Molecular Composition of IMP1 Ribonucleoprotein Granules
2007; Elsevier BV; Volume: 6; Issue: 5 Linguagem: Inglês
10.1074/mcp.m600346-mcp200
ISSN1535-9484
AutoresLars J⊘nson, Jonas Vikesaa, Anders Krogh, Lars K. Nielsen, Thomas van Overeem Hansen, Rehannah Borup, Anders H. Johnsen, Jan Christiansen, Finn Cilius Nielsen,
Tópico(s)RNA Research and Splicing
ResumoLocalized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100–300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons. Localized mRNAs are transported to sites of local protein synthesis in large ribonucleoprotein (RNP) granules, but their molecular composition is incompletely understood. Insulin-like growth factor II mRNA-binding protein (IMP) zip code-binding proteins participate in mRNA localization, and in motile cells IMP-containing granules are dispersed around the nucleus and in cellular protrusions. We isolated the IMP1-containing RNP granules and found that they represent a unique RNP entity distinct from neuronal hStaufen and/or fragile X mental retardation protein granules, processing bodies, and stress granules. Granules were 100–300 nm in diameter and consisted of IMPs, 40 S ribosomal subunits, shuttling heterologous nuclear RNPs, poly(A)-binding proteins, and mRNAs. Moreover granules contained CBP80 and factors belonging to the exon junction complex and lacked eIF4E, eIF4G, and 60 S ribosomal subunits, indicating that embodied mRNAs are not translated. Granules embodied mRNAs corresponding to about 3% of the human embryonic kidney 293 mRNA transcriptome. Messenger RNAs encoding proteins participating in the secretory pathway and endoplasmic reticulum-associated quality control, as well as ubiquitin-dependent metabolism, were enriched in the granules, reinforcing the concept of RNP granules as post-transcriptional operons. Eukaryotic mRNAs are remodeled during their life cycle by attachment of different RNA-binding proteins. Major transitions are the exchange of the nuclear CBP20/80 1The abbreviations used are: CBP, cap-binding protein; ER, endoplasmic reticulum; RNP, ribonucleoprotein; IMP, insulin-like growth factor II mRNA-binding protein; hnRNP, heterogeneous nuclear ribonucleoprotein; ZBP1, zip code-binding protein 1; AFM, atomic force microscopy; G3BP, Ras GTPase-activating protein Src homology 3 domain-binding protein; eIF, eukaryotic translation initiation factor; ELAV, embryonic lethal abnormal vision; ERG1, early growth response 1; FMRP, fragile X mental retardation protein; NCBI, National Center for Biotechnology Information; IP, immunoprecipitation; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; PABP, poly(A)-binding protein; PIG, phosphatidylinositol glycan; SCAMP, secretory carrier membrane protein; VPS, vacuolar protein sorting; RING, really interesting new gene; RNF, RING finger; PSM, proteasome activator complex; NFAR, nuclear factors associated with double-stranded RNA; YB1, Y box-binding protein 1; HEK, human embryonic kidney; mRNP, messenger ribonucleoprotein; P-bodies, processing bodies; RBP, RNA-binding protein; rIMP1, recombinant IMP1; SELEX, systematic evolution of ligands by exponential enrichment; GCOS, GeneChip Operating Software; UTR, untranslated region; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. 1The abbreviations used are: CBP, cap-binding protein; ER, endoplasmic reticulum; RNP, ribonucleoprotein; IMP, insulin-like growth factor II mRNA-binding protein; hnRNP, heterogeneous nuclear ribonucleoprotein; ZBP1, zip code-binding protein 1; AFM, atomic force microscopy; G3BP, Ras GTPase-activating protein Src homology 3 domain-binding protein; eIF, eukaryotic translation initiation factor; ELAV, embryonic lethal abnormal vision; ERG1, early growth response 1; FMRP, fragile X mental retardation protein; NCBI, National Center for Biotechnology Information; IP, immunoprecipitation; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; PABP, poly(A)-binding protein; PIG, phosphatidylinositol glycan; SCAMP, secretory carrier membrane protein; VPS, vacuolar protein sorting; RING, really interesting new gene; RNF, RING finger; PSM, proteasome activator complex; NFAR, nuclear factors associated with double-stranded