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*

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.

RNA quality control machinery to prevent deleterious effects on the cell. Processing and degradation of multiple classes of RNA are performed by RNA endo/exoribonucleases that are recruited to their RNA substrates by specific protein cofactors. These nucleases and their cofactors are highly regulated and evolutionarily conserved.
Air1 and Air2 (45% identical) are predicted to bind RNA because recombinant Air1/2 is essential for Trf4-mediated polyadenylation of hypomodified tRNA i Met in vitro, and air1⌬ air2⌬ cells exhibit loss of polyadenylation of ncRNAs in vivo (14,21,22). Air1/2 proteins contain five adjacent CX 2 CX 4 HX 4 C zinc knuckles (ZnK1-5; Fig. 1A) that could interact with nucleic acids or bind protein (22,45). In retroviral nucleocapsid proteins, one or two CX 2 CX 4 HX 4 C zinc knuckles form metalcoordinated reverse turns that typically contact viral singlestranded tetraloop RNA via the variable (C)X 2 and (H)X 4 residues (14,22,47,48). A recent crystal structure of Trf4 in complex with an Air2 fragment shows that Air2 zinc knuckles 4 and 5 interact with the Trf4 central domain (49). In addition, recombinant Trf4 in complex with Air2 ZnK1-5 preferentially polyadenylates aberrant tRNA in vitro, indicating that Air2 zinc knuckles can also recognize RNA (49). A key question is therefore which zinc knuckles of the Air1/2 proteins contribute to RNA recognition and Air protein function in vivo. At present, studies of Air zinc knuckle function have only been carried out in vitro using recombinant protein, and only mutants of ZnK1, ZnK2, and ZnK3 have been generated in the context of Air2 ZnK1-5 (49). Trf4 complexed with an Air2 ZnK1 mutant, but not a ZnK2 or ZnK3 mutant, exhibits impaired polyadenylation of mutant tRNA, suggesting that Air2 ZnK1 is important for RNA recognition (49). An Air2 ZnK4-5 fragment also supports weak Trf4-mediated polyadenylation of aberrant tRNA, indicating that ZnK4 and ZnK5 also have the capacity to recognize RNA (49). However, these conclusions are all based on biochemical experiments, and the contributions of the individual zinc knuckles to the in vivo function of the Air proteins have not been addressed.
Here, we present the first in vivo functional analysis of fulllength Air1/2 zinc knuckle 1-5 mutants. We find that Air1/2 ZnK4 and ZnK5 are functionally important. In particular, Air1/2 ZnK4 and ZnK5 mutants exhibit temperature-sensitive growth and reduced binding to Trf4. In addition, air1 ZnK4 mutant cells exhibit the highest levels of NEL025c CUT RNA relative to AIR1 cells and the other air1 ZnK mutants, suggesting that Air1 ZnK4 may help facilitate RNA recognition. We also find that Air1/2 and Trf4, but not Trf5 or Mtr4, suppress the temperature-sensitive growth of the air1 ZnK5 mutant. In addition, Air1 interaction with Trf4 and Air1 level are critical for the stability of TRAMP complex components and the integrity of the TRAMP complex. Importantly, our studies also identify a key evolutionarily conserved IWRXY motif in the Air1 ZnK4-5 linker region that is important for Air1 function and interaction with Trf4. A putative human orthologue of the Air proteins, hZCCHC7, which contains the IWRXY motif, local-izes to the nucleolus and binds to both mammalian Trf4-like proteins, PAPD5 and PAPD7, providing evidence that hZC-CHC7 is the Air protein component of a human TRAMP complex. Together, the data suggest that Air1/2 interacts with Trf4 predominantly via the ZnK4-5 linker and ZnK5 and facilitates recognition of RNA via ZnK1-4.
For growth curve analysis, air1⌬ air2⌬ cells harboring air1/2 mutants as the sole copy of Air1/2 were grown overnight at 25°C to saturation in Leu Ϫ media with 2% glucose. Cell concentrations were normalized by A 600 , diluted 100-fold in 100 l of Leu Ϫ media with 2% glucose, and added to wells of a MicroWell F96 microtiter plate (Nunc). Samples were loaded in quintuplicate. Cells in plate wells were grown at 37°C with shaking, and absorbance at A 600 was measured every 30 min for 40 h in an ELX808 Ultra microplate reader with KCjunior software (Bio-Tek Instruments, Inc.). Quintuplicate sample absor- Random Mutagenesis of AIR1/2-To generate random mutants of AIR1/2, the AIR1 and AIR2 genes were subjected to random PCR mutagenesis using the AIR1 and AIR2 template plasmids (pAC1856 and pAC1857), oligonucleotides, 0.4 mM dGTP (0.2 mM dCTP, dATP, and dTTP), 2 mM MgCl 2 , and Taq polymerase (Qiagen). The air mutant PCR products were introduced into linearized AIR1 and AIR2 LEU2 plasmid (pAC1856 and pAC1857) via homologous recombination by transformation into air1⌬ air2⌬ cells (ACY1095) containing an AIR2 URA3 maintenance plasmid (pAC1614) and selection on Ura-Ϫ Leu Ϫ minimal medium plates with 2% glucose at 30°C. Temperature-sensitive air1 and air2 mutants were then identified by replica plating to Leu Ϫ minimal media containing 5-FOA to select for cells that have lost the AIR2 URA3 maintenance plasmid. Colonies that grew at 25°C but not at 37°C were selected. Plasmids containing the air1 and air2 mutants were recovered and fully sequenced to identify the air1 and air2 mutations.
Localization of GFP-tagged Proteins-To localize Air1/2-GFP proteins, air1⌬ air2⌬ cells (ACY1095) harboring an AIR2 URA3 maintenance plasmid (pAC1614) and expressing GFPtagged Air protein (pAC2825-2836, pAC2841-2844, and pAC2863-2866) were grown in Ura Ϫ Leu Ϫ media with 2% glucose overnight at 25°C, transferred to Ura Ϫ Leu Ϫ media with 2% glucose, and grown to log phase at 25°C. GFP fusion proteins were visualized by direct fluorescence microscopy using an Olympus BX60 direct fluorescence microscope equipped with a photometric Quantix digital camera from Roper Scientific (Tucson, AZ) and filters from Chroma Technology (Brattleboro, VT). All images were captured using IP Lab Spectrum software.
Total RNA Isolation-To prepare S. cerevisiae total RNA from cell pellets of 50-ml cultures grown to A 600 ϭ 0.5, 2-3 scoops of glass beads were added to each cell pellet in a 2-ml screw cap tube, 1 ml of TRIzol (Invitrogen) was added, and the cell sample was vigorously disrupted in a Mini Bead Beater 16 cell disrupter (Biospec) for 2 min at 25°C. For each sample, 100 l of 1-bromo-3-chloropropane was added, and the sample was vortexed for 15 s and incubated at 25°C for 2 min. Each sample was then centrifuged at 16,300 ϫ g for 8 min at 4°C, and the upper layer was transferred to a fresh Eppendorf tube. RNA was precipitated with 500 l of isopropyl alcohol, and the sample was vortexed for 10 s to mix. Total RNA was pelleted by centrifugation at 16,300 ϫ g for 8 min at 4°C. Supernatant was decanted, and 1 ml of 75% ethanol was added to wash the RNA pellet. Sample was centrifuged at 16,300 ϫ g for 5 min at 4°C. Supernatant was decanted, remaining ethanol was removed, and the RNA pellet was air-dried for 15 min. Total RNA was resuspended in 50 l of diethylpyrocarbonate (Sigma)-treated water and stored at Ϫ80°C.
