Fig. 3.

The RNA-binding domain of Pum interacts with the hid 3′UTR in vitro. (A) Nucleotides 3847-3925 of the hid mRNA. The NRE sequence is in red and the nucleotides deleted in the ΔNRE probes are underlined. (B) Electrophoretic mobility shift assay to identify an interaction between Pum and the hid 3′UTR. Varying concentrations of purified PumRBD protein (0.1-500 nM) or no protein (–) was incubated with a ³²P-labeled hid 3′UTR probe either containing (hidNRE) or without (hidΔNRE) the NRE. (C) Binding of PumRBD to the ³²P-labeled hidNRE probe was challenged with increasing molar excess (1-, 10-, 100-, 500-, and 1000-fold) of unlabeled competitor hid RNA either containing or without the NRE (hidNRE or hidΔNRE, respectively). The binding reactions contained either no protein (–) or 300 nM purified protein. Unbound free probe indicated by ‘p’; shifts indicated by arrowheads.