To test the functional importance of these molecular interactions in transcript-specific translation initiation, we carried out in vitro translation assays using luciferase reporter genes (Hofbauer et al. 1982; Hussain and Leibowitz 1986; Wang et al. 2013). We generated a monocistronic reporter construct by cloning the 5′UTR of Pab1 mRNA in-frame with the firefly luciferase open reading frame (ORF) under control of the T7 promoter. Cap-dependent and cap-independent translation, the two pathways for the translation initiation of eukaryotic mRNAs, can be distinguished by adding or omitting the 5′ m7G cap to the mRNAs under study. Furthermore, to inhibit ribosome scanning, a stable hairpin upstream of the uncapped 5′UTRPab1 was used to study the cap-independent translation initiation (Wang et al. 2013) in our investigation. The same strategy was used to generate the monocistronic reporter constructs for the 5′UTR of eIF4E mRNA as a control. Additionally, RNAs with nucleotide sequence complementary to 5′UTRPab1 and 5′UTR4E, 5′UTRPab1-complement and 5′UTR4E-complement, were used as controls to rule out nonspecific interactions. The same amount of RNA was added in each translation assay and all measured activities were normalized to the total protein level in cell extracts. Finally, translation of cricket paralysis virus (CrPV) depends on correct folding of the 5′UTR of the CrPV RNA (5′UTRCrPV, also known as the internal ribosome entry site of CrPV) alone and does not require any additional proteins (Thompson et al. 2001). As such, 5′UTRCrPV was included as an experimental control.