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. 2013 May 31;288(22):15959-70.
doi: 10.1074/jbc.M112.445759. Epub 2013 Apr 11.

Assembly of the Yeast Exoribonuclease Rrp6 With Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47

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Free PMC article

Assembly of the Yeast Exoribonuclease Rrp6 With Its Associated Cofactor Rrp47 Occurs in the Nucleus and Is Critical for the Controlled Expression of Rrp47

Monika Feigenbutz et al. J Biol Chem. .
Free PMC article

Abstract

Rrp6 is a key catalytic subunit of the nuclear RNA exosome that plays a pivotal role in the processing, degradation, and quality control of a wide range of cellular RNAs. Here we report our findings on the assembly of the complex involving Rrp6 and its associated protein Rrp47, which is required for many Rrp6-mediated RNA processes. Recombinant Rrp47 is expressed as a non-globular homodimer. Analysis of the purified recombinant Rrp6·Rrp47 complex revealed a heterodimer, suggesting that Rrp47 undergoes a structural reconfiguration upon interaction with Rrp6. Studies using GFP fusion proteins show that Rrp6 and Rrp47 are localized to the yeast cell nucleus independently of one another. Consistent with this data, Rrp6, but not Rrp47, is found associated with the nuclear import adaptor protein Srp1. We show that the interaction with Rrp6 is critical for Rrp47 stability in vivo; in the absence of Rrp6, newly synthesized Rrp47 is rapidly degraded in a proteasome-dependent manner. These data resolve independent nuclear import routes for Rrp6 and Rrp47, reveal a structural reorganization of Rrp47 upon its interaction with Rrp6, and demonstrate a proteasome-dependent mechanism that efficiently suppresses the expression of Rrp47 in the absence of Rrp6.

Keywords: Analytical Ultracentrifugation; Microscopic Imaging; Nuclear Translocation; Protein Cross-linking; Protein Degradation; RNA-binding Protein; Ribonuclease; Yeast Genetics.

