Regulated C-myc gene expression is an important determinant of cell proliferation and differentiation and averts neoplastic cell behavior. Regulation usually occurs at the mRNA level, depends on the marked instability of the mRNA for its rapidity and is frequently post-transcriptional. Preliminary studies show that C2 murine myoblasts express abundant c-myc mRNA and that the turnover of this mRNA is rapid and depends on translation of C-terminal protein-coding sequences. Differentiation of C2 cells is accompanied by a fall in c-myc mRNA levels, which is due to accelerated turnover that also depends on translation of C-terminal protein-coding sequences. The goal of this proposal is to understand these post-transcriptional mechanisms regulating c-myc expression. (I) Delineation of the structural determinant of translation- dependent c-myc mRNA instability will be accomplished by testing myc deletion and fusion mRNAs for induction by cycloheximide, which has been shown to be due to mRNA stabilization by drug inhibition of translation and is indicative of an unstable myc mRNA. Study of point and frame-shift mutants will indicate if the nucleotide sequence or the encoded amino acid sequence of this element is the instability determinant. A similar approach will be used to delineate the structural element targeting c-myc mRNA for regulation during C2 differentiation. This task may be simplified by selecting only the most informative myc genes from the cycloheximide studies for testing in the cumbersome C2 differentiation assay, since preliminary studies indicate that the determinants of regulation and instability may be the same. (II) Identification of these c-myc mRNA structural determinants will be followed by characterization of their significance. The current confusion resulting from the multiplicity of putative destabilizing elements identified in c-myc mRNA to date will be resolved by determining which candidate element actually influences the steady-state level of c-myc mRNA by specifying its rate of turnover. The prevalence of translation-dependent mechanisms for c-myc mRNA instability and regulation during differentiation will be determined by examining their usage in other cell types. If mRNAs other than c-myc are regulated by the same mechanism during differentiation, they may share the myc mRNA regulatory motif and be identifiable on this basis using appropriate search or cloning strategies. (III) Knowledge gained about the cis-acting motifs regulating c-myc mRNA levels will be used to characterize the transacting factors involved. One approach will be to establish an in vitro system of c-myc mRNA degradation that faithfully recapitulates in vivo events and can be used to characterize and purify these factors. If a nucleotide sequence forms the regulatory motif, identification of factors that bind this sequence offers another approach. These studies promise to reveal factors involved in the regulation of cellular mRNA turnover and to disclose the molecular basis of post-transcriptional c-myc mRNA regulation, a key element of the cell's normal response to growth and differentiation stimuli.
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