Stm1p, a G4 Quadruplex and Purine Motif Triplex NucleicAcid-binding Protein, Interacts with Ribosomes and SubtelomericY� DNA in Saccharomyces cerevisiae*

Received for publication, February 23, 2004Published, JBC Papers in Press, March 23, 2004, DOI 10.1074/jbc.M401981200

Michael W. Van Dyke‡, Laura D. Nelson, Rodney G. Weilbaecher, and Dakshesh V. Mehta§

From the Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center,Houston, Texas 77030

The Saccharomyces cerevisiae protein Stm1 was orig-inally identified as a G4 quadruplex and purine motiftriplex nucleic acid-binding protein. However, more re-cent studies have suggested a role for Stm1p in pro-cesses ranging from antiapoptosis to telomere mainte-nance. To better understand the biological role of Stm1pand its potential for G*G multiplex binding, we usedepitope-tagged protein and immunological methods toidentify the subcellular localization and protein andnucleic acid partners of Stm1p in vivo. Indirect immu-nofluorescence microscopy indicated that Stm1p is pri-marily a cytoplasmic protein, although a small percent-age is also present in the nucleus. Conventionalimmunoprecipitation found that Stm1p is associatedwith ribosomal proteins and rRNA. This association wasverified by rate zonal separation through sucrose gradi-ents, which showed that Stm1p binds exclusively tomature 80 S ribosomes and polysomes. Chromatin im-munoprecipitation experiments found that Stm1p pref-erentially binds telomere-proximal Y� element DNA se-quences. Taken together, our data suggest that Stm1p isprimarily a ribosome-associated protein, but one thatcan also interact with DNA, especially subtelomeric se-quences. We discuss the implications of our findings inrelation to prior genetic, genomic, and proteomic stud-ies that have identified STM1 and/or Stm1p as well asthe possible biological role of Stm1p.

It has long been recognized that certain G-rich nucleic acidscan adopt structures other than Watson-Crick base-paired du-plexes (1). Examples include purine motif triple helical DNA(Pu triplex)1 and four-stranded quadruplexes (G4 DNA orRNA) (2, 3). These nucleic acids are characterized by Hoogsteenhydrogen bonding between guanines (G*G) and very high neg-

ative charge densities due to the presence of multiple phos-phate backbones, hence the simpler designation “G*G multi-plex.” Both Pu triplexes and G4 structures can form underphysiological conditions and are both extremely stable speciesonce formed.

Although a considerable amount of information is availableregarding the properties of G*G multiplex structures in vitro,little is known regarding their existence and biological roles invivo (4, 5). Sequences capable of forming these structures arepresent in all eukaryotic organisms. Examples include telo-meric sequences, long oligopurine tracts within several genepromoters, and certain neuronal mRNAs (6–8). G*G multi-plexes have been hypothesized as functional intermediates inmany biological processes, including chromosome condensa-tion, recombination, telomere maintenance, and transcrip-tional and translational control (8–14). By contrast, potentiallydeleterious G*G structures can also form in DNA or RNAduring replication or transcription, with their removal beingcritical for normal cellular function (15, 16).

One line of evidence used to support the hypothesis that G*Gmultiplexes do exist in vivo is the existence of cellular proteinsthat specifically act upon such structures. Proteins have beenidentified in eukaryotes ranging from yeast to humans thatspecifically and avidly bind Pu triplexes and G4 DNAs andRNAs in vitro (8, 10, 17–28, 30–44). In addition, helicases havealso been identified that efficiently unwind these G*G multi-plex structures in vitro (16, 44–47).2

Obviously, biochemical demonstrations of protein-G*G mul-tiplex interactions in vitro do not necessarily prove that suchphenomena actually occur in vivo, let alone that such speciestruly exist. To address this important question, we sought toidentify those nucleic acids bound by G*G multiplex-bindingproteins within living cells. As a model system, we chose theSaccharomyces cerevisiae protein Stm1p, which had previouslybeen shown to be an avid and specific binder of G4 and Putriplex DNAs (22, 32). Stm1p is a moderately abundant (35,000copies/cell), 30-kDa basic (pI � 10.5) protein that has beenimplicated in biological processes ranging from apoptosis totelomere maintenance (48, 49). We constructed epitope-taggedSTM1 strains to determine the cellular localization and in vivobinding partners of this protein. Here we report that Stm1p isprimarily a ribosome-associated protein, although it can also befound specifically associated with subtelomeric Y� elements inyeast chromosomes.

EXPERIMENTAL PROCEDURES

Yeast Strains—Standard yeast genetic techniques were used (50).PCR-based homologous recombination (51) was used to constructstrains in which the chromosomal STM1 gene is tagged with three

* This work was supported by Robert A. Welch Foundation GrantG-1199 (to M. W. V. D.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Dept. of Molecularand Cellular Oncology, The University of Texas M. D. Anderson CancerCenter, Unit 79, 1515 Holcombe Blvd., Houston, TX 77030-4009. Tel.:713-792-8954; Fax: 713-794-0209; E-mail: mvandyke@mdanderson.org.

§ Present address: Aurigene Discovery Technology Ltd., ElectronicCity Phase II, Hosur Rd., 39-40 KIABD Industrial Area, Bangalore 560100, India.

1 The abbreviations used are: Pu triplex, purine motif triple helicalDNA; ARS, autonomous replicating sequence(s); ChIP, chromatin im-munoprecipitation; EMSA, electrophoretic mobility shift assay; G4, G-quartet; G*G, Hoogsteen hydrogen-bonded guanines; HA, influenzahemagglutinin protein epitope; mAb, monoclonal antibody; NTS, non-transcribed sequence; ETS, early transcribed sequence. 2 R. D. Hunt and M. W. Van Dyke, unpublished observations.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 23, Issue of June 4, pp. 24323–24333, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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tandem HA epitopes at either the N or C terminus. PCR screening andWestern blot analysis of protein expression was used to verify strainconstruction. Strain K699 (MATa ade2-1 can1–100 his3–11,-15 leu2-3,-112 ssd1-d trp1-1 ura3) is the isogenic parent of strain STM1-HA3

(DM28). STM1-HA3 (DM28) yeast express Stm1p with a C-terminalHA3 epitope (Stm1p-HA3) under the control of the endogenous STM1promoter. The DNA used to construct strain STM1-HA3 (DM28) wasamplified from plasmid pFA6a-3HA-kanMX6 with primers HAC-F2(5�-GAA CCG TAA CAT TGA CGT TTC TAA CTT GCC ATC TTT GGCTCG GAT CCC CGG GTT AAT TAA-3�) and HAC-R1 (5�-TTA TTG GATTCT TTC AGT TGG AAT TAT TCA TAT ATA AGG CGA ATT CGA GCTCGT TTA AAC-3�). Strain JB811 (MATa leu2-3,112 trp1 ura3–52 prb1prc pep4-3) is the isogenic parent of strain HA3-STM1 (LN12). HA3-STM1 (LN12) yeast express Stm1p with a N-terminal HA3 epitope(HA3-Stm1p) under the control of the GAL1 promoter. The DNA used toconstruct strain HA3-STM1 (LN12) was amplified from plasmid pFA6a-kanMX6-PGAL1–3HA with primers HAN-F4 (5�-TTT CTT TGC AAATTT CTC TTC CCC CCA CAG TAT TCT TTT AGA ATT CGA GCT CGTTTA AAC-3�) and HAN-R3 (5�-CGT CTT CGA CGT CGT TAC CTA ACAAAT CAA ATG GGT TGG AGC ACT GAG CAG CGT AAT CTG-3�. Thestm1-� strain A1454 (MATa aro7 ade8 his3 leu2 ura3 stm1�::ADE8)has been described previously (52). The YKU80-myc18 strain (TVL323)expresses a C-terminal myc18 epitope-tagged Yku80p from the endoge-nous YKU80 promoter. This strain and its untagged isogenic parentused in the chromatin immunoprecipitation experiment are haploidsderived from AVL49 (MAT a/� ura3–52/ura3–52 lys2–801/lys2–801ade2–101/ade2–101 trp1-�1/trp1- �1 his3- �200/his3- �200 leu2- �1/leu2- �1 CF�/� [TRP1-SUP11-CEN4 D8B]). The YKU80-myc18 strain(TVL323) was constructed and provided by A. Bertuch and V. Lundblad(Baylor College of Medicine, Houston, TX).

