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Internal modification of U2 small nuclear (sn)RNA occurs in nucleoli of Xenopus oocytes.

Yu YT, Shu MD, Narayanan A, Terns RM, Terns MP, Steitz JA - J. Cell Biol. (2001)

Bottom Line: The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization.Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization.Together, our results suggest that U2 internal modification occurs within the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA. yitao_yu@urmc.rochester.edu

ABSTRACT
U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.

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(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 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.

Mentions: The procedure used to prepare Xenopus oocyte nuclear spreads and to determine the intranuclear distrution of injected fluorescein-labeled RNAs has been described (Narayanan et al. 1999). In brief, fluorescein-labeled RNAs were generated by in vitro transcription of linearized plasmid DNA in the presence of α[32P]GTP and fluorescein-12-UTP (1:1 ratio with UTP; Boehringer). The fluorescein-labeled RNAs (1 fmol each) were injected into the nuclei of stage V or stage VI oocytes. 32P-labeled U6 snRNA (retention control), U1 snRNA, and tRNA (export controls) were coinjected with each fluorescein-labeled test RNA to help make conclusions about the stability and nucleocytoplasmic distribution of wild-type U2 snRNA and U2 variants. The injected oocytes were incubated at 18°C for 5 h before nuclei were dissected and nuclear spreads were prepared. Images were obtained on a 63× magnification inverted fluorescence microscope (Axiovert S 100; ZEISS) equipped with differential interference contrast (DIC) optics (Thornwood) using a cooled charge-coupled device camera (Quantix-Photometrix) and IPLab Spectrum software (Signal Analytics). Localization analysis was performed using multiple independent sets of RNAs and oocytes, and at least four slides per time point. Representative data are shown (see Fig. 4). To determine the stability and nucleocytoplasmic distribution of the injected RNAs, RNAs present in the nuclear (N) and cytoplasmic (C) fractions were purified 5 h after injection (from the same set of oocytes analyzed by microscopy) and analyzed by 8% denaturing PAGE and autoradiography.


Internal modification of U2 small nuclear (sn)RNA occurs in nucleoli of Xenopus oocytes.

Yu YT, Shu MD, Narayanan A, Terns RM, Terns MP, Steitz JA - J. Cell Biol. (2001)

(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 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.
Mentions: The procedure used to prepare Xenopus oocyte nuclear spreads and to determine the intranuclear distrution of injected fluorescein-labeled RNAs has been described (Narayanan et al. 1999). In brief, fluorescein-labeled RNAs were generated by in vitro transcription of linearized plasmid DNA in the presence of α[32P]GTP and fluorescein-12-UTP (1:1 ratio with UTP; Boehringer). The fluorescein-labeled RNAs (1 fmol each) were injected into the nuclei of stage V or stage VI oocytes. 32P-labeled U6 snRNA (retention control), U1 snRNA, and tRNA (export controls) were coinjected with each fluorescein-labeled test RNA to help make conclusions about the stability and nucleocytoplasmic distribution of wild-type U2 snRNA and U2 variants. The injected oocytes were incubated at 18°C for 5 h before nuclei were dissected and nuclear spreads were prepared. Images were obtained on a 63× magnification inverted fluorescence microscope (Axiovert S 100; ZEISS) equipped with differential interference contrast (DIC) optics (Thornwood) using a cooled charge-coupled device camera (Quantix-Photometrix) and IPLab Spectrum software (Signal Analytics). Localization analysis was performed using multiple independent sets of RNAs and oocytes, and at least four slides per time point. Representative data are shown (see Fig. 4). To determine the stability and nucleocytoplasmic distribution of the injected RNAs, RNAs present in the nuclear (N) and cytoplasmic (C) fractions were purified 5 h after injection (from the same set of oocytes analyzed by microscopy) and analyzed by 8% denaturing PAGE and autoradiography.

Bottom Line: The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization.Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization.Together, our results suggest that U2 internal modification occurs within the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biophysics and Biochemistry, Boyer Center for Molecular Medicine, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06536, USA. yitao_yu@urmc.rochester.edu

ABSTRACT
U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5' trimethylated guanosine cap, 13 pseudouridines, and 10 2'-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.

Show MeSH