RNA; YB1, Y box-binding protein 1; HEK, human embryonic kidney; mRNP, messenger ribonucleoprotein; P-bodies, processing bodies; RBP, RNA-binding protein; rIMP1, recombinant IMP1; SELEX, systematic evolution of ligands by exponential enrichment; GCOS, GeneChip Operating Software; UTR, untranslated region; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. cap-binding complex with the trimeric eIF4F cap-binding complex and the substitution of the nuclear poly(A)-binding protein PABP2 with PABP1. Moreover the exon junction complex, which is deposited during splicing, is removed during the so-called pioneering round of translation (for reviews, see Refs. 1Maquat L.E. Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics.Nat. Rev. Mol. Cell. Biol. 2004; 5: 89-99Crossref PubMed Scopus (935) Google Scholar and 2Moore M. From birth to death: the complex lives of eukaryotic mRNAs.Science. 2005; 309: 1514-1518Crossref PubMed Scopus (785) Google Scholar). Finally a particular mRNA becomes embroidered with nuclear RNA-binding proteins, and the specific ensemble may determine cytoplasmic events such as RNA localization, translation, and stability (for a review, see Ref. 3Hieronymus H. Silver P.A. A systems view of mRNP biology.Genes Dev. 2004; 18: 2845-2860Crossref PubMed Scopus (136) Google Scholar). Cytoplasmic mRNPs may become destined for local translation. In support of this possibility, RNAs have been found in large mRNP granules, which are transported along cytoskeletal structures and anchored at their final destination. Messenger RNA localization has mainly been examined in polarized oocytes and neurons, and it has been proposed that local postsynaptic protein synthesis is required for synaptic plasticity (4Kosik K.S. Krichevsky A.M. The message and the messenger: delivering RNA in neurons.Sci. STKE. 2002; 2002: PE16Crossref PubMed Google Scholar). Previous studies have identified neuronal Staufen (5Krichevsky A.M. Kosik K.S. Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation.Neuron. 2001; 32: 683-696Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) and FMRP granules (6De Diego Otero Y. Severijnen L.A. van Cappellen G. Schrier M. Oostra B. Willemsen R. Transport of fragile X mental retardation protein via granules in neurites of PC12 cells.Mol. Cell. 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A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates.Curr. Biol. 1998; 8: 489-496Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 17Doyle G.A. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.Nucleic Acids Res. 1998; 26: 5036-5044Crossref PubMed Scopus (142) Google Scholar, 18Havin L. Git A. Elisha Z. Oberman F. Yaniv K. Schwartz S.P. Standart N. Yisraeli J.K. RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.Genes Dev. 1998; 12: 1593-1598Crossref PubMed Scopus (180) Google Scholar, 19Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (542) Google Scholar, 20Ross A.F. Oleynikov Y. Kislauskis E.H. Taneja K.L. Singer R.H. Characterization of a β-actin mRNA zipcode-binding protein.Mol. Cell. Biol. 1997; 17: 2158-2165Crossref PubMed Google Scholar, 21Yisraeli J.K. VICKZ proteins: a multi-talented family of regulatory RNA-binding proteins.Biol. Cell. 2005; 97: 87-96Crossref PubMed Scopus (157) Google Scholar). Various post-transcriptional functions of IMPs and their orthologues have been reported. The Xenopus IMP3 orthologue Vg1-RBP/Vera has been implicated in localization of Vg1 mRNA to the vegetal pole of the Xenopus oocyte (16Deshler J.O. Highett M.I. Abramson T. Schnapp B.J. A highly conserved RNA-binding protein for cytoplasmic mRNA localization in vertebrates.Curr. Biol. 1998; 8: 489-496Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 18Havin L. Git A. Elisha Z. Oberman F. Yaniv K. Schwartz S.P. Standart N. Yisraeli J.K. RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.Genes Dev. 1998; 12: 1593-1598Crossref PubMed Scopus (180) Google Scholar), and mouse coding region determinant-binding protein stabilizes the c-myc transcript (17Doyle G.A. Betz N.A. Leeds P.F. Fleisig A.J. Prokipcak R.D. Ross J. The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.Nucleic Acids Res. 