Gel Electrophoretic Analysis of Stm1p—Yeast cells were grown tomidlogarithmic phase (A600 � 1.0) in YPD (1% yeast extract, 2% pep-tone, and 1% dextrose) medium. When required for HA3-Stm1p expres-sion, the final 6 h of growth was performed in YPA-galactose (1% yeastextract, 2% peptone, 0.003% adenine sulfate, and 0.2% galactose) me-dium. Cells were disrupted by vortexing with an equal volume of acid-washed 0.5-mm Glasperlen glass beads (B. Braun Biotech, Allentown,PA) in cold LY buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 6 mM

MgCl2, 2 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM dithiothre-itol, 1 mM phenylmethylsulfonyl fluoride, 1 �M leupeptin, 1 �M pepsta-tin, and 2.5 �g/ml antipain). Cell lysates were clarified by centrifuga-tion at 12,000 � g for 10 min at 4 °C, and the protein concentration wasdetermined using a Bradford assay (Bio-Rad).

To analyze Stm1p using Western blotting, 15 �g of total protein fromthe clarified whole cell extract was heat-denatured in Laemmli samplebuffer and loaded onto an SDS-10% polyacrylamide gel (53). Afterelectrophoretic separation, the proteins were electroblotted onto nitro-cellulose membranes (Schleicher & Schuell). Blocked membranes wereprobed with either the anti-HA.11 epitope-specific mouse mAb 16B12(Covance Research Products, Berkeley, CA) or a rabbit polyclonal an-tibody (University of Texas M.D. Anderson Cancer Center Departmentof Veterinary Medicine and Surgery, Bastrop, TX) generated againstbacterial recombinant Stm1p,3 followed by a sheep anti-mouse or sheepanti-rabbit IgG horseradish peroxidase-conjugated secondary antibody(Amersham Biosciences), depending on the primary antibody used.Antibodies were visualized using an Amersham ECL chemilumines-cence kit and Hyperfilm following the manufacturer’s instructions.Western blots were analyzed by densitometry using an AlphaImager3400 imaging system and AlphaEase FC software (Alpha Innotech, SanLeandro, CA).

To analyze Stm1p binding to triplex DNA by EMSA, 10 �g of totalprotein from the clarified whole cell extract was incubated with 1 nM

radiolabeled X-Pu-T19 probe for 30 min at 24 °C in a 10-�l volume con-taining 25 mM HEPES-Na�, pH 7.9, 50 mM KCl, 1 mM MgCl2, 10%glycerol, 0.5 mM dithiothreitol, and 2 �g poly(dI-dC) carrier DNA aspreviously described (32). The resulting protein-probe complexes wereresolved by nondenaturing PAGE, visualized by autoradiography, andquantitated using a Storm 840 PhosphoImager (Amersham Biosciences).

Immunofluorescence Microscopy—Immunofluorescence microscopywas performed essentially as previously described (48). Yeast cellsgrown to midlogarithmic phase (A600 0.5–1.0) were harvested and fixedin 50 mM potassium phosphate (pH 7.0) and 3.7% formaldehyde.Spheroplasts were made by treating these cells with 5 �g/ml Zymolyase100T (Sigma) and 0.1% �-mercaptoethanol in SP buffer (1.2 M sorbitoland 50 mM potassium phosphate, pH 7). Resulting spheroplasts were

adhered onto poly-L-lysine-coated slides (Sigma). These were treatedwith HA epitope-specific mouse mAb 16B12 (1:200), followed by 5 �g/mlCy3-conjugated affinity-purified goat anti-mouse IgG antibodies (Jack-son Laboratory, Bar Harbor, ME) in phosphate-buffered saline and0.1% bovine serum albumin. The bound cells were finally treated withthe DNA-binding fluorescent dye 4�,6-diamidino-2-phenylindole (1ng/ml in phosphate-buffered saline) and one drop of ProLong antifade(Molecular Probes, Eugene, OR) before mounting and sealing. The cellswere visualized by differential interference contrast microscopy or byfluorescence using a Zeiss Axioplan 2 microscope, and the images wereprocessed with AxioVision version 2.05 software (Carl Zeiss Microim-aging, Thornwood, NY).

Immunoprecipitation—Immunoprecipitations were performed essen-tially according to published protocols but with minor modification (54).Clarified yeast extract (1 mg of total protein), as described above, wasincubated with 5 �g of anti-HA mAb 16B12 in a total volume of 0.5 mlof IPM buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 6 mM MgCl2, 2 mM

EDTA, 0.1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsul-fonyl fluoride, 1 �M leupeptin, 1 �M pepstatin, and 2.5 �g/ml antipain)for 3 h at 4 °C with continual inversion. Suspensions were clarified bycentrifugation (12,000 � g for 10 min at 4 °C), and the supernatantswere added to an equal volume of IPM buffer containing 50 �l of aprotein G-agarose bead suspension (Roche Applied Science). These wereincubated for 3 h at 4 °C with continual inversion to effect binding andisolated by centrifugation.

To investigate immunoprecipitated proteins, thoroughly washedbeads from the above immunoprecipitation were resuspended in La-emmli sample buffer, boiled for 5 min to release bound proteins, andthen clarified by centrifugation before being loaded onto SDS-10% poly-acrylamide gels. After electrophoretic separation, the proteins werevisualized by staining with Coomassie Brilliant Blue R250. Molecularmass analysis and protein quantitation were performed on the dried gelusing an AlphaImager 3400 imaging system and AlphaEase FC soft-ware. Individual protein bands from these gels were excised and sent tothe Protein Chemistry Core Facility at Columbia University for matrix-assisted laser desorption ionization mass spectrometry peptide massmapping. Peptide masses were searched against the National Centerfor Biotechnology Information and/or GenBankTM GenPept databasesusing either the ProFound or the MS-Fit program (55, 56).

To investigate immunoprecipitated nucleic acids, the above men-tioned immunocomplexed beads were resuspended in 0.3 ml of E buffer(50 mM sodium acetate, pH 5.3, 10 mM EDTA, and 1% SDS) andincubated for 5 min at 65 °C to release the bound nucleic acids. Thesenucleic acids were purified by phenol extraction and ethanol precipita-tion, and the purified nucleic acids were electrophoresed through either1.25% agarose TAE or 5% acrylamide/0.13% bisacrylamide TBE gels.Nucleic acids were visualized by staining with ethidium bromide, andtheir analysis and quantitation were performed using an AlphaImager.To verify the identities of the immunoprecipitated RNAs, Northernblotting was performed, essentially as described previously (57, 58).Formaldehyde-treated RNAs were electrophoresed on either a 1% aga-rose MOPS, 3.7% formaldehyde gel or a 7% acrylamide, 0.18% bis-acrylamide/50% urea gel, depending on the RNA size. The RNAs weretransferred to a nitrocellulose membrane by either capillary action(agarose) or electroblotting (acrylamide) and UV cross-linked (Strata-linker 2400, Stratagene, La Jolla, CA). Membrane-bound RNAs werehybridized for 18 h at 42 °C with 5 � 106 cpm of radiolabeled oligonu-cleotide probes specific for S. cerevisiae ribosomal RNAs, including 25 S(5�-CTC TAA TCA TTC GCT TTA CC-3�), 18 S (5�-CAT GGC TTA ATCTTT GAG AC-3�), 5.8 S (5�-CGC ATT TCG CTG CGT TCT TCA TCGATG-3�), and 5 S (5�-GGT CAC CCA CTA CAC TAC TCG G-3�). Hy-bridized RNAs were visualized by autoradiography and analyzed usinga Storm PhosphorImager (Amersham Biosciences).

Ribosome Purification and Analysis—Analysis of S. cerevisiae ribo-somes, ribosomal subunits, and polysomes by sucrose gradient ultra-centrifugation followed published procedures with minor modifications(59). Briefly, yeast cells were grown to midlogarithmic phase in YPDmedium at 30 °C with mild agitation (200 rpm) and then harvested bycentrifugation. For polysome analysis, 50 �g/ml cycloheximide wasadded immediately before harvesting; for monosome and ribosome sub-unit analysis, it was not. Yeast extracts were prepared as previouslydescribed, with the exception that breaking buffer A contained 50 �g/mlcycloheximide for polysome analysis. After processing, five A260 units ofclarified extract was layered onto a 12-ml linear 7–47% sucrose gradi-ent containing 50 mM Tris acetate, pH 7.0, 50 mM NH4Cl, 12 mM MgCl2,and 1 mM dithiothreitol. These gradients were centrifuged in an SW-40rotor (Beckman Coulter, Fullerton, CA) at 38,500 rpm for 2.5 h. Thegradients were recovered using an Auto Densi-Flow IIC gradient frac-3 D. V. Mehta, unpublished observations.

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tionator (Labconco, Kansas City, MO), and 0.6-ml fractions were col-lected. During fraction collection, absorbance at 254 nm was recordedusing a UA-5 absorbance/fluorescence detector (Isco, Lincoln, NE). Pro-teins in these fractions were analyzed using SDS-PAGE, EMSA, andWestern blotting, as described above.

Chromatin Immunoprecipitation and Analysis—Chromatin immu-noprecipitations (ChIPs) were performed essentially as described pre-viously (60). Yeast cultures were grown in 200 ml of YPA-galactose to adensity of 107 cells/ml and fixed with 1% formaldehyde. Cell pelletswere resuspended in 1 ml of lysis buffer and lysed using a BeadBeatercell disrupter (BioSpec Products, Bartlesville, OK) three times for 1 minat 4 °C. Lysates were sonicated (Branson Ultrasonics, Danbury, CT)three times for 20 s (constant output, 1.5 duty cycle) to an average DNAlength of 500 bp, which was confirmed by agarose gel electrophoresisand ethidium staining. Ten percent of the clarified cell lysate was setaside and designated the “input.” Immunoprecipitations were per-formed for 3 h at 4 °C with 500 �l of clarified lysate, 30 �l of proteinA/G-Sepharose (Sigma), and either 5 �g of anti-MYC 9E10 (Covance) or1 �g of anti-HA 16B12 mAbs. Immunoprecipitates were washed, andthe cross-links were reversed at 65 °C for 6 h. The recovered DNA wasfurther purified with QiaQuick (Qiagen, Valencia, CA).

ChIP samples and their corresponding input dilutions were dena-tured by heating to 95 °C for 10 min in the presence of 0.4 M NaOH and10 mM EDTA. Afterward, an equal volume of 2 M ammonium acetate,pH 7.0, was added to neutralize the solution. Denatured DNA wasapplied to a Hybond N� membrane (Amersham Biosciences) using aBio Dot microfiltration apparatus (Bio-Rad). Wells were rinsed twicewith 2� SSC (300 mM NaCl and 30 mM sodium citrate, pH 7.0). Theblotted membrane was rinsed once more with 2� SSC for 5 min, and theDNA was UV cross-linked to the membrane.

Random-prime labeling (Megaprime; Amersham Bioscience) reac-tions with [�-32P]dCTP and [�-32P]dGTP was used to label all probes.The telomere probe and the random genomic DNA probe were gen-erated by random primed labeling of the alternating copolymerpoly(dG-dT/dC-dA) (Amersham Biosciences) or total yeast genomicDNA, respectively. The DNAs for all the other probes were generatedby PCR from yeast genomic DNA. The amplified DNAs, ranging from275 to 450 bp in length, were gel-purified and extracted with Qia-Quick prior to random prime labeling. The telomere probe was hy-bridized for �10 h at 65 °C, whereas all other probes were hybridizedfor �10 h at 60 °C. Blots were washed three times and exposed tophosphoscreens and analyzed with ImageQuant software. The fiveregions of the rDNA repeat used individually and collectively to probe

blots corresponded to a nontranscribed sequence (NTS), an earlytranscribed sequence (ETS), a sequence within the 5 S rRNA gene(5S), and two sequences within the 25 S rRNA gene (25S/1 and 25S/2).The primer sequences used to amplify these five rDNA regions fromgenomic DNA have been described previously (61). The sequences ofprimer pairs used to PCR-amplify various ARS and subtelomericregions for probe preparation included ARS305 (5�-CTC CGT TTTTAG CCC CCC GTG-3�; 5�-GAT TGA GGC CAC AGC AAG ACC G-3�),ARS1 (5�-GGT GAA ATG GTA AAA GTC AAC CCC CTG CG-3�;5�-GCT GGT GGA CTG ACG CCA GAA AAT GTT-3�), ARS306 (5�-GCA AGC ATC TTG TTT GTA ACG CGA-3�; 5�-CCT CAG CGA TGTGCT CGC TC-3�), ARS603 (5�-CTC TTT CCC AGA TGA TAT CTAGAT GG-3�; 5�-CGA GGC TAA ATT AGA ATT TTG AAG TC-3�),ARS501 (5�-ATG CCT GAA AAA GAA TGT GTC T-3�; 5�-GCG TTAGGT AAT GCT CAA TTT T-3�), subtelomere Y� element, telomere-proximal (5�-AAG CTG CAT TTA GCA GGC AT-3�; 5�-TCC AAC TTCTCT GCT CGA ATC-3�), subtelomere Y� element, telomere-distal(5�-CGT TGT TTC AAG TGC ATA CTT T-3�; 5�-TGG GCA TAG ATCACG CTT CA-3�), and subtelomere X element (5�-GAA GCA TGG TTTAGC GTG TT-3�; 5�-AAT GTT TCT GTG TTC TGG TGC G-3�).

RESULTS

Generation and Characterization of Yeast ExpressingEpitope-tagged Stm1p—Immunological assays, includingWestern blotting, immunofluorescence microscopy, and immu-noprecipitation, provide a powerful means of investigating pro-teins and determining their cellular localization and associatedmacromolecules. In yeast, the primary means of immunologicalprotein surveillance involves using epitope-tagged proteins andwell characterized monoclonal antibodies (62). Using the mod-ules of Longtine et al. (51), we constructed strains with theSTM1 gene tagged with three copies of the HA epitope oneither its N or C terminus (HA3-STM1 or STM1-HA3) andlocated at its normal chromosomal location. Expression of theepitope-tagged Stm1p was driven from the endogenous STM1promoter in STM1-HA3 yeast and from the inducible GAL1promoter in HA3-STM1 yeast.

