Skip to main content
. 2006 Mar 22;34(5):1512–1521. doi: 10.1093/nar/gkl027

Figure 2.

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 (5′UTR100) and 583–698 (5′UTR600) 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 5′UTR100 and 5′UTR600 probes in (B)] and prematurely polyadenylated and spliced transcripts [a3 and b3, that are detectable only with the 5′UTR100 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 5′UTR100 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 5′UTR600 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.