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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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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 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).

Mentions: The spliceosomal small nuclear (sn)RNAs (U1, U2, U4, U5, and U6), which are essential for pre-mRNA splicing, are all posttranscriptionally modified (Reddy and Busch 1988; Massenet et al. 1998). Aside from 5′ cap trimethylation, numerous internal nucleotides are pseudouridylated or 2′-O-methylated. U2 is the most extensively modified of all the spliceosomal snRNAs. There are 10 2′-O-methylated residues and 13 pseudouridines in the Xenopus U2 snRNA (Fig. 1; Yu et al. 1998).


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)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199211&req=5

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).
Mentions: The spliceosomal small nuclear (sn)RNAs (U1, U2, U4, U5, and U6), which are essential for pre-mRNA splicing, are all posttranscriptionally modified (Reddy and Busch 1988; Massenet et al. 1998). Aside from 5′ cap trimethylation, numerous internal nucleotides are pseudouridylated or 2′-O-methylated. U2 is the most extensively modified of all the spliceosomal snRNAs. There are 10 2′-O-methylated residues and 13 pseudouridines in the Xenopus U2 snRNA (Fig. 1; Yu et al. 1998).

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