title: Air1 zinc knuckles 4 and 5 and a conserved IWRXY motif are critical for the function and integrity of the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) RNA quality control complex.
abstract: In Saccharomyces cerevisiae, non-coding RNAs, including cryptic unstable transcripts (CUTs), are subject to degradation by the exosome. The Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex in S. cerevisiae is a nuclear exosome cofactor that recruits the exosome to degrade RNAs. Trf4/5 are poly(A) polymerases, Mtr4 is an RNA helicase, and Air1/2 are putative RNA-binding proteins that contain five CCHC zinc knuckles (ZnKs). One central question is how the TRAMP complex, especially the Air1/2 protein, recognizes its RNA substrates. To characterize the function of the Air1/2 protein, we used random mutagenesis of the AIR1/2 gene to identify residues critical for Air protein function. We identified air1-C178R and air2-C167R alleles encoding air1/2 mutant proteins with a substitution in the second cysteine of ZnK5. Mutagenesis of the second cysteine in AIR1/2 ZnK1-5 reveals that Air1/2 ZnK4 and -5 are critical for Air protein function in vivo. In addition, we find that the level of CUT, NEL025c, in air1 ZnK1-5 mutants is stabilized, particularly in air1 ZnK4, suggesting a role for Air1 ZnK4 in the degradation of CUTs. We also find that Air1/2 ZnK4 and -5 are critical for Trf4 interaction and that the Air1-Trf4 interaction and Air1 level are critical for TRAMP complex integrity. We identify a conserved IWRXY motif in the Air1 ZnK4-5 linker that is important for Trf4 interaction. We also find that hZCCHC7, a putative human orthologue of Air1 that contains the IWRXY motif, localizes to the nucleolus in human cells and interacts with both mammalian Trf4 orthologues, PAPD5 and PAPD7 (PAP-associated domain containing 5 and 7), suggesting that hZCCHC7 is the Air component of a human TRAMP complex.
authors: Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA. mfasken@emory.edu
authors: Milo B
authors: MB
authors: Fasken
authors: Sara W
authors: SW
authors: Leung
authors: Ayan
authors: A
authors: Banerjee
authors: Maja O
authors: MO
authors: Kodani
authors: Ramiro
authors: R
authors: Chavez
authors: Elizabeth A
authors: EA
authors: Bowman
authors: Meghan K
authors: MK
authors: Purohit
authors: Max E
authors: ME
authors: Rubinson
authors: Emily H
authors: EH
authors: Rubinson
authors: Anita H
authors: AH
authors: Corbett
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D020125:Mutation, Missense; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability / Q000502:physiology*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C415824:Air1 protein, S cerevisiae; C415825:Air2 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D046912:Multiprotein Complexes; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C545342:ZCCHC7 protein, human; D012316:RNA Nucleotidyltransferases; C567954:TENT4B protein, human; D012321:DNA-Directed RNA Polymerases; C506067:Trf5 protein, S cerevisiae; D004259:DNA-Directed DNA Polymerase; C487545:PAP2 protein, S cerevisiae; C118146:TENT4A protein, human; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases