A tripartite cell-free translation system to study mammalian translation.

Genetic manipulation of cellular systems often leads to the adaptation of gene expression programs, rendering detailed mechanistic insights challenging to isolate and elucidate. The proteome constitutes the ultimate manifestation of gene expression programs with multiple layers of regulation to ensure faithful execution. While current high-throughput techniques to investigate regulation at the level of translation, such as Ribo-Seq and nascent proteomics, can capture nuanced changes in the translational landscape, they suffer from potential confounding factors imposed by adaptation of the cellular states. Cell-free translation systems have been used to elucidate molecular mechanisms for decades, but experimental setups have rigid composition and often rely on non-human model systems and artificially designed mRNA constructs. Here we detail a tripartite cell-free translation system based on the separation of mRNAs, ribosomes and ribosome-depleted cytoplasmic lysate from human cells, allowing for flexible reconstitution of translation reactions, which can be performed in 1-4 days. In this setup, cellular parts such as the cytoplasmic lysate can be kept constant, while ribosome complexes or mRNA can be varied or subjected to treatments or vice versa. We detail how complete mRNA populations can be used as input with subsequent detection of nascent peptides using autoradiography or mass spectrometry. We utilize this protocol to resolve which aspects of the translational machinery are selectively affected by environmental and cellular stress conditions that trigger ribosome stalling and collisions, which have been unresolvable until now.