Quantitative RT-PCR to Detect CUT RNAs-To measure the levels of CUTs, total RNA was isolated from cells and reverse transcribed to cDNA, and cDNA of CUTs was detected by realtime quantitative PCR. Wild type (BY4741), air2⌬ (ACY1091), trf4⌬ (ACY2149), rrp6⌬ (ACY1641), and air1⌬ air2⌬ cells (ACY1095) harboring AIR1 (pAC1856) or air1 mutant (pAC2727-2730 and pAC2012) LEU2 plasmids as the sole copy of air1 were inoculated into 2 ml of YEPD (yeast extract, peptone, dextrose) media and grown overnight at 25°C. An aliquot of 100 l of each overnight culture was used to inoculate 50 ml of YEPD media and grown overnight at 25°C. Cultures were grown to an A 600 ϭ 0.5. Cells were centrifuged at 2,163 ϫ g, transferred to fresh tubes, centrifuged at 16,000 ϫ g, and stored at Ϫ80°C. Following total RNA isolation from each cell pellet, 1 g of RNA was reverse transcribed to first strand cDNA using the QuantiTect reverse transcription kit (Qiagen) according to the manufacturer's protocol. Relative CUT cDNA levels were detected by quantitative PCR on triplicate samples of 50 ng of cDNA using either 2.5 M NEL025c CUT primers or control PGK1 primers and QuantiTect SYBR Green Master Mix (Qiagen) on an iCycler iQ TM real-time PCR detection system (Bio-Rad). Results were analyzed using the iCycler optical system version 3.0a software (Bio-Rad), and data were normalized by the ⌬⌬Ct method to a control PGK1 transcript (53).
Analysis of Protein Expression Levels-For analysis of Myctagged air1/2 mutant protein levels, air1⌬ air2⌬ cells (ACY1095) harboring an AIR2 URA3 maintenance plasmid (pAC1614) and expressing Myc-tagged air1/2 protein (pAC2885-2896) were grown in minimal media overnight at 25°C, the optical density of the cells was measured at 600 nm, and 10-ml cultures with an A 600 ϭ 0.5 were prepared and grown for 4 h at 25°C. The cultures were then split, with one set of 5-ml cultures maintained at 25°C and the other set shifted to 37°C for an additional 2 h. For analysis of Myc-tagged Trf4, trf4 mutant, Trf5, and Mtr4 protein levels, wild type cells (W303) or air1-C178R air2⌬ cells (ACY2020) overexpressing Myc-tagged Trf4 (pAC2910), trf4-DADA (pAC2914), trf4 -378 (pAC3049), Trf5 (pAC3050), or Mtr4 (pAC3051) protein were grown in minimal media overnight at 25°C, and 10-ml cultures with an A 600 ϭ 0.5 were prepared and grown for 4 h at 25°C and then split, with one set of cultures maintained at 25°C and the other set shifted to 30°C for an additional 2 h. For analysis of Myctagged air1-C178R protein levels upon overexpression of Trf4 protein, air1⌬ air2⌬ cells expressing Myc-tagged air1-C178R (pAC2890) and containing vector (pRS423) or overexpressing Trf4 (pAC2940) or trf4-DADA (pAC2946) were grown in minimal media overnight at 25°C, and then 10-ml cultures with an A 600 ϭ 0.5 were prepared and grown for 6 h at 25°C. Whole cell lysates of cells were then prepared, and 40 -50 g of protein was analyzed by immunoblotting with an anti-Myc monoclonal antibody to detect Myc-tagged proteins.
TRAMP Protein Stability Assay-To determine the stability of TRAMP proteins, wild type (W303) cells and air1-C178R air2⌬ (ACY2020) cells overexpressing Trf4 (pAC2910), trf4-DADA (pAC2914), Trf5 (pAC3050), or Mtr4 (pAC3051) were grown in 10 ml of minimal media overnight at 25°C, the optical density of the cells was measured at 600 nm, and 60-ml cultures with an A 600 ϭ 0.5 were prepared and grown for 3 h at 25°C. Cycloheximide (Sigma) was added to the cultures at a final concentration of 100 g/ml. Samples (10 ml) were taken immediately after the cycloheximide addition at the zero time point. Cultures were then grown at 25°C, and 10-ml samples were collected at 30, 60, 90, 120, and 150 min time points after the cycloheximide addition. Protein lysate from each cell sample time point was prepared by resuspension of the cells in 0.5 ml of Lysis Buffer (PBS, 0.1% Nonidet P-40, protease inhibitors (1 mM PMSF, 3 ng/ml pepstatin A, leupeptin, aproptinin, and chymostatin)), the addition of three scoops of glass beads, disruption in a Mini Bead Beater 16 cell disrupter (Biospec) for 4 ϫ 1 min at 25°C, and centrifugation to remove cell debris at 16,000 ϫ g for 20 min at 4°C. Protein lysate concentration was determined by a Bradford assay using the Bio-Rad protein assay. Protein lysate (30 g) from each time point was analyzed by SDS-PAGE and immunoblotting with an anti-Myc monoclonal antibody to detect Myc-tagged TRAMP proteins and an anti-Pgk1 monoclonal antibody to detect 3-phosphoglycerate kinase as a loading control.

