Presence of WT1, the Wilm's Tumor Suppressor Gene Product, in Nuclear Poly(A)+ Ribonucleoprotein
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36520
ISSN1083-351X
AutoresMichael Ladomery, Joan Slight, Sharon Mc Ghee, Nicholas D. Hastie,
Tópico(s)Renal and related cancers
ResumoThe tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C2-H2 zinc fingers of the Krüppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391–401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217–3225) and binds to RNA in vitro(Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562–7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784–1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at ∼60 S, and ∼1.27 g/cm3 on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)+ RNP is a general aspect of WT1 function. The tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C2-H2 zinc fingers of the Krüppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391–401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217–3225) and binds to RNA in vitro(Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562–7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784–1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at ∼60 S, and ∼1.27 g/cm3 on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)+ RNP is a general aspect of WT1 function. small nuclear riboncleoprotein ribonucleoprotein polymerase chain reaction tobacco mosaic virus small nuclear RNA human RNP fluorescein isothiocyanate diethyl pyrocarbonate polyacrylamide gel electrophoresis phosphate-buffered saline tobacco mosaic virus proliferating cell nuclear antigen Wilm's tumor is one of the most common childhood malignancies, affecting 1/10,000 children. In the search for genes involved in this disease, a candidate tumor suppressor gene, named WT1, was identified and cloned, and shown by mutational analysis to be involved in 10–15% of Wilm's tumors (5Call K.M. Glaser T. Ito C.Y. Buckler A.J. Pelletier J. Haber D.A. Rose E.A. Kral A. Yeger H. Lewis W.H. Jones C. Housman D.E. Cell. 1990; 60: 509-520Abstract Full Text PDF PubMed Scopus (1658) Google Scholar, 6Gessler M. Poustka A. Cavenee W. Neve R.L. Orkin S.H. Bruns G.A.P. Nature. 1990; 343: 774-778Crossref PubMed Scopus (1141) Google Scholar). Although described as a tumor suppressor gene, it should be noted that the growth suppressor effects of WT1 are context-dependent (7Menke A.L. Van der Eb A.J. Jochemsen A.G. Int. Rev. Cytol. 1998; 181: 151-212Crossref PubMed Google Scholar). Mutations in the WT1 gene also result in urogenital abnormalities;WT1 therefore provides an excellent opportunity to study the relationship between cancer and development (8Feinberg A.P. J. Cell Sci. 1994; 18 (suppl.): 7-12Crossref Google Scholar, 9Hastie N.D. Annu. Rev. Genet. 1994; 28: 523-558Crossref PubMed Scopus (207) Google Scholar, 10Reddy J.C. Licht J.D. Biochim. Biophys. Acta. 1996; 1287: 1-28PubMed Google Scholar). Given the expression pattern of WT1, the phenotypes associated with a number of genetic syndromes in which it is implicated, and a mouse knockout model (11Kreidberg J.A. Sariola H. Loring J.M. Maeda M. Pelletier J. Housman D. Jaenisch R. Cell. 1993; 74: 679-691Abstract Full Text PDF PubMed Scopus (1643) Google Scholar), it is thought that WT1 plays an essential role in the transition from proliferative mesenchyme to differentiated epithelium (12Pritchard-Jones K.S. Fleming D. Davidson W. Bickmore W. Porteous D. Gosden J. Bard A. Buckler J. Pelletier D. Housman V. van Heyningen V. Hastie N. Nature. 