Recruitment of the RNA Helicase RHAU to Stress Granules via a Unique RNA-binding Domain
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m804857200
ISSN1083-351X
AutoresKateřina Chalupníková, Simon Lattmann, Nives Selak, Fumiko Iwamoto, Yukio Fujiki, Yoshikuni Nagamine,
Tópico(s)RNA and protein synthesis mechanisms
ResumoIn response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain. In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain. Posttranscriptional regulation of gene expression is important and highly regulated in response to developmental, environmental, and metabolic signals (1.Garneau N.L. Wilusz J. Wilusz C.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 113-126Crossref PubMed Scopus (927) Google Scholar). During stress conditions such as heat shock, oxidative stress, ischemia, or viral infection, mRNA translation is reprogrammed and allows the selective synthesis of stress response and repair proteins (2.Anderson P. Kedersha N. J. Cell Sci. 2002; 115: 3227-3234Crossref PubMed Google Scholar). Under these conditions, the translation of housekeeping genes is arrested, and untranslated mRNAs accumulate in cytoplasmic foci known as stress granules (SGs) (3.Anderson P. Kedersha N. Cell Stress Chaperones. 2002; 7: 213-221Crossref PubMed Scopus (210) Google Scholar). SG 3The abbreviations used are: SG, stress granule; RNP, ribonucleoprotein; FRAP, fluorescence recovery after photobleaching; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLIP, cross-linking immunoprecipitation; ARE, AU-rich element; RSM, RHAU-specific motif; uPA, urokinase-type plasminogen activator; PABP-1, poly(A)-binding protein 1; ZBP1, zipcode-binding protein 1; aa, amino acids; TTP, tristetraprolin; HuR, ELAV-like protein 1 (Hu-antigen R); FMRP, Fragile X mental retardation; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GST, glutathione S-transferase; RISP, RNA-Interaction Site Prediction; ROI, regions of interest; EGFP, enhanced green fluorescent protein; Nter, N terminus; HCR, helicase core region; Cter, C terminus; WT, full-length RHAU (wild type); β-gal, β-galactosidase.3The abbreviations used are: SG, stress granule; RNP, ribonucleoprotein; FRAP, fluorescence recovery after photobleaching; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLIP, cross-linking immunoprecipitation; ARE, AU-rich element; RSM, RHAU-specific motif; uPA, urokinase-type plasminogen activator; PABP-1, poly(A)-binding protein 1; ZBP1, zipcode-binding protein 1; aa, amino acids; TTP, tristetraprolin; HuR, ELAV-like protein 1 (Hu-antigen R); FMRP, Fragile X mental retardation; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; GST, glutathione S-transferase; RISP, RNA-Interaction Site Prediction; ROI, regions of interest; EGFP, enhanced green fluorescent protein; Nter, N terminus; HCR, helicase core region; Cter, C terminus; WT, full-length RHAU (wild type); β-gal, β-galactosidase. formation is triggered by translation initiation arrest involving eIF2α phosphorylation or by inhibition of eIF4A helicase function. Phosphorylation of eIF2α can be mediated by several stress-induced protein kinases (for a review, see Ref. 4.Anderson P. Kedersha N. J. Cell Biol. 2006; 172: 803-808Crossref PubMed Scopus (846) Google Scholar), suggesting that the formation of SGs is highly regulated and that eIF2α plays a pivotal role in sensing stress. Upon translational arrest, polysome-free 48 S preinitiation complexes containing initiation factors, small ribosomal subunits, and poly(A)-binding protein 1 (PABP-1) aggregate into SGs (3.Anderson P. Kedersha N. Cell Stress Chaperones. 2002; 7: 213-221Crossref PubMed Scopus (210) Google Scholar, 5.Kedersha N. Chen S. Gilks N. Li W. Miller I.J. Stahl J. Anderson P. Mol. Biol. Cell. 2002; 13: 195-210Crossref PubMed Scopus (427) Google Scholar). Several known SG-associated mRNA-binding proteins have been identified and shown to induce or inhibit SG aggregation when overexpressed. It is presumed that the overexpression of mRNA-binding proteins, which are able to oligomerize, disturb the equilibrium of mRNA distribution between polysomes and polysome-free ribonucleoprotein (RNP) complexes and thus induce SG formation by their aggregation (6.Kedersha N. Stoecklin G. Ayodele M. Yacono P. Lykke-Andersen J. Fritzler M.J. Scheuner D. Kaufman R.J. Golan D.E. Anderson P. J. Cell Biol. 2005; 169: 871-884Crossref PubMed Scopus (1023) Google Scholar). Nevertheless some RNA-binding proteins do not induce SG formation upon overexpression, e.g. a zipcode-binding protein 1 (ZBP1), heterogeneous nuclear RNP A1, or PABP-1 (7.Stohr N. Lederer M. Reinke C. Meyer S. Hatzfeld M. Singer R.H. Huttelmaier S. J. Cell Biol. 2006; 175: 527-534Crossref PubMed Scopus (138) Google Scholar, 8.Kedersha N.L. Gupta M. Li W. Miller I. Anderson P. J. Cell Biol. 1999; 147: 1431-1442Crossref PubMed Scopus (876) Google Scholar, 9.Guil S. Long J.C. Caceres J.F. Mol. Cell. Biol. 2006; 26: 5744-5758Crossref PubMed Scopus (234) Google Scholar). Under normal conditions, most RNA-binding proteins are involved in various aspects of mRNA metabolism, such as translation (TIA-1, TIA-1-related protein, PCBP2, Pumilio 2, and cytoplasmic polyadenylation element-binding protein), degradation (G3BP, TTP, Brf1, rck/p54, KH domain RNA binding protein [KSRP], and PMR1), stability (HuR), and specific intracellular localization (ZBP1, Staufen, Smaug, Caprin-1, and FMRP) (for a review, see Ref. 10.Anderson P. Kedersha N. Trends Biochem. Sci. 2008; 33: 141-150Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar). The differing flux of these SG-associated proteins and poly(A)+ mRNAs revealed by fluorescent recovery after photobleaching (FRAP) analysis suggests that SGs are dynamic foci (6.Kedersha N. Stoecklin G. Ayodele M. Yacono P. Lykke-Andersen J. Fritzler M.J. Scheuner D. Kaufman R.J. Golan D.E. Anderson P. J. 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Lykke-Andersen J. Fritzler M.J. Scheuner D. Kaufman R.J. Golan D.E. Anderson P. J. Cell Biol. 2005; 169: 871-884Crossref PubMed Scopus (1023) Google Scholar).Immediately after transcription, RNA forms with RNA-binding proteins RNPs that are dynamic and take part in RNA metabolism. RNP remodeling, which is essential for the cellular localization, processing, function, and fate of RNA (15.Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell Biol. 2002; 3: 195-205Crossref PubMed Scopus (1104) Google Scholar), is mainly regulated by a large family of proteins called RNA helicases. These enzymes use energy released by ATP hydrolysis to unwind secondary structures of RNA or displace proteins from RNA (16.Linder P. Nucleic Acids Res. 2006; 34: 4168-4180Crossref PubMed Scopus (348) Google Scholar). The majority of RNA helicases are assigned to superfamily 2 (SF2), which is divided into three subfamilies named after the sequence in the helicase motif II: DEAD, DEAH, and DEXH (17.Jankowsky E. Jankowsky A. Nucleic Acids Res. 2000; 28: 333-334Crossref PubMed Scopus (40) Google Scholar). Several DEX(H/D) proteins have been shown to unwind double-stranded RNA in an ATP-dependent manner in vitro (16.Linder P. Nucleic Acids Res. 2006; 34: 4168-4180Crossref PubMed Scopus (348) Google Scholar, 18.Jankowsky E. Gross C.H. Shuman S. Pyle A.M. Science. 