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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 U4 snRNA requires the NHPX/15.5-kD protein binding region but not the Sm protein binding site or the 5′-cap structure. The nucleolar localization assay was performed to compare nucleolar localization of U4 mutated in the Sm binding site (U4 subSm) with U4 substituted in nucleotides essential for binding the NHPX/15.5-kD protein (U4 ΔNHPX/15.5 kD). U4 association with Sm proteins is not required for nucleolar localization. In contrast, mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA. Nucleolar localization is independent of the 5′-cap structure because capping of U4 with a synthetic–A cap instead of a G cap still allowed nucleolar localization of wild-type or mutant U4 snRNA. Bar, 10 μM. Other details as in Fig. 1.
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fig4: Nucleolar localization of U4 snRNA requires the NHPX/15.5-kD protein binding region but not the Sm protein binding site or the 5′-cap structure. The nucleolar localization assay was performed to compare nucleolar localization of U4 mutated in the Sm binding site (U4 subSm) with U4 substituted in nucleotides essential for binding the NHPX/15.5-kD protein (U4 ΔNHPX/15.5 kD). U4 association with Sm proteins is not required for nucleolar localization. In contrast, mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA. Nucleolar localization is independent of the 5′-cap structure because capping of U4 with a synthetic–A cap instead of a G cap still allowed nucleolar localization of wild-type or mutant U4 snRNA. Bar, 10 μM. Other details as in Fig. 1.

Mentions: One outstanding feature of nt 19–55 required for nucleolar localization of U4 is the presence of the binding site for the NHPX/15.5-kD (= yeast Snu13p) protein (Nottrott et al., 1999). We dissected this region further by mutating just the NHPX/15.5-kD protein binding site of U4 by substitution of five nucleotides (Fig. 2), which are essential for binding this protein (Nottrott et al., 1999). The nucleolar localization assay shows that mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA (Fig. 4). In contrast to mutant Δ19–55 (Fig. 3) with a deletion of the entire 5′-proximal stem loop, which completely abolished nucleolar localization, the ΔNHPX/15.5-kD mutant substituted in only five nucleotides exhibited some variability with signals ranging from weak to background staining of nucleoli. In comparison to wild-type U4, however, the ΔNHPX/15.5-kD mutant is clearly impaired in nucleolar localization.


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 U4 snRNA requires the NHPX/15.5-kD protein binding region but not the Sm protein binding site or the 5′-cap structure. The nucleolar localization assay was performed to compare nucleolar localization of U4 mutated in the Sm binding site (U4 subSm) with U4 substituted in nucleotides essential for binding the NHPX/15.5-kD protein (U4 ΔNHPX/15.5 kD). U4 association with Sm proteins is not required for nucleolar localization. In contrast, mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA. Nucleolar localization is independent of the 5′-cap structure because capping of U4 with a synthetic–A cap instead of a G cap still allowed nucleolar localization of wild-type or mutant U4 snRNA. Bar, 10 μM. Other details as in Fig. 1.
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Related In: Results  -  Collection

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fig4: Nucleolar localization of U4 snRNA requires the NHPX/15.5-kD protein binding region but not the Sm protein binding site or the 5′-cap structure. The nucleolar localization assay was performed to compare nucleolar localization of U4 mutated in the Sm binding site (U4 subSm) with U4 substituted in nucleotides essential for binding the NHPX/15.5-kD protein (U4 ΔNHPX/15.5 kD). U4 association with Sm proteins is not required for nucleolar localization. In contrast, mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA. Nucleolar localization is independent of the 5′-cap structure because capping of U4 with a synthetic–A cap instead of a G cap still allowed nucleolar localization of wild-type or mutant U4 snRNA. Bar, 10 μM. Other details as in Fig. 1.
Mentions: One outstanding feature of nt 19–55 required for nucleolar localization of U4 is the presence of the binding site for the NHPX/15.5-kD (= yeast Snu13p) protein (Nottrott et al., 1999). We dissected this region further by mutating just the NHPX/15.5-kD protein binding site of U4 by substitution of five nucleotides (Fig. 2), which are essential for binding this protein (Nottrott et al., 1999). The nucleolar localization assay shows that mutation of the NHPX/15.5-kD protein binding site impaired nucleolar localization of U4 snRNA (Fig. 4). In contrast to mutant Δ19–55 (Fig. 3) with a deletion of the entire 5′-proximal stem loop, which completely abolished nucleolar localization, the ΔNHPX/15.5-kD mutant substituted in only five nucleotides exhibited some variability with signals ranging from weak to background staining of nucleoli. In comparison to wild-type U4, however, the ΔNHPX/15.5-kD mutant is clearly impaired in nucleolar localization.

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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