1990; 346: 194-197Crossref PubMed Scopus (764) Google Scholar). The WT1 gene encodes a protein which includes, at its C terminus, four C2-H2 zinc fingers of the Krüppel-type, with close structural homology to zinc fingers in the early growth response family of transcription factors. At the N terminus, WT1 possesses a proline/glutamine-rich putative transactivation domain. Molecular modeling also suggests the presence of an RNA recognition motif at the N terminus (13Kennedy D. Ramsdale T. Mattick J. Little M. Nat. Genet. 1996; 12: 329-332Crossref PubMed Scopus (95) Google Scholar). Murine WT1 is >95% identical at the amino acid level to its human counterpart (14Buckler A.J. Pelletier J. Haber D.A. Glaser T. Housman D.E. Mol. Cell. Biol. 1991; 11: 1707-1712Crossref PubMed Scopus (229) Google Scholar). The structure of mammalian WT1 protein is complicated by two alternative splicing events as follows: inclusion of exon 5, which inserts 17 amino acids in the middle of the protein, and exon 9, which inserts three amino acids lysine-threonine-serine (KTS) between the third and fourth zinc fingers (15Haber D.A. Sohn R.L. Buckler A.J. Pelletier J. Call K.M. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9618-9622Crossref PubMed Scopus (553) Google Scholar); an RNA editing event (16Sharma P.M. Bowman M. Madden S.L. Rauscher III, F.J. Sukumar S. Genes Dev. 1994; 8: 720-731Crossref PubMed Scopus (152) Google Scholar); and an alternative upstream translation start site (17Bruening W. Pelletier J. J. Biol. Chem. 1996; 271: 8646-8654Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) giving a total of at least 16 possible isoforms. Given its salient features, the immediate assumption was that WT1 protein is a transcription factor. Numerous studies have investigated the effects of WT1 on the expression of candidate target genes, for example IGF2, IGF2R, EGFR, CSF1, TGF-β, PDGFA, Pax-2, Nov,and ODC (7Menke A.L. Van der Eb A.J. Jochemsen A.G. Int. Rev. Cytol. 1998; 181: 151-212Crossref PubMed Google Scholar). These studies are generally based on the expression of reporter constructs in cells co-transfected withWT1; in general, data suggest that WT1 acts as a transcriptional repressor of these growth-associated genes. WT1 binds to a G-rich DNA sequence, similar to the consensus binding site for the early growth response family of transcription factors (18Rauscher F.J. Morris J.F. Tounay O.E. Cook D.M. Currna T. Science. 1990; 250: 1259-1262Crossref PubMed Scopus (469) Google Scholar). In addition, recent evidence suggests that WT1 can also act as a transcriptional activator, up-regulating the anti-apoptotic genebcl-2 (19Mayo M.W. Wang C.Y. Drouin S.S. Madrid L.V. Marshall A.F. Reed J.C. Weissman B.E. Baldwin A.S. EMBO J. 1999; 18: 3990-4003Crossref PubMed Scopus (216) Google Scholar), and as a transcriptional co-factor, modulating SF-1-mediated transactivation in testis development (20Nachtigal M.W. Hirokawa Y. Enyeart-VanHouten D.L. Flanagan J.N. Hammer G.D. Ingraham H.A. Cell. 1998; 93: 445-454Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). Was WT1 therefore a typical transcription factor whose target genes needed to be defined? This picture was complicated when Larssonet al. (1Larsson S.H. Charlieu J.-P. Miyagawa K. Engelkamp D. Rassoulzadegan M. Ross A. Cuzin F. van Heyningen V. Hastie N.D. Cell. 1995; 81: 391-401Abstract Full Text PDF PubMed Scopus (439) Google Scholar) began to look in detail at the subcellular distribution of WT1 protein. They showed that a proportion of WT1 is concentrated in nuclear "speckles," which contain splicing factors, and co-immunoprecipitated WT1 with Sm (snRNP)1 antigens, the splicing factors U170, U2-B″, and p80 coilin. Co-immunoprecipitation of WT1 with Sm antigens was abolished by actinomycin D treatment. Both actinomycin D and injection of antisense snRNAs resulted in the relocation of WT1 to larger nuclear structures. Similarly, in HeLa cells, the microinjection of oligonucleotides and antibodies that disrupt splicing produced rounder and larger interchromatin granule clusters (21O'Keefe R.T. Mayeda A. Sadowski C.L. Krainer A.R. Spector D.L. J. Cell Biol. 1994; 124: 249-260Crossref PubMed Scopus (153) Google Scholar). Larsson et al. (1Larsson S.H. Charlieu J.