FIG. 1.

The U tract sequence in the calcitonin/CGRP gene intron 4 enhancer element is required for exon 4 inclusion. (A) Schematic diagram of the calcitonin/CGRP gene and its alternative RNA processing in thyroid and neuronal cells. A, polyadenylation signal. (B) Diagram showing the calcitonin/CGRP reporter gene and its cell-specific RNA processing patterns. The black oval represents the intron enhancer element. RSV, Rous sarcoma virus promoter. (C) Wild-type and mutant sequences of the three important positively acting motifs within the intron enhancer element. Py mut, pyrimidine mutant; 5′ ss mut, 5′ splice site mutant; U-tract mut, U tract mutant. (D) Results of RT-PCR assay of total RNA from HeLa cells transfected with the diagramed calcitonin/CGRP reporter gene with a wild-type enhancer (lane 1), an enhancer in which the pseudo 5′ splice site was mutated (lane 2), or an enhancer in which the U tract was mutated (lane 3). Amplification bands resulting from exon 4 inclusion or exclusion are indicated. The percent inclusion of exon 4 is indicated below each lane. Higher-molecular-weight products result from precursor RNA or activation of cryptic splicing. The latter product has been characterized previously and results from usage of a cryptic 5′ splice site within exon 4 (36). M, molecular weight markers.