A single band of about 650 bp was detected. Sequence anal- yses of ﬁve independent clones demonstrated that the L1.3 sequence is joined to the sequence of the NeoR gene in the manner consistent with conserved cis elements of mammalian splicing (Figure 1B). Thus, L1.3 contains at least one func- tional SD site that can be utilized with SA sites downstream of its genome. Both SpX and SpX(IN) bands (Figure 1B) require full-length transcription of the L1.3 mRNA prior to splicing. This may represent the primary difference between the levels of full-length transcripts from the L1.3Neo and L1-notag (which mimics endogenous L1 elements) constructs [(17) and Figure 2]. RNA splicing limits production of the full-length L1 mRNA To determine whether there are other functional SD and SA sites in the L1.3 sequence, we probed L1 RNAs with a strand- speciﬁc RNA probe complementary to the ﬁrst 100 bp of the L1.3 50-UTR (50UTR100 probe) (Figure 2A and B). If the SD Nucleic Acids Research, 2006, Vol. 34, No. 5 1515 site in the beginning of the L1.3 50-UTR was utilized for L1 splicing, the 50UTR100 probe would allow quantitative com- parison of the amounts of prematurely terminated transcripts versus spliced products. Northern blot analyses with the 50UTR100 probe detected the SpX band for the L1Neo con- struct and two additional faster-migrating bands (a3 and b3,‘a’ and ‘b’ denote splicing events and the number corresponds to the poly(A) sites used to generate the 30 end of the transcripts) for both L1Neo and L1-notag constructs (Figure 2A and B and Supplementary Figures 1 and 2 that help clarify the nomen- clature of the complex group of RNA species formed by the concurrent use of both variable splicing and polyadenylation). These two smaller bands were consistent with splicing within L1.3 mRNA and were as abundant as the previously reported major, prematurely polyadenylated species (17). A strand- speciﬁc RNA probe complementary to the 600–700 bp region of the L1.3 50-UTR (50UTR600 probe) did not detect bands ‘a3’ and ‘b3’, conﬁrming the loss of this sequence in these bands (Figure 2B). To determine which of the predicted splice Figure 2. L1.3 mRNA undergoes splicing at multiple sites. (A) L1.3Neo splicing. The portions of the L1.3 that are removed in splice products ‘a’ and ‘b’ are annotated above the cartoon of the expression cassette with splice site sequences listed underneath. Strand-specific 100 bp probes corresponding to positions 1–100 (50UTR100) and 583–698 (50UTR600) of the L1.3 sequence are shown under the promoter (Pro) portion of the L1.3 with the arrow denoting the sense of the probes. Strand-specific NeoEx probe is the same as in Figure 1. Underneath the sequences is a schematic representation of the prematurely polyadenylated L1 mRNAs [1–3, that are detected with both 50UTR100 and 50UTR600 probes in (B)] and prematurely polyadenylated and spliced transcripts [a3 and b3, that are detectable only with the 50UTR100 probe in (B)]. Solid black lines represent parts of L1.3 sequence included in the transcripts. Dotted black lines correspond to the L1.3 sequences removed by splicing. SpX(IN) and SpX products are the same as in Figure 1. (B) Northern blot analysis of poly(A)-selected mRNAs from NIH 3T3 cells transfected with vectors expressing either L1.3Neo (L1Neo) or only L1.3 sequences (L1.3-notag). The 50UTR100 strand-specific probe detected premature poly(A) products (bands 1 through 3), as well as the splice products SpX in the L1.3Neo RNA and the ‘a3’ and ‘b3’ products for the L1.3Neo and L1.3-notag constructs [where ‘a’ refers to the splice and 3 to the poly(A) site used to create the transcript]. The SpX, ‘a3’ and ‘b3’ bands were not detected by a strand-specific 50UTR600 probe complementary to the portion of L1.3 expected to be spliced from these transcripts. FL annotates the full-length L1.3 mRNA. (C) RT–PCR analysis of poly(A)- selected mRNAs from NIH 3T3 cells transiently transfected with L1.3 expression cassette. The 48(+) forward primer described in Figure 1B and ORF2( ) reverse primer located in L1 ORF2 were used. Note that ORF2 primer can also anneal at position 1359 of L1.3 sequence therefore products of splices ‘a’ and ‘b’ are smaller than expected when the primer anneals at the position 2038 in ORF2 of L1.3.