To characterize Stm1p expression in these strains, we per-formed Western blot analysis with anti-Stm1p polyclonal anti-bodies (Fig. 1A). We observed the expected expression of Stm1

FIG. 1. Physical and functional characterization of epitope-tagged Stm1p in yeast. Stm1 protein amounts and integrity were charac-terized through Western blotting of SDS-PAGE-resolved whole cell yeast extract proteins with either rabbit polyclonal anti-Stm1p polyclonalantibodies (A) or anti-HA.11 monoclonal antibody (B). Equivalent amounts of whole cell extracts were loaded to compare the HA3-STM1 strain(lanes 2 and 3) with its untagged parent strain (lane 1) and to compare the STM1-HA3 strain (lane 6) with its untagged parent (lane 5) as well aswith an stm1-� strain (lane 4). Stm1 proteins were expressed from the endogenous, constitutive STM1 promoter in all strains except HA3-STM1,which was under the control of the inducible GAL1 promoter. Induction by galactose is indicated by a plus sign. Apparent molecular masses weredetermined by comparison with protein molecular weight markers and are indicated at the left. Stm1p and other yeast Pu triplex-bindingprotein-DNA interactions were characterized through an EMSA with the radiolabeled purine motif triple-helical DNA probe X-PuT19 (C). Laneswere identical to those in A and B, with the addition of a probe alone control (P, lane 0). Locations of the gel well (W), the different yeastprotein-triplex complexes (C0, C1*, C1, C2), the unbound triplex (T), and the unbound duplex (D) are indicated at the left. C1* and C1 correspondto the X-Pu complexes containing Stm1-HA3 and Stm1 proteins, respectively.

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proteins in our strains: e.g. galactose-dependent expression ofHA3-Stm1p (lanes 2 and 3), constitutive expression of Stm1-HA3 (lane 6), and an absence of untagged Stm1p in bothepitope-tagged strains (compare with lanes 1 and 4). Moreimportantly, the absolute levels of epitope-tagged Stm1p pres-ent in the strains were comparable with those of untagged,wild-type Stm1p in the parental strains, with HA3-Stm1p pres-ent at 170% and Stm1-HA3p present at 54% of the wild-typelevels. Western blot analysis was also performed with the an-ti-HA mAb (Fig. 1B). These data confirmed the presence of theHA3 epitope tag as well as the specificity of this antibody andits minimal cross-reactivity with other yeast proteins, as hasbeen observed by others (62).

The functional consequences of epitope-tagging STM1 werealso investigated. Both HA3-STM1 yeast and STM1-HA3 yeasthad growth comparable with their untagged parent strains onrich media containing either dextrose or galactose (data notshown). However, it is known that deletion of STM1 results ina minimal phenotype, especially when the yeasts were grownon rich media, thus limiting the importance of these observa-tions (63). As an additional measure of the functional capabil-ities of epitope-tagged Stm1p, we investigated the binding ofthese proteins to a purine motif triplex (Pu triplex) probe usingEMSA (32). We found that the appearance of a band corre-sponding to an Stm1p-Pu triplex complex coincided with Stm1pprotein expression (i.e. in the presence of galactose for HA3-Stm1p or being absent in the stm1-� extract) (Fig. 1C). Theapparent mobility of the epitope-tagged Stm1p complexes wasslightly more reduced than that of the wild-type untaggedStm1p-Pu triplex complex, as would be expected for the addi-tional mass of the epitope tags. Also, no untagged Stm1p-Putriplex complex was observed in any of the extracts containingepitope-tagged Stm1p. In addition, both epitope-tagged pro-teins yielded single complexes, indicating that their mode oftriplex binding was like that of wild-type, untagged Stm1p.Most importantly, the amount of Stm1p-Pu triplex complexobserved corresponded to the amount of Stm1p protein ex-pressed (Fig. 1, compare C and A), indicating that both HA3-Stm1p- and Stm1-HA3 are functional proteins, with regard totheir capacity to bind a Pu triplex. Finally, we had previouslyfound that yeast extracts contain at least two other activitiesthat bind Pu triplexes and that the levels of these complexesincreased �2-fold when the STM1 gene was deleted (32). Sim-ilar results were observed when HA3-Stm1p expression wasrepressed by growth in dextrose (Fig. 1C, lane 2). However,when HA3-Stm1p was expressed, these endogenous Pu triplex-binding activities were not overexpressed (lane 3), suggestingthat at the level of this response to Stm1p depletion, theepitope-tagged version of Stm1p sufficed. Note that an inter-mediate level of these endogenous triplex-binding proteins wasobserved with Stm1-HA3p (Fig. 1C, see lanes 4–6). We believethat this observation was the result of the reduced cellularconcentration of Stm1-HA3p, below a threshold for the biolog-ical response, and was not due to an incomplete functionality.

Stm1p Is Primarily a Cytoplasmic Protein—Given its veryhigh binding affinity for G4 quadruplex and Pu triplex DNAs invitro, Stm1p was initially thought to be a yeast nuclear protein(22, 32). This localization was supported by the presence of twoconsensus nuclear localization domains within the Stm1p pro-tein sequence (64, 65). However, nuclear localization was notfound with other predictive methods (66, 67). To understandwhy, we sought to empirically determine the cellular localiza-tion of Stm1-HA3p by immunofluorescence microscopy. Usingthe anti-HA mAb 16B12 and Cy3-conjugated anti-mouse IgGpolyclonal antibodies, Stm1-HA3 protein was observed as adiffuse, granular fluorescence throughout formaldehyde-fixed

yeast spheroplasts (Fig. 2, top panels, indicated in red). Most ofthis fluorescence was distributed within the cell cytoplasm,although a significant portion was also present in the nucleus,as evidenced by the overlap between Stm1-HA3 fluorescence(indicated in red) and the fluorescence of a DNA-binding dye,4�,6-diamidino-2-phenylindole (indicated in blue). No substan-tial fluorescence was observed in either untagged wild-type orstm1-� strains (data not shown). Also, no substantial changesin distribution were noted in the Stm1-HA3 fluorescence pat-tern for yeast in different phases of the cell cycle (e.g. see Fig.2, right panels, budding yeast in late mitosis).

Stm1p Is Associated with Ribosomal Proteins and RNAs—The apparent cytoplasmic preponderance of Stm1p was in dis-tinct contrast to its biochemically identified function as a G*Gmultiplex DNA-binding protein. However, Stm1p has also beenshown to bind RNA G4 quadruplexes (22), and G*G multiplexRNA-binding proteins are known to exist in higher eukaryotes(8, 39). Thus, to better understand the biological role of Stm1pin the cell cytoplasm, we sought to determine whether Stm1pwas associated with any proteins or RNAs in vivo. Conven-tional immunoprecipitation assays were performed using ex-tracts from STM1-HA3 yeast and the anti-HA mAb 16B12. Theprotein constituents of the immunoprecipitates were analyzedusing SDS-PAGE and Coomassie Blue staining, whereas theirnucleic acid constituents were analyzed using either nativeagarose or denaturing polyacrylamide gel electrophoresis andethidium bromide staining, depending on their size.

Using SDS-PAGE and Coomassie Blue staining, we observedmore than 20 protein bands that coimmunoprecipitate with theStm1-HA3 protein (Fig. 3, lane 4). These bands varied in inten-sity over a 5-fold range, from 50 to 250 ng of protein, andcorresponded to proteins with apparent molecular massesranging from 50 kDa to less than 12.4 kDa. No protein bandswere observed in a control reaction lacking the anti-HA anti-body (lane 3), and only the heavy and light chains of theanti-HA antibody were observed in immunoprecipitates fromwild-type yeast extracts (lane 2). All protein bands other thanthose from the antibody and Stm1-HA3p were completely lostwhen the immunoprecipitates were washed with 500 mM NaCl

FIG. 2. Stm1p resides primarily in the yeast cytoplasm. STM-HA3 yeast were examined by immunofluorescence microscopy. Stm1-HA3p, indicated by red fluorescence, was visualized using the anti-HA.11 primary antibody followed by Cy3-conjugated secondaryantibody (�-HA). Nuclear chromosomal DNA, indicated by blue fluores-cence, was visualized by its binding to 4�,6-diamidino-2-phenylindole(DAPI). DIC, differential interference contrast microscopy.

