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.
Highlights
RNA quality control machinery to prevent deleterious effects on the cell
Trf4-DADA had a shorter halflife than Trf4 in air1 zinc knuckle 5 (ZnK5) mutant cells and wild type cells (Fig. 6C). These results indicate that the stability of the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex proteins are reduced in air1 ZnK5 mutant cells and suggest that the integrity of the TRAMP complex is compromised in cells with lower levels of Air1 protein
We identify a key evolutionarily conserved IWRXY motif in the Air1 ZnK4-5 linker region that is important for function and Trf4 interaction
Summary
Strains, and Chemicals—All DNA manipulations were performed according to standard methods [50], and all media were prepared by standard procedures [51]. C-terminally GFP-tagged AIR1/2 and air1/2 zinc knuckle mutants (pAC2825–2836, pAC2841–2844, and pAC2863–2866) and C-terminally Myc-tagged AIR1/2 and air1/2 zinc knuckle mutants (pAC2885–2896 and pAC2902– 2909) were constructed by PCR amplification of AIR1/2 genes using oligonucleotides and AIR1/2 or air1/2 templates (pAC1856, pAC2727–2734, pAC2012–2013, pAC2837–2840, pAC2845–2848, and pAC2884) and cloning into pRS415, followed by insertion of GFP or 2xMyc PCR products. To generate single mutants of air or air, air1⌬ air2⌬ cells (ACY1095), containing AIR2 URA3 maintenance plasmid (pAC1614), were transformed with AIR1 (pAC1856), air mutant (pAC2727–2730 and pAC2012), AIR2 (pAC1857), air mutant (pAC2731–2734 and pAC2013) or vector (pRS415) LEU2 plasmids and selected on LeuϪUraϪ minimal media with 2% glucose.
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