Abstract
A central component of Myc's role as a master coordinator of energy metabolism and biomass accumulation is its ability to increase the rate of protein synthesis, driving cell cycle progression, and proliferation. Importantly, Myc-induced alterations in both global and specific mRNA translation is a key determinant of Myc's oncogenic function. Herein, we provide five assays to enable researchers to measure global protein synthesis changes, to identify the translatome uniquely regulated by Myc and to investigate the mechanisms generating the tailored Myc translation network. Metabolic labeling of cells with 35S-containing methionine and cysteine in culture and O-propargyl-puromycin (OP-Puro) incorporation in vivo are presented as methods to measure the overall rate of global protein synthesis. Isolation of polysome-associated mRNAs followed by quantitative real-time PCR (qRT-PCR) and the toeprint assay enable the detection of altered translation of specific mRNAs and isoforms, and visualization of differential ribosomal engagement at start codons uniquely mediated by Myc activation, respectively. Finally, the translation initiation reporter assay is utilized to uncover the molecular mechanism mediating altered translation initiation of a specific mRNA. Together, the protocols detailed in this chapter can be used to illuminate how and to what degree Myc-dependent regulation of translation influences homeostatic cellular functions as well as tumorigenesis.