COOPERATION OF 5' AND 3' PROCESSING SITES AS WELL AS INTRON AND EXON SEQUENCES IN CALCITONIN EXON RECOGNITION

We have previously shown that the calcitonin (CT)-encoding exon 4 of t he human calcitonin/calcitonin gene-related peptide I(CGRP-I) gene (CA LC-I gene) is surrounded by suboptimal processing sites. At the 5' end of exon 4 a weak 3' splice site is present because of an unusual bran ch acceptor nucleotide (U) and a weak poly(A) site is present at the 3 ' end of exon 4. For CT-specific RNA processing two different exon enh ancer elements, A and B, located within exon 4 are required. In this s tudy we have investigated the cooperation of these elements in CT exon recognition and inclusion by transient transfection into 293 cells of CALC-I minigene constructs. Improvement of the strength of the 3' spl ice site in front of exon 4 by the branchpoint mutation U-->A reduces the requirement for the presence of exon enhancer elements within exon 4 for CT-specific RNA processing, irrespective of the length of exon 4. Replacement of the exon 4 poly(A) site with a 5' splice site does n ot result in CT exon recognition, unless also one or more exon enhance r elements and/or the branchpoint mutation U-->A in front of exon 4 ar e present. This indicates that terminal and internal exons are recogni sed in a similar fashion. The number of additional enhancing elements that are required for CT exon recognition depends on the strength of t he 5' splice site. Deletion of a large part of intron 4 also leads to partial exon 4 skipping. All these different elements contribute to CT exon recognition and inclusion. The CT exon is recognised as a whale entity and the sum of the strengths of the different elements determin es recognition as an exon. Curiously, in one of our constructs a 5' sp lice site at the end of exon 4 is either ignored by the splicing machi nery of the cell or recognised as a splice donor or as a splice accept or site.