The production of peptide hormones requires the activities of a number of complex cellular processes involving the transcription and processing of RNA transcripts, the translation and modification of prohormone precursors and the regulated secretion of bioactive peptide products. The long-term objectives of the proposed research are to define the cellular mechanisms involved in the tissue-specific production of peptide hormones in the central and peripheral nervous systems. We propose to precisely identify the cis-active regulatory sequences responsible for dictating tissue-specific patterns of RNA processing for the neuroendocrine gene encoding calcitonin and calcitonin gene- related peptide (CGRP). These studies will take advantage of transfected tissue culture model systems which exhibit RNA processing choices analogous to those observed in vivo. Analyses of RNA from human epithelial and mouse teratocarcinoma cell lines, transfected with a variety of mutant calcitonin/CGRP transcription units, will serve as the primary methodology for these mapping studies. Overexpression of calcitonin/CGRP-derived sequences will also be utilized as a mapping technique by testing the ability of specific RNA sequences to compete for a limiting neuron-specific factor involved in CGRP mRNA production. Isolation and characterization of the cell-specific machinery involved in CGRP messenger RNA production will involve two distinct experimental methodologies: 1) Development of a Xenopus oocyte expression system in which exogenous RNA transcripts encoding putative trans-acting regulatory factors may be microinjected into the oocyte cytoplasm; 2) Isolation of cDNA clones encoding neuron-specific RNA-binding proteins utilizing the polymerase chain reaction with degenerate oligonucleotides corresponding to a characterized """"""""RNA-recognition"""""""" motif. Detailed in situ hybridization and immunocytochemical analyses will be performed to determine the specific pattern of calcitonin/CGRP gene expression in the developing rat central nervous system. Hybridization probes specific for calcitonin and alpha-CGRP mRNAs and antisera specific for the calcitonin and CGRP prohormones will be utilized to determine the onset and location of calcitonin/CGRP gene expression as well as the development pattern of alternative RNA processing. It is anticipated that characterization of the mechanisms regulating the differential production of calcitonin and CGRP mRNAs may provide insights into the cellular processes involved in the post- transcriptional regulation of many peptide hormone systems as well as provide general information regarding mechanisms by which multiple mRNAs are produced from complex transcription units.
