Under cellular stress, tight and coordinated regulation of the gene expression allows to minimize cellular damage, maintains cellular homeostasis, and ensures cell survival. Among stress-induced cellular responses, alteration of translation rates represents one of the most effective and rapid regulatory mechanisms available for cells. Here we report on detailed protocols of mammalian in vitro translation systems. While most of the available in vitro translation methods are based on bacterial or yeast components, tailor-made and robust mammalian systems are sparse. Our protocols allow measuring global translation of the total mRNA pool as well as translation of one specific reporter mRNA. Furthermore, it provides access to measuring translational activity of isolated ribosomes combined with non-ribosomal cytosolic fractions using reduced amounts of biological starting material. The herein described method can be applied to (1) investigate the effects of stress-dependent soluble factors regulating translation (such as tRNA fragments or ribosome-associated ncRNAs), (2) compare translational activity and translational fidelity of different ribosomes supplemented with the same non-ribosomal fractions, and (3) to investigate protein biosynthesis in various mammalian cell lines as well as tissue samples.
Keywords: In vitro translation; Protein synthesis; Ribosomes; Stress response; Translation control.
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.