2001; 291: 121-125Crossref PubMed Scopus (252) Google Scholar), but most are involved in the ATP-dependent remodeling of RNPs (16.Linder P. Nucleic Acids Res. 2006; 34: 4168-4180Crossref PubMed Scopus (348) Google Scholar). Although RNA helicases contain a highly conserved helicase core region, they are involved in all of the RNA processes ranging from transcription, pre-mRNA splicing, ribosome biogenesis, RNA export, and translation initiation to RNA decay (for reviews, see Refs. 19.Bleichert F. Baserga S.J. Mol. Cell. 2007; 27: 339-352Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 20.Abdelhaleem M. Maltais L. 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RNA. 1998; 4: 1216-1229Crossref PubMed Scopus (65) Google Scholar).RHAU is a DEAH box helicase (DHX36) originally identified as an RNA helicase associated with AU-rich element (ARE) of urokinase-type plasminogen activator (uPA) mRNA together with NF90 and HuR (26.Tran H. Schilling M. Wirbelauer C. Hess D. Nagamine Y. Mol. Cell. 2004; 13: 101-111Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). RHAU is a nucleocytoplasmic shuttling protein found predominantly in the nucleus and to a lesser extent in the cytoplasm. As with other helicases (27.Askjaer P. Rosendahl R. Kjems J. J. Biol. Chem. 2000; 275: 11561-11568Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 28.Schmitt C. von Kobbe C. Bachi A. Pante N. Rodrigues J.P. Boscheron C. Rigaut G. Wilm M. Seraphin B. Carmo-Fonseca M. Izaurralde E. EMBO J. 1999; 18: 4332-4347Crossref PubMed Scopus (220) Google Scholar, 29.Wagner J.D. Jankowsky E. Company M. Pyle A.M. Abelson J.N. EMBO J. 1998; 17: 2926-2937Crossref PubMed Scopus (126) Google Scholar), ATPase activity is necessary for RHAU function in the decay of uPA mRNA and for its nuclear localization (26.Tran H. Schilling M. Wirbelauer C. Hess D. Nagamine Y. Mol. Cell. 2004; 13: 101-111Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar). Despite the role of RHAU as a factor destabilizing uPA mRNA, global analysis of gene expression in RHAU knockdown cells revealed that changes in steady-state levels of mRNAs were only partially influenced by mRNA decay regulation (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar). Indeed the nuclear localization of RHAU and its guanine quadruplex (G4) DNA/RNA-resolving activity (31.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) may reflect that RHAU regulates gene expression at various steps other than mRNA decay.Given that SGs are sites at which dynamic changes in protein-RNA interaction determine the fate of mRNAs during and after stress, it is surprising that the role of SG-associated RNA helicases with an essential function in remodeling protein-RNA interactions is largely unknown. Here we show that RHAU is a novel component of SGs and that its recruitment to SGs is mediated by an RNA interaction. Although identified as an ARE-associated protein involved in ARE-mediated mRNA decay of uPA, it was not immediately clear whether RHAU directly binds RNA. Here we show that RHAU physically interacts with RNA in vitro and in vivo via a unique N-terminal RNA-binding domain composed of a G-rich region and an RHAU-specific motif (RSM) that is highly conserved between RHAU orthologs. The same RNA-binding domain is necessary and sufficient for RHAU recruitment to SGs. Finally we show that the ATPase activity of RHAU is involved in the dynamic regulation of RHAU shuttling into and out of SGs.EXPERIMENTAL PROCEDURESPlasmid Constructs—The plasmids pTER-shRHAU and pTER-shLuc were described