GFP reporter constructs are widely used as an expression system for studying the function of regulatory sequence motifs (cis elements) within the 3'-UTRs (3' untranslated regions) of mRNAs. Here we provide details on the characterization of individual sequence motifs, which typically regulate mRNA decay and translation. In addition, we describe methods to identify trans factors required for the function of such elements. To facilitate efficient identification of novel functional 3'-UTR motifs, we describe a screening approach based on dual-color fluorescence reporter constructs. Such screening approaches can be used to test large collections of defined sequence or libraries of random sequences.
Keywords: 5′ and 3′ untranslated regions; Dual-color high-throughput assay; Functional genomics; Lentivirus; Posttranscriptional gene regulation; RNA-seq; Translation; cis-regulatory elements; mRNA decay; shRNAs.