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(lane 5) and were partially lost when 15 mM EDTA was presentduring immunoprecipitation (lane 7). These additional proteinswere also lost when RNase A was present in the immunopre-cipitation buffer (lane 8) but not when DNase I was present(lane 6).

Given that the entire S. cerevisiae genome is known, wesought to identify proteins that co-immunoprecipitated withStm1-HA3p by peptide fingerprinting. Individual bands wereexcised from a Coomassie Blue-stained SDS-polyacrylamide geland digested in situ with trypsin, and the eluted peptides weresubjected to mass spectrometric analysis. Nine bands corre-sponding to molecular masses of 48, 38, 33, 29, 25, 20, 17, 15,and 13 kDa were analyzed. Since several of the bands con-tained more than one protein, we identified 18 proteins thatco-immunoprecipitated with Stm1-HA3p (Fig. 4). Most remark-ably, 16 of these were ribosomal proteins. Of these, 12 wereproteins residing in the large (60 S) ribosomal subunit, and fourwere proteins present in the small (40 S) ribosomal subunit. Inaddition, two other proteins were identified: Ykt6p, a proteininvolved in endoplasmic reticulum-Golgi transport, andYOR051C, an uncharacterized protein associated with the nu-clear pore complex (69).

Immunoprecipitating multiple proteins and RNase sensitiv-ity are strongly suggestive of RNA being present in the immu-noprecipitates. Using agarose gel electrophoresis and ethidiumbromide staining, we observed at least six nucleic acid bands inthe Stm1-HA3 protein immunoprecipitates (Fig. 5A, lane 5).These bands ranged in intensity more than 10-fold, and theycorresponded to nucleic acids with electrophoretic mobilitiesranging from 0.1- to 2.0-kbp double-stranded DNA. The twomain species had molecular sizes corresponding to 0.8 and 1.2kbp and were present in apparently equivalent amounts. Withdenaturing PAGE, we were able to resolve the lower, diffuseband into two species having electrophoretic mobilities compa-rable with 120- and 150-bp double-stranded DNA (Fig. 5B). Nonucleic acid bands were observed in the control reactions lack-

ing the anti-HA mAb (lane 4) or in the immunoprecipitatesfrom untagged wild-type yeast extracts (lane 3). Likewise, nonucleic acids were observed when immunoprecipitates werewashed with a high ionic strength buffer (500 mM NaCl). Allbands were lost in the immunoprecipitates treated with RNaseA (lane 9), but none were lost after treatment with DNase I(lane 7). In addition, a general decrease in the appearance ofthese nucleic acid bands was observed when 15 mM EDTA waspresent in the immunoprecipitation (Fig. 5A, lane 8), with thefastest mobility species being completely lost under these con-ditions (Fig. 5B, lane 8).

One of the most striking observations in our gel electro-phoretic analysis of nucleic acids that co-immunoprecipitatewith Stm1p was that several of these nucleic acids had inten-sities and mobilities identical to those present in a mixture ofwhole yeast RNAs consisting primarily of ribosomal and trans-fer RNAs (Fig. 5, A or B, compare lanes 7 and 12). This matchstrongly suggested that ribosomal RNAs, particularly the 25,18, 5.8, and 5 S rRNAs that constitute the structural RNA inribosomes, are the main nucleic acid constituents in Stm1pimmunoprecipitates. To confirm this supposition, we per-formed Northern blotting with radiolabeled rRNA-specific oli-gonucleotide probes. As shown in Fig. 6A, both the 25 and 18 SrRNAs were present in the Stm1-HA3p immunoprecipitates.For both the 25 and 18 S blots, slower mobility bands wereapparent in the whole yeast extract RNA samples but werepresent to a far lesser extent in the immunoprecipitates. Thefact that these bands corresponded to ribosomal RNA precur-sors suggests that Stm1p is primarily associated with maturerRNAs. Interestingly, several RNA species with apparent sizesbetween 1.5 and 2.5 kbp were observed by ethidium staining inthe Stm1p immunoprecipitates that were not present in thewhole yeast RNA (Fig. 5A, compare lanes 7 and 13). Thus, thisand the Northern blot data suggest that these bands are notimmature rRNA species but rather some other form of RNA.Finally, Northern blotting of the smaller rRNA detected thepresence of both the 5.8 S and the 5 S rRNAs in the Stm1pimmunoprecipitates (Fig. 6B). The 5 S rRNA was less wellimmunoprecipitated than the 5.8 S rRNA. This observation isconsistent with the unique lability of the 5 S rRNA when

FIG. 3. Stm1p is part of a multiprotein complex. Proteins asso-ciating with Stm1-HA3 were examined by immunoprecipitation withthe anti-HA.11 mAb (�-HA) and SDS-PAGE. Shown is a photograph ofa Coomassie Blue-stained gel. Immunoprecipitations were performedunder a variety of conditions, including substitution of a yeast extractfrom the untagged parent strain (wt) and incubations in the presence ofhigh ionic strength (500 Na), a divalent cation chelator (EDTA), and thenonspecific endonucleases DNase I and RNase A. Molecular masses ofthe marker proteins are indicated at the left. The identities of the IgGheavy and light chains and Stm1-HA3 protein are indicated at the right.

FIG. 4. Stm1p is associated with multiple ribosomal proteins.Proteins immunoprecipitated with Stm1-HA3 were resolved by SDS-PAGE, and their identities were determined by peptide fingerprinting.Molecular masses of the marker proteins are indicated at the left. The2 � 8-mm gel slices used for peptide fingerprinting are indicated byboxes. The identities of the IgG heavy (h) and light (l) chains, Stm1-HA3,and the Stm1-HA3-associated proteins are indicated at the right. Rpl,ribosomal protein, large subunit; Rps, ribosomal protein, small subunit.

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ribosomes are exposed to low concentrations of EDTA (70).These data also suggest that Stm1p does not interact withribosomal proteins and rRNAs solely through the 5 S rRNA orthrough ribosomal proteins that associate only with this rRNA.

Stm1p Is an 80 S Ribosome-associated Protein—Taken to-gether, our native immunoprecipitation data indicated that anantibody-accessible Stm1p is associated with one or more ribo-nucleoprotein complexes containing multiple proteins and ri-bosomal RNAs, such as would be found in mature forms of boththe large and the small ribosomal subunits. However, thesedata do not explicitly define what ribosomal species are asso-ciated with Stm1p; nor do they conclusively prove that Stm1pis a ribosome-associated protein. Ribosomes are an extremelyplentiful species in S. cerevisiae, with more than 200,000 cop-ies/cell, and they occupy 30–40% of the cytoplasmic volume(71). Given the basic nature of Stm1p (pI � 10.45) and the largepolyanionic component of ribosomes, it is possible that somedegree of nonspecific interactions occur during the incubationstep required for native immunoprecipitations. To determinewhether this interaction was artifactual and to identify theexact ribosomal species bound by Stm1p, we investigated con-ventionally purified ribosomes for the presence of Stm1p.Whole cell extracts were prepared from either cycloheximide-treated or untreated cells, the treated cells utilizing a transla-tion inhibitor that facilitates retention of polysomes. Theseextracts were subjected to velocity sedimentation through su-

crose gradients, which allowed resolution of individual riboso-mal species. Polysome profiles of these gradients were made bymeasuring sample absorbance at 254 nm during fractionation.Additional analyses included Stm1-HA3p identification byWestern blotting and triplex-binding activity by EMSA. Exam-ples of these analyses are shown in Fig. 7.

