Abstract
Protein synthesis is regulated transcriptionally and posttranscriptionally, with the latter including both the translation and mRNA degradation steps. Eukaryotic mRNAs have a characteristic 7-methyl-G cap structure at their 5' ends and a polyadenylated tail at their 3' ends. These structures, and the sequences of the untranslated regions (UTR) flanking the coding region on the 5' and 3' sides, are recognized by various RNA-binding proteins and determine translational efficiency and mRNA stability. RNA interference is a sequence-specific inhibition of protein synthesis triggered by double-stranded RNA (dsRNA). This process is mediated by RNA-binding proteins named Argonaute. Argonautes incorporate dsRNAs of 21-22 nucleotides (termed short-interfering RNAs or siRNAs) and cleave mRNAs containing sequences complementary to siRNAs. In this chapter, we describe a cell-free translation system from Drosophila Schneider line 2 (S2) cells that recapitulates RNA interference. This system can be programmed with multiple RNA transcripts, a target and a control, and chemically synthesized short-interfering RNA (siRNA). The production of the target protein is reduced in the presence of the target-specific siRNA, in a dose-dependent manner. We also describe a coupled transcription and translation system using the S2 cell lysate.
