title: Assembly of the yeast exoribonuclease Rrp6 with its associated cofactor Rrp47 occurs in the nucleus and is critical for the controlled expression of Rrp47.
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.
authors.affiliation: Molecular Biology and Biotechnology Department, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Monika
authors.initials: M
authors.lastname: Feigenbutz
authors.forename: Rebecca
authors.initials: R
authors.lastname: Jones
authors.forename: Tabot M D
authors.initials: TM
authors.lastname: Besong
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Harding
authors.forename: Phil
authors.initials: P
authors.lastname: Mitchell
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D002467:Cell Nucleus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D059748:Proteolysis; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C494789:LRP1 protein, S cerevisiae; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D063326:Exosome Multienzyme Ribonuclease Complex; C112610:RRP6 protein, S cerevisiae; D046988:Proteasome Endopeptidase Complex
keywords: Analytical Ultracentrifugation; Microscopic Imaging; Nuclear Translocation; Protein Cross-linking; Protein Degradation; RNA-binding Protein; Ribonuclease; Yeast Genetics