Construction of UTR Luciferase Reporters

To generate the c-Jun and BTG1 5′ UTR luciferase reporter plasmids, sections of the 5′ UTR were first amplified from human cDNA. These were then stitched together downstream of a T7 promoter using overlap-extension PCR and Gibson cloning. For the PSMB6 5′ UTR luciferase reporter plasmid, the 5′ UTR was constructed by annealing primers together to create restriction site-compatible overhangs. The 5′ UTRs were then inserted together with Renilla luciferase into pUC19 for c-Jun and pcDNA4 for BTG1 and PSMB6. The eIF3 binding mutants and BTG1 stem loop chimeras were made by inserting annealed primers after cutting the plasmid with enzymes flanking the desired insertion site. The BTG1 overexpression plasmid was constructed by inserting the BTG1 open reading frame, isolated by PCR from human cDNA, into pcDNA4 modified with a Kozak site^{30 (link)}. siRNA pools used were siGENOME Jun (Dharmacon M-003268-03) and siGENOME Non-Targeting siRNA #3 (Dharmacon D-001210-03).