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U4 snRNA nucleolar localization requires the NHPX/15.5-kD protein binding site but not Sm protein or U6 snRNA association.

Gerbi SA, Borovjagin AV, Odreman FE, Lange TS - J. Cell Biol. (2003)

Bottom Line: Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5.Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization.Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.

ABSTRACT
All small nuclear RNAs (snRNAs) of the [U4/U6.U5] tri-snRNP localize transiently to nucleoli, as visualized by microscopy after injection of fluorescein-labeled transcripts into Xenopus laevis oocyte nuclei. Here, we demonstrate that these RNAs traffic to nucleoli independently of one another, because U4 snRNA deleted in the U6 base-pairing region still localizes to nucleoli. Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5. The wild-type U4 transcripts used here are functional: they exhibit normal nucleocytoplasmic traffic, associate with Sm proteins, form the [U4/U6] di-snRNP, and localize to nucleoli and Cajal bodies. The nucleolar localization element (NoLE) of U4 snRNA was mapped by mutagenesis. Neither the 5'-cap nor the 3'-region of U4, which includes the Sm protein binding site, are essential for nucleolar localization. The only region in U4 snRNA required for nucleolar localization is the 5'-proximal stem loop, which contains the binding site for the NHPX/15.5-kD protein. Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization. Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.

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Nucleolar localization of U5 snRNA does not depend on the 5′-cap structure and can occur after mutation of the Sm protein binding site. (a) Mutational analysis of U5 snRNA was performed to determine if the Sm protein binding site is essential for nucleolar localization. No major differences in nucleolar signals from wild-type U5 were observed for U5 subSm, carrying a substitution of the entire Sm binding site. Nucleolar localization was comparable for wild-type and subSm U5 snRNA capped with an A cap instead of a G cap. Thus, the Sm site and nature of the 5′-cap are not essential NoLEs for U5 snRNA, consistent with data for U4 snRNA (Fig. 4). Bar, 10 μM. Other details as in Fig. 1. (b) The ability of the synthetic RNA transcripts used here to associate with Sm proteins was analyzed. U4 or U5 snRNAs (co-labeled with [32P]UTP and fluorescein-UTP) were injected into Xenopus oocyte nuclei. After 4 h of incubation, immunoprecipitation from nuclear lysates was performed with an anti-Sm protein antibody. Coinjection of labeled wild-type U2 snRNA served as an internal control. The equivalent of five nuclei/sample of the immunoprecipitated RNA (pellet) and 0.2 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed on a denaturing gel. Wild-type U2, U4, and U5 snRNAs can be immunoprecipitated with an anti-Sm antibody, unlike U4 and U5 mutants with substitution (subSm) of the Sm site. No immunoprecipitation occurred when using beads coupled to control antibody.
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fig8: Nucleolar localization of U5 snRNA does not depend on the 5′-cap structure and can occur after mutation of the Sm protein binding site. (a) Mutational analysis of U5 snRNA was performed to determine if the Sm protein binding site is essential for nucleolar localization. No major differences in nucleolar signals from wild-type U5 were observed for U5 subSm, carrying a substitution of the entire Sm binding site. Nucleolar localization was comparable for wild-type and subSm U5 snRNA capped with an A cap instead of a G cap. Thus, the Sm site and nature of the 5′-cap are not essential NoLEs for U5 snRNA, consistent with data for U4 snRNA (Fig. 4). Bar, 10 μM. Other details as in Fig. 1. (b) The ability of the synthetic RNA transcripts used here to associate with Sm proteins was analyzed. U4 or U5 snRNAs (co-labeled with [32P]UTP and fluorescein-UTP) were injected into Xenopus oocyte nuclei. After 4 h of incubation, immunoprecipitation from nuclear lysates was performed with an anti-Sm protein antibody. Coinjection of labeled wild-type U2 snRNA served as an internal control. The equivalent of five nuclei/sample of the immunoprecipitated RNA (pellet) and 0.2 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed on a denaturing gel. Wild-type U2, U4, and U5 snRNAs can be immunoprecipitated with an anti-Sm antibody, unlike U4 and U5 mutants with substitution (subSm) of the Sm site. No immunoprecipitation occurred when using beads coupled to control antibody.

Mentions: As shown in Fig. 1 b, injection of U4 or U5 transcripts into oocyte nuclei results in specific fluorescent nucleolar signals 1.5 h later, despite the depletion of endogenous U6 snRNA (Fig. 1 a), whereas injection of a control RNA does not label nucleoli (Fig. 1 b). The signal strength is strong for U4 and moderate for U5 snRNA, similar to nondepleted oocytes (compare with Figs. 3 and 8 and Gerbi and Lange, 2002), indicating that the nucleolar localization of U4 and U5 snRNAs is not impaired by the absence of U6 snRNA. Therefore, the data presented in Fig. 1 demonstrate that U4 and U5 snRNAs localize to nucleoli independently of U6 snRNA.


U4 snRNA nucleolar localization requires the NHPX/15.5-kD protein binding site but not Sm protein or U6 snRNA association.

