1516 Nucleic Acids Research, 2006, Vol. 34, No. 5 sites are used, we performed an RT–PCR analysis of RNA species produced by the L1.3-notag construct in NIH 3T3 cells with primers located at the beginning of the L1.3 sequence and at the 50 end of ORF2. Sequence analysis of the bands pro- duced in this experiment conﬁrmed usage of splices ‘a’ and ‘b’ (Figure 2C and Supplementary Figures 1 and 2) and detected an additional functional SD site at position 54 of the L1.3 element and ﬁve SA sites (Figures 2C and 1, splice sites are marked by an asterisk). One of the functional SA sites is located at position 1837 of the L1.3 sequence. Any mRNA resulting from the usage of this splice site would completely lack ORF1 sequence but would have the potential to produce ORF2 protein. To determine whether L1.3 splicing detected in NIH 3T3 cells is supported by human cells, the L1.3 expression cassette was transiently transfected in transformed (HeLa and MCF7) and normal (HME) human cells. Northern blot analysis of poly(A)-selected RNAs with the 50UTR100 strand-speciﬁc RNA probe detected mRNA proﬁles identical to those char- acterized in the mouse cells (Figure 3A). To evaluate RNA proﬁles of the endogenous human L1 elements, we performed northern blot analysis of RNAs extracted from human Ntera2 (38) and Sk-Br-3 cancer cells that express naturally high levels of L1 elements. The 50UTR100 probe detected RNA species consistent with ‘a’ and ‘b’ splice products detected in transient transfection of mouse and human cells in both cell types (Figure 3B). Figure 3. Transiently transfected and endogenously expressed L1s undergo splicing in human cells. (A) Northern blot analyses of RNA species produced by the L1.3-notag expression cassette transiently transfected in HeLa, MCF7 and HME cells. Poly(A)-selected total mRNAs from the cell lines were detected with the strand-specific RNA probe complementary to the first 100 bp of the L1.3 50UTR (50UTR100). FL corresponds to the full-length L1.3 element, a3 and b3 and c4 and b4 indicate spliced and prematurely polyadenylated products. Note that spliced mRNAs are detected in both normal (HME) and cancer cells transfected with L1.3 expression cassette. The star denotes bands with an uncharacterized combination of splicing and polyadenylation. (B) Northern blot analyses of RNA species produced by the L1.3-notag expression cassette transiently transfected in NIH 3T3 cells (Lane 3T3/+L1) or endogenously expressed human L1s from Ntera2 (Nt.2/End.) and Sk-Br-3 (Sk/End.) cells. Poly(A)-selected total mRNAs from the cell lines were detected with the strand- specific RNA probe complementary to the first 100 bp of the L1.3 50UTR (50UTR100). FL corresponds to the full-length L1.3 element detected in NIH 3T3 cells or to the endogenous L1s detected in human cell lines. a3 and b3 indicate spliced and prematurely polyadenylated products. Black aster- isks mark truncated mRNA products in human cells exhibiting similar migra- tion patterns to the spliced mRNAs identified in NIH 3T3 cells. White asterisks point out truncated mRNA products in Ntera2 and Sk-Br-3 cells that are not detected in transiently transfected mouse cells. Black horizontal arrows corre- spond to the position of the size markers. Additional faster-migrating bands that were not detected in transient transfections were observed in Ntera2 and Sk-Br-3 cells. These bands are consistent with the expected heterogeneity of the endogenous L1 elements; they could also be tissue- or cancer-speciﬁc splice and/or polyadenylation products. To identify additional functional splice sites in the human L1, and to conﬁrm that endogenous L1 elements undergo splicing, we used a pair of primers located in the beginning and the end of the L1.3 sequence for RT–PCR analysis of poly(A)-selected RNAs from NIH 3T3 cells transfected with the L1.3-notag construct, and endogenous RNAs from HeLa cells (Figure 4). Although there were some variations consistent with the expected heterogeneity of endogenous L1 elements, sequence analysis of some of the bands detected a common functional SA site at the end of the L1 element (position 5721) that was used with SD sites in the beginning of the 50-UTR by both transfected and endogenous L1 elements (Figure 1A). RT–PCR targeting of other regions of the L1 sequence produced bands consistent with splicing, suggest- ing that there are almost certainly many other functional L1 SD and SA sites (data not shown). The relationship between splicing and premature polyadenylation within LINE-1 It has been reported previously that there is competition among, and between (39–41), different splice sites (42,43) and poly(A) signals (44). It appears that the L1 sequence is riddled with both splice and poly(A) sites. To determine the relationship between these signals, we compared RNA species produced by the wild type (WT) and mutant of the strongest functional internal poly(A) site (1M) for both L1.3Neo and L1- notag (17). This mutant is biologically relevant because one of the ‘hot’ L1 elements, AL137845, (23) is lacking this poly(A) site. We performed a northern blot analysis with the strand- speciﬁc 50UTR100 probe of RNAs from NIH 3T3 cells trans- fected with WT and 1M L1.3-notag elements. In the WT background, splice variants ‘a3’ and ‘b3’ are prematurely terminated at the strongest poly(A) site at the end of ORF1 (Figure 5A and B). When the strongest poly(A) site is not Figure 4. Endogenous L1 elements expressed in HeLa cells undergo splicing. RT–PCR analysis of poly(A)-selected RNAs from HeLa cells and L1-notag transfected NIH 3T3 cells was carried out with 48(+) upstream primer and 30- UTR( ) downstream primer located in the L1.3 30-UTR. RT(+) and ( ) in- dicate reactions with and without reverse transcriptase.