Air1 ZnK4 and ZnK5 Required for TRAMP Complex
Quantitation of Immunoblots-The protein band intensities/ areas from all immunoblots were quantitated using ImageJ version 1.4 software (National Institute of Health, Bethesda, MD), and relevant percentages of protein were calculated in Microsoft Excel for Mac 2008 (Microsoft Corp.). In binding assays, all bound air1/2-Myc intensities were normalized to Trf4-TAP intensities, bound trf4-Myc intensities were normalized to Air1-TAP intensities, and input air1/2/trf4-Myc intensities were normalized to Pgk1 intensities. In TRAMP protein level and stability assays, all Myc-tagged Trf4, trf4-DADA, Trf5, and Mtr4 intensities were normalized to Pgk1. In the Trf4-TAP and Air1-TAP binding assays, to quantitate the percentage of bound air1/2-Myc or trf4-Myc proteins relative to the amount of input protein and wild type Air1/2 or Trf4, the normalized bound air1/2/trf4 intensities were divided by the normalized input air1/2/trf4 intensities, and these values were divided by the wild type Air1/2/Trf4 intensity. For the percentage of bound and input air1 ZnK1-5 mutants, the S.E. value (S.D. divided by the square root of n, where n ϭ 3) from triplicate binding experiments was calculated. To quantitate the percentage of input air1/2/trf4 proteins relevant to wild type Air1/2/ Trf4, the normalized input air1/2/trf4 intensities were divided by the input wild type Air1/2/Trf4 intensities. To quantitate the percentage decrease in Myc-tagged TRAMP proteins in wild type and air1-C178R air2⌬ cells at 30°C compared with 25°C, the normalized TRAMP protein intensities at 30°C were divided by normalized intensities at 25°C. To quantitate the -fold increase in air1-C178R in cells overexpressing Trf4 or trf4-DADA relevant to cells containing vector alone, the air1-C178R intensities in vector alone and Trf4/trf4 samples were normalized to Pgk1 intensities, and the normalized air1-C178R intensities in Trf4/trf4 samples were divided by that in the vector alone sample. In the TRAMP protein stability assay, to quantitate the percentages of Myc-tagged TRAMP proteins in wild type and air1-C178R air2⌬ cells at each time point relevant to time 0, the normalized TRAMP protein intensities at each time point were divided by the normalized TRAMP protein intensity at time 0. These percentages were graphed using Microsoft Excel. To generate the graphs with least-squares fit lines through the log-transformed TRAMP protein intensities at time points 0, 60, and 90 min, the natural logarithms (ln) of normalized TRAMP protein intensities at each time point were graphed, and the best fit lines were calculated using Microsoft Excel. All best fit lines had good correlation coefficients (R 2 ϭ 0.98 -0.99). The slopes of the best fit lines were calculated to determine the decay rate constants (k) for the TRAMP proteins, and the half-lives (T1 ⁄ 2 ) of the TRAMP proteins were calculated using the equation, T1 ⁄ 2 ϭ ln(2)/k as previously used to determine protein half-lives (54).
Localization of hZCCHC7-Endogenous hZCCHC7 was visualized in HeLa cells by indirect immunofluorescence microscopy using an anti-hZCCHC7 polyclonal rabbit antibody (HPA021088, Sigma) according to the protocol from the Human Protein Atlas (available on the World Wide Web) (55). Cells were fixed with 4% formaldehyde (EM Science) in growth medium supplemented with 10% fetal bovine serum (FBS) for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 3 ϫ 5 min, washed with PBS, and incubated at 4°C overnight with primary rabbit anti-hZCCHC7 antibody (1:1000; HPA021088, Sigma) and mouse anti-fibrillarin antibody (1:1000; EnCore) and in PBS supplemented with 4% FBS. Cells were then washed with PBS for 4 ϫ 5 min and incubated with secondary antirabbit fluorescein-conjugated antibody or anti-mouse Texas Red-conjugated antibody (1:1000; Jackson ImmunoResearch Laboratories). Cells were incubated with Hoechst stain to mark nuclear DNA. Images were obtained using an Olympus IX81 microscope with a 0.3 numerical aperture, ϫ60 Zeiss Plan-Neofluor objective. Images were captured using a Hamamatsu digital camera with Slidebook software (version 5) and globally processed for brightness using Adobe Photoshop.
Immunoprecipitation of mPAPD5/hPAPD7 and hZCCHC7-To immunoprecipitate mPAPD5/hPAPD7, HEK293 cells were transiently transfected with Myc-tagged mPAPD5 (pAC2998) or Myc-tagged hPAPD7 (pAC2999) plasmid using Lipofectamine 2000 (Invitrogen), and cells were collected 48 h after transfection. Control untransfected HEK293 cells or cells containing Myc-tagged mPAPD5 or hPAPD7 were lysed in binding buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 0.3% Nonidet P-40, 32 mM NaF, protease inhibitors (1 mM PMSF, 3 ng/ml pepstatin A, leupeptin, aprotinin, and chymostatin)). Cell lysates (0.5 mg) in 0.5 ml of binding buffer were incubated with 40 l of agarose-conjugated monoclonal mouse c-Myc (9E10) antibody beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 2 h at 4°C with mixing. Beads were pelleted by centrifugation, unbound supernatant was removed, and beads were washed three times with 0.5 ml of binding buffer for 5 s each. Input, unbound (25 g), and bound Myc bead (one-quarter the total amount) samples were analyzed by SDS-PAGE and immunoblotting with anti-hZCCHC7 polyclonal antibody (Sigma) to detect hZCCHC7 protein and anti-Myc monoclonal antibody (Cell Signaling) to detect mPAPD5 and hPAPD7. To immunoprecipitate hZCCHC7, HEK293 cells were transiently transfected with Myc-tagged mPAPD5 or hPAPD7 and lysed in binding buffer as before. HEK293 cell lysates (0.5 mg) containing Myc-tagged mPAPD5 or hPAPD7 in 0.5 ml of lysis buffer were incubated with 5 l of anti-hZCCHC7 polyclonal antibody (Sigma) or a control rabbit IgG antibody (GE Healthcare) alone overnight at 4°C with mixing. Lysates were further incubated with 20 l of Dynabeads coupled to Protein G (Invitrogen) for 2 h at 4°C with mixing. Beads were then washed three times as before, and input, unbound, and bound hZCCHC7bead or IgG bead samples were analyzed by SDS-PAGE and immunoblotting with anti-Myc monoclonal antibody (Cell Signaling) to detect mPAPD5 and hPAPD7.

Air1 ZnK4 and ZnK5 Required for TRAMP Complex
an analogous amino acid substitution in zinc knuckle 5 (air1-C178R), generated by site-directed mutagenesis, also exhibited profoundly slow growth at 37°C (Fig. 1D). This result suggests that zinc knuckle 5 of the Air proteins is functionally important.
Given that random mutagenesis of AIR1/2 identified a mutation in zinc knuckle 5 (ZnK5) that confers temperature-sensitive growth, we systematically altered the corresponding second cysteine in each of the other four zinc knuckles (ZnK1-4) of Air1/2 to arginine (Fig. 1A) and assessed the impact on protein function by examining the growth of air1⌬ air2⌬ cells expressing each air mutant as the sole cellular copy of the Air protein. Cell growth for each air mutant was monitored at 16, 25, 30, and 37°C. The air1 and air2 ZnK1-3 mutants showed growth comparable with control AIR1/2 cells at all temperatures tested ( Fig. 1D; summarized in Table 2). In contrast, air1 ZnK4 mutant (air1-C139R) and air2 ZnK4 mutant (air2-C126R) are temperature-sensitive and exhibit severely impaired growth at 37°C compared with AIR1/2 wild type cells ( Fig. 1D; summarized in Table 2). We confirmed these results by measuring the optical density of the cells in liquid culture over time and generating quantitative growth curves for the cells (Fig. 1E). The growth analysis highlights the fact that growth of the air1/2 ZnK4 mutants is not as severely impaired as that of the air1/2 ZnK5 mutants at 37°C. In addition, the curves indicate that air1 ZnK4/5 mutants are more growthimpaired/thermosensitive than the air2 ZnK4/5 mutants at 37°C. Growth analysis of air1/2 zinc knuckle 1-5 mutants therefore suggests that both zinc knuckle 4 and 5 of the Air proteins are functionally important.
