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

• Type of Material: Article
• Authors: Milo Fasken, Emory University; Sara Leung, Emory University; Ayan Banerjee, Emory University; Maja O. Kodani, Emory University; Ramiro Chavez, Emory University; Elizabeth A. Bowman, Emory University; Meghan K. Purohit, Emory University; Max E. Rubinson, Emory University; Emily H. Rubinson, Emory University; Anita Corbett, Emory University
• Language: English
• Date: 2011-10-28
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://www.jbc.org/content/286/43/37429
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 286
• Issue: 43
• Start Page: 37429
• End Page: 37445
• Grant/Funding Information: This work was supported, in whole or in part, by a National Institutes of Health grant (to A. H. C.).
• Keywords: TRAMP Complex; Trf4; Protein-Protein Interactions; Zinc Knuckle Protein; RNA Processing; Yeast Genetics; RNA Turnover; RNA Quality Control; Air1; RNA-binding Proteins
• Research Categories: Chemistry, Biochemistry

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.