FIG. 4.

VM1 site mutations eliminate direct binding of both PTB/hnRNP I and Vg1RBP/vera to VLE RNA. (A) UV cross-linking analysis was performed using radiolabeled VM1 mutant (ΔVM1; lanes 1 and 2) and wild-type (lanes 3 and 4) VLE RNA to test the ability to be directly bound by partially purified preparations of Vg1RBP/vera (top panels) or PTB/hnRNP I (bottom panels). Specificity of in vitro binding was assessed by challenging the binding reactions with unlabeled specific (sp.; lanes 1 and 3) or nonspecific (nsp.; lanes 2 and 4) competitor RNAs. (B) Radiolabeled VM1 mutant (ΔVM1; lane 1) and wild-type (wt; lane 2) VLE RNA transcripts were injected into the nuclei of stage III oocytes. After culture for 16 h, oocyte lysates were prepared, cross-linked by UV irradiation, and treated with RNase. Proteins interacting with the injected RNA were resolved by SDS-PAGE and autoradiography. The position of Vg1RBP/vera is indicated at the right, and molecular mass markers are shown on the left. (C) Stage III oocytes were injected with Alexa 546-labeled VM1 mutant (ΔVM1; left) and wild-type (wt; right) VLE RNA transcripts. VM1 mutant VLE RNA (left) shows no detectable localization after culture, whereas vegetal localization of the injected RNA (red) is evident in oocytes injected with wild-type VLE RNA (right). The oocytes are oriented with the vegetal hemisphere toward the bottom. Scale bar, 100 μm.