-P. Miyagawa K. Engelkamp D. Rassoulzadegan M. Ross A. Cuzin F. van Heyningen V. Hastie N.D. Cell. 1995; 81: 391-401Abstract Full Text PDF PubMed Scopus (439) Google Scholar) also showed that isoforms of WT1 that included exon 9 (+KTS) preferentially associated with nuclear speckles, whereas (−KTS) isoforms were enriched in areas where the transcription factor Sp1 was more abundant. Thus, the possibility arose that WT1, and in particular the +KTS isoforms, may be involved in post-transcriptional events, specifically splicing. To extend these findings, Davies et al. (2Davies R.C. Calvo C. Larsson S.H. Lamond A.I. Hastie N.D. Genes Dev. 1998; 12: 3217-3225Crossref PubMed Scopus (202) Google Scholar) subsequently described an interaction between WT1 and the ubiquitous splicing factor U2AF65. This interaction was defined and analyzed using the yeast two-hybrid approach, coupled to in vitro binding andin vivo co-immunoprecipitation. Moreover, both WT1 and the splicing factor U2-B″ incorporated in vitro into large molecular weight complexes associated with the sense, but not the antisense strand, of a biotinylated adenoviral pre-mRNA. In the same study, +KTS isoforms preferentially associated with U2AF65. Significantly, the presence of +/−KTS isoforms, and their correct ratio, is evolutionarily conserved throughout vertebrates (22Kent J. Coriat A.-M. Sharpe P.T. Hastie N.D. van Heyningen V. Oncogene. 1995; 11: 1781-1792PubMed Google Scholar). It has recently been shown that Frasier syndrome, which is characterized by slow progressive nephropathy and streak gonads, can arise when this alternative splicing event is perturbed (23Barbaux S. Niaudet M.-C. Grünfield J.-P. Jaubert F. Kuttenn C.C. Fekete N. Souleyreau-Therville E. Thibaud M. Fellous M. McElreavy K. Nat. Genet. 1997; 17: 467-470Crossref PubMed Scopus (563) Google Scholar, 24Kikuchi H. Takata A. Akasaka Y. Fukuzawa R. Yoneyama H. Kurosawa Y. Honda M. Kamiyama Y. Hata J. J. Med. Genet. 1998; 35: 45-48Crossref PubMed Scopus (74) Google Scholar, 25Klamt B. Koziell A. Poulat F. Wieacker P. Scambler P. Berta P. Gessler M. Hum. Mol. Gen. 1998; 7: 709-714Crossref PubMed Scopus (283) Google Scholar). In addition, WT1 was shown to bind RNA in vitro. In one study, the WT1 zinc fingers, particularly the first out of the four, bound an RNA sequence encoded by exon 2 of the IGF2 gene (3Caricasole A. Duarte A. Larsson S.H. Hastie N.D. Little M. Holmes G. Todorov I. Ward A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7562-7566Crossref PubMed Scopus (191) Google Scholar), which overlaps with a putative DNA target sequence. A more recent paper describes a SELEX (in vitro ligand selection) experiment that used the zinc fingers of WT1 to define three candidate RNA target sequences (4Bardeesy N. Pelletier J. Nucleic Acids Res. 1998; 26: 1784-1792Crossref PubMed Scopus (75) Google Scholar). The significance of these findings remains to be investigated. Almost a decade after its discovery, the molecular function of WT1, in particular at the post-transcriptional level, is still unclear. Understanding the biochemistry of WT1 is an urgent priority in the field. In particular, its association with the splicing machinery is still controversial, lacking functional data. The aim of