NUFIP and the HSP90/R2TP chaperone bind the SMN complex and facilitate assembly of U4-specific proteins

Abstract

The Sm proteins are loaded on snRNAs by the SMN complex, but how snRNP-specific proteins are assembled remains poorly characterized. U4 snRNP and box C/D snoRNPs have structural similarities. They both contain the 15.5K and proteins with NOP domains (PRP31 for U4, NOP56/58 for snoRNPs). Biogenesis of box C/D snoRNPs involves NUFIP and the HSP90/R2TP chaperone system and here, we explore the function of this machinery in U4 RNP assembly. We show that yeast Prp31 interacts with several components of the NUFIP/R2TP machinery, and that these interactions are separable from each other. In human cells, PRP31 mutants that fail to stably associate with U4 snRNA still interact with components of the NUFIP/R2TP system, indicating that these interactions precede binding of PRP31 to U4 snRNA. Knock-down of NUFIP leads to mislocalization of PRP31 and decreased association with U4. Moreover, NUFIP is associated with the SMN complex through direct interactions with Gemin3 and Gemin6. Altogether, our data suggest a model in which the NUFIP/R2TP system is connected with the SMN complex and facilitates assembly of U4 snRNP-specific proteins.

Figure 1.

Identification and Y2H characterization of yeast Prp31p mutants. (A) Schematic representation of the insertional mutagenesis screen. Random insertion of MuA and excision by NotI digestion and religation leave a 5 nucleotide insertion at site of integration. (B) Schematic representation of Prp31p domains and location of the insertions that were identified in primary two-hybrid screen. (C) Summary of the two-hybrid validation screen. ‘+’ and ‘-’ indicate the presence or absence of interactions. The numbers of the pACTII plasmids indicate the amino-acid after which the 5 nucleotide insertion occurred in yPRP31. The proteins encoded by pAS2 plasmids are indicated, with in parenthesis the name of the human homologs. (D) Two-hybrid interactions in WT yeast strain or in yeast strains deleted for Rsa1 (ΔRSA1) or Pih1 (ΔPIH1). The pACTII-yPRP31 plasmid was introduced in the indicated strain and tested against the indicated proteins. ‘na’: not applicable.

Figure 2.

PRP31 A216P and PRP31 K243A-A246R are mostly cytoplasmic in human cells. (A) 3D Structure of human PRP31 with various mutations indicated. In yellow, amino-acids corresponding to MuA insertion sites leading to the loss of interaction with Pih1, Rsa1 and Hit1 (numbers correspond to the yeast protein). In blue, amino-acids corresponding to MuA insertion sites related to Hit1 (numbers correspond to the yeast protein). In red, amino acids mutated to prevent binding of 15.5K (numbers correspond to the human protein). (B) Micrographs showing the localization of wild-type and mutant PRP31 fused to GFP in U2OS cells. Scale bar is 10 μm. Blue: DAPI staining corresponding to nuclei; green: GFP-tagged PRP31 proteins. (C) PRP31 K243A-A246R does not interact with 15.5K in human cells. Western blotting of inputs and pellets of anti-Flag immuno-precipitates of 293T cells co-transfected with 3xFlag-PRP31 (wild-type and indicated mutants), GFP-15.5K or untransfected (293T). Western blots were probed with the indicated antibodies. Input: 5% of pellet.

Figure 3.

Association of PRP31 and 15.5K with wild-type and mutant U4 snRNAs. (A) RNA gel showing the products of an RNase protection assay made with samples from anti-GFP immuno-precipitation of 293T cells, co-transfected with U4-MS2 and GFP-PRP31 (wild-type and indicated mutants). The products corresponding to endogenous U4 and to U4-MS2 are indicated at the left of the gel. Star: additional doublet produced with the U4-MS2 probe. 293T: RNAs from untransfected 293T cells; I: input (5% of pellets); Ct: control immuno-precipitation of the extracts with unconjugated agarose beads; IP: immuno-precipitation of the extracts with GFP-Trap agarose beads. (B) Schematic depicting the mutations in U4-MS2 that prevent binding to PRP31 or 15.5K. The MS2 stem-loop (nucleotides 99–117) was inserted in the stem-loop upstream of the Sm site. The mutations affecting binding of PRP31 and 15.5K are indicated and described with small and large arrows, respectively. The region covered by the probe used for the RNase protection assays is indicated (nucleotides 43–160). (C) RNA gels showing the products of an RNase protection assay made with samples from GST affinity purification of 293T cells co-transfected with GST-15.5K (left gel) or GST-PRP31 (right gel), and U4-MS2 (wild-type or the indicated mutants). Legends as in (A). An image of higher contrast (expo +) is shown to illustrate the interaction of GST-15.5K with U4-MS2-mutPRP31. 293T: RNAs from untransfected 293T cells; I: input (5% of pellets); Ct: control precipitation of the extracts with unconjugated sepharose 4B beads; IP: GST precipitation of the extracts with sepharose 4B glutathione beads.

Figure 4.

