FIG. 2.
Interaction between Vg1RBP/vera and VLE RNA is remodeled during localization. (A) The experimental strategy for identifying proteins directly bound to RNA versus RNAs contained in RNP complexes is diagrammed. Stage III Xenopus oocytes are microinjected with RNA transcripts encoding FLAG-tagged proteins. After overnight culture to allow protein expression, 32P-labeled VLE RNA transcripts are injected into the oocyte nuclei. Oocyte lysates are prepared and split into two aliquots. One aliquot is analyzed by “cross-link IP,” in which cross-linking by UV irradiation is followed by anti-FLAG IP and RNase treatment. The RNA-bound proteins, labeled by covalent attachment of RNA oligonucleotides, are resolved by SDS-PAGE and visualized by autoradiography. The other aliquot is analyzed by “RNA IP,” in which anti-FLAG IP is carried out directly, and RNAs associated with the FLAG-tagged proteins are detected by autoradiography following PAGE. (B) Cross-link IP analysis. 32P-labeled VLE RNA was injected into nuclei of 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). Oocyte lysates were prepared either 1 h (lanes 1, 3, 5, and 7) or 16 h (lanes 2, 4, 6, and 8) postinjection and subjected to cross-link IP as detailed for panel A (above). Autoradiograms of cross-linked proteins resolved by SDS-PAGE are shown, and the positions of Vg1RBP/vera-FLAG (lanes 1 to 4) and PTB/hnRNP I-FLAG (lanes 4 to 8) are indicated to the right. (C) RNA IP analysis. 32P-labeled VLE RNA was injected into nuclei of oocytes expressing Vg1RBP/vera-FLAG (lanes 1 to 3), PTB/hnRNP I-FLAG (lanes 4 to 6), XStau-FLAG (lanes 7 to 9), or control oocytes without expression of FLAG-tagged proteins (lanes 10 to 12). Oocyte lysates were prepared either 1 h (lanes 2, 5, 8, and 11) or 16 h (lanes 3, 6, 9, and 12) postinjection and subjected to RNA IP as detailed for panel A (above). Autoradiograms of isolated VLE RNA resolved by PAGE are shown, and the position of VLE RNA is indicated at the left. For each panel, samples were run on the same gel, except for lanes 7 to 9 (XStau) in panel C, which were run together on a separate gel. Lane order was changed for clarity of presentation.