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. 2007 Oct;27(19):6782-93.
doi: 10.1128/MCB.01097-07. Epub 2007 Jul 16.

A Dynamic Scaffold of pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

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Free PMC article

A Dynamic Scaffold of pre-snoRNP Factors Facilitates Human Box C/D snoRNP Assembly

Kenneth Scott McKeegan et al. Mol Cell Biol. .
Free PMC article

Abstract

The box C/D small nucleolar RNPs (snoRNPs) are essential for the processing and modification of rRNA. The core box C/D proteins are restructured during human U3 box C/D snoRNP biogenesis; however, the molecular basis of this is unclear. Here we show that the U8 snoRNP is also restructured, suggesting that this may occur with all box C/D snoRNPs. We have characterized four novel human biogenesis factors (BCD1, NOP17, NUFIP, and TAF9) which, along with the ATPases TIP48 and TIP49, are likely to be involved in the formation of the pre-snoRNP. We have analyzed the in vitro protein-protein interactions between the assembly factors and core box C/D proteins. Surprisingly, this revealed few interactions between the individual core box C/D proteins. However, the novel biogenesis factors and TIP48 and TIP49 interacted with one or more of the core box C/D proteins, implying that they mediate the assembly of the pre-snoRNP. Consistent with this, we show that NUFIP bridges interactions between the core box C/D proteins in a partially reconstituted pre-snoRNP. Restructuring of the core complex probably reflects the conversion of the pre-snoRNP, where core protein-protein interactions are maintained by the bridging biogenesis factors, to the mature snoRNP.

Figures

FIG. 1.
FIG. 1.
Nuclear and nucleolar core U8 box C/D snoRNPs exhibit different salt stabilities. U8 snoRNPs present in either nuclear (upper panel) or nucleolar (lower panel) extract were immunoprecipitated with nonimmune serum (NIS) or antibodies to either NOP56 or NOP58. Bound particles were incubated with buffer containing 250, 500, or 750 mM NaCl, as indicated. The associated U8 snoRNAs were isolated, separated on an 8% polyacrylamide-7 M urea gel, and analyzed by Northern blotting. The U8 pre-snoRNAs (I to IV) and mature snoRNA (m) present in nuclear extracts are indicated on the right of the panel. Input represents 10% of the starting material. The antibody used is indicated at the top of the panel.
FIG. 2.
FIG. 2.
Protein-protein interactions between the known pre-snoRNP factors. (A) Recombinant GST, GST-15.5K, GST-fibrillarin, GST-TIP48, and GST-TIP49 were separated on an SDS-12% polyacrylamide gel and visualized by Coomassie staining. The identity of the protein loaded is indicated at the top of the gel. M, molecular weight marker. The sizes of the molecular mass marker bands are indicated at the left of the panel in kilodaltons. (B and C). Equal amounts of GST, GST-15.5K, and GST-fibrillarin (B) or GST, GST-TIP48, and GST-TIP49 (C) were bound to glutathione-Sepharose and incubated with in vitro-translated [35S]methionine-labeled 15.5K, fibrillarin, NOP56, and NOP58. GST-TIP48 and GST-TIP49 immobilized on glutathione-Sepharose were incubated with the 35S-labeled proteins. Bound proteins were purified, resolved on an SDS-PAGE gel, and visualized by autoradiography. The identity of the GST-tagged protein used is indicated above each lane. The identity of the radiolabeled protein is indicated on the left of the panel. The full-length protein is indicated by an asterisk. Input, 10% of the 35S-labeled input material. Note that a single exposure of one gel is used for each individual protein translate. (D) Recombinant, purified GST or GST-15.5K was bound to glutathione-Sepharose and then incubated with either recombinant TIP48 or TIP49 either alone or in the presence of ATP or ADP. Bound proteins were then separated by SDS-PAGE and visualized by Coomassie blue staining. “Input” indicates the proteins added to the assay. The GST-tagged protein and nucleotide used are indicated at the top of each lane. The migration of the individual proteins is indicated on the right of each panel.
FIG. 3.
FIG. 3.
Four novel factors associated with U3 and U8 pre-snoRNP complexes. Nuclear (A) or nucleolar (C) extract was immunoprecipitated with either protein-specific antibodies or control nonimmune serum (NIS). Bound RNAs were isolated and separated on an 8% polyacrylamide-7 M urea gel, and the U3 and U8 snoRNAs were revealed by Northern blotting. The antibody used is indicated at the top of each lane. The RNA analyzed is indicated to the left of each panel. The precursor U3 (p) and U8 (I to IV) and mature length transcripts (m) are indicated on the right of each panel. Input, 10% of the material used for immunoprecipitation. (B) NOP58-containing complexes were immunopurified from nuclear extract using anti-NOP58 or control IgG antibodies, and the bound material was released using an excess of the NOP58 epitope peptide. The bound material was then separated by SDS-PAGE and analyzed by Western blotting. The antibody used is indicated above each lane. Input, 10% of the starting nuclear extract. The antibodies used for Western blotting are indicated on the right.
FIG. 4.
FIG. 4.
BCD1 and NOP17 are essential for the maintenance of box C/D snoRNA levels. HeLa cells were treated with siRNA duplexes targeting either NOP58, fibrillarin, BCD1, NOP17, hPRP31, or the control siRNA targeting luciferase (GL2). Cells were analyzed 60 h after transfection. (A) Material derived from equal numbers of cells was analyzed by Western blotting to confirm protein depletion. The protein targeted is indicated above each panel. The antibodies used are indicated on the right. (B) Total RNA was extracted from an equal number of HeLa cells and analyzed by Northern blotting to determine the relative levels of the U3, U8, and U14 box C/D snoRNAs and the 7SL RNA. The specific probe used is indicated to the right of each panel. The protein targeted is indicated at the top of the panel. (C) Transfected HeLa cells were hybridized with fluorescent oligonucleotides complementary to the U3 snoRNA and the U2 snRNA and images captured by fluorescence microscopy. The same exposure time was used for each fluorescent probe to allow the direct comparison of the subcellular distribution of the RNA after the depletion of specific factors. The protein targeted is indicated at the top of each column of images. The first and second row of images from the top of the figure represent short and long exposures of pictures of the U3 snoRNA, respectively. The third row shows the U2 snRNA. The fourth row shows an overlay of the U2 snRNA and the long exposure of the U3 snoRNA images, respectively, for each series of cells. White arrows indicate examples of Cajal bodies. The inset panels in the bottom row of images shows a magnified view of a single Cajal body. (D) HeLa cells were transfected with siRNAs targeting hPRP31 or the control GL2 siRNA and hybridized with fluorescent oligonucleotides complementary to the U3 snoRNA and the U4 snRNA, and images were captured by fluorescence microscopy as described above. White arrows indicate examples of Cajal bodies. The protein targeted is indicated at the top of each column of images.
FIG. 5.
FIG. 5.
Interactions between pre-snoRNP and core box C/D proteins. (A) SDS-PAGE analysis of recombinant protein inputs GST-NUFIP, GST, TIP48, TIP49, GST-BCD1(1-360), and GST-NOP17. Proteins were visualized by Coomassie blue staining. The protein loaded is indicated at the top of the gel. M, molecular weight marker. The sizes are indicated in kilodaltons to the left of the panel. Where appropriate, full-length proteins are indicated by an asterisk. (B) 35S-labeled BCD1, NOP17, NUFIP, and TAF9 were incubated with immobilized GST, GST-BCD1(1-360), GST-NUFIP, or GST-TAF9. (C) 35S-labeled TIP48 and TAF9 were incubated with GST, GST-TIP48, or GST-TIP49. (D) 35S-labeled TIP48 and TIP49 were incubated with GST-NUFIP immobilized on glutathione-Sepharose. (E and F) Recombinant, purified GST, GST-BCD1(1-360) (E), or GST-NOP17 (F) were bound to glutathione-Sepharose and then incubated with either recombinant TIP48 or TIP49 (indicated below each panel) either alone or in the presence of ATP or ADP. Bound proteins were then separated by SDS-PAGE and revealed by Coomassie blue staining. The GST-tagged protein and nucleotide used are indicated at the top of each lane. The migration of the individual proteins is indicated on the right of the panels. (G) 35S-labeled 15.5K, fibrillarin, NOP56, and NOP58 were incubated with immobilized GST, GST-BCD1(1-360), GST-NUFIP, GST-TAF9, or GST-NOP17. Bound proteins were eluted, resolved by SDS-PAGE, and revealed by autoradiography. The GST-protein used is indicated above each lane. The identity of the radiolabeled protein is indicated on the left of the panel. The asterisk denotes nonspecific material that is sometimes seen with the NOP56 and NOP58 in vitro-translated proteins. Input, 10% of the input material. Note that a single exposure of one gel was used for each individual translated protein.
FIG. 6.
FIG. 6.
NUFIP bridges interactions between both NOP56 and NOP58 and 15.5K. (A) Recombinant thioredoxin-tagged NOP56(1-458) and NOP58(1-435) were separated by SDS-PAGE and visualized by Coomassie blue staining. Full-length proteins are indicated by an asterisk. The protein loaded is indicated at the top of the gel. M, molecular weight marker. The sizes are indicated to the left in kilodaltons. (B and C) In vitro assembly of a partial pre-snoRNP complex. Thioredoxin-tagged (A) NOP56(1-458) or (B) NOP58(1-435) were immobilized onto MagZ beads with GST-NUFIP and 15.5K added successively. The beads were then incubated with 32P-labeled U14 wild-type or U14 mutC transcripts. Bound RNA was recovered, separated on an 8% polyacrylamide-7 M urea gel, and visualized by autoradiography. The RNA used is indicated on the left of each panel. The proteins added to each reaction are indicated above each lane. Input, 10% of the input material.
FIG. 7.
FIG. 7.
Schematic representation of the box C/D pre-snoRNP interaction network. The protein-protein interaction data produced during the present study are represented schematically. Black lines represent interactions between individual proteins. Gray lines indicate protein-protein interactions that are altered by the nucleotide bound to either TIP48 or TIP49.

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