Development of an In Vitro mRNA Decay System in Insect Cells

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Summary

Cytoplasmic extracts have proven to be a versatile system for assaying the mechanisms and interactions of RNA metabolism. Using Aedes albopictus (C6/36) cells adapted to suspension culture, we have been able to faithfully reproduce and manipulate all aspects of mRNA decay in vitro. Described in this chapter are the processes for both producing an active cytoplasmic extract and the subsequent applications of the extract with respect to mRNA decay. The following protocol for the production of cytoplasmic extracts from C6/36 cells can be altered to encompass a wide variety of cell types, including mammalian cell lines. In addition, a method for designing and implementing an in vitro transcription template to produce specific products are described in detail. Applications of the in vitro transcripts, specifically the deadenylation and exosome assays by which the decay of reporter transcripts is observed, are also examined in detail.