previously (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar). Plasmids EGFP-RHAU, EGFP-Nter, EGFP-HCR, and EGFP-Cter were based on pEGFP-C1 (Clontech) and EGFP-E335A (also termed DAIH in this study), which was derived from EGFP-RHAU by point mutation in motif II as described previously (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar). Plasmids RHAU-EGFP, (50-1008)-EGFP, (105-1008)-EGFP, (1-130)-EGFP, and (1-105)-EGFP were based on pEGFP-N1 (Clontech) by inserting corresponding fragments into the BglII (XhoI for RHAU full length) and AgeI sites of the vector. Plasmids EGFP-(50-1008) and EGFP-(105-1008) were prepared by inserting the corresponding fragments between BglII and BamHI sites of pEGFP-C1. RHAU-FLAG, (50-1008)-FLAG, (105-1008)-FLAG, (1-105)-FLAG, and (1-130)-FLAG were prepared by inserting corresponding RHAU fragments into the BglII (NheI for RHAU full length) and AgeI sites of pIRES.EGFP-N1-FLAG. The vector was prepared by insertion of IRES.EGFP between AgeI and NotI of pEGFP-N1 (Clontech). The IRES.EGFP insert was designed by PCR using the pIRES.ECMV-EGFP vector, which was kindly provided by D. Schmitz Rohmer and B. A. Hemmings, as a template. The PCR product of the IRES.EGFP insert contained FLAG sequence with the AgeI site on the 5′ end and the NotI site on the 3′ end. Plasmids β-gal-(1-52)-EGFP, β-gal-(1-130)-EGFP, and β-gal-(52-200)-EGFP were prepared by inserting corresponding RHAU fragments into EcoRI and SalI sites of pβ-gal-EGFP. 4F. Iwamoto and Y. Fujiki, unpublished data. The GST-RHAU vector was designed as described previously (26.Tran H. Schilling M. Wirbelauer C. Hess D. Nagamine Y. Mol. Cell. 2004; 13: 101-111Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). GST-Nter was prepared by inserting the fragment (1-200 aa) between BamHI and EcoRI sites of pGEX-2T (Amersham Biosciences). The oligonucleotides used in this work and detailed descriptions of the plasmid construction are available upon request.Antibodies—Mouse monoclonal anti-RHAU antibody (12F33) was generated against a peptide corresponding to the C terminus of RHAU, 991-1007 aa, as described previously (31.Vaughn J.P. Creacy S.D. Routh E.D. Joyner-Butt C. Jenkins G.S. Pauli S. Nagamine Y. Akman S.A. J. Biol. Chem. 2005; 280: 38117-38120Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Commercially obtained antibodies were: mouse anti-green fluorescent protein (B-2, sc-9996), goat anti-TIA-1 (sc-1751), goat anti-eIF3b (N-20, sc-16377), mouse anti-HuR (3A2, sc-5261), and rabbit anti-eIF2α (FL-315, sc-11386) (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-actin (pan Ab-5, Thermo Fisher Scientific, Fremont, CA); rabbit monoclonal anti-eIF2α-P (Ser-51, 119A11, Cell Signaling Technology, Danvers, MA); and mouse anti-FLAG M2 (Sigma-Aldrich). The mouse antibodies were all monoclonal.Cell Culture, Transfection, and Stress Treatments—HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in the presence of 5% CO2. T-REx-HeLa cells stably transfected with pTER-shRHAU were maintained as described previously (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar). To induce short hairpin RNA expression and consequent depletion of endogenous RHAU, cells were treated with 1 μg/ml doxycycline (Sigma-Aldrich) for 7 days as described in supplemental Fig. 1. For immunofluorescence analysis, transient transfection of DNA plasmids using FuGENE 6 (Roche Applied Science) was performed according to the manual provided using 1 μg of plasmid DNA and 3 μl of FuGENE 6/35-mm dish. For immunoprecipitation analysis, cells were transfected by Lipofectamine 2000 (Invitrogen) in Opti-MEM I medium (Invitrogen). Briefly HeLa cells were seeded at 0.8 × 106 cells/35-mm dish and 24 h later were transfected with 4 μg of plasmid DNA and 10 μl of Lipofectamine 2000. The cells were used 24 h later for the following experiments. RNA interference silencing was induced by transient transfection of small interfering RNAs with INTERFERin (Polyplus Transfection, New York, NY) following the manual instructions. Small interfering RNA was added to give a final concentration of 2.5 nm in 2 ml of medium and 8 μl of INTERFERin for transfection of 40% confluent cells in each 35-mm dish. To test SG formation, 0.5 mm sodium arsenite (Sigma-Aldrich) or 1 μm hippuristanol (kindly provided by J. Tanaka (32.Mazroui R. Sukarieh R. Bordeleau M.E. Kaufman R.J. Northcote P. Tanaka J. Gallouzi I. Pelletier J. Mol. Biol. Cell. 2006; 17: 4212-4219Crossref PubMed Scopus (241) Google Scholar)) was added in conditioned medium for 45 or 30 min, respectively. To induce SGs with the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), cells were washed with 1× PBS- (PBS without Ca2+ and Mg2+) and cultured in glucose- and pyruvate-free Dulbecco's modified Eagle's medium containing 1 μm CCCP. Heat shock was performed at 44 °C in a 5% CO2 incubator for 45 min.Immunocytochemistry and Image Processing—At 24 h after transfection by FuGENE 6, HeLa cells were replated in 12-well dishes with coverslips coated with 0.2% gelatin. The next day, cells were treated with the indicated stimuli, fixed with 3.8% paraformaldehyde in 1× PBS- for 10 min, permeabilized with 0.2% Triton X-100 in PHEM buffer (25 mm HEPES, 10 mm EGTA, 60 mm PIPES, 2 mm MgCl2, pH 6.9) for 30 min and blocked with 5% horse serum in PHEM buffer for 1 h. All steps were performed at room temperature. Samples were incubated with primary antibodies (goat anti-TIA-1 (1:200), mouse anti-HuR (1:200), and goat anti-eIF3b (1:200)) diluted in the blocking buffer overnight at 4 °C. Coverslips with fixed cells were washed three times with 0.2% Triton X-100 in PHEM buffer and incubated in the dark with the secondary antibody and 500 ng/ml DAPI (Santa Cruz Biotechnology) to identify the nuclei for 40 min at room temperature. Cy2-, Cy3-, or Cy5-conjugated donkey secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used at dilutions of 1:200, 1:2,000, or 1:200 with 2.5% horse serum in PHEM buffer, respectively. The cells were mounted in FluoroMount reagent (SouthernBiotech, Birmingham, AL). Fluorescent images were captured with a confocal microscope (LSM 510 META, Carl Zeiss, Jena, Germany) as described previously (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar) except that a Plan-NeoFluar 100×/1.3 oil differential interference contrast objective was used. The data obtained were processed using standard image software (Bitplane Imaris 5.7.1, Adobe Photoshop, and Adobe Illustrator). To quantify association of RHAU with SGs, at least 100 transfected cells were analyzed under the wide spectrum microscope (Axioplan 2, Carl Zeiss) and scored as positive when the green fluorescent protein signal was enriched and co-localized with TIA-1 in SGs. Three (n = 3) independent transfections were analyzed to calculate mean percentages and ±S.E.Protein Extraction and Western Blotting—To prepare total cell lysates, cells were lysed with Nonidet P-40 buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 1% Nonidet P-40, 1 mm EDTA, 5 mm Na3VO4, 5 mm NaF, 0.5 μg/ml aprotinin, 1 μg/ml leupeptin) on ice for 10 min and centrifuged at 20,000 × g for 15 min at 4 °C to remove cell debris. Typically 30 μg of the total cell lysate was loaded for Western blotting. The protein bands were visualized with the direct infrared fluorescence method or the chemiluminescence method as described previously (30.Iwamoto F. Stadler M. Chalupnikova K. Oakeley E. Nagamine Y. Exp. Cell Res. 2008; 314: 1378-1391Crossref PubMed Scopus (39) Google Scholar).Cross-linking Immunoprecipitation (CLIP)—RHAU and RNA interaction was determined by the previously reported CLIP method with slight modifications (33.Ule J. Jensen K. Mele A. Darnell R.B. Methods. 