Figure 5.

Characterization of PTB binding to oskar RNA. (A) RNA affinity pull-down assay using biotinylated RNAs covering the entire oskar mRNA, and w ovarian extracts. The coding sequence of y14 was used as an unrelated RNA control. (Top) Diagram showing the three different probes used in the assay. Proteins in the bound or unbound fractions were visualized after Western blot analysis using the indicated antibodies. Bruno exclusively binds to the oskar 3′UTR, whereas Khc, a component of the oskar transport machinery without RNA-binding capacity, is not detected in the precipitated fraction. (B–D) EMSA analysis using 50 nM fluorescently labeled oskar M1M2 region (B), oskar 3′UTR (C), or y14 coding sequence (D) in the presence of increasing amounts of MBP-PTB (from 0 to 1500 nM) and of 5 μM of tRNA. The position of the unbound RNA (Free-RNA) and of the discrete RNP complexes is marked on the left and right, respectively.