To further characterize the Air1/2 zinc knuckle mutants, we examined the steady-state protein levels of C-terminally Myctagged wild type Air1/2 and air1/2 ZnK1-5 mutant proteins. air1⌬ air2⌬ cells containing AIR2 URA3 maintenance plasmid and expressing Myc-tagged wild type Air1/2 protein or air1/2 ZnK1-5 mutant protein were grown at 25 and 37°C, and whole cell lysates prepared from these cells were analyzed by SDS-PAGE and immunoblotting with anti-Myc antibody. At 25°C, air1/2 ZnK1-4 mutants were expressed at a level similar to that of wild type Air1/2, whereas the air1/2 ZnK5 mutant showed decreased expression compared with wild type Air1/2 (Fig. 1F). At 37°C, the expression of air1/2 ZnK1-3 mutants was similar to that of wild type Air1/2; however, the expression of air1/2 ZnK4 and -5 mutants was profoundly decreased relative to wild type Air1/2 (Fig. 1F). These data indicate that the air1/2 ZnK4 and -5 mutant proteins are unstable at 37°C and suggest that the thermosensitive growth of the air1/2 ZnK4 and -5 mutants could result from a decrease in the steady-state level of the mutant proteins at 37°C. However, unlike the air1/2 ZnK5 mutants, the steady-state level of the air1/2 ZnK4 mutant proteins is not greatly reduced at 25°C.
To examine the cellular localization of the air1/2 zinc knuckle mutants, we localized C-terminally GFP-tagged Air1/2 and air1/2 ZnK1-5 mutants in live cells by direct fluorescence microscopy. Like wild type Air1, air1 ZnK1-5 mutants localized predominantly to the nucleolus and to a lesser extent to the nucleoplasm, whereas, similarly to wild type Air2, air2 ZnK1-5 mutants localized mostly to the nucleoplasm with a degree of nucleolar localization (supplemental Fig. S1). These results indicate that the air1/2 zinc knuckle mutant proteins were correctly targeted to the nucleus.

Residues in the Air1 Zinc Knuckle 4-5 Linker and Zinc Knuckle 5 Are Critical for Air1
Function-Our results with air1/2 ZnK1-5 mutants indicate that ZnK4 and ZnK5 play a role in the structural integrity of the Air proteins, which complicates analysis but does not exclude the possibility that ZnK4 and ZnK5 also play an important functional role. To gain further insight into the specific functional requirements of the ZnK4-5 region, we targeted residues within the linker region between Air1 ZnK4 and ZnK5 that would be less likely to impact the structural integrity of the protein. To identify conserved residues in the Air1 ZnK domain, we performed a BLAST search of the NCBI RefSeq protein data base with the Air1 ZnK1-5 amino acid sequence to identify putative Air1 orthologues and aligned the ZnK domains of these Air1 proteins (Fig. 1B). The alignment of ZnK domains from S. cerevisiae Air1 and Air2, S. pombe Air1, and human hZCCHC7 and  hZCCHC9 highlights conserved residues within and flanking Air1 ZnK5 (Fig. 1B). In addition, the alignment reveals a conserved IWRXY motif adjacent to Air1 ZnK4 within the Air1 ZnK4-5 linker region (Fig. 1B). Notably, one putative human Air1 orthologue, hZCCHC7, a 542-residue protein with four intact CCHC zinc knuckles (35% identity to Air1 ZnK1-5), contains the IWRXY motif, but the other potential human Air1 orthologue, hZCCHC9, a 271-residue protein with four CCHC zinc knuckles (19% identity to Air1 ZnK1-5), lacks this IWRXY motif (Fig. 1B). Given the importance of Air1 ZnK4 and 5, we wished to determine if the IWRXY motif in the Air1 ZnK4-5 linker region and the evolutionarily conserved residues in Air1 ZnK5 are functionally important. To this end, we mutated the IWRXY motif and ZnK5 residues to alanine in Air1 and tested the function of these air1 mutants by examining the growth of air1⌬ air2⌬ cells expressing each air mutant at different temperatures as described under "Experimental Procedures." The air1 IWRXY mutants, air1-W153A and air1-Y156A, grew slowly at 30°C and exhibited severely impaired growth at 37°C compared with AIR1 cells (Fig. 2; summarized in Table 2). The slow growth of air1-W153A and air-Y156A cells at 30 and 37°C was similar to that of air1-C178R cells, but, unlike air1-C178R cells, air1-W153A and air-Y156A cells grow normally at 25°C. The air1 IWRXY mutants air1-I152A and air1-R154A grew similarly to AIR1 cells at 30°C but showed slower growth at 37°C relative to AIR1 cells. These results confirm that the IWRXY motif residues in the Air1 ZnK4-5 linker, particularly Trp-153 and Tyr-156, are functionally important. The air1 ZnK5 mutant air1-F174A and air1-R192A cells grew similarly to AIR1 cells at all temperatures, suggesting that Air1 residues Phe-174 and Arg-192 that flank the Air1 ZnK5 are not critical for function and importantly do not impact protein stability ( Fig. 2; summarized in Table 2). In contrast, air1 ZnK5 mutants air1-Y176A, air1-F184A and air1-D186A grew similarly to AIR1 cells at 30°C but exhibited profoundly temperature-sensitive growth at 37°C (Fig. 2; summarized in Table 2). The slow growth of these air1 ZnK5 mutants at 37°C was similar to that of air1 ZnK4 mutant air1-C139R cells. This result suggests that Air1 residues Tyr-176, Phe-184, and Asp-186 within Air1 ZnK5 are also functionally important. Critically, these air1 IWRXY and ZnK5 mutants predominantly localize, like wild type Air1, to the nucleolus and to a lesser extent to the nucleoplasm (supplemental Fig. S1).
Air1 Zinc Knuckle Mutants Exhibit Elevated CUT RNA Levels-Because retroviral CCHC type zinc knuckles bind to single-stranded tetraloop RNA and Air1/2 protein is required for Trf4-mediated polyadenylation of tRNA and RNA oligomers in vitro (14,21,22,47,48), Air1/2 CCHC zinc knuckles are hypothesized to facilitate recognition of RNA. In support of a key role for Air proteins in mediating TRAMP function, CUTs, normally polyadenylated by TRAMP and degraded by the exosome, are stabilized in air1⌬ air2⌬ cells (14). To determine whether CUTs are stabilized in air1 zinc knuckle 1-5 mutants, we analyzed CUT RNA levels by quantitative RT-PCR. Total RNA was isolated from wild type cells, control TRAMP/exosome mutant cells (air2⌬ and trf4⌬/rrp6⌬), and air1⌬ air2⌬ cells containing only AIR1 or air1 ZnK1-5 mutant plasmids grown at 25°C and reverse transcribed, and cDNA was measured by quantitative PCR using gene-specific primers for the CUT NEL025c. Relative to wild type cells, the air2⌬ and trf4⌬ TRAMP mutants exhibited a 3-fold increase in NEL025c RNA levels, whereas the rrp6⌬ exosome mutant showed a 22-fold increase in NEL025c RNA levels (Fig. 3). Like air2⌬ cells, air1⌬ air2⌬ cells expressing AIR1 exhibited a 4-fold increase in NEL025c RNA levels relative to wild type cells. In contrast, air1⌬ air2⌬ cells expressing air1 ZnK1-3 and air1 ZnK5 mutants showed a 13-20-fold increase in NEL025c RNA levels, whereas the air1⌬ air2⌬ cells expressing air1 ZnK4 mutant cells showed a 28-fold increase in NEL025c RNA levels relative to wild type cells ( Fig. 3; summarized in Table 2). Because air1 ZnK1-4 mutant proteins were expressed at levels similar to those of wild type Air1 protein at 25°C (Fig. 1E), these results suggest that Air1 zinc knuckles 1-4 are important for the recognition/degradation of CUT RNA. In particular, Air1 zinc knuckle 4, which when mutated causes the greatest increase in   air1-C79R, -C97R, -C117R, -C139R, and -C178R) show elevated levels of NEL025c CUT RNA relative to wild type cells. Total RNA from wild type, air2⌬, trf4⌬, rrp6⌬, and air1⌬ air2⌬ cells containing only AIR1 or air1 ZnK1-5 mutant plasmid grown at 25°C was measured by quantitative RT-PCR using gene-specific primers for the CUT NEL025c as described under "Experimental Procedures." Relative NEL025c RNA levels were measured in triplicate biological samples, normalized to a control PGK1 transcript by the ⌬⌬Ct method, averaged, and graphically shown as -fold increase relative to wild type cells. Error bars, S.E.
NEL025c RNA levels, may be critical for CUT RNA recognition/degradation.

Levels of TRAMP Complex Components Are Reduced in air1 Zinc Knuckle 5 Mutant Cells-
The previous data reveal that air1 ZnK5 mutant air1-C178R cells are temperature-sensitive at 37°C. To examine genetic interactions with air1-C178R, we integrated the air1-C178R allele at the genomic AIR1 locus in air2⌬ cells to create an air1-C178R air2⌬ mutant strain. Like air1⌬ air2⌬ cells harboring air1-C178R on a plasmid, the integrated air1-C178R air2⌬ strain was highly thermosensitive and exhibited slow growth at 30°C and profoundly impaired growth at 37°C (Fig. 4A). To determine whether components of the TRAMP complex can suppress the temperature-sensitive growth of the air1-C178R mutant, we overexpressed Air1/2, Trf4/5, trf4-DADA, a catalytically inactive mutant of Trf4 (D236A/D238A in the catalytic domain; see Fig. 8) (22), and Mtr4 in the integrated air1-C178R air2⌬ cells and examined cell growth at different temperatures as described under "Experimental Procedures." We also overexpressed trf4 -378 (E378A/E381A in the central domain; see Fig. 8), a nonfunctional mutant of Trf4 containing alanine substitutions of Glu-378 and Glu-381 residues (56), which are close to Air2 residues in the Trf4-Air2 structure and could mediate interactions with the Air proteins (49). As expected, overexpression of Air1 or Air2 completely suppressed the slow growth of air1-C178R cells at 30°C and 37°C (Fig. 5A). In addition, we overexpressed air1 ZnK1-5 mutants in air1-C178R cells and found that air1 ZnK1-4 mutants, but not the air1 ZnK5 mutant, fully suppress the slow growth of air1-C178R cells at 30°C (supplemental Fig.  S2). Overexpression of Trf4 partially suppressed the slow growth of air1-C178R cells at 30°C but not at 37°C (Fig. 4A). Interestingly, overexpression of the catalytically inactive Trf4 mutant, trf4-DADA, suppressed the slow growth of air1-C178R cells at 30°C only weakly if at all and had no effect at 37°C, suggesting that the catalytic function of Trf4 is required or that the expression level of the trf4-DADA mutant is reduced in air1-C178R cells (Fig. 4A). Overexpression of Trf5 suppressed the slow growth of air1-C178R cells at 30°C only weakly and had no effect at 37°C (Fig. 5A). Overexpression of Mtr4 or trf4 -378, however, did not suppress the slow growth of air1-C178R cells at 30°C or 37°C (Fig. 5A). These data indicate that Trf4, but not Trf5 or Mtr4, can suppress air1-C178R thermosensitive cell growth.
The variation in suppression of air1-C178R cell growth by the TRAMP components could reflect differences in the expression levels of the proteins. To address this point, we examined the expression levels of Myc-tagged Trf4, trf4-DADA, trf4 -378, Trf5, and Mtr4 proteins in air1-C178R cells grown at 25 and 30°C by immunoblotting. As a control, we also examined the levels of these proteins in wild-type cells. Surprisingly, the level of the trf4-DADA mutant was greatly reduced compared with wild-type Trf4 in air1-C178R cells at 25°C but not in wild-type cells (Fig. 5B). In addition, the levels of Trf5 and Mtr4 are reduced in air1-C178R cells at 25°C compared with wild type cells (Fig. 5B). Moreover, the levels of trf4-DADA, trf4 -378, Trf5, and Mtr4 were reduced (23-100%) in air1-C178R cells shifted to 30°C compared with the same cells at 25°C (Fig. 5B). These data indicate that the steady-state levels of the trf4-DADA mutant, Trf5, and Mtr4 are decreased in air1-C178R cells and suggest that the integrity of the TRAMP complex is compromised in these mutant cells because they express reduced levels of Air1 protein.
The most logical explanation for suppression of air1-C178R thermosensitive cell growth by Trf4 is that Trf4 stabilizes the air1-C178R mutant protein. To address this possibility, we Air1 ZnK4 and ZnK5 Required for TRAMP Complex OCTOBER 28, 2011 • VOLUME 286 • NUMBER 43 overexpressed Trf4 and trf4-DADA in air1⌬ air2⌬ cells expressing Myc-tagged air1-C178R protein and examined the level of air1-C178R protein by immunoblotting. Compared with cells containing vector alone, cells overexpressing Trf4 and trf4-DADA showed a 3.1-3.8-fold increase in the level of the air1-C178R mutant protein (Fig. 5C). This result indicates that Trf4 can stabilize the air1 ZnK5 mutant.
TRAMP Complex Proteins Are Unstable in air1 Zinc Knuckle 5 Mutant Cells-The previous results show that the steadystate levels of TRAMP proteins are reduced in air1-C178R air2⌬ cells relative to wild type cells. One potential explanation for the decreased TRAMP protein levels in air1 ZnK5 mutant cells could be that the TRAMP proteins are more unstable in these cells. To address the question of TRAMP protein stability in a rigorous manner, we measured the levels of Myc-tagged Trf4, trf4-DADA, Trf5, and Mtr4 in wild type and air1-C178R air2⌬ cells over time following translation inhibition with cycloheximide. We found that the TRAMP proteins were indeed more unstable in air1 ZnK5 mutant cells compared with wild type cells (Fig. 6). Quantitation of the immunoblots (Fig.  6A) showed that the half-lives of TRAMP proteins were reduced 2-4-fold in air1 ZnK5 mutant cells compared with wild type cells (Fig. 6B). Notably, trf4-DADA had a shorter halflife than Trf4 in air1 ZnK5 mutant cells and wild type cells (Fig.  6C). Together, these results indicate that the stability of the 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.
