1. The recombinant Puf4 pumilio domain (Puf4-PD) binds to PUF consensus elements in HXL1 mRNA.
The recombinant Puf4 pumilio domain (Puf4-PD) binds to PUF consensus elements in HXL1 mRNA. Native electrophoretic mobility shift assays demonstrate recombinant Puf4 pumilio domain binding to either the 5′ UTR HXL1 oligonucleotide (A) or the 3′ UTR HXL1 oligonucleotide (B). Reaction mixtures to which labeled oligonucleotide probe was added are marked with a plus sign in the RNA row. Reaction mixtures to which the pumilio domain of Puf4 was added are indicated by a plus sign in the Puf4 row. Increasing concentrations of an unlabeled 5′ UTR (A) or 3′ UTR (B) HXL1 oligonucleotide competitor (Unlabeled comp.) were also added to the reaction mixtures to confirm competition. Unlabeled 5′ UTR (A) or 3′ UTR (B) HXL1 oligonucleotides with a mutated PUF competitor element (Mutant UL comp.) were also used in a competition assay to confirm the specificity of the interaction.
Virginia E. Glazier et al. Eukaryotic Cell 2015; doi: /EC