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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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U2 internal modifications occur exclusively in the nucleus. α[32P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[32P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.
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Figure 2: U2 internal modifications occur exclusively in the nucleus. α[32P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[32P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.

Mentions: Given the fact that U2 snRNA cycles through the cytoplasm before returning to the nucleus, we first tested whether the internal modification (pseudouridylation and 2′-O-methylation) occurs in the nucleus and/or in the cytoplasm. In these experiments, we exploited the fact that after injection into the cytoplasm of Xenopus oocytes, GpppG-capped U2 is hypermethylated and rapidly transported into the nucleus, whereas ApppG-capped U2 is not trimethylated and remains in the cytoplasm (Fischer et al. 1991). 5 h after injection of differentially capped, uniformly α[32P]UTP-labeled U2 snRNA into the cytoplasm, the cytoplasm and the nucleus were manually separated. The RNA in each cellular compartment was assayed for pseudouridylation by complete nuclease P1 digestion followed by TLC analysis (Fig. 2 A) and for 2′-O-methylation by RNase H site-specific cleavage directed by 2′-O-methyl RNA–DNA chimeras (Fig. 2 B; Yu et al. 1997, Yu et al. 1998). The G-capped U2 recovered from nuclei was efficiently pseudouridylated (Fig. 2 A, lane 1) and 2′-O-methylated (Fig. 2 B, lanes 1–3). In contrast, the A-capped U2 recovered from the cytoplasm was not detectably modified (Fig. 2A and Fig. B, lanes 2 and 4–6, respectively). To ensure that the cap structure does not affect the ability to serve as a substrate for internal modification, we injected both A-capped and G-capped U2 RNA directly into nuclei and found that both were modified to a comparable extent (Fig. 2 A, compare lanes 4 and 5, and 2 B, compare lanes 10–12 and 13–15).


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)

U2 internal modifications occur exclusively in the nucleus. α[32P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[32P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.
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Figure 2: U2 internal modifications occur exclusively in the nucleus. α[32P]UTP uniformly labeled G- or A-capped U2 was injected into the cytoplasm of oocytes (A, lanes 1 and 2; B, lanes 1–6) or directly into nuclei (A, lanes 4 and 5; B, lanes 10–15). After cytoplasmic injection, only G-capped U2 entered the nucleus; after nuclear injection, both G- and A-capped U2 RNAs were retained in the nucleus. RNAs recovered from nuclei (A, lanes 1, 4, and 5; B, lanes 1–3, 10–15) or from cytoplasm (A, lane 2; B, lanes 4–6) were assayed for pseudouridylation (A) and 2′-O-methylation (B). In the 2′-O-methylation assay, two different chimeras were used to test two positions (G11 and A30). The control is uninjected U2 RNA. (C) Oocyte nuclei were separated from the cytoplasm under oil. α[32P]UTP uniformly labeled U2 was then injected into the isolated nuclei (lane 1) or enucleated oocytes (lane 2). After a 5-h incubation, U2 RNAs were recovered and assayed for pseudouridylation. The positions of uridylate, pseudouridylate, uncleaved U2, and 3′ fragments generated by RNase H site-specific cleavage are indicated on the side of each gel. In these experiments, >80% of the expected level of pseudouridylation and 2′-O-methylation was observed.
Mentions: Given the fact that U2 snRNA cycles through the cytoplasm before returning to the nucleus, we first tested whether the internal modification (pseudouridylation and 2′-O-methylation) occurs in the nucleus and/or in the cytoplasm. In these experiments, we exploited the fact that after injection into the cytoplasm of Xenopus oocytes, GpppG-capped U2 is hypermethylated and rapidly transported into the nucleus, whereas ApppG-capped U2 is not trimethylated and remains in the cytoplasm (Fischer et al. 1991). 5 h after injection of differentially capped, uniformly α[32P]UTP-labeled U2 snRNA into the cytoplasm, the cytoplasm and the nucleus were manually separated. The RNA in each cellular compartment was assayed for pseudouridylation by complete nuclease P1 digestion followed by TLC analysis (Fig. 2 A) and for 2′-O-methylation by RNase H site-specific cleavage directed by 2′-O-methyl RNA–DNA chimeras (Fig. 2 B; Yu et al. 1997, Yu et al. 1998). The G-capped U2 recovered from nuclei was efficiently pseudouridylated (Fig. 2 A, lane 1) and 2′-O-methylated (Fig. 2 B, lanes 1–3). In contrast, the A-capped U2 recovered from the cytoplasm was not detectably modified (Fig. 2A and Fig. B, lanes 2 and 4–6, respectively). To ensure that the cap structure does not affect the ability to serve as a substrate for internal modification, we injected both A-capped and G-capped U2 RNA directly into nuclei and found that both were modified to a comparable extent (Fig. 2 A, compare lanes 4 and 5, and 2 B, compare lanes 10–12 and 13–15).

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