title: Cooperation of 5' and 3' processing sites as well as intron and exon sequences in calcitonin exon recognition.
abstract: We have previously shown that the calcitonin (CT)-encoding exon 4 of the human calcitonin/calcitonin gene-related peptide I (CGRP-I) gene (CALC-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 branch 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 enhancer elements, A and B, located within exon 4 are required. In this study 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' splice 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 not result in CT exon recognition, unless also one or more exon enhancer elements and/or the branchpoint mutation U-->A in front of exon 4 are present. This indicates that terminal and internal exons are recognised in a similar fashion. The number of additional enhancing elements that are required for CT exon recognition depends on the strength of the 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 whole entity and the sum of the strengths of the different elements determines recognition as an exon. Curiously, in one of our constructs a 5' splice site at the end of exon 4 is either ignored by the splicing machinery of the cell or recognised as a splice donor or as a splice acceptor site.
authors.affiliation: Laboratory for Physiological Chemistry, Utrecht University, Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Zandberg
authors.forename: T C
authors.initials: TC
authors.lastname: Moen
authors.forename: P D
authors.initials: PD
authors.lastname: Baas
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002116:Calcitonin / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005091:Exons; D005914:Globins / Q000235:genetics; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D011817:Rabbits; D014162:Transfection
chemical_list: D005914:Globins; D002116:Calcitonin