title: Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation.
abstract: Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation.
authors.affiliation: CEITEC-Central European Institute of Technology, Masaryk University, Kamenice 5, CZ-62500 Brno, Czech Republic.
authors.forename: Peter
authors.initials: P
authors.lastname: Holub
authors.forename: Jana
authors.initials: J
authors.lastname: Lalakova
authors.forename: Hana
authors.initials: H
authors.lastname: Cerna
authors.forename: Josef
authors.initials: J
authors.lastname: Pasulka
authors.forename: Marie
authors.initials: M
authors.lastname: Sarazova
authors.forename: Kristyna
authors.initials: K
authors.lastname: Hrazdilova
authors.forename: Maria Sanudo
authors.initials: MS
authors.lastname: Arce
authors.forename: Fruzsina
authors.initials: F
authors.lastname: Hobor
authors.forename: Richard
authors.initials: R
authors.lastname: Stefl
authors.forename: Stepanka
authors.initials: S
authors.lastname: Vanacova
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C415825:Air2 protein, S cerevisiae; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae; D012260:Ribonucleases; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases