FIG. 1.

9G8 activates α-TM exon 2 inclusion. (A) Exons 1 to 4 of α-TM and splicing regulatory elements are shown, encompassing the branchpoint/pyrimidine tracts of exons 2 (B2P2) and 3 (B3P3), upstream and downstream regulatory elements (URE and DRE), and purine-rich enhancers in exon 2 (denoted by vertical lines). A portion of the exon 2 sequence is shown below, with the four enhancers underlined, denoted B, A, X, and M (20). Previous work showed that only the two central enhancers are needed for activity (5, 20). (B) A minigene (400 ng) encompassing the first four exons of α-TM (pSVpA α-TM 1-4) were cotransfected with 800 ng of eight individual SR protein expression vectors. Transfections were carried out in PAC1 cells, and RT-PCR products were analyzed by dideoxy termination primer extension. The graph below shows averages and standard errors derived from at least three independent transfections. (C) Mutation of the A and X elements (AAAAGAGAAG to AAAACTTAAG, GGAGGAC to GGAGCTT) abolished activation by 9G8. Samples were analyzed and quantitated as described above.