This project proposes to study the mechanisms by which cell- specific expression of the neuro- and endocrine peptides calcitonin and calcitonin gene-related peptide (CGRP) is determined at a transcriptional and post-transcriptional level. Cell-specific alternative RNA processing events direct the preferential expression of CGRP mRNA from the rat calcitonin gene in neurons of the peripheral and cental nervous system, where the encoded peptide appears to function in sensory, motor and integrative systems. Four areas of research are proposed: 1) Regulation of alternative splicing: gene transfer studies. The rat calcitonin gene will be mutagenized to assess which portions of the pre-mRNA are required to maintain cell-specific patterns of RNA processing. In transfection studies, minigenes comprised of the alternatively spliced calcitonin/CGRP exons 3-5 will be tested to see if they exhibit normal patterns of cell-specific alternative splicing, and mutations of alternatively used 3' splice sites of calcitonin/CGRP exons will test whether these 3' splice sites simply complete for a single 5' splice donor. 2) Somatic cell fusions testing potential factor dominance. Stable cell hybrids and transient heterokaryons of calcitonin and CGRP mRNA producing cells will be analyzed to test the hypothesis that a neuron-specific factor or machinery is required to produce CGRP mRNA. 3) Analysis of in vitro nuclear extracts. The ability of factors in nuclear extracts from tissue culture cell lines to direct alternative splicing of the calcitonin gene will be studied by using in vitro splicing assays, in vitro RNA-factor binding gel retardation assays or fusion of extract-loaded erythrocyte ghosts with cultured cells. 4) Identification of cell-specific regulatory elements/enhancers. Sequences associated with the calcitonin gene and its flanking DNA will be studied to identify elements that promote enhanced transcription in medullary thyroid carcinoma cells (MTCs) and ultimately, sensory neurons. Methods will include transfection of promoter/reporter fusion genes, DNAse hypersensitivity studies, DNAse footprinting of cellular proteins to cis-active elements and in vitro transcription analysis. This information and these techniques will be used to conduct comparative analyses between enhancer elements and factors of the calcitonin/CGRP gene and other neuropeptide genes that are coexpressed in MTCs and some sensory neurons such as preprotachykinin, somatostatin and cholecystokinin.
| Roesser, J R; Liittschwager, K; Leff, S E (1993) Regulation of tissue-specific splicing of the calcitonin/calcitonin gene-related peptide gene by RNA-binding proteins. J Biol Chem 268:8366-75 |
