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

J Biol Chem. 2011 Oct 28;286(43):37429-45. doi: 10.1074/jbc.M111.271494. Epub 2011 Aug 30.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism*
  • Amino Acid Motifs
  • Amino Acid Substitution
  • Cell Nucleolus / genetics
  • Cell Nucleolus / metabolism
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • DNA-Directed RNA Polymerases / genetics
  • DNA-Directed RNA Polymerases / metabolism*
  • Humans
  • Multiprotein Complexes / genetics
  • Multiprotein Complexes / metabolism*
  • Mutagenesis
  • Mutation, Missense
  • RNA Nucleotidyltransferases / genetics
  • RNA Nucleotidyltransferases / metabolism
  • RNA Stability / physiology*
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Adaptor Proteins, Signal Transducing
  • Air1 protein, S cerevisiae
  • Air2 protein, S cerevisiae
  • Chromosomal Proteins, Non-Histone
  • Multiprotein Complexes
  • RNA, Fungal
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • ZCCHC7 protein, human
  • RNA Nucleotidyltransferases
  • TRF4-2 protein, human
  • DNA-Directed RNA Polymerases
  • Trf5 protein, S cerevisiae
  • DNA-Directed DNA Polymerase
  • PAP2 protein, S cerevisiae
  • PAPD7 protein, human
  • MTR4 protein, S cerevisiae
  • DEAD-box RNA Helicases