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. 2014 Nov 24;207(4):463-80.
doi: 10.1083/jcb.201404160. Epub 2014 Nov 17.

Proteomic and 3D Structure Analyses Highlight the C/D Box snoRNP Assembly Mechanism and Its Control

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Free PMC article

Proteomic and 3D Structure Analyses Highlight the C/D Box snoRNP Assembly Mechanism and Its Control

Jonathan Bizarro et al. J Cell Biol. .
Free PMC article

Abstract

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

Figures

Figure 1.
Figure 1.
hBCD1 identifies a new snoRNP assembly factor. (A) GFP-hBCD1 was purified from more and less extractable fractions of U2OS cells, and pellets were analyzed by SILAC proteomic. X axis: protein abundance (Log10); Y axis: SILAC ratios (specific vs. control IP). LC, liquid chromatography; H/L, heavy/light; MW, molecular weight. (B) Yeast two-hybrid assays with ZNHIT3, hBCD1, and snoRNP assembly factors and core proteins. Alix is used as a negative control. Fib, Fibrillarin; Nter, N terminal; Cter, C terminal. (C) Co-IP assays between NUFIP and ZNHIT3. Extracts from 293T cells stably expressing GST-NUFIP and GST-ZNHIT3 were purified on glutathione beads and analyzed by Western blots (WB). (D) Intracellular localization of GFP-ZNHIT3. Microscopy images of HeLa cells transfected with a GFP-ZNHIT3 expression vector and labeled with DAPI to stain nuclei. Bar, 10 µm.
Figure 2.
Figure 2.
ZNHIT3 forms a complex containing Nop58 and 15.5K as well as five assembly factors. (A) Proteomic analysis of GFP-ZNHIT3. X axis: protein abundance (Log10); Y axis: SILAC ratios (specific vs. control IP). LC, liquid chromatography; M/L, medium/light; MW, molecular weight. (B) Proteomic analysis of GFP-ZNHIT3, in the presence and absence of RNase treatment. X axis: SILAC ratios (specific vs. control IP) in the presence of RNase treatment; Y axis: SILAC ratios (specific vs. control IP) in the absence of RNase treatment. H/L, heavy/light. (C) Co-IP assays with GFP-NUFIP, GFP-hBCD1, and GFP-15.5K. 293T cells were transiently transfected with the indicated proteins, extracts were purified on GFP-TRAP beads, and pellets were analyzed by Western blots with the indicated antibodies. When indicated, extracts were treated with 0.6 µg/ml RNase. Pellets: 5% of inputs.
Figure 3.
Figure 3.
ZNHIT3 associates preferentially with assembly defective U3 snoRNAs. (A) Schematic of the U3mut6 mutant. (B) Intracellular localization of U3mut6. HeLa cells were transfected with the U3wt and U3mut6 gene and hybridized in situ with a probe specific for the transfected rat U3 gene. Arrows on the merged image point to a Cajal body that is zoomed in the insets. Bars: (main images) 10 µm; (insets) 0.6 µm. (C–F) Binding of NUFIP (C), ZNHIT3 (D), RuvBL1 (E), and hBCD1 (F) to U3 snoRNAs. HeLa cells were transfected with the indicated plasmids, extracts were immunopurified on glutathione beads, and RNAs in the pellet were analyzed by RNase protection with a probe covering the 3′ end of the transfected rat U3 gene. Ct: control IP with empty beads. Pre-U3-I, pre-U3-II, and pre-U3-III: precursor forms of U3 snoRNA. U3m: mature form of U3. Pellets: 5% of inputs.
Figure 4.
Figure 4.
SILAC proteomic analysis of GFP-NUFIP reveals binding to snoRNAs. (A) Proteomic analysis of GFP-NUFIP. X axis: protein abundance (Log10); Y axis: SILAC ratios (Log10 specific vs. control IP). CTL, control; LC, liquid chromatography; H/L, heavy/light; MW, molecular weight. (B) Co-IP assays with GFP-NUFIP. U2OS cells were extracted in HNTG, extracts were purified on GFP-TRAP beads, and pellets were analyzed by Western blots (WB) with the indicated antibodies. Pellets: 5% of inputs.
Figure 5.
Figure 5.
SILAC proteomic analyses of GFP-Nop58 and GFP-PIH1D1. (A) Proteomic analysis of GFP-Nop58. X axis: protein abundance (Log10); Y axis: SILAC ratios (specific vs. control IP). (B) Proteomic analysis of GFP-PIH1D1. X axis: protein abundance (Log10); Y axis: SILAC ratios (Log10 specific vs. control IP). Legend as in A. CTL, control; LC, liquid chromatography; H/L, heavy/light; M/L, medium/light; Fib, Fibrillarin; MW, molecular weight.
Figure 6.
Figure 6.
Meta-analysis of the proteomic data. (A) Network analysis of the factors involved in snoRNP biogenesis. The interactions detected by SILAC proteomics were used to create an interaction network using Cytoscape. (B) Clustering analysis of the proteomic data. SILAC ratio was used to perform a clustering analysis. Columns: baits, −1 is the less extractable fraction and −2 is the more extractable fraction. Rows: preys.
Figure 7.
Figure 7.
The PEP domain of Rsa1p lies in a groove at the surface of Snu13p. (A and B) Ribbon representation (A) and molecular surface (B) of the complex between Snu13p and Rsa1239–265, obtained from the crystal structure of the complex. A and B are in the same orientation. (C) Superimposition of Snu13p bound to Rsa1p with Snu13p in free state (PDB no. ZWZ). Upon interaction with Rsa1239–265, a rigid-body movement of ∼3 Å of the α5 helix is observed in Snu13p. (D–G) Electrostatic properties of protein Snu13p (D and E) and Rsa1239–265 (F and G). The electrostatic potentials were computed by using the algorithm of Boltzmann available on the Swiss-PdbViewer. Blue, white, and red regions correspond to positive, neutral, and negative electrostatic potentials, respectively. (D and E) Electrostatic potential mapped on the molecular surface of Snu13p and viewed in two different orientations (rotation by 90°). Rsa1239–265 is shown in magenta in a ribbon representation. (F and G) Electrostatic potential mapped on the molecular surface of Rsa1239–265 and viewed in two opposite directions.
Figure 8.
Figure 8.
Hydrogen bonds, ionic interaction, and hydrophobic contacts at the Snu13p–Rsa1p interface. (A) Network of hydrogen bonds between Snu13p and Rsa1p. (B) Hydrophobic contacts and ionic interaction between Snu13p and Rsa1p. (C and D) The residue W253 of Rsa1p tightly binds in a hydrophobic pocket at the molecular surface of Snu13p.
Figure 9.
Figure 9.
Binding of Rsa1p to Snu13p prevents formation of the catalytically active structure of box C/D snoRNP. Model of the human box C/D snoRNP comprising the core proteins 15.5K, Nop58, and Fibrillarin in inactive (left) and active (right) states. Both states were shown in two orthogonal views (up and down representations). The PEP domain of the protein factor NUFIP is represented in magenta. Whereas the presence of NUFIP233–258 is compatible with the structure of the inactive form (comprising Nop58, Fibrillarin, and 15.5K), the rotation of the catalytic module of the box C/D snoRNP (including Fibrillarin and the N-terminal domain of Nop58) leads to a clash between the second α helix of NUFIP233–258 and the N-terminal domain of Nop58. The region of spatial hindrance between NUFIP and Nop58 is indicated with a red dashed circle. Model was built on the basis of the known 3D structures of the box C/D sRNP in an inactive state from P. furiosus ((Xue et al., 2010); PDB no. 3NMU), in an active state from S. solfataricus (Lin et al., 2011; PDB no. 3PLA), and the crystal structure of Snu13p–Rsa1239–265 (our work). The snoRNA is not depicted on these pictures.
Figure 10.
Figure 10.
An assembly scheme for human box C/D snoRNPs. Complexes identified in the SILAC experiments were ordered according to the presence or absence of RNA and the presence of increasing numbers of core snoRNP proteins. The putative role of the R2TP complex is represented. Fib, Fibrillarin.