1998; 26: 5036-5044Crossref PubMed Scopus (142) Google Scholar). Chicken ZBP1 regulates localization of β-actin mRNA to the leading edge of fibroblasts (22Farina K.L. Huttelmaier S. Musunuru K. Darnell R. Singer R.H. Two ZBP1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment.J. Cell Biol. 2003; 160: 77-87Crossref PubMed Scopus (187) Google Scholar), and finally IMPs play a role in H19 and tau mRNA transport (23Atlas R. Behar L. Elliott E. Ginzburg I. The insulin-like growth factor mRNA binding-protein IMP-1 and the Ras-regulatory protein G3BP associate with tau mRNA and HuD protein in differentiated P19 neuronal cells.J. Neurochem. 2004; 89: 613-626Crossref PubMed Scopus (122) Google Scholar, 24Runge S. Nielsen F.C. Nielsen J. Lykke-Andersen J. Wewer U.M. Christiansen J. H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein.J. Biol. Chem. 2000; 275: 29562-29569Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and IGF2 mRNA translation (19Nielsen J. Christiansen J. Lykke-Andersen J. Johnsen A.H. Wewer U.M. Nielsen F.C. A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development.Mol. Cell. Biol. 1999; 19: 1262-1270Crossref PubMed Scopus (542) Google Scholar, 25Liao B. Hu Y. Herrick D.J. Brewer G. The RNA-binding protein IMP-3 is a translational activator of insulin-like growth factor II leader-3 mRNA during proliferation of human K562 leukemia cells.J. Biol. Chem. 2005; 280: 18517-18524Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). IMPs are expressed during early embryogenesis and at midgestation in the mouse, and disruption of IMP1 leads to dwarfism and impaired gut development (26Hansen T.V. Hammer N.A. Nielsen J. Madsen M. Dalbaeck C. Wewer U.M. Christiansen J. Nielsen F.C. Dwarfism and impaired gut development in insulin-like growth factor II mRNA-binding protein 1-deficient mice.Mol. Cell. Biol. 2004; 24: 4448-4464Crossref PubMed Scopus (173) Google Scholar). In the same vein, the IMP3 orthologue Vg1-RBP/Vera is required for normal pancreatic development (27Spagnoli F.M. Brivanlou A.H. The RNA-binding protein, Vg1RBP, is required for pancreatic fate specification.Dev. Biol. 2006; 292: 442-456Crossref PubMed Scopus (32) Google Scholar) and promotes migration of neural crest cells (28Yaniv K. Fainsod A. Kalcheim C. Yisraeli J.K. The RNA-binding protein Vg1 RBP is required for cell migration during early neural development.Development. 2003; 130: 5649-5661Crossref PubMed Scopus (79) Google Scholar). In the cytoplasm, IMPs are distributed in large 200–700-nm (optical diameter) RNP granules, and in motile cells granules are found around the nucleus and transported toward the leading edge where they dock at the cortical region of the lamellipodia. Granules travel at a speed of 0.2 μm/s, and cells are able to switch from a delocalized to a localized pattern within 15–20 min (29Nielsen F.C. Nielsen J. Kristensen M.A. Koch G. Christiansen J. Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains.J. Cell Sci. 2002; 115: 2087-2097Crossref PubMed Scopus (51) Google Scholar). In neuronal cells, ZBP1 granules localize to dendrites, and reduced levels of ZBP1 lead to impaired growth of dendritic filopodia (30Tiruchinapalli D.M. Oleynikov Y. Kelic S. Shenoy S.M. Hartley A. Stanton P.K. Singer R.H. Bassell G.J. Activity-dependent trafficking and dynamic localization of zipcode binding protein 1 and β-actin mRNA in dendrites and spines of hippocampal neurons.J Neurosci. 2003; 23: 3251-3261Crossref PubMed Google Scholar). The function of IMP1 and other RNP granules is incompletely understood. The number of different types of granules is unknown, and it is uncertain whether the particles are heterogeneous and how they are assembled and dismantled, although recent data have indicated that local signaling events may play a role in mRNA release (31Huttelmaier S. Zenklusen D. Lederer M. Dictenberg J. Lorenz M. Meng X. Bassell G.J. Condeelis J. Singer R.H. Spatial regulation of β-actin translation by Src-dependent phosphorylation of ZBP1.Nature. 