Methods in Molecular Biology Volume 1012, 2013, pp 201-212
Date: 05 Aug 2013

Investigating Myc-Dependent Translational Regulation in Normal and Cancer Cells

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Abstract

There is an increasing realization that a primary role for Myc in driving cellular growth and cell cycle progression relies on Myc’s ability to increase the rate of protein synthesis. Myc induces myriad changes in both global and specific mRNA translation. Herein, we present three assays that allow researchers to measure changes in protein synthesis at the global level as well as alterations in the translation of specific mRNAs. Metabolic labeling of cells with 35S-containing methionine and cysteine is presented as a method to measure the overall rate of global protein synthesis. The bicistronic reporter assay is employed to determine levels of cap-dependent and cap-independent translation initiation in the cell. Finally, isolation of polysome-associated mRNAs followed by next-generation sequencing, microarray or quantitative real-time PCR (qRT-PCR) analysis is utilized to detect changes in the abundance of specific mRNAs that are regulated upon Myc hyperactivation. The protocols described in this chapter can be used to understand how and to what extent Myc-dependent regulation of translation influences normal cellular functions as well as tumorigenesis.