2005; 37: 376-386Crossref PubMed Scopus (440) Google Scholar). HeLa cells (0.8 × 106/35-mm dish) were rinsed twice with ice-cold PBS and then UV irradiated (400 mJ/cm2) to induce cross-linking between protein and RNA. Cells were then lysed with 200 μl of RIPA buffer (1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 50 mm NaCl, 10 mm sodium phosphate, pH 7.2, 2 mm EDTA, 50 mm NaF, 0.2 mm sodium vanadate, 100 units/ml aprotinin)/well in a 6-well dish and shaken for 15 min at 4 °C. Pooled lysates from 6 wells were treated with 30 μl of RQ1 RNase-free DNase (1 unit/μl; Promega, Madison, WI) and with 31 units of RNase A (31 units/μl; USB Corp.) as described in the CLIP protocol (33.Ule J. Jensen K. Mele A. Darnell R.B. Methods. 2005; 37: 376-386Crossref PubMed Scopus (440) Google Scholar). Treated samples were centrifuged at 20,000 × g for 20 min at 4 °C. The supernatants (600 μg) were incubated with 4.5 μg of a mouse anti-RHAU monoclonal antibody (12F33) or 10 μl (bed volume) of anti-FLAG M2 affinity gel (A2220, Sigma-Aldrich) with rotation for 2 h at 4 °C. Beads were washed twice with RIPA buffer, twice with high salt washing buffer (5× PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% Nonidet P-40) and twice with 1× PNK buffer (50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 0.5% Nonidet P-40). The associated nucleic acids were radiolabeled with [γ-32P]ATP using T4 polynucleotide kinase (Roche Applied Science) as described in the CLIP protocol (33.Ule J. Jensen K. Mele A. Darnell R.B. Methods. 2005; 37: 376-386Crossref PubMed Scopus (440) Google Scholar), and RHAU-RNA complexes were resolved in a NuPAGE 4-12% bis-Tris gel (Invitrogen). Immunoprecipitated RHAU was detected by Coomassie Blue staining and in-gel Western blotting according to the manual of Odyssey In-Gel Western detection (LI-COR Biosciences). Half of the samples were transferred to a polyvinylidene difluoride membrane to facilitate better protein detection by Western blotting and to remove free RNA. The proteins were detected by the Odyssey infrared imager as described above. Radiolabeled RNA was detected by a phosphorimaging system, Typhoon 9400 (GE Healthcare), and analyzed using the ImageQuant TL program. To test whether RHAU associates with RNA, bound nucleic acids were isolated and radiolabeled according to the CLIP protocol (33.Ule J. Jensen K. Mele A. Darnell R.B. Methods. 2005; 37: 376-386Crossref PubMed Scopus (440) Google Scholar). Nucleic acids were mixed with increasing amounts of RNase A (0.015, 0.15, 1.5, and 15 units) in H2O and 1 unit of RQ1 DNase in 1× RQ1 DNase reaction buffer. Reactions were incubated for 30 min at 37 °C and resolved by denaturing 8% PAGE in 1× Tris borate-EDTA buffer.Protein Purification—Escherichia coli BL21 (DE3) transformed with glutathione S-transferase (GST) or GST-Nter proteins were induced by 1 mm isopropyl 1-thio-β-d-galactopyranoside for 12 h at 25 °C and purified by affinity chromatography with glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. GST-RHAU protein was expressed in Sf9 cells according to the supplier's