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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.

Show MeSH
Sequence and mutations of U4 snRNA. The structure of chicken UB4 snRNA is shown with sites of 2'-O-methylation and pseudouridylation indicated (modified after Tycowski et al., 1998 for human U4A). The 3′-end is extended by three nucleotides not removed by processing from some U4 isoforms (Hoffman et al., 1986). Most mutations designed for this work were deletions covering the nucleotides indicated by lines. The site of base pairing with U6 in the di-snRNP (dashed line) was deleted in mutant Δ1–18/56–63. Nucleotides within the binding domain (dotted) for the NHPX/15.5-kD protein that are essential for NHPX/15.5-kD protein interaction (Nottrott et al., 1999) are shaded and were substituted in the present work (ΔNHPX/15.5 kD) as indicated. The Sm protein-binding site was mutated either by substitution of two nucleotides (3/4Sm) or the entire sequence (subSm).
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fig2: Sequence and mutations of U4 snRNA. The structure of chicken UB4 snRNA is shown with sites of 2'-O-methylation and pseudouridylation indicated (modified after Tycowski et al., 1998 for human U4A). The 3′-end is extended by three nucleotides not removed by processing from some U4 isoforms (Hoffman et al., 1986). Most mutations designed for this work were deletions covering the nucleotides indicated by lines. The site of base pairing with U6 in the di-snRNP (dashed line) was deleted in mutant Δ1–18/56–63. Nucleotides within the binding domain (dotted) for the NHPX/15.5-kD protein that are essential for NHPX/15.5-kD protein interaction (Nottrott et al., 1999) are shaded and were substituted in the present work (ΔNHPX/15.5 kD) as indicated. The Sm protein-binding site was mutated either by substitution of two nucleotides (3/4Sm) or the entire sequence (subSm).

Mentions: This result was also supported by mutational analysis of U4 snRNA that demonstrated that sites of base pairing between U4 and U6 snRNA are not essential for nucleolar localization. Fig. 2 depicts all the U4 snRNA mutations studied here. Deletion of nt 1–18 and 56–63 removed the sequences of U6 snRNA that base pair with U4 snRNA; nevertheless, fluorescent transcripts of Δ1–18/56–63 U4 still localized to nucleoli as efficiently as wild-type U4 (Fig. 3).


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)

Sequence and mutations of U4 snRNA. The structure of chicken UB4 snRNA is shown with sites of 2'-O-methylation and pseudouridylation indicated (modified after Tycowski et al., 1998 for human U4A). The 3′-end is extended by three nucleotides not removed by processing from some U4 isoforms (Hoffman et al., 1986). Most mutations designed for this work were deletions covering the nucleotides indicated by lines. The site of base pairing with U6 in the di-snRNP (dashed line) was deleted in mutant Δ1–18/56–63. Nucleotides within the binding domain (dotted) for the NHPX/15.5-kD protein that are essential for NHPX/15.5-kD protein interaction (Nottrott et al., 1999) are shaded and were substituted in the present work (ΔNHPX/15.5 kD) as indicated. The Sm protein-binding site was mutated either by substitution of two nucleotides (3/4Sm) or the entire sequence (subSm).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172826&req=5

fig2: Sequence and mutations of U4 snRNA. The structure of chicken UB4 snRNA is shown with sites of 2'-O-methylation and pseudouridylation indicated (modified after Tycowski et al., 1998 for human U4A). The 3′-end is extended by three nucleotides not removed by processing from some U4 isoforms (Hoffman et al., 1986). Most mutations designed for this work were deletions covering the nucleotides indicated by lines. The site of base pairing with U6 in the di-snRNP (dashed line) was deleted in mutant Δ1–18/56–63. Nucleotides within the binding domain (dotted) for the NHPX/15.5-kD protein that are essential for NHPX/15.5-kD protein interaction (Nottrott et al., 1999) are shaded and were substituted in the present work (ΔNHPX/15.5 kD) as indicated. The Sm protein-binding site was mutated either by substitution of two nucleotides (3/4Sm) or the entire sequence (subSm).
Mentions: This result was also supported by mutational analysis of U4 snRNA that demonstrated that sites of base pairing between U4 and U6 snRNA are not essential for nucleolar localization. Fig. 2 depicts all the U4 snRNA mutations studied here. Deletion of nt 1–18 and 56–63 removed the sequences of U6 snRNA that base pair with U4 snRNA; nevertheless, fluorescent transcripts of Δ1–18/56–63 U4 still localized to nucleoli as efficiently as wild-type U4 (Fig. 3).

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