Endogenous hZCCHC7 Localizes to the Nucleolus and Interacts with Mammalian PAPD5 and PAPD7-Like S. cerevisiae Air1, a putative human Air1 orthologue should possess CCHC zinc knuckles, localize to the nucleus, and have the capacity to interact with a Trf4-like protein. The human protein, hZC-CHC7, which possesses four intact CCHC zinc knuckles, contains the conserved Air1 ZnK4-5 linker IWRXY motif residues and Air1 ZnK5 residues that are important for interaction with Trf4 (see Fig. 1B). To examine endogenous hZCCHC7, we immunoblotted HeLa cell lysates with a commercially available anti-hZCCHC7 polyclonal antibody raised to the N terminus of hZCCHC7. This antibody detected a single band of ϳ85 kDa in HeLa cell lysate (supplemental Fig. S3). We localized endogenous hZCCHC7 in HeLa cells by indirect immunofluorescence with the anti-hZCCHC7 antibody as described under "Experimental Procedures." This immunolocalization revealed that hZCCHC7 colocalized with the nucleolar protein fibrillarin to FIGURE 5. Overexpression of TRAMP complex components can suppress the thermosensitive growth of air1-C178R ZnK5 mutant cells, and TRAMP component levels are reduced in air1-C178R cells. A, overexpression of Air1/2 and Trf4, but not Trf5 or Mtr4, can effectively suppress air1-C178R thermosensitive growth. air1-C178R air2⌬ cells containing vector, AIR1, AIR2, TRF4, trf4-DADA mutant, TRF5, MTR4, or trf4 -378 mutant 2 URA3 plasmids were grown to saturation, serially diluted and spotted on plates, and grown at the indicated temperatures. B, steady-state levels of TRAMP components are reduced in air1-C178R cells relative to wild-type cells. Lysates of wild type and air1-C178R air2⌬ cells overexpressing Myc-tagged Trf4, trf4-DADA mutant, trf4 -378 mutant, Trf5, or Mtr4 grown at 25 and 30°C were analyzed by SDS-PAGE and immunoblotting with an anti-Myc antibody (␣-Myc) to detect Trf4-Myc proteins and anti-Pgk1 antibody (␣-Pgk1) to detect 3-phosphoglycerate kinase as a loading control. In air1-C178R air2⌬ cells, relative to the amounts of the TRAMP proteins at 25°C, the amounts of the TRAMP proteins at 30°C show the following percentage decreases: Trf4 (23%), trf4-DADA (50%), trf4 -378 (63%), Trf5 (100%), and Mtr4 (79%). In wild type cells, relative to the amounts of the TRAMP proteins at 25°C, the amounts of the TRAMP proteins at 30°C show the following percentage decreases: Trf4 (0%), trf4-DADA (8%), trf4 -378 (18%), Trf5 (8%), and Mtr4 (0%). C, overexpression of Trf4 increases the steady-state level of air1-C178R mutant protein. Lysates of air1⌬ air2⌬ cells expressing Myc-tagged air1-C178R protein and containing vector alone or overexpressing Trf4 or trf4-DADA grown at 25°C were analyzed by immunoblotting with an anti-Myc antibody to detect air1-C178R-Myc protein and anti-Pgk1 antibody to detect 3-phosphoglycerate kinase as a loading control. Relative to cells containing vector alone, cells overexpressing Trf4 show a 3.8-fold increase in air1-C178R, whereas cells overexpressing trf4-DADA show a 3.1-fold increase in air1-C178R protein. D, nonfunctional Trf4 mutant, trf4 -378, shows decreased binding to Air1 relative to wild-type Trf4. TAP-tagged Air1 was precipitated from lysates of AIR1-TAP cells expressing Myc-tagged Trf4 or trf4 mutants, trf4-DADA or trf4 -378, and bound (B), unbound (U), and input fractions were analyzed by immunoblotting with an anti-Myc antibody to detect Air1/2-Myc proteins and an anti-Pgk1 antibody to detect 3-phosphoglycerate kinase as a loading control. The percentage of bound trf4 protein relative to the amount of input protein and wild type Trf4 (% Bound) is shown below the bound fractions. The percentage of input trf4 protein relative to the amount of wild type Trf4 protein (% Input) is shown below the input fractions. The percentages of protein were calculated by measuring the intensities of the protein bands as described under "Experimental Procedures." Air1 ZnK4 and ZnK5 Required for TRAMP Complex OCTOBER 28, 2011 • VOLUME 286 • NUMBER 43 the nucleolus in HeLa cells (Fig. 7A) similar to the observed nucleolar localization of Air1 in S. cerevisiae cells.
To determine if hZCCHC7 interacts with one or both of the putative mammalian Trf4 orthologues, PAPD5 and PAPD7, we immunoprecipitated Myc-tagged murine PAPD5, which exhibits 94% sequence identity to human PAPD5 and Myc-tagged human PAPD7, from lysates of HEK293 cells expressing these tagged proteins and examined the binding to endogenous hZCCHC7 by immunoblotting with an anti-hZCCHC7 antibody. Endogenous hZCCHC7 bound to both mPAPD5 and hPAPD7 but showed little or no binding to control Myc beads alone (Fig. 7B). To confirm the interactions between hZCCHC7 and PAPD5/7, we immunoprecipitated endogenous hZCCHC7 from lysates of HEK293 cells expressing Myc-tagged mPAPD5 or hPAPD7 and immunoblotted the samples with an anti-Myc antibody. As observed previously, both mPAPD5 and hPAPD7 bound to hZCCHC7 but did not bind to control IgG beads (Fig.  7C). Nucleolar hZCCHC7 thus has the capacity to bind both FIGURE 6. TRAMP components are unstable in air1-C178R ZnK5 mutant cells. A, protein levels of Trf4, trf4-DADA, Trf5, and Mtr4 decrease more rapidly over time in air1-C178R air2⌬ cells relative to wild type cells following cycloheximide treatment. Wild type and air1-C178R air2⌬ cells overexpressing Myc-tagged Trf4, trf4-DADA, Trf5, or Mtr4 were treated with translation inhibitor cycloheximide, and cell samples were collected every 30 min. Cell sample lysates from each time point were analyzed by SDS-PAGE and immunoblotting with an anti-Myc antibody to detect Myc-tagged proteins (Trf4-Myc, trf4-DADA-Myc, Trf5-Myc, and Mtr4-Myc) and an anti-Pgk1 antibody (Pgk1) to detect 3-phosphoglycerate kinase as a loading control. B, the immunoblots shown in A were quantitated to plot the percentage of Trf4, trf4-DADA, Trf5, and Mtr4 proteins at each time point relative to inhibition of translation at time 0 in wild type and air1-C178R air2⌬ cells. The inset graphs show the log-transformed intensities of Trf4, trf4-DADA, Trf5, and Mtr4 protein bands at time points 0, 60, and 90 min fitted with linear least-squares fit lines. These best fit lines were used to determine the decay rate constant (k) for each protein. C, the table shows the half-lives (T1 ⁄2 ) of the TRAMP proteins in wild type and air1-C178R air2⌬ cells that were calculated from each decay rate constant using the equation T1 ⁄2 ϭ ln(2)/k (54). Further details on the measurement