title: The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase.
abstract: The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.
authors.affiliation: Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuch
authors.affiliation: Molecular Biology and Biotechnology Department, The University of Sheffield, Sheffield, UK.
authors.forename: Monika
authors.initials: M
authors.lastname: Feigenbutz
authors.affiliation: Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Debora L
authors.initials: DL
authors.lastname: Makino
authors.affiliation: Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Falk
authors.affiliation: Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany.
authors.forename: Claire
authors.initials: C
authors.lastname: Basquin
authors.affiliation: Molecular Biology and Biotechnology Department, The University of Sheffield, Sheffield, UK p.j.mitchell@sheffield.ac.uk conti@biochem.mpg.de.
authors.forename: Phil
authors.initials: P
authors.lastname: Mitchell
authors.affiliation: Structural Cell Biology Department, Max Planck Institute of Biochemistry, Martinsried, Germany p.j.mitchell@sheffield.ac.uk conti@biochem.mpg.de.
authors.forename: Elena
authors.initials: E
authors.lastname: Conti
mesh_terms: D015153:Blotting, Western; D002151:Calorimetry; D002850:Chromatography, Gel; D003460:Crystallization; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D063326:Exosome Multienzyme Ribonuclease Complex / Q000737:chemistry / Q000378:metabolism*; D005454:Fluorescence Polarization; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012394:Rosaniline Dyes; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C494789:LRP1 protein, S cerevisiae; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D016601:RNA-Binding Proteins; D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins; C048139:Coomassie blue; D063326:Exosome Multienzyme Ribonuclease Complex; C112610:RRP6 protein, S cerevisiae; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: RNA degradation; X‐ray crystallography; nuclear exosome; yeast genetics