As shown by a trace of the UV absorbance (Fig. 7A), the small(40 S) and large (60 S) ribosomal subunits, as well as 80 Smonosomes and higher sedimentation coefficient polysomes,could be well resolved by sucrose gradient sedimentation. Abetter recovery of individual polysome species was obtainedwith cycloheximide-treated yeast (Fig. 7A, left panel), whereasin its absence, more monosomes were recovered (Fig. 7A, rightpanel). As shown by the Western blots with anti-HA mAbs,Stm1-HA3p was primarily found in 80 S monosome and poly-some fractions (Fig. 7B), with a small percentage present in the60 S ribosomal subunit fraction for the cycloheximide-treatedextracts. No Stm1-HA3p was present in any lower sedimenta-tion coefficient species, suggesting that in vivo most Stm1p isassociated with ribosomes or ribosomal subunits. Finally, weused EMSA to analyze these sucrose gradient fractions for Putriplex binding activities. As shown in Fig. 7D, complex C1*,which corresponded to the Pu triplex probe bound by Stm1-HA3p, was present primarily in the 80 S monosome fractions 9and 10 of the sucrose gradient from untreated cells. Thesefractions corresponded to those containing the most Stm-HA3p(Fig. 7B, right panel). Only a very small amount of C1* complexwas observed with the low sedimentation fractions (e.g. fraction1), which is consistent with the fact that most Stm1p is asso-ciated with ribosomes. Notably, the proteins responsible for thelow mobility complexes C0 and C0* were present only in theselow sedimentation fractions. However, a complex having anelectrophoretic mobility comparable with that of the yeast

FIG. 5. Stm1p is associated with multiple RNAs. RNAs associat-ing with Stm1-HA3 were examined by immunoprecipitation with theanti-HA.11 mAb (�-HA) and either nondenaturing agarose (A) or dena-turing polyacrylamide (B) gel electrophoresis. Shown are photographsof ethidium bromide-stained gels. Immunoprecipitations were per-formed under a variety of conditions, including substitution of extractfrom the untagged parent strain (wt) and incubations in the presence ofhigh ionic strength (500 Na), a divalent cation chelator (EDTA), and thenonspecific endonucleases DNase I and RNase A. Lengths of duplexDNA markers in A and single-stranded RNA markers in B are indicatedat the left. A titration of yeast RNAs (lanes 10–13) are included forquantitation purposes. The identities of the ribosomal RNAs (25S, 18S,and 5S) and tRNA are indicated at the right.

FIG. 6. Stm1p is associated with multiple ribosomal RNAs.RNAs that coimmunoprecipitate with Stm1-HA3 were resolved by ei-ther denaturing agarose (A) or polyacrylamide gel electrophoresis (B),and their identities were determined by Northern blotting. RNA-spe-cific probes used are indicated at the left of each autoradiogram. Inputmaterial includes RNAs that coimmunoprecipitate in the presence of aMg2�-containing buffer (M) or EDTA-containing buffer (E) buffer and atitration of total yeast RNA for comparison.

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whole cell extract C2 complex (see Fig. 1C) was obtained withfractions corresponding to the 60 S large ribosomal subunit, 80S monosomes, and polysomes. These data are consistent withthe fact that the protein responsible for the C2 Pu triplexcomplex is associated with the large ribosomal subunit.

Stm1p Associates Specifically with Y� Element SequencesProximal to Yeast Telomeres—Although most Stm1p is appar-ently located in the cytoplasm (Fig. 2) and is associated withribosomes (Fig. 7), there also is a fraction of Stm1p visible inthe nucleus (Fig. 2). Stm1p has been shown to specifically andavidly interact with both RNA- and DNA-containing G*G mul-tiplex structures in vitro (22, 32). Thus, to address whetherStm1p interacts with genomic DNA sequences in vivo, we per-formed ChIPs with HA3-STM1 yeast. In this technique, DNA-binding proteins can be cross-linked to their cognate DNAbinding sites during a brief exposure of live yeast to formalde-hyde (72). The cross-link between protein and DNA facilitatesrecovery of the DNA site during immunoprecipitation of theDNA-binding protein. PCR or probe hybridization is typicallyused to analyze the recovered DNA sequences. For our purpose,probe hybridization was preferable, since most of the G/C-richgenomic regions we were initially interested in examining re-

side in naturally repetitive sequences, which are more difficultto analyze by PCR.

The specificity of our ChIP and probe hybridization analysiswas first determined by ChIP using the control yeast strainYKU80-myc18 or its isogenic untagged parent (Fig. 8, rightcolumn, and Table I). Yku80p and Hdf1p comprise the yeastKu70/Ku80 heterodimer, a well known DNA end-binding com-plex present at the yeast telomere (73). We observed thatYku80-myc18 preferentially associates with TG1–3 telomeretracts (Fig. 8A) and with telomere-proximal regions of thesubtelomeric Y� element (Table I). “-Fold enhanced association”values were calculated by normalizing specific hybridizationsignals against nonspecific background hybridization signal. Inthe first of two controls to assess nonspecific hybridization,primary antibody (�-myc) was not added during immunopre-cipitation of Yku80-myc18 extracts. In the second backgroundcontrol, ChIP was performed on the untagged isogenic parentstrain to assess dependence on the epitope-tagged protein.Higher backgrounds were consistently observed in the mockChIP experiments lacking primary antibody. Consequently, all-fold enhanced association values were calculated normalizingto this background control. Thus, Yku80-myc18 was found to

FIG. 7. Stm1p is associated with 80S ribosomes and polysomes. Yeast were treated either with (left panels) or without (right panels) thetranslation inhibitor cycloheximide (CHX), and their ribosomes were fractionated on a 7–47% sucrose gradient and scanned for absorbance at 254nm (A254). A, UV absorbance trace of the fractionated gradient. Locations of different ribosomal species (40 and 60 S subunits, 80 S monosomes,and polysomes) are indicated above each trace. Fraction numbers are indicated below. B, Western blots of SDS-PAGE-separated Stm1-HA3ppresent in each fraction probed with anti-HA.11 mAb. C, EMSA analysis of triplex binding activities present in sucrose gradient fractions fromuntreated yeast. Fraction numbers are indicated below. Locations of the gel well (W), the different yeast protein-triplex complexes (C0, C0*, C1*,and C2), the unbound triplex (T), and the unbound duplex (D) are indicated at the left. C1* corresponds to the X-PuT19 complex containingStm1-HA3p.

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exhibit 30- and 22-fold enhanced association for telomere andtelomere-proximal Y� element sequences. Probing sequencesprogressively further from the telomere, we did not detect asubstantial association of Yku80-myc18 at the telomere-distalend of the Y� element or at the subtelomeric X element. Asexpected, we did not observe any Yku80-myc18 association withARS or with randomly distributed sequences throughout the

yeast genome (Table I). A somewhat unexpected finding wasthe association of Yku80-myc18 with certain rDNA sequences(Fig. 8B). However, taken as a whole, our analysis of the controlYKU80-myc18 strain suggests that the specificity of our ChIPand probe hybridization analysis was quite reasonable.

