Regulation of c-myc mRNA Decay by Translational Pausing in a Coding Region Instability Determinant

Abstract

A 249-nucleotide coding region instability determinant (CRD) destabilizes c-myc mRNA. Previous experiments identified a CRD-binding protein (CRD-BP) that appears to protect the CRD from endonuclease cleavage. However, it was unclear why a CRD-BP is required to protect a well-translated mRNA whose coding region is covered with ribosomes. We hypothesized that translational pausing in the CRD generates a ribosome-deficient region downstream of the pause site, and this region is exposed to endonuclease attack unless it is shielded by the CRD-BP. Transfection and cell-free translation experiments reported here support this hypothesis. Ribosome pausing occurs within the c-myc CRD in tRNA-depleted reticulocyte translation reactions. The pause sites map to a rare arginine (CGA) codon and to an adjacent threonine (ACA) codon. Changing these codons to more common codons increases translational efficiency in vitro and increases mRNA abundance in transfected cells. These data suggest that c-myc mRNA is rapidly degraded unless it is (i) translated without pausing or (ii) protected by the CRD-BP when pausing occurs. Additional mapping experiments suggest that the CRD is bipartite, with several upstream translation pause sites and a downstream endonuclease cleavage site.

We thank Andreas Kuhn and Brad Berberet for critical reading of the manuscript, Jia Qian for assistance with the initial phases of this study, and Brad Berberet, Anisa Kaenjak-Angeletti, and Charles Tessier for helpful suggestions. We also thank John Sedivy for providing the HO-15 cells.

This work was supported by National Cancer Institute grants R01-CA78710 (to J.R.) and P30-CA07175 (Cancer Center Support Grant to the McArdle Laboratory) and by a grant from the University of Wisconsin Robert Draper Technology Innovation Fund (to J.R.).