Nucleic Acids Research, 2006, Vol. 34, No. 5 1517 Figure 5. The relationship between polyadenylation and splicing of L1 transcripts. (A) A diagram of the L1.3-notag construct. Diagrams of the major splice variants detected by northern blot analysis of the wild type (WT) and mutant of the strongest poly(A) site (1M) L1.3 elements are shown underneath the construct. Solid black lines represent L1.3 sequences included into RNA transcripts. Dotted lines represent regions of L1.3 sequence removed by splicing. Some ‘a’ and ‘b’ splicing events may use poly(A) sites at the end of the L1 genome (a,bFL in WT and 1M elements), while others may prematurely terminate at the internal L1 poly(A) signals (‘a3’ and ‘b3’ in the WT and ‘a4’ and ‘b4’ in the 1M L1.3). (B) Northern blot analysis of the WT and 1M L1.3-notag constructs transiently transfected in NIH 3T3 cells with 50UTR100 and 50UTR600 strand-specific probes. Full-length L1.3 mRNA and spliced L1.3 mRNA terminated at the end of the L1 genome are marked as FL and a,bFL, respectively. Spliced and prematurely truncated mRNAs are marked as ‘a3’, ‘b3’, a4, b4. The star denotes bands with an as yet uncharacterized combination of splicing and polyadenylation that arise in the 1M mutant. (C) Northern blot analysis of the WT and 1M L1.3Neo constructs transiently transfected in NIH 3T3 cells with 50UTR100 strand-specific probe. Full-length L1.3Neo mRNA is labeled FL1.3Neo. The splice variant specific to this vector is shown as SpX. Spliced and prematurely truncated mRNAs are identified as described in (B). (D) Northern blot analysis of the full-length and faster-migrating L1.3 mRNAs produced by the WT, 1M and DSV40 (L1.3 with no pA site at the end of the element) L1.3 constructs with 50UTR100 and 50UTR600 strand-specific probes. TRpA stands for the truncated polyadenylated L1.3 mRNA. Other products are marked as described above. present in the L1.3 sequence, the 50UTR100 probe detects a slower-migrating doublet (Figure 5A and B, a4 and b4). This doublet is consistent with the ‘a’ and ‘b’ splice products util- izing poly(A) sites (4) located further downstream in the L1.3 sequence (Figure 5A). Additionally, two new products occur in the 1M mutant for both the WT L1.3 (Figure 5B) and the L1.3Neo constructs (Figure 5C). The small size of these new L1-related RNA species and the fact that they are not detected with the 50UTR600 strand-speciﬁc probe (data not shown) is consistent with the usage of alternative SA/poly(A) sites and/ or an increase in production of the splice variants that are made by the WT L1.3 in much lower quantities. It appears that mutations of functional poly(A) sites result in not only increased utilization of the poly(A) signals nearby (17) but also in quantitative alterations in the use of speciﬁc splice sites. Some human and mouse L1 splice products are retrotranspositionally active The 50UTR100 probe also detected a slightly faster-migrating product than the full-length L1.3 mRNA (Figure 5B and D, a,bFL). The relative amount of this band increased in the 1M mutant of L1.3-notag. The 50UTR600 strand-speciﬁc probe failed to identify the a,bFL band, but the truncated prematurely polyadenylated product (TRpA) produced by the L1.3DSV40 construct was detected (Figure 3D). The size of the a,bFL RNA is consistent with either splice ‘a’ and/or ‘b’ that ter- minated at the poly(A) site at the end of the L1.3 element (Figure 5A). Splice ‘a’ would result in L1 mRNA containing both ORFs and could potentially be retrotranspositionally active. Using a BLAST search with the splice junctions cor- responding to the splice ‘a’ (Figure 2A), we identiﬁed four sequences on chromosome #1 (AL031985), #3 (AC093006), #9 (AL137022) and #11 (AP00560) that were ﬂanked by target-site duplications, a hallmark of endonuclease- dependant L1 retrotransposition. Alignment of these sequences demonstrated that AC093006 belongs to the Ta family while the others were from older subfamilies (Supple- mentary Figure 3). Splice ‘b’ would produce a L1 mRNA that could make a truncated ORF1 protein, by utilizing an in-frame AUG downstream of the wt translation initiation codon (Figure 2A). A BLAST search of the human genome with the sequence corresponding to the splice junction ‘b’ identiﬁed at least 10 matching hits (Supplementary Table 1). Alignment