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Figure 1. Primary and secondary structure of Xenopus U2 snRNA. Internal modified nucleotides are highlighted by circles (pseudouridines) and squares (2′-O-methylated residues). The Sm binding site is indicated by a hatched gray box. Sequences known or predicted to be involved in intermolecular interactions are indicated: the thick line denotes bases interacting with the branch site of pre-mRNA; sequences boxed in dark gray, medium gray, and light gray are involved in U2-U6 interactions called Helix I, Helix II, and Helix III, respectively (Nilsen 1998). The arrows indicate mutations introduced into the Sm binding site (Sm-Mut).
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Figure 3. Binding of Sm proteins is required for U2 internal modification. [α32P]UTP uniformly labeled wild-type (lanes 2, 4, 5, and 6) or Sm-mutant U2 (lanes 3 and 7; see Fig. 1) was injected into isolated nuclei under oil. After 5 h (lanes 1–5) or 10 h (lanes 6 and 7) at room temperature, RNAs were recovered by phenol-chloroform-isoamylalcohol extraction and ethanol precipitation and subjected to the pseudouridylation assay (lanes 1–3, 6, and 7). For the wild-type U2, ∼40% (lane 2, 5 h) or 50% (lane 6, 10 h) of the expected level of modification was observed. Alternatively, wild-type U2 was recovered by anti-Sm (Y-12 antibody) immunoprecipitation after 5 h at room temperature and then subjected to the pseudouridylation assay (lanes 4 and 5). More than 85% of the expected level of modification was observed in immunoprecipitated RNA (lane 5). S, supernatant; P, pellet. The control is an uninjected U2 RNA.
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Figure 2. U2 internal modifications occur exclusively in the nucleus. α[32P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[32P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.
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Figure 5. Introduction of a snoRNA nucleolar localization signal restores internal modification of Sm-mutant U2. (A) Structures of the wild-type U2-C/D motif chimera, the Sm-mutant U2-C/D motif chimera, the 5′ 1/2U2-C/D motif chimera, the 3′ 1/2U2-C/D motif chimera, and the 5′ 1/2U2-C/DΔC motif chimera, each containing a nucleolar localization signal derived from U14 snoRNA (box C, box D, and a short terminal stem), are shown schematically. The two arrows in the Sm-mutant U2-C/D chimera depict the double point mutation in the Sm binding site. The two arrows in 5′ 1/2U2-C/DΔC indicate the sequence alterations in box C. (B) [α32P]UTP uniformly labeled wild-type U2-C/D motif chimera (lane 2), Sm-mutant U2-C/D motif chimera (lane 3), 5′ 1/2U2-C/D motif chimera (lane 4), 3′ 1/2U2-C/D motif chimera (lane 5), or 5′ 1/2U2-C/DΔC motif chimera (lane 6) was injected into isolated nuclei under oil. 5 h later, RNAs were recovered and assayed for modification. 40–50% of the calculated level of modification was observed in lanes 2–4. The positions of uridylate and pseudouridylate are indicated.
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Figure 4. (A) Intranuclear localization of wild-type U2 and Sm-mutant U2 (U2sm−) snRNAs. 1 fmol of 32P- and fluorescently labeled, in vitro–transcribed U2, Sm-mutant U2, U1, or U65 was injected into Xenopus oocyte nuclei. Nuclear spreads were prepared 5 h later. U1 was included as a positive control for Cajal body localization and as a negative control for nucleolar localization (Narayanan et al. 1999). U65 snoRNA served as a positive control for nucleolar localization and as a negative control for Cajal body localization (Narayanan et al. 1999). A nuclear spread prepared from an uninjected oocyte was included to control for background fluorescence of the preparations. The nuclear spreads were analyzed by DIC and fluorescence (FL) microscopy. Each panel includes several nucleoli and a few Cajal bodies. Cajal bodies are indicated by arrowheads in the DIC panels. (B) Nucleocytoplasmic distribution of U2 and Sm-mutant U2 (U2sm−) snRNAs. Injected oocytes from the same batch analyzed above were dissected into nuclear and cytoplasmic fractions after 5 h. Samples were analyzed by denaturing PAGE and autoradiography to determine the stability and nucleocytoplasmic distribution of the injected RNAs. tRNA was used as a positive control for export and U6 served as a nuclear retention control (Narayanan et al. 1999; Speckmann et al. 1999). Nuclear (N) RNAs are in lanes 2 and 5, cytoplasmic (C) RNAs are in lanes 3 and 6, and marker (M) lanes 1 and 4 show RNAs before injection. Bar, 10 μm.
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Figure 6. (A) Intranuclear localization of U2-C/D motif chimeras. 1 fmol of fluorescein-labeled wild-type U2-C/D motif chimera (U2+CD), Sm-mutant U2-C/D motif chimera (U2sm−-CD), 5′ 1/2U2-C/D motif chimera (5′ 1/2U2-CD), or 5′ 1/2U2-C/DΔC motif chimera (5′ 1/2U2+CDΔC) was injected into Xenopus oocyte nuclei, and nuclear spreads were prepared 5 h later. DIC and fluorescence (FL) panels are shown for each field. The arrowheads in the DIC panels indicate Cajal bodies. (B) Nucleocytoplasmic distribution of the U2-C/D motif chimeras. The distribution of the injected RNAs within the nuclear and cytoplasmic oocyte compartments was determined as described in the legend to Fig. 4 B. Bar, 10 μm.
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Figure 7. (A) Structures of Xenopus wild-type U7 snRNA and MIDU2-U7 chimera are shown schematically. The U7 Sm binding site is indicated by a hatched box. Thin lines represent the U7 RNA chain. In the MID U2-U7 chimera, the first 18 nucleotides of U7 are substituted with a Xenopus U2 sequence (nucleotides 19–46) indicated by a thicker line. (B) The U7 Sm binding site also supports U2 internal modification. [α32P]UTP uniformly labeled wild-type U7 (lane 1), U2-U7 chimera (MID U2-U7; lane 2), or wild-type U2 (lane 3) was injected into isolated nuclei under oil. 5 h later, RNAs were recovered and assayed for modification. The positions of uridylate and pseudouridylate are indicated. (C) Intranuclear localization of U7 and the U2-U7 chimera. 32P- and fluorescently labeled wild-type U7 and MID U2-U7 chimera were synthesized by in vitro transcription and 1 fmol of each RNA was microinjected into the nuclei of Xenopus oocytes. Nuclei were isolated 5 h later and nuclear spreads were prepared. Spreads were also prepared from uninjected oocytes as controls. DIC and fluorescence (FL) images are shown for each field. The arrowheads in the DIC panels point to Cajal bodies. Prominent Cajal body labeling was observed for both U7 and the MID U2-U7 chimera. Weak but above background nucleolar signal was also detected for the MID U2-U7 chimera. (D) The nucleocytoplasmic distribution of U7 and the U2-U7 chimera RNAs was determined as described in the legend to Fig. 4 B. Bar, 10 μm.
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