1 elements had been seen as capable of retrotransposition (20), it had been widely assumed that L1 makes only a single RNA species (31). This was called into question with the demonstration that the majority of L1 RNAs are truncated by premature polyadenylation (17). Our current data demonstrate that L1 RNAs are also involved in extensive RNA splicing that would radically alter the diversity of expressed RNA forms from these elements, as well as inﬂuence their impact on gene expression upon genomic insertion. Relevance to the L1 life cycle The presence of extensive and complex splicing of the L1 mRNA has many potential impacts on the life cycle of