Generating Biotinylated and Radiolabeled RNA

The DNA sequences encoding the VCP mRNA 5’ UTR and random 5’ UTR were PCR amplified from plasmid P1 and plasmid P4, respectively, using the appropriate forward and reverse primer. The forward primers contained a flanking T7 promoter at the 5’ terminus. As previously reported [46 (link)], overlapping PCR was used to incorporate a point mutation in the VCP mRNA 5’ UTR at 545 nucleotides from the 5’ terminus by converting the AUG codon to AUC. The DNA segments encoding other deletion mutants used in Fig 8 were generated using similar approach. The PCR products were gel purified and used as template in an in vitro T7 transcription reaction. RNA synthesis was carried out using the T7 RiboMax kit (Promega), following the manufacturer’s instructions. The RNA was either biotinylated or radiolabeled with [α³²P] GTP during synthesis as previously reported [35 (link),47 (link),48 (link)].