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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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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 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.

Mentions: The Sm binding site is critical for U2 snRNP biogenesis (Luhrmann 1988; Mattaj 1988) and is located in the 3′ half of the RNA, where essentially no modified nucleotides are present. We mutated this element by changing the second and the third uridines in the Sm binding site to guanosines (Fig. 1). 5 h after injection of RNA that was uniformly labeled with α[32P]UTP into isolated nuclei, total RNAs were recovered and modification assays were performed. Although the wild-type U2 was pseudouridylated (Fig. 3, lane 2) and 2′-O-methylated (data not shown), no modified nucleotides were detected in the Sm-mutant RNA (Fig. 3, lane 3 and data not shown), even after a prolonged (10-h) incubation (Fig. 3, lane 7), suggesting that the Sm site is required for internal modification. The Sm site is important as a site for binding of Sm proteins, which normally occurs in the cytoplasm; however, assembly of the Sm RNP can occur in the nucleus under some conditions, apparently via exchange of proteins with endogenous snRNPs (Terns and Dahlberg 1994; Terns et al. 1995). Assembly of the injected wild-type U2 into particles in our experiments was confirmed by anti-Sm immunoprecipitation: ∼50% of injected RNAs were immunoprecipitated (Fig. 3, compare lanes 4 and 5). The U2 RNA bound by Sm proteins was pseudouridylated (Fig. 3, lane 5), whereas only a trace amount of pseudouridylate was detected in the supernatant (unbound) fraction (Fig. 3, lane 4), presumably because of incomplete immunoprecipitation. We conclude that the Sm binding site, and likely the binding of Sm proteins, is required for U2 internal modification.


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)

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 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.
Mentions: The Sm binding site is critical for U2 snRNP biogenesis (Luhrmann 1988; Mattaj 1988) and is located in the 3′ half of the RNA, where essentially no modified nucleotides are present. We mutated this element by changing the second and the third uridines in the Sm binding site to guanosines (Fig. 1). 5 h after injection of RNA that was uniformly labeled with α[32P]UTP into isolated nuclei, total RNAs were recovered and modification assays were performed. Although the wild-type U2 was pseudouridylated (Fig. 3, lane 2) and 2′-O-methylated (data not shown), no modified nucleotides were detected in the Sm-mutant RNA (Fig. 3, lane 3 and data not shown), even after a prolonged (10-h) incubation (Fig. 3, lane 7), suggesting that the Sm site is required for internal modification. The Sm site is important as a site for binding of Sm proteins, which normally occurs in the cytoplasm; however, assembly of the Sm RNP can occur in the nucleus under some conditions, apparently via exchange of proteins with endogenous snRNPs (Terns and Dahlberg 1994; Terns et al. 1995). Assembly of the injected wild-type U2 into particles in our experiments was confirmed by anti-Sm immunoprecipitation: ∼50% of injected RNAs were immunoprecipitated (Fig. 3, compare lanes 4 and 5). The U2 RNA bound by Sm proteins was pseudouridylated (Fig. 3, lane 5), whereas only a trace amount of pseudouridylate was detected in the supernatant (unbound) fraction (Fig. 3, lane 4), presumably because of incomplete immunoprecipitation. We conclude that the Sm binding site, and likely the binding of Sm proteins, is required for U2 internal modification.

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