of the percentages of protein using protein band intensities and calculation of the protein half-lives are described under "Experimental Procedures." FIGURE 7. Endogenous hZCCHC7 localizes to the nucleolus in HeLa cells and interacts with both mammalian Trf4 orthologues, PAPD5 and PAPD7, in HEK293 cells. A, endogenous hZCCHC7 localizes to the nucleolus in HeLa cells. hZCCHC7 was localized in HeLa cells by indirect immunofluorescence with anti-hZCCHC7 antibody (␣-hZCCHC7). The nucleolar protein fibrillarin was detected with an anti-fibrillarin antibody (␣-Fibrillarin) to show the position of the nucleolus. The merge shows that hZCCHC7 colocalizes with fibrillarin in the nucleolus. Hoechst dye was used to stain DNA and show the position of the nucleus. B, endogenous hZCCHC7 interacts with both mammalian Trf4 homologues, PAPD5 and PAPD7. Myc-tagged mPAPD5 and Myc-tagged hPAPD7 were immunoprecipitated (IP) from lysates of HEK293 cells expressing these tagged proteins. As a control, Myc beads alone were used with untransfected HEK293 cells. Input, unbound (U), and bound (B) fractions were analyzed by SDS-PAGE and immunoblotting with an anti-hZC-CHC7 antibody. hZCCHC7 binds to both mPAPD5 and hPAPD7, but no binding to Myc beads alone is detected. C, both PAPD5 and PAPD7 interact with endogenous hZCCHC7. Endogenous hZCCHC7 was immunoprecipitated from lysates of HEK293 cells expressing Myc-tagged mPAPD5 (mPAPD5) or Myc-tagged hPAPD7 (hPAPD7), and input, unbound (U) and bound (B) fractions were analyzed by SDS-PAGE and immunoblotting with an anti-Myc antibody. As a control, IgG beads alone were also used for immunoprecipitation. Both mPAPD5 and hPAPD7, but not IgG beads alone, bind to hZCCHC7. putative mammalian Trf4 orthologues, PAPD5 and PAPD7, and could be the Air component of a human TRAMP complex.

DISCUSSION
Here we present the first in vivo analysis of full-length Air1 and Air2 zinc knuckle mutant proteins, which reveals that Air1/2 zinc knuckles 4 and 5 are critical for cell function. In particular, our data indicate that Air1/2 ZnK5 is important for interaction with Trf4 and suggest that Air1 ZnK4 may help facilitate recognition of RNA targets. Furthermore, interaction of Air1 with Trf4 and the level of Air1 are critical for the integrity of the TRAMP complex. We also identify a key evolutionarily conserved IWRXY motif in the Air1 ZnK4-5 linker region that is important for function and Trf4 interaction. The presence of the IWRXY motif, a potential signature for Air orthologues, in the human CCHC zinc knuckle protein, hZCCHC7, strongly suggests that this is the human orthologue of S. cerevisiae Air1. In support of the idea, we find that hZCCHC7 localizes to the nucleolus in human cells and binds to both mammalian Trf4 orthologues, PAPD5 and PAPD7, consistent with it being a component of a human TRAMP complex that performs nuclear RNA surveillance. Taken together, the data suggest that Air1/2 forms a stable complex with Trf4 via the ZnK4-5 linker and ZnK5 and targets CUT RNAs for degradation using the ZnK1-4.
Our results support the recent structural model of Trf4 in complex with an Air2 fragment reported by Hamill and colleagues (49), which shows that Air2 ZnK4 and -5 interact with the Trf4 central domain. In Fig. 8, we use the Trf4-Air2 structural model (49) to highlight the positions of the Air2 ZnK4, ZnK5, and IWRXY motif residues and Trf4 central and catalytic domain residues. Importantly, the structural model reveals that Air2 ZnK5 is buried against the surface of Trf4, but Air2 ZnK4 makes little contact with the Trf4 surface and remains tethered to Trf4 via an adjacent linker (49). Our data showing that several Air1 mutants of the ZnK4-5 linker-located IWRXY motif and ZnK5 bind more weakly to Trf4 compared with Air1 ZnK4 mutants are consistent with these structural data. In particular, we note that the Air1 IWRXY motif residues Trp-153 and Tyr-156 and the ZnK5 CX 2 residue Tyr-176 and HX 4 residues Phe-184 and Asp-186 (in CX 2 CX 4 HX 4 C) are critical for interaction with Trf4 (1-7% bound, Fig. 4B). Importantly, in the Trf4-Air2 structure, the equivalent Air2 IWRXY residues Trp-140 and Tyr-143 and ZnK5 residues Tyr-165, Phe-173, and Asp-175 are located close to Trf4 residues and thus have the potential to interact with Trf4. The Trf4-Air2 structure highlights an interaction between Air2 and the Trf4 helix H8 (residues 373-386) in the central domain (49) (Fig. 8). The proximity of Air2 IWRXY residues to Trf4 helix H8 residues, especially Ile-377, Glu-378, and Glu-381, suggests that they could interact. In particular, Air2 IWRXY residues Trp-140, Arg-141, and Tyr-143 could interact with Trf4 helix H8 residues Ile-377, Glu-381, and Glu-378, respectively, based on interresidue distances of 2.5-4 Å (Fig. 8). In support of this idea, we find that the Trf4 mutant, trf4 -378, containing alanine substitutions of helix H8 residues Glu-378 and Glu-381, which is nonfunctional (56) and cannot suppress the air1-C178R thermosensitive cell growth (Fig. 5A), shows decreased binding to Air1 (15% bound; Fig. 5D). These results indicate that Trf4 residues Glu-378 and Glu-381 are functionally important and that altering these residues can impair the Trf4-Air interaction.
The data presented here indicate that the Air1-Trf4 interaction and the steady-state level of the Air1 protein contribute to the stability of TRAMP complex proteins and integrity of the TRAMP complex. The air1-C178R ZnK5 mutant protein shows decreased binding to Trf4 (69 Ϯ 4%) and a lower steadystate level (26 Ϯ 4%) compared with wild-type Air1 protein (Fig.  4A). In air1 ZnK5 mutant cells, Trf4, trf4-DADA, Trf5, and Mtr4 proteins all exhibit reduced stability (T1 ⁄ 2 reduced 2-4fold) compared with wild-type cells (Fig. 6). Such instability of the TRAMP proteins in air1 ZnK5 mutant cells may account for the lower steady-state levels of TRAMP proteins in these cells (Fig. 5B). However, we note that the initial (time 0) TRAMP protein levels are reduced in air1 ZnK5 cells relative to wild type cells (Fig. 6), suggesting that additional mechanisms operate to regulate TRAMP protein levels. One possibility is that TRF4/5/MTR4 RNA levels are regulated by the TRAMP complex itself in a feedback mechanism. Thus, in air1 ZnK5 cells, decreased TRAMP complex could result in reduced TRF4/5/MTR4 RNA levels, leading to reduced TRAMP protein levels. Consistent with this proposal, TRF4 RNA levels are reduced in trf5⌬ cells (34).
