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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 and U5 snRNAs does not depend on U6 snRNA. Fluorescein-labeled U4 or U5 snRNA were injected into the nuclei of Xenopus oocytes that were depleted of endogenous U6 snRNA using antisense oligonucleotides. (a) Northern blot analysis of RNA from U6-depleted oocyte nuclei demonstrated the absence of endogenous U6 snRNA, in contrast to the presence of endogenous U3 snoRNA (control). (b) Injection of U4 or U5 snRNAs into U6-depleted oocyte nuclei resulted in nucleolar labeling (FL, green) 1.5 h later, showing that U4 and U5 nucleolar localization is independent from U6. The multiple nucleoli present in Xenopus oocyte nuclei are visualized in nucleolar preparations by phase contrast (PC) and can be distinguished from other nuclear bodies by the staining of rDNA (DAPI, blue) located only in nucleoli. Bar, 10 μM.
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fig1: Nucleolar localization of U4 and U5 snRNAs does not depend on U6 snRNA. Fluorescein-labeled U4 or U5 snRNA were injected into the nuclei of Xenopus oocytes that were depleted of endogenous U6 snRNA using antisense oligonucleotides. (a) Northern blot analysis of RNA from U6-depleted oocyte nuclei demonstrated the absence of endogenous U6 snRNA, in contrast to the presence of endogenous U3 snoRNA (control). (b) Injection of U4 or U5 snRNAs into U6-depleted oocyte nuclei resulted in nucleolar labeling (FL, green) 1.5 h later, showing that U4 and U5 nucleolar localization is independent from U6. The multiple nucleoli present in Xenopus oocyte nuclei are visualized in nucleolar preparations by phase contrast (PC) and can be distinguished from other nuclear bodies by the staining of rDNA (DAPI, blue) located only in nucleoli. Bar, 10 μM.

Mentions: Previously, we reported that U4, U5, and U6 transiently localize to nucleoli of Xenopus laevis oocytes (Lange and Gerbi, 2000; Gerbi and Lange, 2002). To address if U4 and U5 have their own NoLEs and do not depend on U6 snRNA as a carrier, fluorescein-labeled in vitro transcripts of U4 snRNA, U5 snRNA, or a control RNA were injected into Xenopus oocyte nuclei that were depleted of endogenous U6 snRNA (Fig. 1 a). This assay allows direct visualization of the labeled RNA in nucleolar preparations (Fig. 1 b) and has been used in nondepleted oocytes to monitor nucleolar localization of small nucleolar RNAs (snoRNAs) from various families as well as snRNAs (for review see Lange, 2003). Here, the assay was combined with disruption of endogenous U6 snRNA through RNase H–mediated degradation by nuclear injections of anti-U6 snRNA antisense oligonucleotides (Vankan et al., 1990, 1992; Gerbi and Lange, 2002).


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 and U5 snRNAs does not depend on U6 snRNA. Fluorescein-labeled U4 or U5 snRNA were injected into the nuclei of Xenopus oocytes that were depleted of endogenous U6 snRNA using antisense oligonucleotides. (a) Northern blot analysis of RNA from U6-depleted oocyte nuclei demonstrated the absence of endogenous U6 snRNA, in contrast to the presence of endogenous U3 snoRNA (control). (b) Injection of U4 or U5 snRNAs into U6-depleted oocyte nuclei resulted in nucleolar labeling (FL, green) 1.5 h later, showing that U4 and U5 nucleolar localization is independent from U6. The multiple nucleoli present in Xenopus oocyte nuclei are visualized in nucleolar preparations by phase contrast (PC) and can be distinguished from other nuclear bodies by the staining of rDNA (DAPI, blue) located only in nucleoli. Bar, 10 μM.
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Related In: Results  -  Collection

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

fig1: Nucleolar localization of U4 and U5 snRNAs does not depend on U6 snRNA. Fluorescein-labeled U4 or U5 snRNA were injected into the nuclei of Xenopus oocytes that were depleted of endogenous U6 snRNA using antisense oligonucleotides. (a) Northern blot analysis of RNA from U6-depleted oocyte nuclei demonstrated the absence of endogenous U6 snRNA, in contrast to the presence of endogenous U3 snoRNA (control). (b) Injection of U4 or U5 snRNAs into U6-depleted oocyte nuclei resulted in nucleolar labeling (FL, green) 1.5 h later, showing that U4 and U5 nucleolar localization is independent from U6. The multiple nucleoli present in Xenopus oocyte nuclei are visualized in nucleolar preparations by phase contrast (PC) and can be distinguished from other nuclear bodies by the staining of rDNA (DAPI, blue) located only in nucleoli. Bar, 10 μM.
Mentions: Previously, we reported that U4, U5, and U6 transiently localize to nucleoli of Xenopus laevis oocytes (Lange and Gerbi, 2000; Gerbi and Lange, 2002). To address if U4 and U5 have their own NoLEs and do not depend on U6 snRNA as a carrier, fluorescein-labeled in vitro transcripts of U4 snRNA, U5 snRNA, or a control RNA were injected into Xenopus oocyte nuclei that were depleted of endogenous U6 snRNA (Fig. 1 a). This assay allows direct visualization of the labeled RNA in nucleolar preparations (Fig. 1 b) and has been used in nondepleted oocytes to monitor nucleolar localization of small nucleolar RNAs (snoRNAs) from various families as well as snRNAs (for review see Lange, 2003). Here, the assay was combined with disruption of endogenous U6 snRNA through RNase H–mediated degradation by nuclear injections of anti-U6 snRNA antisense oligonucleotides (Vankan et al., 1990, 1992; Gerbi and Lange, 2002).

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
Related in: MedlinePlus