FIG. 5.

Binding of PTB/hnRNP I to VLE RNA is required for direct interaction between Vg1RBP/vera and VLE RNA. (A) Cross-link IP analysis (as detailed in Fig. 2A) was carried out using oocytes expressing Vg1RBP/vera-FLAG (lanes 3 and 4), PTB/hnRNP I-FLAG (lanes 7 and 8), or control oocytes without expression of FLAG-tagged proteins (lanes 1, 2, 5, and 6). ³²P-labeled VM1 mutant (ΔVM1; lanes 1, 3, 5, and 7) or wild-type (wt; lanes 2, 4, 6, and 8) VLE RNA transcripts were injected into the oocyte nuclei, and lysates were prepared after 16 h of culture. After cross-linking by UV irradiation, RNase treatment, and anti-FLAG IP, proteins directly bound to the injected RNA transcripts were resolved by SDS-PAGE and detected by autoradiography. The positions of Vg1RBP/vera-FLAG (lanes 1 to 4) and PTB/hnRNP I-FLAG (lanes 4 to 8) are indicated to the right. (B) RNA IP analysis (as diagrammed in Fig. 2A) was carried out using oocytes expressing Vg1RBP/vera-FLAG (lanes 1 and 2), PTB/hnRNP I-FLAG (lanes 3 and 4), or control oocytes without expression of FLAG-tagged proteins (lanes 5 and 6). ³²P-labeled VM1 mutant (ΔVM1; lanes 1, 3, 5, and 7) or wild-type (wt; lanes 2, 4, 6, and 8) VLE RNA transcripts were injected into the oocyte nuclei, and lysates were prepared either immediately (input RNA; lanes 7 and 8) or after 16 h of culture (lanes 1 to 6). RNA was isolated after anti-FLAG IP and resolved by PAGE; the position of input VLE RNA is indicated at the right. (C) UV cross-linking analysis was performed using radiolabeled VLE RNA and stage III oocyte lysates. In vitro binding reactions included a 500-fold molar excess of nonspecific RNA (nsp.; lane 1) or a 500-fold binding site excess of wild-type VM1 RNA (VM1; lane 2) or RNAs containing mutated VM1 sites (mutVM1; lane 3). VLE-bound Vg1RBP/vera is at the top, and VLE-bound PTB/hnRNP I is at the bottom. (D) Quantitation of UV cross-linking results (as shown in panel C). Binding of Vg1RBP/vera to VLE RNA was quantitated by phosphorimage analysis, and the level of VLE-bound Vg1RPB/vera obtained in the presence of a 500- to 1,000-fold molar excess of nonspecific RNA (nsp.) was set to 1. The results of three experiments are graphed, and the levels of VLE-bound Vg1RBP/vera averaged 0.44 (standard deviation = 0.07) in the presence of 500- to 1,000-fold binding site excesses of unlabeled wild-type VM1 RNA (VM1) and averaged 0.78 (standard deviation = 0.12) in the presence of mutant VM1 RNA (mutVM1). For panels A to C, samples were run on the same gel, but lane order was changed for presentation in the figure.