ChIP experiments with HA3-STM1 yeast and its isogenicuntagged parent strain were performed in parallel with thecontrol YKU80 strains. The recovered DNA was blotted to thesame nitrocellulose membranes to directly compare hybridiza-tion signal and background levels between the Yku80 controlstrain and the Stm1 experimental strain. We observed HA3-Stm1p preferentially associated with the telomere-proximal Y�element (23-fold) and to a lesser extent with the telomere-distalY� element (6-fold) as well as with terminal TG1–3 telomerictracts (8.5-fold) (Fig. 8A, Table I). However, our analysis re-vealed that a lower but significant level (3-fold) of HA3-Stm1pwas associated with every genomic DNA sequence we tested. Inaddition to 3-fold enhanced association dispersed randomlythroughout S. cerevisiae genomic DNA, we observed 2–5-foldenhanced association with several specific regions includingARS, subtelomeric X element, and certain rDNA regions (TableI). This contrasts with the specificity of Yku80p and suggeststhat either HA3-Stm1p associates globally and relatively non-specifically with the bulk of yeast genomic DNA or that underour conditions there is a propensity for HA3-Stm1p to nonspe-cifically interact with genomic DNA that is not biologicallyrelevant. In this regard, it is worthwhile to note that Yku80p isnot a sequence-specific DNA-binding protein but rather bindsDNA ends and some unusual DNA junctions (74), making it aparticularly useful control for comparison with Stm1p. How-ever, in contrast to Stm1p, Yku80p exhibited specific associa-tion in our analysis similar to what has been reported previ-ously (73). Whereas we have not determined the basis for thisglobal or nonspecific binding, HA3-Stm1p immunoprecipitateswere still enriched in telomere-proximal Y� element sequencesby 7.7-fold relative to this elevated background. In addition,significant amounts of 25 S rDNA (21-fold) were associatedwith HA3-Stm1p but not neighboring 5 S rDNA (3.2-fold) andETS (2.5-fold) sequences. However, one caveat for the apparentassociation with 25 S rDNA is the aforementioned Stm1p as-sociation with ribosomes. Whereas exhaustive RNase treat-ment is used in the ChIP protocol, intact 80 S ribosomes arerather RNase-resistant species, and the 25 S rRNA present inStm1p-immunoprecipitating ribosomes could provide the ob-served enhancement of hybridization for 25 S rDNA.

DISCUSSION

Stm1p is a moderately abundant (35,000–46,800 proteins/cell) S. cerevisiae protein that has been investigated for avariety of reasons over the last 10 years. Originally identifiedby biochemical means as one of the primary G4 quadruplexnucleic acid-binding proteins in yeast whole cell extracts,Stm1p or its corresponding gene has been identified in a num-ber of genetic or biochemical screens. Although these studieshave implicated Stm1p in several biological processes, rangingfrom apoptosis to telomere maintenance, they have not conclu-sively determined the molecular functions of Stm1p or itsbiological roles. With the development of multiple highthroughput technologies and their applications in studies ofS. cerevisiae, there now exists a wealth of information regard-ing almost every yeast protein, including Stm1p. Thus, we areat a point where the actual role of Stm1p can now be betterdetermined.

Using immunofluorescence microscopy, we found that Stm1-HA3p was primarily localized in the yeast cell cytoplasm, ex-hibiting a granular pattern of immunofluorescence, with asmall percentage of the Stm1-HA3p also present in the cell

FIG. 8. Stm1p associates with subtelomeric Y� DNA. ChIPs wereperformed with HA3-STM1 yeast and its untagged parental strainusing anti-HA.11 antibody (�-HA, left column of spots). For comparison,a control ChIP was performed with YKU80-myc18 yeast and its un-tagged parental strain using anti-myc 9E11 antibody (�-myc, rightcolumn of spots). The ChIP samples and dilutions representing 1.0 and0.1% of the of the corresponding cell extract used for immunoprecipita-tion (“input”) were applied to a nitrocellulose membrane and hybridizedwith several different probes, including a telomere probe (A) and anrDNA probe (B) (25S/1, 25S/2, 5S, ETS, and NTS, as described under“Experimental Procedures.” To ascertain a relative measure of -foldenhanced chromatin immunoprecipitation, two controls were includedto evaluate specificity and background levels. First, we performed amock ChIP (ChIP �Ab) in which the appropriate primary antibody wasomitted from an otherwise standard chromatin immunoprecipitationusing extract prepared from the epitope-tagged strains. Second, weperformed a standard chromatin immunoprecipitation with primaryantibody on extracts of the untagged isogenic parent strains. In allcases, higher background hybridizations were observed with the for-mer, ChIP �Ab control experiment. Therefore, we used this control asthe background reference point to calculate the -fold enhanced chroma-tin immunoprecipitations as shown in Table I.

TABLE IChromatin immunoprecipitations

Genomic regiona HA3-Stm1 Yku80-myc18

Telomere 8.5b 30.0Subtelomere: Y� element telomere proximal 23.0 22.0Subtelomere: Y� element telomere distal 6.0 1.0Subtelomere: X element 2.8 1.0rDNA: 25S/1, 25S/2, 5S, ETS, NTS 6.8 6.7rDNA: 25S/1 21.0 1.0rDNA: 5S 3.2 4.0rDNA: ETS 2.5 1.0ARS: 1, 305, 306, 501, 603 4.2 1.0Random S. cerevisiae genomic probes 3.0 1.0

a Probes used to detect these genomic regions are described under‘‘Experimental Procedures.’’

b Values shown correspond to the amount of hybridization observedfor appropriate specific antibody immunoprecipitations (e.g., ChIP�-HA for HA3-Stm1p) divided by hybridization levels observed for mockimmunoprecipitations (ChIP�Ab).

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nucleus. Our results concur nicely with those obtained in agenome-wide transposon-tagging survey, in which a C-termi-nal truncated Stm1(�178–273)p with a 93-amino acid HAT tagyielded a cytoplasmic/nuclear granular staining pattern of im-munofluorescence with moderate intensity (75). Similar resultswere obtained using direct fluorescence microscopy with a full-length Stm1p bearing a C-terminal Aequorea victoria greenfluorescent protein, which showed a primarily cytoplasmic lo-calization (76). These data, however, contrast with those of Ligret al. (48), who found a predominantly perinuclear localizationof Stm1p C-terminally tagged with a single IRS epitope. Theyfurther determined through chromosome spreading experi-ments that Stm1p may also be directly associated with thechromatin that is present on the periphery of nucleolids. Thereasons for this difference in the subcellular location betweenthe work of Ligr et al. and others is not known but may reflectthe choice of epitope tag or yeast strain used. In all cases, thesetagged proteins were expressed from the endogenous STM1promoter and from integrants at the normal chromosome XIIlocation. Regardless, the consensus is that Stm1p is primarilya cytoplasmic protein, although some nuclear involvement can-not be completely ruled out.

Using conventional immunoprecipitation, we found thatStm1p was primarily associated with ribosomal proteins andrRNAs. This result was confirmed by fractionation of solubleyeast proteins using sucrose gradient ultracentrifugation andWestern blotting, which found almost all of the Stm1-HA3p tobe primarily associated with 80 S monosomes and polysomes.Such results were not observed in a systematic analysis of S.cerevisiae protein complexes using tandem affinity purification,which found Stm1p present in three complexes obtained withTAP-tagged Sec31p, Las17p, or Hek2p (77). Interestingly, boththe TAP-tagged Sec31p and Hek2p complexes contained someribosomal proteins and/or translation initiation proteins. How-ever, these observations most likely reflect the focus of theseinvestigations, which were directed toward identifying proteinsin 100–900-kDa complexes (78). Stm1p has been identified asan abundant, salt-labile ribosome-associated protein using anoverexpressed C-terminal FLAG-His6 epitope-tagged riboso-mal protein Rpl25p and immunoaffinity purification (79). How-ever, these authors did not determine which ribosomal specieswere associated with Stm1p or that most soluble Stm1p isribosome-associated. Likewise, an Stm1p-related protein,Ray38p, was found to be released from ribosomes isolated fromcycloheximide-treated wild-type Candida maltosa yeast (80).The association of Ray38p with ribosomes was verified by sizeexclusion chromatography and Western blotting, although theidentity of the exact ribosomal species was not determined.Note that we also examined the association of Stm1p withribosomes isolated from cycloheximide-treated S. cerevisiaecells and found that although polysome integrity was main-tained as expected, little to no Stm1p was released.

