Polysome Fractionation for Transcriptome-Wide Studies of mRNA Translation

Protein synthesis and degradation determine the relationship between mRNA and corresponding protein amounts. This relationship can change in a dynamic and selective fashion when translational efficiencies of transcript subsets are altered downstream of, for example, translation factors and/or RNA binding proteins. Notably, even transcription factors such as estrogen receptor alpha (ERα) can modulate mRNA translation in a transcript-selective manner. Yet, despite ample evidence suggesting a key role for mRNA translation in shaping the proteome in health and disease, it remains largely unexplored. Here, we present a guide for the extraction of mRNA engaged in translation using polysome fractionation with linear and optimized sucrose gradients. The isolated polysome-associated RNA is then quantified, in parallel with total mRNA from the same conditions, using methods such as RNA sequencing; and the resulting data set is analyzed to derive transcriptome-wide insights into how mRNA translation is modulated. The methods we describe are applicable to cultured cells, small numbers of FACS-isolated primary cells, and small tissue samples from biobanks or animal studies. Accordingly, this approach can be applied to study in detail how ERα and other factors control gene expression by selectively modulating mRNA translation both in vitro and in vivo.