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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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U4 wild-type snRNA but not the NoLE mutants associate with the endogenous counterpart of the human NHPX/15.5-kD protein in Xenopus. The ability of U4 transcripts (labeled with [32P]UTP) to bind to the NHPX/15.5-kD protein was analyzed by immunoprecipitation from either (a) HeLa cell nuclear lysate or (b) Xenopus oocyte nuclear lysate using an anti– human NHPX/15.5-kD protein antiserum. After immunoprecipitation, either the total precipitate or 1/100 of the supernatant (control for equal amounts injected) were analyzed by PAGE. The NHPX/15.5-kD protein from Hela cells as well as from Xenopus can bind to wild-type U4 snRNA (U4 WT) in vitro transcripts and with slightly less efficiency to U4 mutated in the Sm site (subSm). In contrast, the NoLE mutants with a deletion of the entire 5′-proximal stem loop (Δ19–55) or with substitution in five conserved nucleotides (ΔNHPX/15.5 kD) shown to be essential for binding the NHPX/15.5-kD protein (Nottrott et al., 1999) are not precipitated. U4 snRNA after deletion of sequences that base pair with U6 snRNA (Δ1–18/56–63), which still carried the 5′-proximal stem loop, could still be recognized and precipitated by the antibody although the signals obtained were slightly reduced (b, bottom). Only background signals were observed with control beads coupled with preimmune serum. The supernatant lanes show that equal amounts of U4 were used in the various immunoprecipitations. (c) Immunostaining of a nuclear preparation of Xenopus oocyte nuclei was performed using rabbit anti-NHPX/15.5-kD antiserum, rabbit anticoilin antiserum (as a control for staining of Cajal bodies) or rabbit preimmune serum (as a negative control). The goat anti–rabbit secondary antibody was coupled to Alexa 594 (red signals). Nucleoli contain rDNA (DAPI, blue) and can be distinguished from Cajal bodies that lack rDNA and, thus, are not stained by DAPI. Moreover, Cajal bodies are often associated with B-snurposomes (Gall et al., 1999 and references therein). Coilin was detected in Cajal bodies, whereas the anti–NHPX/15.5-kD antiserum stained Cajal bodies uniformly and also stained nucleoli (arrowheads in phase contrast [PC] and immunofluorescence [FL] panels) in a more spotted manner, suggesting that the endogenous counterpart of the human NHPX/15.5-kD protein is located in Xenopus Cajal bodies and nucleoli. The control serum did not stain any structure in the nuclear spread. Bar, 10 μm.
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fig7: U4 wild-type snRNA but not the NoLE mutants associate with the endogenous counterpart of the human NHPX/15.5-kD protein in Xenopus. The ability of U4 transcripts (labeled with [32P]UTP) to bind to the NHPX/15.5-kD protein was analyzed by immunoprecipitation from either (a) HeLa cell nuclear lysate or (b) Xenopus oocyte nuclear lysate using an anti– human NHPX/15.5-kD protein antiserum. After immunoprecipitation, either the total precipitate or 1/100 of the supernatant (control for equal amounts injected) were analyzed by PAGE. The NHPX/15.5-kD protein from Hela cells as well as from Xenopus can bind to wild-type U4 snRNA (U4 WT) in vitro transcripts and with slightly less efficiency to U4 mutated in the Sm site (subSm). In contrast, the NoLE mutants with a deletion of the entire 5′-proximal stem loop (Δ19–55) or with substitution in five conserved nucleotides (ΔNHPX/15.5 kD) shown to be essential for binding the NHPX/15.5-kD protein (Nottrott et al., 1999) are not precipitated. U4 snRNA after deletion of sequences that base pair with U6 snRNA (Δ1–18/56–63), which still carried the 5′-proximal stem loop, could still be recognized and precipitated by the antibody although the signals obtained were slightly reduced (b, bottom). Only background signals were observed with control beads coupled with preimmune serum. The supernatant lanes show that equal amounts of U4 were used in the various immunoprecipitations. (c) Immunostaining of a nuclear preparation of Xenopus oocyte nuclei was performed using rabbit anti-NHPX/15.5-kD antiserum, rabbit anticoilin antiserum (as a control for staining of Cajal bodies) or rabbit preimmune serum (as a negative control). The goat anti–rabbit secondary antibody was coupled to Alexa 594 (red signals). Nucleoli contain rDNA (DAPI, blue) and can be distinguished from Cajal bodies that lack rDNA and, thus, are not stained by DAPI. Moreover, Cajal bodies are often associated with B-snurposomes (Gall et al., 1999 and references therein). Coilin was detected in Cajal bodies, whereas the anti–NHPX/15.5-kD antiserum stained Cajal bodies uniformly and also stained nucleoli (arrowheads in phase contrast [PC] and immunofluorescence [FL] panels) in a more spotted manner, suggesting that the endogenous counterpart of the human NHPX/15.5-kD protein is located in Xenopus Cajal bodies and nucleoli. The control serum did not stain any structure in the nuclear spread. Bar, 10 μm.