Using chromatin immunoprecipitation, we found that HA3-Stm1p preferentially associated with telomere-proximal Y� el-ement sequences. Some prior evidence had suggested thatStm1p might interact with the ends of chromosomes. The afore-mentioned chromosome spreading data of Ligr et al. (48) isconsistent with a telomeric localization for Stm1p, given thattelomeres are overrepresented on the periphery of nucleolids(81). Ito et al. (82) documented a two-hybrid interaction be-tween a MEC3 bait and an STM1 prey in a comprehensivetwo-hybrid analysis of yeast protein-protein interactions.Mec3p is a DNA-binding protein involved in DNA damage-de-pendent checkpoints, telomere silencing, and telomere lengthmaintenance (83). Further evidence was obtained in a directedtwo-hybrid screen using CDC13 as bait, which isolated a single

STM1 clone out of 17,000 total colonies (49). Cdc13p is a te-lomere-binding protein specific for (TG1–3)n single-strandedDNA and is directly involved in regulating the replication oftelomeres and protecting the integrity of telomeres (84). Thephysical interaction between the N-terminal portion of Cdc13pand Stm1p was verified using a glutathione S-transferase pull-down experiment (49). In addition, STM1 in multicopy par-tially suppressed the temperature-sensitive growth and longtelomere phenotypes of cdc13-1 yeast but failed to suppress theelevated level of single-stranded G-rich tails. Interestingly, afusion between STM1 and the N terminus of CDC13, whichlacks the DNA binding domain required for Cdc13p function(85), complemented the viability of cdc13-� yeast, albeit growthwas weak (49). Last, the partial suppression of cdc13-1 tem-perature sensitivity by STM1 multicopy expression was abol-ished by multicopy expression of Sgs1p, a primarily nucleolarRecQ family helicase that is involved in maintaining genomestability and is of special interest because of its ability tounwind G-quadruplex and Pu-triplex DNAs (16).2

Genetic analyses have proven a highly powerful means ofelucidating the biological functions of proteins in S. cerevisiae,and STM1 has appeared in several genetic screens. STM1 hasbeen identified as a multicopy suppressor of temperature-sen-sitive tom1 and htr1 mutants (63), staurosporine-sensitivepop2, ccr4, and pkc1 mutants, and a caffeine-sensitive mpt5mutant (52). None of these genes have an obvious direct rela-tionship with either ribosome or telomere function. STM1 hasmore recently been identified in a screen for yeast cDNAswhose overexpression caused growth arrest in proteasome-de-fective pre1-1 pre4-1 cells in a fashion consistent with an apo-ptosis-like cell death (48). STM1 deletion has been reported tohave a minimal phenotype (52, 63), although subsequent in-vestigators found significant growth defects for stm1-�1 yeastwhen grown in the presence of caffeine, following UV irradia-tion, or when grown at elevated temperatures in the presenceof the radiomimetic drug bleomycin, but not when grown withstaurosporine or the alkylating agent methyl methanesulfon-ate (48). Most recently, STM1 was identified in a large scalesynthetic genetic array analysis with deletions of the querygenes CTF4 and KRE9, genes that are involved in sister chro-matid cohesion and cell surface (136)-�-glucan assembly, re-spectively (87). Taken together, these diverse genetic data sug-gest that Stm1p is involved in multiple, seemingly unrelated,biological processes. Alternatively, they may also reflect theinvolvement of Stm1p in a very general process (e.g. transcrip-tion or translation) that impinges on many more specific bio-logical processes.

Complete sequencing of the S. cerevisiae genome, coupledwith advances in microarray technology, has allowed the faciledetermination of genome-wide changes in gene expression as afunction of different genotypic changes or environmental stim-uli. Changes in Stm1 mRNA levels have been observed in manyof these (88). Potentially more useful is the ability to identifyyeast genes whose expression pattern mirrors that of STM1 fora given stimulus, since such coordinate regulation can be in-dicative of a shared function. Only a few of the microarraystudies are easily amenable to comparative analysis, and ofthese only a subset demonstrated significant changes in STM1expression (89). However, among these studies, including ex-pression changes in response to DNA-damaging agents (90),environmental changes (91), varying zinc levels (92), histonedepletion (93), the diauxic shift (94), and sporulation (29), 62 ofthe 113 genes reported as having expression profiles similar tothat of STM1 were known structural components of the ribo-some, with the next most commonly represented genes beingeither those of unknown function (9 of 113) or those involved in

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translation (6 of 113). This coordinate regulation was mostaptly demonstrated in two studies, response to DNA-damagingagents and environmental changes, which reported 32 of the 34genes that responded like STM1 as those encoding ribosomalproteins and the remaining genes being either involved intranslation or of unknown function. Taken together, these datastrongly suggest that Stm1p is involved in either ribosomestructure or function.

What is the actual role of Stm1p in yeast, and does it involveinteraction with G*G multiplex nucleic acids? Stm1p associa-tion with the ribosome strongly suggests a role in translation oran associated process (e.g. protein folding or export). In addi-tion, the large ribosomal RNAs contain multiple G-richstretches that have the potential for G*G multiplex formation.Telomere-proximal Y� element DNA sequences include a regionof 36-bp repeats, an ARS consensus sequence, and multipleTbf1p-binding sites (86). Many of these sequences are G/T-richand have the potential for interesting DNA structures. Theexact role of these sequences is unknown. Nonetheless, theinteraction of Stm1p with both RNA- and DNA-containing mac-romolecular complexes is intriguing and suggests a possibletype of communication between protein synthesis and variousDNA-dependent phenomena. We hypothesize that under nor-mal growth conditions, Stm1p is primarily associated withfunctional ribosomes, where it plays an ancillary role in theprocess of translation. A small fraction of the cellular Stm1p isnormally associated with chromosomal DNA, particularly sub-telomeric sequences, where it may be involved in some aspect oftelomere biosynthesis. However, under circumstances of stress,especially stresses that compromise ribosome function, theStm1p binding equilibrium will be shifted toward higher con-centrations of free protein. If not degraded through the proteo-somal pathway, these free Stm1p can then promiscuously in-teract with DNA and impede various DNA template-dependentprocesses. Given effective checkpoint controls, these impededprocesses could result in the delay of cell cycle progression.Such a delay could be used to rectify conditions of stress and/orto respond to their consequences (e.g. through postreplicativerepair or sporulation, both of which have been shown to involveG*G multiplex-acting proteins) (31, 68).3 However, under cir-cumstances where this delay cannot be achieved, or if sufficientconcentrations of Stm1p are present, events may then tran-spire resulting in an apoptosis-like death. Determining theprecise Stm1p-binding sites on both ribosomes and subtelo-meric Y� DNA as well as experiments directed toward elucidat-ing the functions of Stm1p in translation and DNA-dependentprocesses under normal and stress conditions should ulti-mately aid in understanding of the biological roles of Stm1p.

Acknowledgments—We thank John Pringle for the pFA6a series ofplasmids used in making the deletion and epitope-tagged STM1 strains,Christian Koch for S. cerevisiae strain K699, and A. Bertuch and V.Lundblad for strain YKU80-myc18. We also thank Mary Ann Gawinow-icz, Natasha Van Dyke, and Miles Wilkerson for assistance and com-ments and the Saccharomyces Genome Database for its wealth of freelyaccessible information.

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Michael W. Van Dyke, Laura D. Nelson, Rodney G. Weilbaecher and Dakshesh V. MehtaSaccharomyces cerevisiae DNA in ′Interacts with Ribosomes and Subtelomeric Y

Quadruplex and Purine Motif Triplex Nucleic Acid-binding Protein,4Stm1p, a G

doi: 10.1074/jbc.M401981200 originally published online March 23, 20042004, 279:24323-24333.J. Biol. Chem. 

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