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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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Stability of wild-type and mutated U4 snRNA. 32P-labeled U4 snRNA (mutants or wild type) were injected into oocyte nuclei; nuclear RNA was isolated and analyzed by 8% polyacrylamide, 8 M urea gel electrophoresis (PAGE). The top panel shows the controls (sample recovery immediately after injection, 0 h), the bottom panel shows the short-term stability at 1.5 h (the time when localization assays were performed). To determine the stability of the various RNAs after nuclear injection, 32P-labeled U2 snRNA was coinjected and served as an internal control to normalize for any differences in injection or recovery of the samples. The relative RNA stability is the ratio [(U4 RNA transcript/U2 after incubation)/(U4 RNA transcript/U2 at 0 h)]. All the mutants are stable at the 1.5-h time point used for analysis of nucleolar localization.
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fig5: Stability of wild-type and mutated U4 snRNA. 32P-labeled U4 snRNA (mutants or wild type) were injected into oocyte nuclei; nuclear RNA was isolated and analyzed by 8% polyacrylamide, 8 M urea gel electrophoresis (PAGE). The top panel shows the controls (sample recovery immediately after injection, 0 h), the bottom panel shows the short-term stability at 1.5 h (the time when localization assays were performed). To determine the stability of the various RNAs after nuclear injection, 32P-labeled U2 snRNA was coinjected and served as an internal control to normalize for any differences in injection or recovery of the samples. The relative RNA stability is the ratio [(U4 RNA transcript/U2 after incubation)/(U4 RNA transcript/U2 at 0 h)]. All the mutants are stable at the 1.5-h time point used for analysis of nucleolar localization.

Mentions: To guard against the possibility that failure of a mutant to localize to nucleoli might simply be due to its degradation, stability assays using 32P-labeled transcripts were performed. All transcripts were stable 1.5 h after injection into oocyte nuclei, the time when the localization assays were performed (Fig. 5). This included U4 mutants Δ19–55 and ΔNHPX/15.5 kD that failed to localize to nucleoli.


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)

Stability of wild-type and mutated U4 snRNA. 32P-labeled U4 snRNA (mutants or wild type) were injected into oocyte nuclei; nuclear RNA was isolated and analyzed by 8% polyacrylamide, 8 M urea gel electrophoresis (PAGE). The top panel shows the controls (sample recovery immediately after injection, 0 h), the bottom panel shows the short-term stability at 1.5 h (the time when localization assays were performed). To determine the stability of the various RNAs after nuclear injection, 32P-labeled U2 snRNA was coinjected and served as an internal control to normalize for any differences in injection or recovery of the samples. The relative RNA stability is the ratio [(U4 RNA transcript/U2 after incubation)/(U4 RNA transcript/U2 at 0 h)]. All the mutants are stable at the 1.5-h time point used for analysis of nucleolar localization.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172826&req=5

fig5: Stability of wild-type and mutated U4 snRNA. 32P-labeled U4 snRNA (mutants or wild type) were injected into oocyte nuclei; nuclear RNA was isolated and analyzed by 8% polyacrylamide, 8 M urea gel electrophoresis (PAGE). The top panel shows the controls (sample recovery immediately after injection, 0 h), the bottom panel shows the short-term stability at 1.5 h (the time when localization assays were performed). To determine the stability of the various RNAs after nuclear injection, 32P-labeled U2 snRNA was coinjected and served as an internal control to normalize for any differences in injection or recovery of the samples. The relative RNA stability is the ratio [(U4 RNA transcript/U2 after incubation)/(U4 RNA transcript/U2 at 0 h)]. All the mutants are stable at the 1.5-h time point used for analysis of nucleolar localization.
Mentions: To guard against the possibility that failure of a mutant to localize to nucleoli might simply be due to its degradation, stability assays using 32P-labeled transcripts were performed. All transcripts were stable 1.5 h after injection into oocyte nuclei, the time when the localization assays were performed (Fig. 5). This included U4 mutants Δ19–55 and ΔNHPX/15.5 kD that failed to localize to nucleoli.

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.

Show MeSH