instructions (BD Biosciences Pharmingen) and purified as above. The purity of recombinant proteins was analyzed by 10% SDS-PAGE and Coomassie Blue staining as reported in supplemental Fig. 2.Double Filter RNA Binding Assay—5 μg of total RNA isolated from HeLa cells was alkali-treated with 0.1 m NaOH on ice for 10 min and then EtOH-precipitated. Redissolved RNA or poly(rU) (P9528, Sigma-Aldrich) was 5′-end-labeled using [γ-32P]ATP (Hartmann Analytic GmbH, Braunschweig, Germany) and T4 polynucleotide kinase (Roche Applied Science) at 37 °C for 30 min and passed through a G-50 column (GE Healthcare) to remove free nucleotides. Reaction mixtures (50 μl) containing varying amounts of recombinant proteins (0-150 nm as specified in text), radiolabeled RNA (poly(rU), 10,000 cpm; total RNA, 5,000 cpm) and 2 units of RNase inhibitor (RNAguard, Roche Applied Science) in the binding buffer (50 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 5 mm NaCl) were incubated for 30 min at 37 °C. The double filter RNA binding assay was performed with a slot-blot apparatus using a 0.45-μm nitrocellulose (Protran, Whatman) and nylon membranes (positively charged; Roche Diagnostics) that was presoaked in different buffers as described previously (34.Tanaka N. Schwer B. Biochemistry. 2005; 44: 9795-9803Crossref PubMed Scopus (60) Google Scholar). Loaded samples were washed three times with 200 μl of the binding buffer. Retained RNA was detected with a phosphorimaging system, Typhoon 9400, and analyzed using the ImageQuant TL program. The nitrocellulose membrane retains only RNA-protein complexes, and free RNAs are captured on the nylon membrane. The ratio of RNA that was bound to GST-RHAU or GST-Nter was calculate using the following formula: bound RNA (%) = 100((signalnitrocellulose)/(signalnitrocellulose + signalnylon)).Bioinformatics—The program RNABindR (35.Terribilini M. Sander J.D. Lee J.H. Zaback P. Jernigan R.L. Honavar V. Dobbs D. Nucleic Acids Res. 2007; 35: W578-W584Crossref PubMed Scopus (162) Google Scholar) was used to predict RNA binding potential in amino acid sequences. Programs RISP (RNA-Interaction Site Prediction) and BindN+ were used to confirm the reliability of the RNABindR program: RISP (36.Tong J. Jiang P. Lu Z.H. Comput. Methods Programs Biomed. 2008; 90: 148-153Crossref PubMed Scopus (47) Google Scholar) runs with 72.2% RNA binding prediction accuracy, and BindN+ (37.Jeong E. Chung I.F. Miyano S. Genome Inform. 2004; 15: 105-116PubMed Google Scholar) runs with 68% RNA binding prediction accuracy. For multiple sequence alignments of N termini, RHAU orthologs were identified by a BLASTP (version 2.2.18+) search of non-redundant protein entries in the NCBI data base using the entire sequence of RHAU as a query. Multiple sequence alignment was carried out with ProbCons (version 1.12) (38.Do C.B. Mahabhashyam M.S. Brudno M. Batzoglou S. Genome Res. 2005; 15: 330-340Crossref PubMed Scopus (868) Google Scholar). Similarity of groups was generated using GeneDoc (version 2.7) with the BLOSUM62 scoring matrix.Fluorescence Recovery after Photobleaching—HeLa cells (1.5 × 105/35-mm dish) were plated and transfected by FuGENE 6 on glass-bottomed dishes (Micro-Dish 35 mm, Fisher Scientific). FRAP experiments were carried out with a confocal microscope (LSM 510, Carl Zeiss) using a 63×/1.4 numerical aperture oil differential interference contrast objective. Bleaching was performed using the 488-nm lines from a 40-milliwatt argon laser operating at 75% laser power. Bleaching of circular regions of interest
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