RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts.

Removal of the poly(A) tail, or deadenylation, is a crucial step in destabilizing mRNAs in eukaryotes. In this chapter, we describe a cell-free deadenylation assay that uses cytoplasmic cell extracts from human HEK293 cells transiently transfected with DNA encoding RNA-binding proteins (RBP), and in vitro-transcribed, radiolabeled, RNA probes. We include methods to evaluate the effects of RBPs or deadenylases on various in vitro-transcribed probes, with or without poly(A) tails. Finally, we also demonstrate the adaptability of these assays to test purified protein components in our cell-free deadenylation assay. In our experience, these methods are well suited for the initial assessment of the effects of RBPs on the deadenylation of mRNAs. © 2024. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.

Citation

Wi S Lai, Stephanie N Hicks, Perry J Blackshear. RNA-Binding Protein-Mediated mRNA Deadenylation in Mammalian Cell Extracts. Methods in molecular biology (Clifton, N.J.). 2024;2723:173-191

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PMID: 37824071

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