Figure 1.
Figure 1. Transport of Cy3-labeled U1ΔSm RNA microinjected into the Xenopus oocyte nuclei. (A) In vitro–transcribed Cy3-labeled m7G-capped U1ΔSm RNA was microinjected into Xenopus oocyte nuclei that had been cytoplasmically preinjected with Cy5-labeled U7 RNA as a marker of Cajal Bodies 12 h earlier. The nuclei were manually isolated under mineral oil at 1 (left), 3 (middle), and 6 h (right) after injection. The nuclei were observed by fluorescent microscopy as described in Materials and methods. Red, Cy3-U1ΔSm; yellow, Cy5-U7. The arrows and the dotted lines represent Cajal bodies and the nuclear envelope, respectively. Bars, 20 µm. (B) A mixture of in vitro–transcribed Cy3-labeled U1ΔSm and FITC-labeled U6Δss RNAs was microinjected into the Xenopus oocyte nuclei. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions 1, 3, and 6 h after microinjection and analyzed by 8% denaturating PAGE followed by Typhoon scan. (C) Xenopus oocytes were fractionated into Nsup, Nppt, and cyctoplasmic fractions as described in Materials and methods. Western blotting was performed with total oocyte (total), Nsup, Nppt, and cytoplasmic fractions using antibodies against coilin, PHAX, and U2B”. Each lane was loaded with materials from 2.5 oocytes. (D) Northern blotting was performed with RNA prepared from total, Nsup, Nppt, and cytoplasmic fractions using probes against U3 snoRNA and 5.8S rRNA. Each lane was loaded with RNA from 2.5 oocytes. nt, nucleotide. (E) A mixture of 32P-labeled m7G-capped U1ΔSm, m7G-capped U5ΔSm, U6Δss, and initiator methionyl tRNA was injected into the nucleus of Xenopus oocytes. RNA was extracted from Nsup, Nppt, and cytoplasmic fractions immediately at 0 (lanes 1–3) 1 (lanes 4–6), 2 (lanes 7–9), 3 (lanes 10–12), and 6 h (lanes 13–15) after microinjection and analyzed by 8% denaturating PAGE followed by autoradiography. (F and G) Quantification of the radioactivity of U1 and U5 bands in E, respectively. Yellow, C fraction; red, Nsup fraction; blue, Nppt fraction. Black lines indicate that intervening lanes have been spliced out.

Transport of Cy3-labeled U1ΔSm RNA microinjected into the Xenopus oocyte nuclei. (A) In vitro–transcribed Cy3-labeled m7G-capped U1ΔSm RNA was microinjected into Xenopus oocyte nuclei that had been cytoplasmically preinjected with Cy5-labeled U7 RNA as a marker of Cajal Bodies 12 h earlier. The nuclei were manually isolated under mineral oil at 1 (left), 3 (middle), and 6 h (right) after injection. The nuclei were observed by fluorescent microscopy as described in Materials and methods. Red, Cy3-U1ΔSm; yellow, Cy5-U7. The arrows and the dotted lines represent Cajal bodies and the nuclear envelope, respectively. Bars, 20 µm. (B) A mixture of in vitro–transcribed Cy3-labeled U1ΔSm and FITC-labeled U6Δss RNAs was microinjected into the Xenopus oocyte nuclei. RNA was extracted from nuclear (N) and cytoplasmic (C) fractions 1, 3, and 6 h after microinjection and analyzed by 8% denaturating PAGE followed by Typhoon scan. (C) Xenopus oocytes were fractionated into Nsup, Nppt, and cyctoplasmic fractions as described in Materials and methods. Western blotting was performed with total oocyte (total), Nsup, Nppt, and cytoplasmic fractions using antibodies against coilin, PHAX, and U2B”. Each lane was loaded with materials from 2.5 oocytes. (D) Northern blotting was performed with RNA prepared from total, Nsup, Nppt, and cytoplasmic fractions using probes against U3 snoRNA and 5.8S rRNA. Each lane was loaded with RNA from 2.5 oocytes. nt, nucleotide. (E) A mixture of 32P-labeled m7G-capped U1ΔSm, m7G-capped U5ΔSm, U6Δss, and initiator methionyl tRNA was injected into the nucleus of Xenopus oocytes. RNA was extracted from Nsup, Nppt, and cytoplasmic fractions immediately at 0 (lanes 1–3) 1 (lanes 4–6), 2 (lanes 7–9), 3 (lanes 10–12), and 6 h (lanes 13–15) after microinjection and analyzed by 8% denaturating PAGE followed by autoradiography. (F and G) Quantification of the radioactivity of U1 and U5 bands in E, respectively. Yellow, C fraction; red, Nsup fraction; blue, Nppt fraction. Black lines indicate that intervening lanes have been spliced out.

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