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. 2002 Dec;22(23):8342-52.
doi: 10.1128/MCB.22.23.8342-8352.2002.

Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP

Nicholas J Watkins  1 , Achim Dickmanns, Reinhard Lührmann
Affiliations

Conserved stem II of the box C/D motif is essential for nucleolar localization and is required, along with the 15.5K protein, for the hierarchical assembly of the box C/D snoRNP

Nicholas J Watkins et al. Mol Cell Biol. 2002 Dec.

Abstract

The 5' stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5' stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.

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Figures

FIG. 1.
FIG. 1.
The binding of box C/D snoRNP-specific proteins is solely dependent on the box C/D motif. (A) Schematic representation of the U14 box C/D snoRNA. The conserved nucleotides of the box C/D motif are shown in white on a black background for the internal loop and white on a grey background for conserved stem II. The tentative box C′/D′ motif is indicated. Base-pairing interactions necessary for 18S rRNA modification and processing are shown. An arrow indicates the 2′ O methylation site directed by the U14 snoRNA. The three regions of the U14 snoRNA, namely, A, B, and V, are labeled (49). The mutations ΔAV and ΔABV involve the deletion of regions A and V and of A, B, and V, respectively. Mutations U14 mutC and U14 mutD involve the replacement of the conserved GA dinucleotides in boxes C and D, respectively, with CC (50). (B) Coimmunoprecipitation of in vitro-assembled snoRNPs with antibodies specific to the box C/D snoRNP proteins. SnoRNP complexes were assembled onto 32P-radiolabeled RNAs in HeLa nuclear extract (see Materials and Methods). RNP complexes formed during this reaction were then immunoprecipitated, and the copurifying RNAs were analyzed on an 8% polyacrylamide-7 M urea gel. The RNA used is indicated above each lane. Antibodies used for immunoprecipitation are indicated on the right. Input, 10% of the RNA after incubation in nuclear extract. NIS, nonimmune serum. (C) U14 snoRNA transcripts undergo limited trimming upon incubation in nuclear extract. U14 RNA before (lane 1, Input) or after (lane 2, Extract) incubation in nuclear extract. RNA was recovered from the nuclear extract and separated on an 8% polyacrylamide-7 M urea gel.
FIG. 2.
FIG. 2.
The binding of the 15.5K protein is an essential first step in the assembly of the box C/D snoRNP. (A) Sequence and structure of the two U4 5′ stem-loop oligonucleotides used to block 15.5K binding. The conserved internal loop nucleotides are shown in white on a black background. U4-SL1 is the wild-type sequence, while U4-SL17 contains a point mutation in the internal loop that inhibits 15.5K binding (33). (B) An excess of a U4 5′ stem-loop oligonucleotide can specifically block the assembly of the box C/D snoRNP. Radiolabeled U14 snoRNA was incubated in HeLa nuclear extract that had been preincubated with increasing amounts of either U4-SL1 or U4-SL17 RNA oligonucleotides. The binding of individual snoRNP proteins was then assayed by immunoprecipitation (see Materials and Methods). Bound RNAs were recovered and then separated on an 8% polyacrylamide-7 M urea gel. Antibodies used for immunoprecipitation are indicated on the left of the figure. Input, 10% of the RNA after incubation in nuclear extract. The amount as well as the identity of the RNA oligonucleotide used is indicated above each lane. (C) Rescue of 15.5K depletion by the addition of recombinant 15.5K. HeLa nuclear extract was preincubated with either 800 pmol of U4-SL1 RNA oligonucleotide (+) or buffer (−). Radiolabeled U14 snoRNA was subsequently added in the presence (10 pmol in lane 3, 20 pmol in lane 4, and 40 pmol in lane 5) or absence (lanes 1 and 2) of recombinant 15.5K. The binding of individual snoRNP proteins was assayed as for panel B.
FIG. 3.
FIG. 3.