The reduced stability of the air1/2 ZnK5 mutant proteins could, in part, be caused by disruption of the Air1/2-Trf4 interaction. In support, cysteine to arginine substitutions within the zinc-liganding residues in these air1/2 ZnK mutants should disrupt ZnK5, and Air2 ZnK5 is buried against the surface of Trf4 in the structure (49) (Fig. 8). In addition, overexpression of Trf4 causes a 3.8-fold increase in the steady-state level of air1-C178R protein (Fig. 5C). Furthermore, Trf4 protein and trf4-DADA mutant protein, which contains alanine substitutions of catalytic domain residues Asp-236 and Asp-238 that are quite distant from the Trf4 Air1/2-interacting central domain (Fig. 8), are more unstable in air1 ZnK5 cells compared with wild-type cells (T1 ⁄ 2 reduced 4-fold; Fig. 6), suggesting that Trf4 proteins are normally stabilized by interaction with Air1/2 in wild type cells. The combined results thus suggest that Air1/2 is probably in constant partnership with Trf4/5 in S. cerevisiae cells, consistent with biochemical purification of the complex, and, furthermore, that the Air-Trf4/5 interaction plays an important role in the integrity of the TRAMP complex. Interestingly, we find that overexpression of Trf4, but not Trf5 or Mtr4, can effectively suppress the thermosensitive growth of air1-C178R ZnK5 mutant cells (Fig. 5A). Trf4 most likely suppresses air1-C178R cell growth by stabilizing the air1-C178R protein because Trf4 overexpression increases the steady-state level of air1-C178R protein (Fig. 5C). The observation that Trf5 only weakly suppresses air1-C178R cell growth is somewhat surprising, given the functional redundancy between Trf5 and Trf4; however, the steady-state level of Trf5 is 4-fold less than Trf4 in wild type cells (57,58), suggesting that a lower Trf5 level may not be sufficient to stabilize the air1-C178R mutant. The weak suppression by Trf5 may also be explained by the fact that Trf5 is more unstable in air1-C178R cells compared with wild-type cells and exhibits the shortest half-life of the TRAMP components (Fig. 6). Surprisingly, Mtr4 does not suppress air1-C178R cell growth. Like Trf5, the reduced stability of Mtr4 in air1-C178R cells relative to wild-type cells may contribute to the lack of suppression (Fig. 6). In addition, Mtr4 may not directly contact Air1 to stabilize the air1-C178R mutant protein.
The data presented here begin to address the question of which Air1/2 zinc knuckles are important for recognition of RNA. We find that, relative to AIR1 cells, air1 ZnK1-3 and air1 ZnK5 mutant cells show a 2-3-fold increase in NEL025 CUT RNA levels, whereas air1 ZnK4 mutant cells show a 5-fold increase in NEL025 RNA levels (Fig. 3). These results suggest that Air1 ZnK1-5 are important for the recognition/degradation of CUT RNA and suggest an important role for ZnK4 in CUT RNA recognition because the air1 ZnK4 mutant shows the greatest level of NEL025c RNA. Recent work by Hamill et al. (49) using recombinant air2 ZnK1, ZnK2, or ZnK3 mutants in Trf4 polyadenylation assays showed that Air2 ZnK1 is important for polyadenylation of tRNA, suggesting that ZnK1 helps to recognize RNA. This study reported that recombinant Air2 ZnK4-5 alone can weakly support Trf4 polyadenylation of tRNA, suggesting that Air2 ZnK4 and/or ZnK5 may help recognize RNA (49). Although the Trf4-Air2 structure showed that Air2 ZnK4 does not make many contacts with Trf4 and would be available to interact with RNA (Fig. 8), recombinant air2 ZnK4 mutant could not be made to directly test the contribution of ZnK4 in Trf4 polyadenylation. Our observation that the level of NEL025c CUT RNA is increased to the greatest extent in air1-C139R ZnK4 mutant cells is consistent with the idea that Air1/2 ZnK4 facilitates recognition of RNA. Potentially, one or more Air1/2 ZnKs may interact with the RNA substrate nonspecifically, and another Air1/2 ZnK, such as ZnK4, may recognize the RNA substrate specifically. Further Air1/2-RNA binding studies will be required to confirm which zinc knuckles interact directly with RNA.
Our combined results support the notion that the human CCHC zinc knuckle protein, hZCCHC7, is a putative human Air1 orthologue. First, hZCCHC7 ZnK1-5, which show 35% sequence identity to Air1 ZnK1-5, contain the ZnK4-5 linkerlocated IWRXY motif and key ZnK5 residues (e.g. CX 2 residue Tyr-350) that are conserved in the yeast Air1/2 ZnK domains and important for Trf4 interaction (see Fig. 1B). Second, like Air1, hZCCHC7 localizes to the nucleolus in HeLa cells, consistent with hZCCHC7 serving a nuclear function, such as RNA processing/degradation. Third, hZCCHC7 interacts with both putative mammalian Trf4 orthologues, PAPD5 and PAPD7, suggesting that hZCCHC7 could be the Air component in a human TRAMP complex. Although the human CCHC zinc knuckle protein, hZCCHC9, has also been proposed to be a putative human Air1 orthologue, based on the fact that its four zinc knuckles show 19% sequence identity to Air1 ZnK1-5 and it localizes to the nucleolus (46,59), this seems less likely because hZCCHC9 lacks the conserved IWRXY motif and ZnK5 residues important for Trf4 interaction (see Fig. 1B). In addition, hZCCHC9 ZnK1 has less sequence similarity within Air1 ZnK1, which may be important for tRNA interaction (49), and the hZCCHC9 ZnK3-4 linker (13 residues) is much shorter than the Air1 ZnK4-5 linker (25 residues) (see Fig. 1B).
Future analysis of the function of hZCCHC7, hPAPD5/7, and the human TRAMP complex will be intriguing. Because promoter upstream transcripts are polyadenylated and degraded in an exosome-dependent manner in human cells (20) and aberrant pre-rRNAs have been shown to be polyadenylated by the Trf4 orthologue, PAPD5, in mouse cells (60), it seems highly likely that a human TRAMP complex will act as an exosome cofactor in the processing/degradation of ncRNAs in human cells. Very recently, hZCCHC7 and hPAPD5 were identified in hMTR4 and hRRP6 precipitates and shown to interact with each other and play a role in the polyadenylation of pre-rRNAs (61), providing strong support for a human TRAMP complex containing ZCCHC7 and PAPD5 and also supporting the data presented here. Notably, ZCCHC7 is disrupted in t(9;20) chromosomal translocations in acute lymphoblastic leukemia, and ZCCHC7 expression is up-regulated in early B-cells (62,63), suggesting that hZCCHC7 plays roles in lymphoblast and B-cell development. hZCCHC7 and the human TRAMP complex may thus help target ncRNAs for degradation to regulate gene expression and cell development.