Association of box C/D snoRNP assembly factors with PRP31 and U4 snRNA. (A) Western blotting of inputs and pellets of anti-GFP Trap immuno-precipitates of 293T cells, transfected with the indicated GFP-PRP31 fusion proteins. Membrane was cut to separate proteins of different molecular weights and probed with the indicated antibodies. The control corresponding to untransfected 293T cells (293T) was present on the same gel and is shown using a vertical division to remove non-relevant lanes present in the original gel. Input: 5% of pellet. (B-D) RNA gels showing the products of an RNase protection assay made with samples from GST precipitations of 293T cells, co-transfected with U4-MS2 (wild-type or indicated mutants), and either GST-RuvBL1 (B), GST-NUFIP (C), or GST-ZNHIT3 (D). The products corresponding to endogenous U4 (Endo U4) and to U4-MS2 are indicated. 293T: RNAs from untransfected 293T cells; I: input (5% of pellets); Ct: control immuno-precipitation of the extracts with sepharose 4B beads; IP: precipitation of the extracts with sepharose 4B glutathione beads.

Figure 5.

PRP31 accumulates in Cajal bodies following NUFIP depletion. (A) Microscopy images of HeLa cells treated with control (Ct) or NUFIP siRNAs. GFP-PRP31 is visible in green, anti-coilin antibody in red, and DAPI in blue. Scale bar is 10 μm. Insets show zoom of selected CBs. (B) Microscopy images of HeLa cells treated with control or NUFIP siRNAs. GFP-PRP31 is visible in green, FISH labeling of U85 in red, and DAPI in blue. Scale bar is 10 μm. (C) Graph showing the quantification of CB localization of GFP-PRP31, coilin, and U85. Histograms represent an average of signal intensities in CB divided by that of nucleoplasm. (D) Western blot of HeLa extracts treated with control and NUFIP siRNAs, and blotted with anti-NUFIP and anti-tubulin antibodies. Star: non-specific band of the NUFIP antibody. (E) Same as in (D) but anti-GFP and anti-GAPDH antibodies were used for western blot.

Figure 6.

GFP-PRP31 associates less efficiently with U4 snRNA in NUFIP depleted cells. (A) RNA gels showing the products of an RNase protection assay made with samples from anti-GFP immuno-precipitations of HeLa cells stably expressing GFP-PRP31, and treated with NUFIP or control (Ct) siRNAs. H9: control cells that do not express GFP-PRP31; IP anti-GFP: pellets after immune-precipitation with agarose GFP-trap beads. (B) Graph displaying the quantification of the association of GFP-PRP31 with U4 snRNA. Values are averages of triplicate experiments measuring the ratio of U4 RNA levels in pellets over inputs (± STD). Probability that the two siRNA samples are not different is less than 0.1 (two-sided Student t-test). (C) Depletion of NUFIP does not affect the efficiency of GFP-PRP31 immuno-precipitation as compared to control. Western blotting of inputs and pellets of anti-GFP immuno-precipitates of cells expressing GFP-PRP31 transfected with siControl or siNUFIP. Western blot was probed with anti-GFP and anti-GAPDH antibodies. Inputs: 5% of pellet.

Figure 7.

Association of PRP31, NUFIP and ZNHIT3 with the SMN complex. (A) Western blotting of inputs and pellets of anti-GFP immuno-precipitates of 293T cells transfected with the indicated GFP constructs. Left gel: no RNase treatment; right gel: RNase treated samples. Membranes are probed with the indicated antibodies. (B) Autoradiogram of a gel with input and pellets of immune-precipitates of in vitro translated [³⁵S]methionine-labeled Ran, SMN and NUFIP (Total; 10%), tested for binding to immobilized SMN complex (SMN complex; 100%). Specific binding was tested with anti-Flag beads pre-incubated with HeLa cell extracts expressing Flag-Gemin2. Non-specific binding was assessed with anti-Flag beads incubated with parental cell extracts that do not express Flag-Gemin2 (Control). (C) Micrographs of yeast strains grown in drops on yeast-two hybrid selective media. pACTII-NUFIP was used as prey and the components of the SMN complex used as bait (left panel). Controls were performed with empty pACT2 vector (right panel). Growth on medium lacking histidine indicates an interaction. Increasing amount of 3-Amino-1,2,4-triazol (3-AT), was added to the medium to evaluate the strength of the interaction. (D) Western blots of inputs (Total; 10%) and immuno-precipitates (100%) of E. coli total extracts expressing recombinant Gemin3, 4, 6 or 15.5K as control, and blotted with the indicated antibodies. Immuno-precipitation was performed in presence of RNase and with beads containing immobilized recombinant GST-NUFIP or GST as control.

Figure 8.

NUFIP associates with Gemin6 in intact HeLa cells. (A) Micrographs of HeLa cells fixed and labeled with the PLA (green), using antibodies against the indicated proteins. In controls, one of the primary antibodies was omitted panels on the second and third line. Scale bar, 20 μm. Blue signal demarcate nuclei visualized by TO-PRO-3 staining. (B) U2OS LacO cells were co-transfected with GFP-NUFIP and either mRFP–LacI–Gemin6 or mRFP–LacI–KpnA2 used as a negative control. When mRFP–LacI–Gemin6 accumulated at the LacO sites, GFP-NUFIP was also enriched there. In contrast, mRFP–LacI–KpnA2 failed to recruit GFP-NUFIP. Scale bar is 20 μm.

Figure 9.

Model for the assembly of U4-specific proteins. The Sm-core containing U4 pre-snRNP is assembled by the SMN complex in the cytoplasm. After its transport into CB, U4 pre-snRNP bind SMN complexes associated with NUFIP, ZNHIT3 and PRP31. Binding of 15.5K then allows stable assembly of PRP31 on U4, and subsequent formation of the di- and tri-snRNP (see the text).