Lysate and cell-based assays to probe the translational role of RNA helicases.

Abstract

Translation initiation is the first step in protein synthesis, during which the small subunit of the ribosome scans the 5' untranslated region (5'UTR) of an mRNA to identify a start codon and commence translation elongation. By unwinding and modulating secondary structures and other RNA features present in the 5'UTR, RNA helicases can regulate ribosome scanning and start codon selection. This chapter presents an approach to measure the effect of RNA helicases on mRNA translation initiation. 5'UTR luciferase reporters are transcribed in vitro and employed in either of two assays. The in vitro assay translates the reporters in a cell-free whole-cell lysate system, which allows for greater biochemical manipulation and tighter control over confounding effects. In the alternative cell-based approach, the reporters are transfected and translated in living cells, which provides a more physiological setup. Either method can be used to investigate how the perturbation of a helicase, such as changes in protein levels or mutations, affects translation initiation at the 5'UTR level. The chapter also discusses alternative approaches, troubleshooting, and further applications of these methods. These assays will provide insights on the role of helicases and other translational factors as regulators of the proteome both in physiological and diseased settings.