The primary RNA transcript of the calcitonin (CT) gene is alternatively processed. In C-cells, the primary transcript is processed by polyadenylation after the 4th exon with constitutive splicing of exons 1 through 4. Processing in neuronal cells results in polyadenylation at the end of exon 6 and skipping of exon 4 to produce a five-exon mRNA (exons 1,2,3,5 and 6) encoding for calcitonin gene-related peptide (CGRP). A minigene construct consisting of the first adenovirus exon fused to exons 3, 4, and 5 and of the CT gene is processed in a CT-specific pattern by HeLa cells and in a """"""""skip"""""""" or CGRP-specific pattern in F9 teratocarcinoma cells. We used this truncated construct to develop an in vitro RNA processing system. In the in vitro model system, nuclear extract from F9 cell line processed RNA derived from the CT minigene construct in a CGRP- specific pattern. Nuclear extracts the HeLa cell line processed the same construct by recognizing and polyadenylating after exon 4, but did not remove the intron immediately preceding the CT exon. We determined this failure to remove intron 3 was caused by a weak 3' CT branch/splice site; a single base mutation at the CT 3' branch point resulted in production of a CT-specific splice in the HeLa cell system and a correct cell-specific splice when processed in the F9 system. Complementation of HeLa extract with F9 extract produced a CGRP-specific splice, indicating the presence of a dominant factor in the F9 nuclear extract. Studies in the in vitro system have defined 2 regions of regulatory significance: first, the terminal portion of intron 3 (the intron preceding the calcitonin exon); and second, a 10 bp sequence in the proximal 4th (CT) exon. In preliminary studies we have identified a 66 kK protein (exon binding protein) which binds to the exon 4 sequence. We hypothesize the presence of an F9 factor which interacts at the 3' CT branch/splice or within exon 4 to prevent binding of the exon binding protein thereby causing a """"""""skip"""""""" splice. We propose further mapping of these regulatory regions and utilization of this in vitro system to purify, sequence and clone the exon binding protein and putative F9 factor. Purification of the exon binding protein will be monitored by its specific crosslinking to the 10 nucleotide exon 4 sequence. Purification of the F9 factor will be monitored by its dominant ability to switch the pattern of splicing from a CT to a CGRP pattern or by its ability to prevent crosslinking of the exon binding protein to the 10 nucleotide exon 4 sequence.
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