Gerbi SA, Borovjagin AV, Odreman FE, Lange TS - J. Cell Biol. (2003)

Nucleolar localization of U5 snRNA does not depend on the 5′-cap structure and can occur after mutation of the Sm protein binding site. (a) Mutational analysis of U5 snRNA was performed to determine if the Sm protein binding site is essential for nucleolar localization. No major differences in nucleolar signals from wild-type U5 were observed for U5 subSm, carrying a substitution of the entire Sm binding site. Nucleolar localization was comparable for wild-type and subSm U5 snRNA capped with an A cap instead of a G cap. Thus, the Sm site and nature of the 5′-cap are not essential NoLEs for U5 snRNA, consistent with data for U4 snRNA (Fig. 4). Bar, 10 μM. Other details as in Fig. 1. (b) The ability of the synthetic RNA transcripts used here to associate with Sm proteins was analyzed. U4 or U5 snRNAs (co-labeled with [32P]UTP and fluorescein-UTP) were injected into Xenopus oocyte nuclei. After 4 h of incubation, immunoprecipitation from nuclear lysates was performed with an anti-Sm protein antibody. Coinjection of labeled wild-type U2 snRNA served as an internal control. The equivalent of five nuclei/sample of the immunoprecipitated RNA (pellet) and 0.2 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed on a denaturing gel. Wild-type U2, U4, and U5 snRNAs can be immunoprecipitated with an anti-Sm antibody, unlike U4 and U5 mutants with substitution (subSm) of the Sm site. No immunoprecipitation occurred when using beads coupled to control antibody.
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fig8: Nucleolar localization of U5 snRNA does not depend on the 5′-cap structure and can occur after mutation of the Sm protein binding site. (a) Mutational analysis of U5 snRNA was performed to determine if the Sm protein binding site is essential for nucleolar localization. No major differences in nucleolar signals from wild-type U5 were observed for U5 subSm, carrying a substitution of the entire Sm binding site. Nucleolar localization was comparable for wild-type and subSm U5 snRNA capped with an A cap instead of a G cap. Thus, the Sm site and nature of the 5′-cap are not essential NoLEs for U5 snRNA, consistent with data for U4 snRNA (Fig. 4). Bar, 10 μM. Other details as in Fig. 1. (b) The ability of the synthetic RNA transcripts used here to associate with Sm proteins was analyzed. U4 or U5 snRNAs (co-labeled with [32P]UTP and fluorescein-UTP) were injected into Xenopus oocyte nuclei. After 4 h of incubation, immunoprecipitation from nuclear lysates was performed with an anti-Sm protein antibody. Coinjection of labeled wild-type U2 snRNA served as an internal control. The equivalent of five nuclei/sample of the immunoprecipitated RNA (pellet) and 0.2 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed on a denaturing gel. Wild-type U2, U4, and U5 snRNAs can be immunoprecipitated with an anti-Sm antibody, unlike U4 and U5 mutants with substitution (subSm) of the Sm site. No immunoprecipitation occurred when using beads coupled to control antibody.
Mentions: As shown in Fig. 1 b, injection of U4 or U5 transcripts into oocyte nuclei results in specific fluorescent nucleolar signals 1.5 h later, despite the depletion of endogenous U6 snRNA (Fig. 1 a), whereas injection of a control RNA does not label nucleoli (Fig. 1 b). The signal strength is strong for U4 and moderate for U5 snRNA, similar to nondepleted oocytes (compare with Figs. 3 and 8 and Gerbi and Lange, 2002), indicating that the nucleolar localization of U4 and U5 snRNAs is not impaired by the absence of U6 snRNA. Therefore, the data presented in Fig. 1 demonstrate that U4 and U5 snRNAs localize to nucleoli independently of U6 snRNA.

Bottom Line: Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5.Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization.Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Medicine, Brown University, Providence, RI 02912, USA.

ABSTRACT
All small nuclear RNAs (snRNAs) of the [U4/U6.U5] tri-snRNP localize transiently to nucleoli, as visualized by microscopy after injection of fluorescein-labeled transcripts into Xenopus laevis oocyte nuclei. Here, we demonstrate that these RNAs traffic to nucleoli independently of one another, because U4 snRNA deleted in the U6 base-pairing region still localizes to nucleoli. Furthermore, depletion of endogenous U6 snRNA does not affect nucleolar localization of injected U4 or U5. The wild-type U4 transcripts used here are functional: they exhibit normal nucleocytoplasmic traffic, associate with Sm proteins, form the [U4/U6] di-snRNP, and localize to nucleoli and Cajal bodies. The nucleolar localization element (NoLE) of U4 snRNA was mapped by mutagenesis. Neither the 5'-cap nor the 3'-region of U4, which includes the Sm protein binding site, are essential for nucleolar localization. The only region in U4 snRNA required for nucleolar localization is the 5'-proximal stem loop, which contains the binding site for the NHPX/15.5-kD protein. Even mutation of just five nucleotides, essential for binding this protein, impaired U4 nucleolar localization. Intriguingly, the NHPX/15.5-kD protein also binds the nucleolar localization element of box C/D small nucleolar RNAs, suggesting that this protein might mediate nucleolar localization of several small RNAs.

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