Abstract
The levels of mRNA and protein encoded by the c-myc protooncogene set the balance between proliferation and differentiation of mammalian cells. Thus, it is essential for the cell to tightly control c-myc expression. Indeed, cells utilize many mechanisms to control c-myc expression, including transcription, RNA processing, translation, and protein stability. We have focused on turnover of c-myc mRNA as a key modulator of the timing and level of c-myc expression. c-myc mRNA is labile in cells, and its half-life is controlled by multiple instability elements located within both the coding region and the 3'-untranslated region (3'-UTR). Much work has focused on the protein factors that bind the instability elements, yet little is known about the enzymatic activities that effect the degradation of c-myc mRNA. Here I have utilized a novel cell-free mRNA decay system to characterize the c-myc mRNA decay machinery. This machinery consists of 3' to 5' mRNA decay activities that are Mg2+-dependent, require neither exogenous ATP/GTP nor an ATP-regenerating system, and act independently of a 7mG(5')ppp(5')G cap structure to deadenylate an exogenous mRNA substrate in a c-myc 3'-UTR-dependent fashion. Following deadenylation, nucleolytic decay of the 3'-UTR occurs generating 3' decay intermediates via a ribonucleolytic activity that can assemble on the c-myc 3'-UTR in a poly(A)-independent manner.
Highlights
The neoplastic phenotype is often characterized by the uncoupling of the normally opposing processes of cellular proliferation and differentiation
A major focus toward understanding the control of mRNA turnover in mammalian cells has been the identification of proteins that bind to cis-acting instability elements
I have described a novel cell-free system that utilizes soluble enzymatic activities to examine 3Ј to 5Ј decay using c-myc mRNA as a model
Summary
The neoplastic phenotype is often characterized by the uncoupling of the normally opposing processes of cellular proliferation and differentiation (reviewed in Refs. 1–3). The major decay pathway for c-myc mRNA in exponentially growing cells involves gradual removal of the poly(A) tract followed by degradation of the mRNA body [13,14,15]. In vitro mRNA decay reactions were performed for the indicated times at 37 °C with polysomes prepared by the standard low salt procedure.
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