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References

    1. Aittaleb M., Rashid R., Chen Q., Palmer J.R., Daniels C.J., and Li H. 2003. Structure and function of archaeal box C/D sRNP core proteins. Nat. Struct. Biol. 10:256–263 10.1038/nsb905 - DOI - PubMed
    1. Back R., Dominguez C., Rothé B., Bobo C., Beaufils C., Moréra S., Meyer P., Charpentier B., Branlant C., Allain F.H., and Manival X. 2013. High-resolution structural analysis shows how Tah1 tethers Hsp90 to the R2TP complex. Structure. 21:1834–1847 10.1016/j.str.2013.07.024 - DOI - PubMed
    1. Bardoni B., Schenck A., and Mandel J.L. 1999. A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein. Hum. Mol. Genet. 8:2557–2566 10.1093/hmg/8.13.2557 - DOI - PubMed
    1. Battle D.J., Lau C.K., Wan L., Deng H., Lotti F., and Dreyfuss G. 2006. The Gemin5 protein of the SMN complex identifies snRNAs. Mol. Cell. 23:273–279 10.1016/j.molcel.2006.05.036 - DOI - PubMed
    1. Boulon S., Verheggen C., Jady B.E., Girard C., Pescia C., Paul C., Ospina J.K., Kiss T., Matera A.G., Bordonné R., and Bertrand E. 2004. PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Mol. Cell. 16:777–787 10.1016/j.molcel.2004.11.013 - DOI - PubMed

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