Mentions: Immunoprecipitation assays confirmed that the NoLE mutants of U4 snRNA cannot associate with the NHPX/15.5-kD protein anymore, supporting the implication of this protein in nucleolar localization. The functional ability of U4 to bind to the NHPX/15.5-kD protein was first analyzed using HeLa cell nuclear lysate incubated with various U4 transcripts and anti–human NHPX/15.5-kD protein antiserum coupled to protein A–Sepharose beads (Fig. 7 a). The NHPX/15.5-kD protein can bind to wild-type U4 snRNA (U4 WT) and with a slightly lesser efficiency to U4 mutated in the Sm site (subSm), but not to NoLE mutants with either deletion of the entire 5′-proximal stem loop (Δ19–55) or substituted in the five conserved nucleotides (ΔNHPX/15.5 kD) known to be essential for binding to human NHPX/15.5-kD protein (Nottrott et al., 1999).


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)

U4 wild-type snRNA but not the NoLE mutants associate with the endogenous counterpart of the human NHPX/15.5-kD protein in Xenopus. The ability of U4 transcripts (labeled with [32P]UTP) to bind to the NHPX/15.5-kD protein was analyzed by immunoprecipitation from either (a) HeLa cell nuclear lysate or (b) Xenopus oocyte nuclear lysate using an anti– human NHPX/15.5-kD protein antiserum. After immunoprecipitation, either the total precipitate or 1/100 of the supernatant (control for equal amounts injected) were analyzed by PAGE. The NHPX/15.5-kD protein from Hela cells as well as from Xenopus can bind to wild-type U4 snRNA (U4 WT) in vitro transcripts and with slightly less efficiency to U4 mutated in the Sm site (subSm). In contrast, the NoLE mutants with a deletion of the entire 5′-proximal stem loop (Δ19–55) or with substitution in five conserved nucleotides (ΔNHPX/15.5 kD) shown to be essential for binding the NHPX/15.5-kD protein (Nottrott et al., 1999) are not precipitated. U4 snRNA after deletion of sequences that base pair with U6 snRNA (Δ1–18/56–63), which still carried the 5′-proximal stem loop, could still be recognized and precipitated by the antibody although the signals obtained were slightly reduced (b, bottom). Only background signals were observed with control beads coupled with preimmune serum. The supernatant lanes show that equal amounts of U4 were used in the various immunoprecipitations. (c) Immunostaining of a nuclear preparation of Xenopus oocyte nuclei was performed using rabbit anti-NHPX/15.5-kD antiserum, rabbit anticoilin antiserum (as a control for staining of Cajal bodies) or rabbit preimmune serum (as a negative control). The goat anti–rabbit secondary antibody was coupled to Alexa 594 (red signals). Nucleoli contain rDNA (DAPI, blue) and can be distinguished from Cajal bodies that lack rDNA and, thus, are not stained by DAPI. Moreover, Cajal bodies are often associated with B-snurposomes (Gall et al., 1999 and references therein). Coilin was detected in Cajal bodies, whereas the anti–NHPX/15.5-kD antiserum stained Cajal bodies uniformly and also stained nucleoli (arrowheads in phase contrast [PC] and immunofluorescence [FL] panels) in a more spotted manner, suggesting that the endogenous counterpart of the human NHPX/15.5-kD protein is located in Xenopus Cajal bodies and nucleoli. The control serum did not stain any structure in the nuclear spread. Bar, 10 μm.
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Related In: Results  -  Collection

