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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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Localization of U4 snRNA to nucleoli and Cajal bodies after cytoplasmic and nuclear injections. Fluorescein-labeled synthetic transcripts of wild-type U4 snRNA (WT U4), a U4 mutant unable to base pair with U6 snRNA (Δ1–18/56–63), the NoLE mutant of U4 (ΔNHPX/15.5 kD), or wild-type U3 snoRNA (WT U3) were injected into Xenopus oocytes. Either 5 h after cytoplasmic injection or 1.5 h after nuclear injection, nuclear spreads were prepared and analyzed by phase contrast (PC) or fluorescence microscopy (FL, green). Nucleoli can be distinguished from other nonchromosomal nuclear bodies because they contain rDNA visualized by staining (DAPI, blue). Cajal bodies are indicated by arrows; they do not contain rDNA (DAPI negative) and often are associated with B-snurposomes (Gall et al., 1999 and references therein). After cytoplasmic injection, both wild-type U4 as well as mutant Δ1–18/56–63 localize weakly to nucleoli (compare DAPI with FL) and exhibit a much stronger preference to localize to Cajal bodies (arrows). The same observations were made for U5 snRNA (Lange, 2003). In contrast, U4 NoLE mutant ΔNHPX/15.5 kD does not reveal any signals in either nuclear compartment. U3 snoRNA after cytoplasmic injection strongly stained nucleoli but not Cajal bodies, unlike the pattern for U4 or U5. After nuclear injection, as shown before (Fig. 3), wild-type U4 and mutant Δ1–18/56–63 preferentially localized to nucleoli which are stained strongly (compare DAPI with FL); Cajal bodies are stained weakly and indicated by arrows (no DAPI signal). U3 snoRNA, similar to U4 snRNA after injection into Xenopus oocyte nuclei, can weakly stain Cajal bodies but strongly localizes to nucleoli (Lange and Gerbi, 2000). A synthetic negative control RNA after nuclear or cytoplasmic injection did not stain either nucleoli or Cajal bodies (Lange and Gerbi, 2000; Lange, 2003). Bar, 10 μm.
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fig9: Localization of U4 snRNA to nucleoli and Cajal bodies after cytoplasmic and nuclear injections. Fluorescein-labeled synthetic transcripts of wild-type U4 snRNA (WT U4), a U4 mutant unable to base pair with U6 snRNA (Δ1–18/56–63), the NoLE mutant of U4 (ΔNHPX/15.5 kD), or wild-type U3 snoRNA (WT U3) were injected into Xenopus oocytes. Either 5 h after cytoplasmic injection or 1.5 h after nuclear injection, nuclear spreads were prepared and analyzed by phase contrast (PC) or fluorescence microscopy (FL, green). Nucleoli can be distinguished from other nonchromosomal nuclear bodies because they contain rDNA visualized by staining (DAPI, blue). Cajal bodies are indicated by arrows; they do not contain rDNA (DAPI negative) and often are associated with B-snurposomes (Gall et al., 1999 and references therein). After cytoplasmic injection, both wild-type U4 as well as mutant Δ1–18/56–63 localize weakly to nucleoli (compare DAPI with FL) and exhibit a much stronger preference to localize to Cajal bodies (arrows). The same observations were made for U5 snRNA (Lange, 2003). In contrast, U4 NoLE mutant ΔNHPX/15.5 kD does not reveal any signals in either nuclear compartment. U3 snoRNA after cytoplasmic injection strongly stained nucleoli but not Cajal bodies, unlike the pattern for U4 or U5. After nuclear injection, as shown before (Fig. 3), wild-type U4 and mutant Δ1–18/56–63 preferentially localized to nucleoli which are stained strongly (compare DAPI with FL); Cajal bodies are stained weakly and indicated by arrows (no DAPI signal). U3 snoRNA, similar to U4 snRNA after injection into Xenopus oocyte nuclei, can weakly stain Cajal bodies but strongly localizes to nucleoli (Lange and Gerbi, 2000). A synthetic negative control RNA after nuclear or cytoplasmic injection did not stain either nucleoli or Cajal bodies (Lange and Gerbi, 2000; Lange, 2003). Bar, 10 μm.

Mentions: This observation and other data (see Discussion) suggest that U4 and U5 snRNAs can localize to nucleoli without cytoplasmic passage. Accordingly, we compared the localization of fluorescein-labeled synthetic U4 transcripts after injection into either Xenopus oocyte nuclei (and incubation for 1 h) or the cytoplasm (and incubation for over 5 h). Interestingly, after nuclear injection wild-type U4 stains nucleoli strongly and Cajal bodies weakly, but after cytoplasmic injection the pattern is reversed and U4 exhibits a stronger preference to localize to Cajal bodies than to nucleoli, which are weakly labeled (Fig. 9). The same observations were made for U5 snRNA (Lange, 2003). Moreover, U4 NoLE mutant NHPX/15.5 kD cannot localize to Cajal bodies or nucleoli after cytoplasmic injection.


