Kress TL , Yoon YJ , Mowry KL .

5-T32-GM07601 NIGMS NIH HHS , R01 HD30699 NICHD NIH HHS , R01 GM071049 NIGMS NIH HHS , T32 GM007601 NIGMS NIH HHS , R01 HD030699 NICHD NIH HHS

	Figure 1. Subcellular distribution of hnRNP I during oogenesis. (A) Immunoblot analysis of nuclear (lanes 1, 3, and 5) and cytoplasmic (lanes 2, 4, and 6) lysates from stage I/II (lanes 1 and 2), stage III/IV (lanes 3 and 4), and stage V/VI (lanes 5 and 6) oocytes using anti-hnRNP I (top) and anti-tubulin (bottom). (B–G) Whole-mount immunofluorescence using anti-hnRNP I to detect distribution of hnRNP I in stage I/II (B and E), stage III/IV (C and F), and stage V/VI (D and G) oocytes. E, F, and G are higher magnification images of the vegetal pole of oocytes from B, C, and D, respectively. Confocal images were obtained on a microscope (LSM 410; Carl Zeiss MicroImaging, Inc.). Bars, 50 μm.
	Figure 2. hnRNP I and Vg1RBP/vera associate with Vg1 and VegT RNAs in the nucleus and cytoplasm. (A) Stage III/IV oocytes were injected with RNA encoding hnRNP I-FLAG, and S10 lysates were prepared from manually isolated nuclei and cytoplasm. Vg1 (top) or VegT (bottom) RNAs in the nucleus (lane 1) or cytoplasm (lane 2) were detected using RT-PCR. Immunoprecipitations were performed using S10 lysates with Sepharose beads (lanes 3 and 6), anti-FLAG beads in the absence of lysate (lanes 4 and 7), and anti-FLAG beads in the presence of nuclear S10 (lane 5) or cytoplasmic S10 (lane 8) lysate. Vg1 and VegT RNAs were detected in each immunoprecipitate by RT-PCR. All samples were run on the same gel, but lane order was changed for presentation in the figure. (B) Stage III/IV oocytes were injected with RNA encoding Vg1RBP/vera-FLAG and S10 lysates were prepared as in A. Vg1 (top) or VegT (bottom) RNAs in the nucleus (lane 1) or cytoplasm (lane 2) were detected by RT-PCR. Immunoprecipitations were performed using S10 lysates with Sepharose beads (lanes 3 and 6), anti-FLAG beads in the absence of lysate (lanes 4 and 7), and anti-FLAG beads in the presence of nuclear (lane 5) or cytoplasmic (lane 8) S10 lysate. Vg1 RNA and VegT RNA were detected in each immunoprecipitate by RT-PCR; all samples were separated on the same gel, but lane order was changed for presentation in the figure.
	Figure 3. Vg1RBP/vera and hnRNP I interact in vitro and in vivo. (A) Anti-hnRNP I was used to immunoprecipitate endogenous hnRNP I from oocyte S10 lysate. Bound Vg1RBP/vera was detected by immunoblotting (lane 4). Shown in lane 1 is the total Vg1RBP/vera in the lysate, and immunoprecipitation with protein G beads with or without lysate is shown in lanes 2 and 3, respectively. (B) In vitro–translated Vg1RBP/vera-FLAG was incubated with oocyte S10 lysate. Immunoprecipitations were performed using anti-FLAG beads with S10 lysate (lane 2), anti-FLAG beads with in vitro–translated Vg1RBP/vera-FLAG (lane 3), Sepharose beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG (lane 4), anti-FLAG beads alone (lane 5), and anti-FLAG beads with S10 lysate plus in vitro–translated Vg1RBP/vera-FLAG in the absence (lane 6) or presence (lane 7) of RNase A. Total (lane 1) and bound (lanes 2–7) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. (C) Recombinant Vg1RBP/vera and hnRNP I were translated in vitro and mixed; shown in lane 1 is the input amount of in vitro–translated hnRNP I. Immunoprecipitation reactions were performed using protein G beads in the presence of recombinant Vg1RBP/vera and hnRNP I (lane 2) and anti-Vg1RBP/vera bound to protein G beads either alone (lane 3) or with recombinant Vg1RBP/vera and hnRNP I (lane 4). Total (lane 1) and bound (lanes 2–4) proteins were separated by 10% SDS-PAGE, and were immunoblotted with anti-hnRNP I. For each panel (A–C), samples were run on the same gel, but lane order was changed for presentation in the figure; adjacent lanes are boxed.
