The cellular oncogene c-myc is expressed in many cell types. c- myc expression is highest in proliferating cells and it is turned off in differentiating cells. A human c-myc mRNA half-life of 30-60 minutes has been reported for several cell lines. In these studies, however, poly(a+) RNA prepared from whole cells was used as a source of c-myc RNA and not total cellular RNA. We have investigated the turnover of c-myc mRNA using total cellular RNA from the human promyelocytic leukemia cell line, HL-60. We find two classes of c-myc RNA, a relatively labile, polyadenylated population (which binds to oligo(dT)-cellulose) and a more stable c-myc RNA population lacking long polyadenylate (which does not bind to oligo(dT)-cellulose). Further structural analysis of these two RNA populations demonstrates that both have similar structures except that the labile, polyadenylated c- myc mRNA is approximately 200nt larger than the stable c-myc RNA which does not bind to oligo(dT)-cellulose. Taken together data suggest that poly(A) is a structural feature of c-myc mRNA that regulates its steady-state level. We will further study these two c-myc mRNA populations for their role in c-myc gene regulation. Subcellular localization of these RNAs will be accomplished by fractionating nuclei and cytoplasm. Questions we will ask are the following: 1) Are (A-) c-myc RNAs found in the nucleus or cytoplasm? 2) If found in the cytoplasm, are the (A) c-myc RNA species translated? We will also investigate the transcriptional and post-transcriptional regulation of both the (A-) and (A+) c-myc RNAs during mitogenic stimulation of fibroblasts (up regulation) and differentiation of HL-60 cells (down regulation). To investigate the nucleotide sequences involved in the regulation of c-myc expression fusion genes have been created. These fusion genes have structure, 5' c-myc/3' beta-globlin or 5' beta-globin/3' c-myc These fusion genes as well as various deletions and site- specific mutants will be introduced into cultured cells. c-myc RNA half-life studies and analysis of adenylation status will be performed. The role of poly(A) in c-myc RNA will be directly tested with fusion genes which have a transcribed poly(dT) tract inserted in the 3' untranslated region of the terminal c-myc exon. Resultant c-myc RNAs will be assayed for polyadenylation and RNA half-life. Transcriptional activation will be studied using a c-myc promoter/CAT gene fusion genes as well as 5' myc/3' beta- globin constructs.
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