In vitro splicing analysis of mini-gene constructs of the alternatively processed human calcitonin/CGRP-I pre-mRNA.
The human calcitonin/CGRP-I (CALC-I) gene can be alternatively expressed into calcitonin mRNA in thyroid C-cells and into CGRP-I mRNA in particular nerve cells. Formation of calcitonin mRNA requires splicing of exons 1, 2, 3 and 4 and addition of poly(A) at exon 4, whereas splicing of exons 1, 2, 3, 5 and 6 and addition of poly(A) at exon 6 yields CGRP-I mRNA. The calcitonin and CGRP-I mRNA-specific splicing reactions were investigated in vitro, in nuclear extracts of HeLa cells, using model precursor RNAs containing the exon 3 to exon 5 region of the gene. A precursor RNA containing the full-length exon 3 to exon 5 region was only poorly spliced in vitro. Therefore, a systematic analysis was performed of the effect of deletions introduced in the intron 3, exon 4 and intron 4 of this precursor RNA on calcitonin/CGRP mRNA-specific splicing. The deletions increased the efficiency of splicing considerably. In all cases CGRP mRNA-specific splicing is strongly favoured over calcitonin mRNA-specific splicing. In addition, splicing reactions using cryptic 5' splice sites were detected which interfered with the usage of processing signals for calcitonin and CGRP mRNA-specific splicing. The results imply a major regulatory role for the exon 4 poly(A) addition reaction in the generation of calcitonin mRNA.
Bovenberg
R A
RA
Institute of Molecular Biology and Medical Biotechnology, University of Utrecht, The Netherlands.
Moen
T C
TC
Jansz
H S
HS
Baas
P D
PD
D002116:Calcitonin / Q000235:genetics*; D015740:Calcitonin Gene-Related Peptide; D002872:Chromosome Deletion; D005091:Exons; D006801:Humans; D066298:In Vitro Techniques; D007438:Introns; D009479:Neuropeptides / Q000235:genetics*; D009841:Oligonucleotides / Q000494:pharmacology; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*
D009479:Neuropeptides; D009841:Oligonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D002116:Calcitonin; D015740:Calcitonin Gene-Related Peptide