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fig7: U4 wild-type snRNA but not the NoLE mutants associate with the endogenous counterpart of the human NHPX/15.5-kD protein in Xenopus. The ability of U4 transcripts (labeled with [32P]UTP) to bind to the NHPX/15.5-kD protein was analyzed by immunoprecipitation from either (a) HeLa cell nuclear lysate or (b) Xenopus oocyte nuclear lysate using an anti– human NHPX/15.5-kD protein antiserum. After immunoprecipitation, either the total precipitate or 1/100 of the supernatant (control for equal amounts injected) were analyzed by PAGE. The NHPX/15.5-kD protein from Hela cells as well as from Xenopus can bind to wild-type U4 snRNA (U4 WT) in vitro transcripts and with slightly less efficiency to U4 mutated in the Sm site (subSm). In contrast, the NoLE mutants with a deletion of the entire 5′-proximal stem loop (Δ19–55) or with substitution in five conserved nucleotides (ΔNHPX/15.5 kD) shown to be essential for binding the NHPX/15.5-kD protein (Nottrott et al., 1999) are not precipitated. U4 snRNA after deletion of sequences that base pair with U6 snRNA (Δ1–18/56–63), which still carried the 5′-proximal stem loop, could still be recognized and precipitated by the antibody although the signals obtained were slightly reduced (b, bottom). Only background signals were observed with control beads coupled with preimmune serum. The supernatant lanes show that equal amounts of U4 were used in the various immunoprecipitations. (c) Immunostaining of a nuclear preparation of Xenopus oocyte nuclei was performed using rabbit anti-NHPX/15.5-kD antiserum, rabbit anticoilin antiserum (as a control for staining of Cajal bodies) or rabbit preimmune serum (as a negative control). The goat anti–rabbit secondary antibody was coupled to Alexa 594 (red signals). Nucleoli contain rDNA (DAPI, blue) and can be distinguished from Cajal bodies that lack rDNA and, thus, are not stained by DAPI. Moreover, Cajal bodies are often associated with B-snurposomes (Gall et al., 1999 and references therein). Coilin was detected in Cajal bodies, whereas the anti–NHPX/15.5-kD antiserum stained Cajal bodies uniformly and also stained nucleoli (arrowheads in phase contrast [PC] and immunofluorescence [FL] panels) in a more spotted manner, suggesting that the endogenous counterpart of the human NHPX/15.5-kD protein is located in Xenopus Cajal bodies and nucleoli. The control serum did not stain any structure in the nuclear spread. Bar, 10 μm.
Mentions: Immunoprecipitation assays confirmed that the NoLE mutants of U4 snRNA cannot associate with the NHPX/15.5-kD protein anymore, supporting the implication of this protein in nucleolar localization. The functional ability of U4 to bind to the NHPX/15.5-kD protein was first analyzed using HeLa cell nuclear lysate incubated with various U4 transcripts and anti–human NHPX/15.5-kD protein antiserum coupled to protein A–Sepharose beads (Fig. 7 a). The NHPX/15.5-kD protein can bind to wild-type U4 snRNA (U4 WT) and with a slightly lesser efficiency to U4 mutated in the Sm site (subSm), but not to NoLE mutants with either deletion of the entire 5′-proximal stem loop (Δ19–55) or substituted in the five conserved nucleotides (ΔNHPX/15.5 kD) known to be essential for binding to human NHPX/15.5-kD protein (Nottrott et al., 1999).

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