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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 mutated in various positions throughout the molecule. Wild-type or mutated fluorescein-labeled U4 snRNA transcripts were injected into the nuclei of Xenopus oocytes. U4 snRNA deleted in the sites for base pairing with U6 (Δ1–18/56–63), carrying other deletions (Δ64–84, Δ85–117), or deleted in the 3′-area containing the Sm protein binding site (Δ118–145), retained the ability to localize to nucleoli (FL-green). In contrast, a deletion of the 5′-proximal stem loop (Δ19–55) completely abolished nucleolar localization, indicating that this sequence included a nucleolar localization element (NoLE). Bar, 10 μM. Other details as in Fig. 1.
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fig3: Nucleolar localization of U4 snRNA mutated in various positions throughout the molecule. Wild-type or mutated fluorescein-labeled U4 snRNA transcripts were injected into the nuclei of Xenopus oocytes. U4 snRNA deleted in the sites for base pairing with U6 (Δ1–18/56–63), carrying other deletions (Δ64–84, Δ85–117), or deleted in the 3′-area containing the Sm protein binding site (Δ118–145), retained the ability to localize to nucleoli (FL-green). In contrast, a deletion of the 5′-proximal stem loop (Δ19–55) completely abolished nucleolar localization, indicating that this sequence included a nucleolar localization element (NoLE). Bar, 10 μM. Other details as in Fig. 1.

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 U4 snRNA mutated in various positions throughout the molecule. Wild-type or mutated fluorescein-labeled U4 snRNA transcripts were injected into the nuclei of Xenopus oocytes. U4 snRNA deleted in the sites for base pairing with U6 (Δ1–18/56–63), carrying other deletions (Δ64–84, Δ85–117), or deleted in the 3′-area containing the Sm protein binding site (Δ118–145), retained the ability to localize to nucleoli (FL-green). In contrast, a deletion of the 5′-proximal stem loop (Δ19–55) completely abolished nucleolar localization, indicating that this sequence included a nucleolar localization element (NoLE). Bar, 10 μM. Other details as in Fig. 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172826&req=5

fig3: Nucleolar localization of U4 snRNA mutated in various positions throughout the molecule. Wild-type or mutated fluorescein-labeled U4 snRNA transcripts were injected into the nuclei of Xenopus oocytes. U4 snRNA deleted in the sites for base pairing with U6 (Δ1–18/56–63), carrying other deletions (Δ64–84, Δ85–117), or deleted in the 3′-area containing the Sm protein binding site (Δ118–145), retained the ability to localize to nucleoli (FL-green). In contrast, a deletion of the 5′-proximal stem loop (Δ19–55) completely abolished nucleolar localization, indicating that this sequence included a nucleolar localization element (NoLE). Bar, 10 μM. Other details as in Fig. 1.
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.

Show MeSH