	Figure 4. Vg1RBP/vera and hnRNP I interact in both the nucleus and cytoplasm. (A) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes, and nuclear (lanes 1 and 3–6) or cytoplasmic (lanes 2 and 7–10) lysates were prepared. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), anti-FLAG beads with Vg1RBP/vera-FLAG, and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate in the presence (lanes 6 and 10) or absence (lanes 5 and 9) of RNase A. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by 10% SDS-PAGE and immunoblotted with anti-hnRNP I. All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together. (B) Nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) S10 lysates were prepared and were subjected to digestion with RNase A (lanes 2 and 4) or mock digestion (lanes 1 and 3). Vg1 (top) and VegT (bottom) RNAs present in the lysates were detected by RT-PCR. All samples were run on the same gel; adjacent lanes are grouped together. (C) Nuclear (lanes 1 and 3–6) and cytoplasmic (lanes 2 and 7–10) lysates were prepared from oocytes expressing hnRNP I-FLAG. Immunoprecipitations were performed using Sepharose beads plus lysate (lanes 3 and 7), anti-FLAG beads alone (lanes 4 and 8), and anti-FLAG beads and nuclear (lanes 5 and 6) or cytoplasmic (lanes 9 and 10) S10 lysate either with (lanes 6 and 10) or without (lanes 5 and 9) RNase A treatment. Total (lanes 1 and 2) and bound (lanes 3–10) proteins were separated by SDS-PAGE. Bound Vg1RBP/vera was detected by immunoblot with anti-Vg1RBP/vera (top panels), and immunoprecipitated hnRNP I was detected by immunoblot with anti-hnRNP I (bottom panels). All samples were run on the same gel, but lane order was changed for presentation in the figure, with adjacent lanes boxed together.
	Figure 5. Prrp and XStau interact with the localization RNP in the cytoplasm. (A) Stage III/IV oocytes were injected with RNA encoding Prrp-myc, and S10 lysates were prepared from isolated nuclear (lane 1 and lanes 3–5) and cytoplasmic (lane 2 and lanes 6–8) fractions. Immunoprecipitations were performed using control antibody plus S10 lysates (lanes 3 and 6), anti-myc in the absence of lysate (lanes 4 and 7), and anti-myc in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and immunoprecipitated (lanes 3–8) Vg1 (top) and VegT (bottom) RNAs were detected by RT-PCR. All samples were run on the same gel, but lane order was changed for presentation in the figure. (B) After expression of Prrp-myc in stage III/IV oocytes, immunoprecipitations were performed using control antibody with Prrp-myc cytoplasmic S10 lysate (lane 2), anti-myc alone (lane 3), and anti-myc plus lysate without (lane 4) or with (lane 5) RNase A treatment. Total (lane 1) and bound proteins (lanes 2–5) were separated by 10% SDS-PAGE and were immunoblotted for Vg1RBP/vera (top), hnRNP I (middle), or XStau (bottom). For each panel all samples were run on the same gel, and adjacent lanes are boxed together. (C) Vg1RBP/vera-FLAG was expressed in stage III/IV oocytes and S10 lysates were prepared from isolated nuclear (lanes 1 and 3–5) and cytoplasmic (lanes 2 and 6–8) fractions. Immunoprecipitations were performed using Sepharose beads plus Vg1RBP/vera S10 lysates (lanes 3 and 6), anti-FLAG beads alone (lanes 4 and 7), and anti-FLAG beads in the presence of nuclear (lane 5) or cytoplasmic (lane 8) lysates. Total (lanes 1 and 2) and bound (lanes 3–8) proteins were separated by 10% SDS-PAGE and immunoblotted for XStau. All samples were run on the same gel, with adjacent lanes boxed together for presentation in the figure.
	Figure 6. Model for the vegetal RNA localization pathway. Vegetal RNA localization begins in the nucleus (gray) where hnRNP I (red) and Vg1RBP/vera (blue) bind to the RNA, forming a core nuclear RNP. Upon export to the cytoplasm, the RNP complex is remodeled such that hnRNP I and Vg1RBP/vera no longer directly interact. In the cytoplasm, Prrp (green) and XStau (yellow) associate with the RNP, creating the cytoplasmic localization RNP. In this model, a motor protein promotes transport to the vegetal cortex, where anchoring could occur via an interaction of Prrp with factors that promote actin dynamics.

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