2005; 438: 512-515Crossref PubMed Scopus (477) Google Scholar). It is anticipated that associated mRNAs are translationally quiescent during transport, but there are limited data to support this assumption. With the exception of FMRP granules, which have been demonstrated to contain about 2% of the transcriptome (32Brown V. Jin P. Ceman S. Darnell J.C. O'Donnell W.T. Tenenbaum S.A. Jin X. Feng Y. Wilkinson K.D. Keene J.D. Darnell R.B. Warren S.T. Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.Cell. 2001; 107: 477-487Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar), no global picture of human mRNA cargo is available. An intriguing proposal is that RNP granules, or more generally mRNP particles, represent post-transcriptional operons (33Keene J.D. Tenenbaum S.A. Eukaryotic mRNPs may represent posttranscriptional operons.Mol. Cell. 2002; 9: 1161-1167Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 34Niehrs C. Pollet N. Synexpression groups in eukaryotes.Nature. 1999; 402: 483-487Crossref PubMed Scopus (297) Google Scholar), and the finding that RNA-binding Puf proteins in yeast associate with mRNAs encoding proteins with common functions and subcellular localization lends strong support for this proposal (35Gerber A.P. Herschlag D. Brown P.O. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.PLoS Biol. 2004; 2: E79Crossref PubMed Scopus (503) Google Scholar). To deepen our understanding of human RNP granules or "locasomes" (36Darzacq X. Powrie E. Gu W. Singer R.H. Zenklusen D. RNA asymmetric distribution and daughter/mother differentiation in yeast.Curr. Opin. Microbiol. 2003; 6: 614-620Crossref PubMed Scopus (40) Google Scholar), we determined the molecular composition of IMP1-containing RNP granules. The results demonstrate that IMP1 granules represent a unique RNP entity containing untranslated mRNAs, corresponding to ∼3% of the transcriptome. Messenger RNAs encoding proteins of the secretory pathway and ER-associated quality control, as well as ubiquitin-dependent metabolism, were enriched reinforcing the concept of post-transcriptional operons. Human embryonic kidney (HEK) 293 cells stably expressing 3×FLAG-IMP1 were generated as described by the manufacturer (Invitrogen). Briefly Flp-In T-Rex-293 cells (Invitrogen) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 mg/ml streptomycin, 5 μg/ml blasticidin, and 100 μg/ml Zeocin. Cells were co-transfected with pcDNA5/FRT/TO-3×FLAG-IMP1 and pOG44 (Invitrogen). Integration was selected by exchanging the Zeocin with 100 μg/ml hygromycin. HEK293 cells stably expressing FLAG-ERG1 were a kind gift from Bo Porse (Rigshospitalet, Copenhagen, Denmark). The expression of 3×FLAG-IMP1 and FLAG-ERG1 was induced by the addition of 1 μm tetracycline. The cells were harvested 48 h later after the addition of tetracycline, and 12.5 μg/ml cycloheximide was added 10 min before harvest. HEK293 cells were transiently transfected with FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer's instructions. Briefly 20,000 cells/cm2 were seeded on fibronectin-coated glass plates or 10-cm plastic dishes 24 h prior to transfection. Cells were transfected with a 2 μg/ml concentration of the indicated plasmid and left for 48 h before they were examined. The distribution of CFP-IMP1, YFP-hStaufen, YFP-FMRP, YFP-IMP3, YFP-ELAV, and YFP in living and fixed cells (see below) was examined with a Zeiss LSM 510 confocal laser scanning microscope. The Drosophila S2 cells were maintained in Drosophila Schneider medium (Invitrogen) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin at 25 °C. HEK293 cells were seeded on fibronectin-coated (25 μg/cm2) glass bottom dishes. Cells were fixed with 4% formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 3 min. After blocking with 1% BSA in PBS for 1 h at room temperature, the cells were incubated with primary antibody overnight at 4 °C, washed 3 × 5 min in PBS, and visualized with Alexa Fluor 488- or Alexa Fluor 555-conjugated anti-mouse or anti-rabbit antibodies. Protein extracts from HEK293 cells were separated in SDS-polyacrylamide gels and transferred to Hybond-P membranes (Amersham Biosciences). After blocking, membranes were incubated overnight with primary antibody in blocking solution at 4 °C before they were washed and incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG for 1 h at room