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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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The NoLE mutant of U4 snRNA can form a [U4/U6] snRNP. U4 snRNA transcripts (labeled with fluorescein-UTP) and U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) were coinjected into Xenopus oocyte nuclei depleted of endogenous U4 and U6 snRNA. After 4 h of incubation, the ability of the in vitro transcripts to form a [U4/U6] snRNP was analyzed by immunoprecipitation from nuclear lysate with anti-Sm protein antibody. Equivalents of 10 nuclei/sample of the precipitated RNA (pellet) and 0.1 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed by PAGE. U4/U6 snRNP assembly occurs between wild-type U4 snRNA (U4 WT) and wild-type U6 snRNA (U6 WT), and even the U4 NoLE mutant (ΔNHPX/15.5 kD) retains the ability to coprecipitate U6 snRNA. Di-snRNP formation was disrupted in mutant U4 (Δ1–18/ 56–63) or mutant U6 (Δ43–81) that lack the sites for U4-U6 base pairing. No immunoprecipitation occurred when using beads coupled to control antibody. The supernatant lanes demonstrate that equal amounts of U6 transcript were used in the various immunoprecipitations.
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fig6: The NoLE mutant of U4 snRNA can form a [U4/U6] snRNP. U4 snRNA transcripts (labeled with fluorescein-UTP) and U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) were coinjected into Xenopus oocyte nuclei depleted of endogenous U4 and U6 snRNA. After 4 h of incubation, the ability of the in vitro transcripts to form a [U4/U6] snRNP was analyzed by immunoprecipitation from nuclear lysate with anti-Sm protein antibody. Equivalents of 10 nuclei/sample of the precipitated RNA (pellet) and 0.1 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed by PAGE. U4/U6 snRNP assembly occurs between wild-type U4 snRNA (U4 WT) and wild-type U6 snRNA (U6 WT), and even the U4 NoLE mutant (ΔNHPX/15.5 kD) retains the ability to coprecipitate U6 snRNA. Di-snRNP formation was disrupted in mutant U4 (Δ1–18/ 56–63) or mutant U6 (Δ43–81) that lack the sites for U4-U6 base pairing. No immunoprecipitation occurred when using beads coupled to control antibody. The supernatant lanes demonstrate that equal amounts of U6 transcript were used in the various immunoprecipitations.

Mentions: Control experiments confirmed that in vitro transcripts of U4 and U6 snRNAs retained their functional activity to form a [U4/U6] di-snRNP. U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) was coinjected together with U4 snRNA transcripts (labeled with fluorescein-UTP) into U6- and U4-depleted Xenopus oocytes, and subsequently immunoprecipitated from the nuclear extract by an anti-Sm antibody. Because Sm proteins are bound to U4 and not to U6 snRNA, the immunoprecipitation of radioactive U6 indicates that it is associated with U4 snRNA (Vankan et al., 1990). As shown in Fig. 6, wild-type U4 and U6 in vitro transcripts are functionally able to form a [U4/U6] di-snRNP, as they are coimmunoprecipitated by the anti-Sm antibody but not by a control antibody. Mutation of the base-pairing site in either U4 (Δ1–18/56–63) or U6 (Δ43–81) prevented coimmunoprecipitation (Fig. 6); nonetheless, both the U4 and U6 base-pairing mutants could still localize to nucleoli (Fig. 3; Gerbi and Lange, 2002). Importantly, the NoLE mutant of U4 (ΔNHPX/15.5 kD) still retained the ability to base pair with U4 and form a di-snRNP, even though it was unable to localize to nucleoli. Therefore, nucleolar localization and di-snRNP formation are separable properties of U4 and U6 snRNAs.


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)

The NoLE mutant of U4 snRNA can form a [U4/U6] snRNP. U4 snRNA transcripts (labeled with fluorescein-UTP) and U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) were coinjected into Xenopus oocyte nuclei depleted of endogenous U4 and U6 snRNA. After 4 h of incubation, the ability of the in vitro transcripts to form a [U4/U6] snRNP was analyzed by immunoprecipitation from nuclear lysate with anti-Sm protein antibody. Equivalents of 10 nuclei/sample of the precipitated RNA (pellet) and 0.1 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed by PAGE. U4/U6 snRNP assembly occurs between wild-type U4 snRNA (U4 WT) and wild-type U6 snRNA (U6 WT), and even the U4 NoLE mutant (ΔNHPX/15.5 kD) retains the ability to coprecipitate U6 snRNA. Di-snRNP formation was disrupted in mutant U4 (Δ1–18/ 56–63) or mutant U6 (Δ43–81) that lack the sites for U4-U6 base pairing. No immunoprecipitation occurred when using beads coupled to control antibody. The supernatant lanes demonstrate that equal amounts of U6 transcript were used in the various immunoprecipitations.
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Related In: Results  -  Collection

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fig6: The NoLE mutant of U4 snRNA can form a [U4/U6] snRNP. U4 snRNA transcripts (labeled with fluorescein-UTP) and U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) were coinjected into Xenopus oocyte nuclei depleted of endogenous U4 and U6 snRNA. After 4 h of incubation, the ability of the in vitro transcripts to form a [U4/U6] snRNP was analyzed by immunoprecipitation from nuclear lysate with anti-Sm protein antibody. Equivalents of 10 nuclei/sample of the precipitated RNA (pellet) and 0.1 nuclei/sample of the supernatant (control for equal amounts injected) were analyzed by PAGE. U4/U6 snRNP assembly occurs between wild-type U4 snRNA (U4 WT) and wild-type U6 snRNA (U6 WT), and even the U4 NoLE mutant (ΔNHPX/15.5 kD) retains the ability to coprecipitate U6 snRNA. Di-snRNP formation was disrupted in mutant U4 (Δ1–18/ 56–63) or mutant U6 (Δ43–81) that lack the sites for U4-U6 base pairing. No immunoprecipitation occurred when using beads coupled to control antibody. The supernatant lanes demonstrate that equal amounts of U6 transcript were used in the various immunoprecipitations.
Mentions: Control experiments confirmed that in vitro transcripts of U4 and U6 snRNAs retained their functional activity to form a [U4/U6] di-snRNP. U6 snRNA (co-labeled with [32P]UTP and fluorescein-UTP) was coinjected together with U4 snRNA transcripts (labeled with fluorescein-UTP) into U6- and U4-depleted Xenopus oocytes, and subsequently immunoprecipitated from the nuclear extract by an anti-Sm antibody. Because Sm proteins are bound to U4 and not to U6 snRNA, the immunoprecipitation of radioactive U6 indicates that it is associated with U4 snRNA (Vankan et al., 1990). As shown in Fig. 6, wild-type U4 and U6 in vitro transcripts are functionally able to form a [U4/U6] di-snRNP, as they are coimmunoprecipitated by the anti-Sm antibody but not by a control antibody. Mutation of the base-pairing site in either U4 (Δ1–18/56–63) or U6 (Δ43–81) prevented coimmunoprecipitation (Fig. 6); nonetheless, both the U4 and U6 base-pairing mutants could still localize to nucleoli (Fig. 3; Gerbi and Lange, 2002). Importantly, the NoLE mutant of U4 (ΔNHPX/15.5 kD) still retained the ability to base pair with U4 and form a di-snRNP, even though it was unable to localize to nucleoli. Therefore, nucleolar localization and di-snRNP formation are separable properties of U4 and U6 snRNAs.

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