Stems I and II of the box C/D motif are essential for 15.5K binding. (A) Sequence and structure of U14 snoRNA stem I and stem II mutations. The conserved nucleotides of the box C/D motif are represented as in Fig. 1A. (B) Gel mobility shift analysis of the interaction of recombinant 15.5K with the stem I and stem II mutants. The wild-type (wt) U14 as well as the mutant transcripts outlined in panel A were incubated with recombinant 15.5K, and the resulting RNA-protein complexes were resolved on a 6% native polyacrylamide gel. The presence (+) or absence (−) of 15.5K is indicated above each lane. The position of the protein-RNA complex (RNP) and the free RNA is indicated on the right. The RNA used is indicated above each lane.
FIG. 4.
FIG. 4.
The conserved sequence of stem II is essential for the specific assembly of the box C/D snoRNP. (A) Sequence and structure of the U14 snoRNA stem II mutants. The conserved nucleotides of the box C/D motif are represented as in Fig. 1A. (B) Role of conserved stem II in snoRNP formation in vitro. The binding of individual snoRNP proteins to the mutant transcripts outlined in panel A was assayed by immunoprecipitation (see Materials and Methods) with the bound RNAs being separated on an 8% polyacrylamide-7 M urea gel. The RNA mutant used is indicated above each lane. Antibodies used for immunoprecipitation are indicated on the right. Input, 10% of the RNA after incubation in nuclear extract; NIS, nonimmune serum.
FIG. 5.
FIG. 5.
The conserved sequence of stem II of the box C/D motif is required for nucleolar localization. (A) Time-dependent localization of snoRNPs in HeLa nuclei. Fluorescent U14 snoRNA transcripts were microinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C, and images were taken at 2, 30, 60, and 90 min after injection. Three examples of each time point are shown. The time point is indicated above each set of pictures. (B) Injected fluorescent U14 transcripts and 70-kDa dextran differentially localize upon injection into HeLa nuclei. Fluorescent U14 snoRNA transcripts and FITC-labeled 70-kDa dextran were coinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C, and fluorescent images of the labeled RNA and dextran were taken at 2 and 90 min after injection. Three examples at each time point are shown. The time point is indicated above the panels. The Alexa 546-labeled snoRNA is shown in red, and the FITC-labeled dextran image is shown in green. The overlay of the snoRNA and dextran images is shown at the bottom of the figure. (C) The internal loop and conserved stem II of the box C/D motif are both essential for nucleolar localization. Fluorescent snoRNA and snRNA transcripts were microinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C for 90 min, and fluorescent images were taken. Three examples for each injected RNA are shown. The injected RNA is indicated to the right of the images. (D) Mutant U14 snoRNAs injected into HeLa nuclei are stable. Unlabeled U14 mutC and FITC-labeled 70-kDa dextran were coinjected into the nuclei of cultured HeLa cells. The cells were then incubated at 37°C for 90 min. The U14 snoRNA was subsequently detected by fluorescence in situ hybridization. The Cy3 in situ-hybridized U14 snoRNA (U14 FISH) is shown in red, and the FITC-labeled dextran image is shown in green. The injected cell is shown on the right, and the control, uninjected cell is on the left-hand side of the panel. The overlay of the snoRNA and dextran image is shown at the bottom of the figure.
FIG. 6.
FIG. 6.
Summary of box C/D snoRNP assembly. The box C/D snoRNA, bound to an rRNA target sequence (methylation site is indicated by a circle), is shown here schematically with the conserved nucleotides of the box C/D motif represented as in Fig. 1A. The specific sequence requirements for the hierarchical assembly of the box C/D snoRNP are indicated. The 15.5K protein, the primary snoRNA binding protein, binds to the internal loop, while the remaining box C/D snoRNP proteins bind the 15.5K-snoRNA complex, specifically recognizing the conserved sequence of stem II. Fibrillarin (fibr.) and NOP56 and NOP58, TIP48, and TIP49 are grouped to indicate the connected nature of their association with the snoRNP.
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