Fluorescence-Based mRNA Translation Reporter.

Herein, we describe a fluorescence reporter assay for investigating regulatory elements in the 5'-UTR of mRNA that modulate translational initiation. The canonical translation initiation process generally begins with the assembly of the eIF4F complex at the cap structure, the formation of the ternary complex, which is loaded with the initiator tRNA, and finally, the preinitiation complex. The preinitiation complex then scans the 5'-UTR to identify the authentic translation start site, which is typically characterized by an AUG codon within a sequence context known as the Kozak sequence. It is well established that the 5'-UTR varies extensively in length and contains regulatory, structural, and sequence elements. To investigate these elements, we use a green fluorescence protein (GFP)- and a luciferase-based mRNA reporter. These reporters can be quickly generated by ordering gBlocks or by PCR-based template generation for in vitro transcription, capping and polyadenylation, to produce synthetic mRNAs. These mRNAs can be transfected into cells, and expression can be monitored via GFP fluorescence or by measuring luciferase activity. In this chapter, we provide a step-by-step protocol for designing gBlocks, generating the reporter mRNA, transfecting the cells, and analyzing the results.