THE EXON-4 POLY(A) SITE OF THE HUMAN CALCITONIN CGRP-I PRE-MESSENGER-RNA IS A WEAK SITE IN-VITRO

The human calcitonin/CGRP-I (CALC-I) pre-mRNA is processed in a tissue -specific alternative way into either calcitonin (CT) or calcitonin ge ne-related peptide-I (CGRP,I) mRNA. The exons 1 to 3 are common exons. They are spliced to exon 4, which becomes polyadenylated to form CT m RNA, or to exon 5 and the polyadenylated exon 6 to form CGRP-I mRNA. P olyadenylation at exon 4 and splicing of exon 3 to exon 5 are mutually exclusive processing reactions. Only splicing of exon 3 to exon 5 was detected in vitro, with a minigene containing the exon 3 to exon 5 re gion. No polyadenylation at the exon 4 poly(A) site could be observed. Investigation of the properties of the exon 4 poly(A) site in vitro s hows that it is inefficiently used in vitro. Cleavage and polyadenylat ion of short RNAs containing only the exon 4 poly(A) site is strongly dependent on the 3' length of the RNA. Downstream sequences located wi thin 39 nucleotides from the cleavage site are required for optimal cl eavage and polyadenylation. When the exon 4 poly(A) site in the minige ne is replaced with the strong adenovirus L3 or rabbit beta-globin pol y(A) sites, these sites can be efficiently used in vitro.