Figures

FIGURE 1.
FIGURE 1.
Recombinant Rrp47 is expressed as a homodimer. A, sedimentation velocity molecular weight distribution analysis of Rrp47. Rrp47 at 0.25 mg/ml (∼10 μm) was centrifuged at 38,000 rpm and at 20.0 °C. The amount of Rrp47 found in the main peak is indicated, along with the corresponding molecular mass of the protein in daltons. B, glutaraldehyde cross-linking analysis of full-length Rrp47 and the Rrp47NT domain. Reaction products were resolved by SDS-PAGE and analyzed by Western blotting. The electrophoretic migration of molecular mass markers (sizes in kDa) are given. Cross-linked products (XL) are indicated.
FIGURE 2.
FIGURE 2.
Rrp47 forms a stoichiometric heterodimer with the Rrp6NT domain. A, Purification of the recombinant Rrp47·Rrp6NT complex. Aliquots of the cell extract (CXT; lane 1), nickel-nitrilotriacetic acid (Ni-NTA) flow-through (FT; lane 2), and eluate (E; lane 3) fractions, the eluate upon GST-Sepharose affinity chromatography (GST eluate; lane 4), and the peak fraction after gel filtration (GF peak; lane 5) were resolved by SDS-PAGE, and proteins were visualized by staining with colloidal Coomassie Blue. A densitometric scan of the peak fraction in lane 5 is shown on the right. B, Western analyses of the purified Rrp47·Rrp6NT complex. Images are shown after probing a blot of the GF peak fraction (A, lane 5) for His-Rrp47 and after reprobing for GST-Rrp6NT. C, glutaraldehyde cross-linking analysis of the Rrp47·Rrp6NT complex. Reactants were resolved by SDS-PAGE and analyzed by Western blotting using the penta-His antibody. Lane 1, non-treated sample. Lane 2, prequenched sample. Lanes 3–5, samples cross-linked for the times indicated. The positions of molecular mass markers (sizes in kDa) are shown for each panel.
FIGURE 3.
FIGURE 3.
Rrp47-GFP depletion in rrp6 mutants correlates with accumulation of a degradation intermediate. A, Western analysis of Rrp47-GFP in rrp6 mutants. Extracts from a wild-type RRP6 strain (lane 1) and an isogenic rrp47-GFP rrp6Δ strain transformed with either the control vector (lane 2), a plasmid encoding non-tagged Rrp6 (lane 3), or plasmids encoding zz epitope-tagged Rrp6 fusions (lanes 4–7) were analyzed. The D238N mutant is a full-length, catalytically inactive protein. The ΔNT fusion lacks the first 211 residues of Rrp6. The 197X mutant encodes the first 196 residues of Rrp6. The Western blot was initially probed for Rrp47-GFP (top) and subsequently reprobed with anti-Pgk1 antibody (bottom). B, Western analysis of the relative expression levels of zz-Rrp6 proteins. Extracts were analyzed from an rrp47-GFP rrp6Δ strain transformed with the control vector (lane 1) and zz-Rrp6 fusions (lanes 2–6), as indicated. Blots were first probed with the Pgk1 antibody (bottom) and then with the PAP antibody (top) to detect the zz fusion proteins.
FIGURE 4.
FIGURE 4.
Rrp47 protein is unstable in the absence of Rrp6. A, Rrp47 stability assays in an isogenic wild-type, RRP6 strain and an rrp6Δ mutant bearing a GAL-regulated Rrp47-zz fusion protein. The strains were pregrown in raffinose-based medium, and Rrp47 expression was then induced to normal levels by the addition of galactose. New protein synthesis was subsequently blocked by the addition of cycloheximide and the reduction of Rrp47 levels followed by Western analyses. The lower band, indicated with an asterisk, is a proteolytic Rrp47-zz degradation product. The Western blots were also probed for Pgk1. B, relative expression levels of RRP47 mRNA and protein observed in a wild-type RRP6 strain and an isogenic rrp6Δ mutant strain. RRP47 mRNA levels were determined by quantitative RT-PCR and standardized to SCR1 levels. Western analyses were performed on alkaline-lysed cells and standardized to Pgk1 levels. Error bars, positive range of the S.E.
FIGURE 5.
FIGURE 5.
Rrp47 levels in rrp6Δ mutants increase upon treatment with MG132. Western analyses of Rrp47 in isogenic RRP6 and rrp6Δ strains upon treatment with either MG132, PMSF, or vehicle solvent. Cells were harvested before treatment and 30, 60, and 90 min thereafter. Cell extracts were resolved by SDS-PAGE and analyzed by Western blotting, using the PAP antibody to detect the Rrp47-zz fusion protein and using an anti-Pgk1 antibody as a loading control. A, wild-type RRP6 strain treated with MG132. B, rrp6Δ mutant treated with MG132. C, RRP6 strain treated with PMSF. D, rrp6Δ mutant treated with PMSF. The sample to the left in D labeled RRP6 CXT is a native cell extract from an RRP6 strain. The sample to the right in D labeled RRP6 AL is an alkaline lysate from an RRP6 strain.
FIGURE 6.
FIGURE 6.
Nuclear localization of Rrp47 and Rrp6 is independent. Shown is localization of Rrp47 and Rrp6 GFP fusion proteins in strains either expressing the other protein or bearing mutant alleles of the corresponding gene. GFP, DAPI, and merged images are shown for representative cells of strains expressing no GFP fusion (a), Rrp47-GFP (b), Rrp47-GFP and lacking Rrp6 (c), Rrp47-GFP and full-length Rrp6 (d), Rrp47-GFP and the Rrp6NT domain (e), Rrp6-GFP (f), and Rrp6-GFP and lacking Rrp47 (g).
FIGURE 7.
FIGURE 7.
Rrp47 is not associated with the Rrp6·Srp1 complex. Western blot analysis of pull-downs on extracts from yeast strains expressing epitope-tagged zz fusions of Rrp6 or Rrp47, using an Srp1-specific antibody. A non-tagged, wild-type strain was used as a negative control. Cell extracts (CXT) and the flow-through (FT) and eluate (EL) fractions obtained upon fractionation using IgG-Sepharose beads were resolved through 12% SDS-polyacrylamide gels and analyzed by Western blotting. The top panel shows a Western blot using an Srp1-specific antibody. The bottom panel shows a Western blot using the PAP antibody. Rrp6 and Rrp47 proteolytic fragments are denoted with asterisks.
FIGURE 8.
FIGURE 8.
Model for assembly of the Rrp6·Rrp47 complex. The Rrp47 homodimer is imported independently of Rrp6, the latter protein being translocated across the nuclear membrane by the importin heterodimer Srp1·Kap95. The Rrp6·Rrp47 heterodimer is assembled in the nucleus, generating the RNA-processing/degradation-competent ribonuclease complex. In the absence of Rrp6, the Rrp47 homodimer is degraded by the nuclear proteasome.

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