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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 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 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.

Mentions: Visual evidence of nucleolar localization conferred on these chimeric RNAs by the box C/D motif was obtained by fluorescence microscopy using nuclear spreads prepared 5 h after injection of fluorescently labeled chimeric RNAs into oocyte nuclei. Fig. 6 shows that introduction of the C/D motif indeed resulted in targeting of the Sm-mutant U2-C/D motif chimera and the 5′ half U2-C/D motif chimera to nucleoli (U2sm−-CD and 5′ half U2-CD). The nucleolar localization of these chimeric RNAs was dependent on the presence of the C/D motif, as point mutations within the motif resulted in loss of localization to nucleoli but not Cajal bodies (Fig. 6, Fig. 5′ half U2-CDΔC). Introduction of the C/D motif into wild-type U2 did not alter its nucleolar targeting (compare Fig. 6, U2-CD and Fig. 4 A, U2).


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 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 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.
Mentions: Visual evidence of nucleolar localization conferred on these chimeric RNAs by the box C/D motif was obtained by fluorescence microscopy using nuclear spreads prepared 5 h after injection of fluorescently labeled chimeric RNAs into oocyte nuclei. Fig. 6 shows that introduction of the C/D motif indeed resulted in targeting of the Sm-mutant U2-C/D motif chimera and the 5′ half U2-C/D motif chimera to nucleoli (U2sm−-CD and 5′ half U2-CD). The nucleolar localization of these chimeric RNAs was dependent on the presence of the C/D motif, as point mutations within the motif resulted in loss of localization to nucleoli but not Cajal bodies (Fig. 6, Fig. 5′ half U2-CDΔC). Introduction of the C/D motif into wild-type U2 did not alter its nucleolar targeting (compare Fig. 6, U2-CD and Fig. 4 A, U2).

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
Related in: MedlinePlus