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)

Localization of U4 snRNA to nucleoli and Cajal bodies after cytoplasmic and nuclear injections. Fluorescein-labeled synthetic transcripts of wild-type U4 snRNA (WT U4), a U4 mutant unable to base pair with U6 snRNA (Δ1–18/56–63), the NoLE mutant of U4 (ΔNHPX/15.5 kD), or wild-type U3 snoRNA (WT U3) were injected into Xenopus oocytes. Either 5 h after cytoplasmic injection or 1.5 h after nuclear injection, nuclear spreads were prepared and analyzed by phase contrast (PC) or fluorescence microscopy (FL, green). Nucleoli can be distinguished from other nonchromosomal nuclear bodies because they contain rDNA visualized by staining (DAPI, blue). Cajal bodies are indicated by arrows; they do not contain rDNA (DAPI negative) and often are associated with B-snurposomes (Gall et al., 1999 and references therein). After cytoplasmic injection, both wild-type U4 as well as mutant Δ1–18/56–63 localize weakly to nucleoli (compare DAPI with FL) and exhibit a much stronger preference to localize to Cajal bodies (arrows). The same observations were made for U5 snRNA (Lange, 2003). In contrast, U4 NoLE mutant ΔNHPX/15.5 kD does not reveal any signals in either nuclear compartment. U3 snoRNA after cytoplasmic injection strongly stained nucleoli but not Cajal bodies, unlike the pattern for U4 or U5. After nuclear injection, as shown before (Fig. 3), wild-type U4 and mutant Δ1–18/56–63 preferentially localized to nucleoli which are stained strongly (compare DAPI with FL); Cajal bodies are stained weakly and indicated by arrows (no DAPI signal). U3 snoRNA, similar to U4 snRNA after injection into Xenopus oocyte nuclei, can weakly stain Cajal bodies but strongly localizes to nucleoli (Lange and Gerbi, 2000). A synthetic negative control RNA after nuclear or cytoplasmic injection did not stain either nucleoli or Cajal bodies (Lange and Gerbi, 2000; Lange, 2003). Bar, 10 μm.
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fig9: Localization of U4 snRNA to nucleoli and Cajal bodies after cytoplasmic and nuclear injections. Fluorescein-labeled synthetic transcripts of wild-type U4 snRNA (WT U4), a U4 mutant unable to base pair with U6 snRNA (Δ1–18/56–63), the NoLE mutant of U4 (ΔNHPX/15.5 kD), or wild-type U3 snoRNA (WT U3) were injected into Xenopus oocytes. Either 5 h after cytoplasmic injection or 1.5 h after nuclear injection, nuclear spreads were prepared and analyzed by phase contrast (PC) or fluorescence microscopy (FL, green). Nucleoli can be distinguished from other nonchromosomal nuclear bodies because they contain rDNA visualized by staining (DAPI, blue). Cajal bodies are indicated by arrows; they do not contain rDNA (DAPI negative) and often are associated with B-snurposomes (Gall et al., 1999 and references therein). After cytoplasmic injection, both wild-type U4 as well as mutant Δ1–18/56–63 localize weakly to nucleoli (compare DAPI with FL) and exhibit a much stronger preference to localize to Cajal bodies (arrows). The same observations were made for U5 snRNA (Lange, 2003). In contrast, U4 NoLE mutant ΔNHPX/15.5 kD does not reveal any signals in either nuclear compartment. U3 snoRNA after cytoplasmic injection strongly stained nucleoli but not Cajal bodies, unlike the pattern for U4 or U5. After nuclear injection, as shown before (Fig. 3), wild-type U4 and mutant Δ1–18/56–63 preferentially localized to nucleoli which are stained strongly (compare DAPI with FL); Cajal bodies are stained weakly and indicated by arrows (no DAPI signal). U3 snoRNA, similar to U4 snRNA after injection into Xenopus oocyte nuclei, can weakly stain Cajal bodies but strongly localizes to nucleoli (Lange and Gerbi, 2000). A synthetic negative control RNA after nuclear or cytoplasmic injection did not stain either nucleoli or Cajal bodies (Lange and Gerbi, 2000; Lange, 2003). Bar, 10 μm.
Mentions: This observation and other data (see Discussion) suggest that U4 and U5 snRNAs can localize to nucleoli without cytoplasmic passage. Accordingly, we compared the localization of fluorescein-labeled synthetic U4 transcripts after injection into either Xenopus oocyte nuclei (and incubation for 1 h) or the cytoplasm (and incubation for over 5 h). Interestingly, after nuclear injection wild-type U4 stains nucleoli strongly and Cajal bodies weakly, but after cytoplasmic injection the pattern is reversed and U4 exhibits a stronger preference to localize to Cajal bodies than to nucleoli, which are weakly labeled (Fig. 9). The same observations were made for U5 snRNA (Lange, 2003). Moreover, U4 NoLE mutant NHPX/15.5 kD cannot localize to Cajal bodies or nucleoli after cytoplasmic injection.

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