temperature. Immunoreactive proteins were detected with SuperSignal chemiluminescence reagents (Pierce) according to the manufacturer's instructions. Quantitation was performed on a LAS-1000 luminescence imager (Fuji) using Image Gauge 4.0 software (Fuji). Cells were lysed in ice-cold lysis buffer (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.5 μg/ml PMSF, and 0.5% protease inhibitor mixture (Sigma-Aldrich)) by sonication. The cell lysate was cleared by centrifugation at 8200 × g for 10 min at 4 °C before 250 units of Protector RNase inhibitor (Roche Applied Science)/ml of lysis buffer was added. Immunoprecipitations were performed using FLAG-specific monoclonal antibody M2 covalently coupled agarose beads (Sigma) using 30 million cells/50 μl of beads. The beads were prewashed three times in TBS (50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm EDTA, 0.5 μg/ml PMSF, and 0.5% protease inhibitor mixture) and resuspended in lysis buffer containing 25 μg/ml synthetic FLAG peptide (Sigma-Aldrich). The suspension was rotated for 1 h at 4 °C before the supernatant was exchanged with cleared cell lysate and rotated for an additional 2 h. The immunoprecipitate was washed four times in TBS, and the proteins were eluted by addition of 50 μg of synthetic FLAG peptide/ml of TBS followed by rotation for 2 h at 4 °C. Eluted proteins used for silver staining were precipitated by addition of 10% TCA followed by five washes in acetone. Purification of the 3×FLAG-IMP1 granule in the presence of Drosophila S2 cells was performed as described above with the exception that 1.7 times S2 cells were added to the HEK293 3×FLAG-IMP1 cells before lysis so equal amounts of RNA would be extracted. RNA isolation was performed as described above with a few modifications. Prior to the addition of the cleared lysate, 10 μg of yeast ribosomal RNA was added. The mRNA was isolated using oligo(dT)-agarose beads (Roche Applied Science). After washing of the beads mRNA was retrieved from the agarose beads by addition of 1 ml of TRIzol with 5 μg of yeast ribosomal RNA. Total RNA from HEK293 cells, HEK293 cells stably transfected with the 3×FLAG-IMP1 (no tetracycline), HEK293 cells stably expressing 3×FLAG-IMP1 by the inclusion of 1 μm tetracycline, or Drosophila S2 cells was isolated in TRIzol as described by the manufacturer (Invitrogen). Immunoprecipitation (IP) was performed on 120 million HEK293 cells, HEK293 cells stably expressing 3×FLAG-IMP1, and HEK293 cells stably expressing FLAG-ERG1. After lysis in 3.6 ml of lysis buffer, 10 μl was removed and used as total protein extract of the cells. The IPs were TCA-precipitated and dissolved in 50 μl of 2× SDS loading buffer. The proteins were separated on a 10% SDS-polyacrylamide gel and silver-stained using Silverquest (Invitrogen) as described by the manufacturer. Slices were cut from the gel and digested with trypsin. The gel piece was cut into cubes and washed for 1 h in 100 mm NH4HCO3 before excess liquid was removed. Proteins were reduced for 30 min at 60 °C in 4 mm dithiothreitol in 100 mm NH4HCO3, cooled, and alkylated by addition of 100 mm iodoacetamide for 30 min in the dark. Excess liquid was removed, and the gel pieces were washed for 1 h in 50% acetonitrile in 100 mm NH4HCO3 followed by shrinkage in acetonitrile and vacuum centrifugation. The dry gel pieces were placed on ice and allowed to swell in 25 mm NH4HCO3 including 2 μg/100 μl modified trypsin (Promega). The supernatant was removed, and buffer was added to cover the gel pieces during overnight incubation at 37 °C. The next day, the supernatant was removed and combined with a second extract from the gel piece, obtained by incubation for 30 min in 50 μl of 0.5% formic acid. The combined extracts were loaded onto a C18 ZipTip (Millipore), washed with 0.5% formic acid, and eluted with 10 μl of 0.5% formic acid in 50% acetonitrile. A 0.5-μl aliquot was analyzed by MALDI-TOF using an AutoFlex instrument with TOF-TOF facility (Bruker Daltonics). An aliquot of 2 μl of the purified peptide mixture was introduced into a Q-TOF-2 tandem mass spectrometer (Micromass) using the nanospray interface and analyzed in MS mode as well as in