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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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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 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.

Mentions: The observation that a double point mutation in the Sm binding site resulted both in a loss of nucleolar localization and internal modification of U2 RNA (Sm-mutant U2) prompted us to test whether targeting of this RNA to nucleoli can restore internal modification. Therefore, we introduced a well-characterized nucleolar localization signal, the snoRNA Box C/D motif (containing boxes C and D and the terminal stem; Samarsky et al. 1998; Narayanan et al. 1999; Speckmann et al. 1999), into Sm-mutant U2 as well as into wild-type U2 RNA. Specifically, the 5′-terminal sequence of U14 snoRNA, including box C and one strand of the terminal stem, was fused to the 5′ terminus of U2, while the 3′-terminal sequence of U14, including box D and the other strand of the terminal stem, was linked to the 3′ terminus of U2 (Fig. 5 A). In addition, the 5′ half (nucleotides 1–100) and the 3′ half (101–188) of U2 were each inserted into the same box C/D motif (Fig. 5 A). These chimeric RNAs were injected into isolated nuclei, incubated for 5 h, and assayed for 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)

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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Related In: Results  -  Collection

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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.
Mentions: The observation that a double point mutation in the Sm binding site resulted both in a loss of nucleolar localization and internal modification of U2 RNA (Sm-mutant U2) prompted us to test whether targeting of this RNA to nucleoli can restore internal modification. Therefore, we introduced a well-characterized nucleolar localization signal, the snoRNA Box C/D motif (containing boxes C and D and the terminal stem; Samarsky et al. 1998; Narayanan et al. 1999; Speckmann et al. 1999), into Sm-mutant U2 as well as into wild-type U2 RNA. Specifically, the 5′-terminal sequence of U14 snoRNA, including box C and one strand of the terminal stem, was fused to the 5′ terminus of U2, while the 3′-terminal sequence of U14, including box D and the other strand of the terminal stem, was linked to the 3′ terminus of U2 (Fig. 5 A). In addition, the 5′ half (nucleotides 1–100) and the 3′ half (101–188) of U2 were each inserted into the same box C/D motif (Fig. 5 A). These chimeric RNAs were injected into isolated nuclei, incubated for 5 h, and assayed for 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
Related in: MedlinePlus