MS/MS mode for a number of fragments to obtain sequence information. For the MALDI-TOF results, the flexAnalysis program (Bruker Daltonics) was used to create the peak lists, and internal calibration was performed using trypsin peptides. Profound version 4.10 was used for the database searches with a mass accuracy of 100–200 ppm. Mascot was used for database search on the MS/MS results. The nonredundant NCBI (NCBInr) database was searched, and the search results were accepted when the scores indicated identity or extensive homology with p values less than 0.05. Default values of the score threshold were used. When needed, keratin peaks were excluded prior to a new search. Often several names were indicated that appeared to be the same protein. Overall we used the Swiss-Prot names as the most appropriate. Atomic force microscopy (AFM) measurements were carried out using a MultiMode atomic force microscope on a Nanoscope III controller (Veeco). The atomic force microscope was calibrated in all axes using certified reference standards to an accuracy below 5%. For the measurements in liquid, silicon nitride cantilevers were used (OMCL-TR400PSA, Olympus), and for measurements in air, silicon cantilevers were used (Point-Probe-Plus, Nanosensors). For all samples mica was used as substrate (Moscovite Clear Ruby, Goodfellow). Mica pieces were glued onto metal discs using Araldite 2011 (Huntsman). 3×FLAG-IMP1 RNP and FLAG-ERG1 proteins were immunoprecipitated from 3 × 107 cells as described above. The eluted proteins were diluted 1000× in TBS, and 100 μl was pipetted onto the freshly cleaved mica surface. After 15 min of incubation, the samples were rinsed three time in TBS buffer. The samples were kept under TBS buffer at all times and mounted in the atomic force microscope. The dimerization of IMP1 on RNA was performed by mixing a 1 nm concentration of an RNA SELEX target with 2 nm recombinant IMP1 (rIMP1) in UV cross-linking buffer (24Runge S. Nielsen F.C. Nielsen J. Lykke-Andersen J. Wewer U.M. Christiansen J. H19 RNA binds four molecules of insulin-like growth factor II mRNA-binding protein.J. Biol. Chem. 2000; 275: 29562-29569Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). The sample was incubated for 25 min at room temperature and stored at −20 °C. 50 μl of the rIMP1 SELEX sample was allowed to adsorb to freshly cleaved mica for 15 min. The samples were rinsed briefly in TBS and water, dried, and mounted in the atomic force microscope. Thermal equilibration of the atomic force microscope and sample was allowed for at least 1 h before imaging was started. All samples were investigated in at least three separate areas. Image analysis was done using Scanning Probe Image Processor (SPIP) (Image Metrology). All images were subjected to first order linewise leveling before further analysis. Heights of the various features in the images were determined using cross-section plots. 2 μg of purified total RNA from HEK293 cells (HEK293 total), 0.3–1 μg of RNA from immunoprecipitated beads containing HEK293 cell lysates (HEK293 IP), 15 μg of RNA from immunoprecipitated beads containing lysates from HEK293 cells expressing FLAG-IMP1 (HEK293 FLAG-IMP1 IP), 2 μg of total RNA from Drosophila S2 cells (S2 total), 0.3–1 μg of RNA from immunoprecipitated beads containing Drosophila S2 cell lysates (S2 IP), and 15 μg of RNA from immunoprecipitated beads containing a mixture of lysates from HEK293 cells expressing FLAG-IMP1 and Drosophila S2 cells (HEK293 FLAG-IMP1/S2 IP) were used to synthesize double-stranded cDNA with the Superscript Choice system (Invitrogen). The cDNA was used as template in an in vitro transcription reaction to generate biotin-labeled antisense cRNA (BioArray high yield RNA transcript labeling kit, Enzo Diagnostics). After fragmentation at 94 °C for 35 min in 40 mm Tris, 30 mm magnesium acetate, 10 mm potassium acetate, 1–3 μg of labeled HEK293 IP cRNA, 15 μg of labeled HEK293 FLAG-IMP1 IP, or 15 μg of labeled HEK293 total cRNA was hybridized for 16 h to Affymetrix HG-U133plus2 arrays (Affymetrix Inc.) containing 54,613 probe sets, whereas 1–3 μg of labeled S2 I
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