this study was to use established fractionation techniques to see if WT1 is present in nuclear RNP particles. It is hoped that these fractionation techniques can, in the near future, help determine the molecular function of WT1 and identify its RNA targets in a physiological context. Extracts were obtained from two expressing mouse cell lines as follows: M15, derived from mesonephros (26Rassoulzadegan M. Paquis-Flucklinger V. Bertino B. Sage J. Jasin M. Miyagawa K. van Heyningen V. Besmer P. Cuzin F. Cell. 1993; 75: 997-1006Abstract Full Text PDF PubMed Scopus (146) Google Scholar), and AC29, derived from an asbestos-induced mesothelioma (27Davis M.R. Manning L.S. Whitaker M.J. Garlepp M.J. Robinson B.W.S. Int. J. Cancer. 1992; 52: 881-886Crossref PubMed Scopus (158) Google Scholar); also from whole embryonal E17 kidneys; and COS7 cells transfected with constructs expressing WT1. AC29 mouse mesothelioma cells were cultured in RPMI 1640 (Life Technologies, Inc.) with 10% fetal calf serum (Life Technologies, Inc.). M15 cells, established from mouse mesonephros, transgenically expressing the large T protein of polyoma virus, under control of the early viral enhancer, were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% fetal calf serum. COS7 and HeLa cells were similarly cultured in Dulbecco's modified Eagle's medium. Cell lines were cultured at 37 °C with 5% CO2 in air. Nuclei from AC29 and M15 cells were isolated according to the method of Lee and Green (68Lee K.A.W. Green M.R. Methods Enzymol. 1990; 181: 20-30Crossref PubMed Scopus (63) Google Scholar). 17-Day fetal kidneys were dissected and immediately frozen in liquid nitrogen. When required, fetal kidneys, dissected manually, were thawed on ice and homogenized in 2× 1-ml volumes of phosphate-buffered saline (PBS) in a Dounce homogenizer, 10 strokes with a loose fitting pestle. The resulting cell suspension was pelleted by centrifugation at 3000 rpm for 10 min. Briefly, cells were suspended in Buffer "A" (10 mm KCl, 1.5 mm MgCl2, 10 mm Hepes, pH 8.0) for 15 min on ice. Following 10 passages through a 25-gauge needle, greater than 90% of cells were lysed. Nuclei were lysed in Buffer "C" (1.5 mmMgCl2, 20 mm Hepes, pH 8.0, 420 mmNaCl, 0.2 mm EDTA pH 8.0, 25% (v/v) glycerol). The resulting lysate was clarified by centrifugation. Both the cell lysis Buffer A and nuclear lysis Buffer C contained protease inhibitor mixture (Roche Molecular Biochemicals). Buffer A contained DNase I, 1000 units/ml, and RNase inhibitor, 400 units/ml. Buffer C contained DNase I, 200 units/ml, and RNase inhibitor, 80 units/ml (Roche Molecular Biochemical). Extracts to be treated with RNase A or T1 were prepared in the absence of RNase inhibitor. For subsequent fractionation, nuclear extracts were dialyzed against the appropriate buffer in a Microdialyzer System 100 using a 50,000 molecular weight cut-off dialysis membrane (Pierce). 500 mg of oligo(dT)-cellulose (Amersham Pharmacia Biotech) was pre-swollen in DEPC-treated distilled H2O and then applied to a 10-ml syringe in column binding buffer (CBB: 10 mm Tris-HCl, pH 7.5, 0.3 m KCl, 1.5 mm MgCl2, 1 mm dithiothreitol, 0.2% Nonidet P-40). Total cell extracts were prepared as follows: cells were lysed in low salt Buffer A as above, and nuclei were pelleted at 3,000 rpm for 5 min. Nuclei were lysed in 2× CBB, debris spun out 2 × 20 min at 13,000 rpm, and the supernatant combined either with an equal volume of cytoplasmic supernatant to make a total cell extract or with DEPC-treated water, when only nuclear extract was desired. Up to 5 ml of extract was applied to the column and left to cycle for 4 h at 4 °C via a peristaltic pump. The poly(A)− fraction (flow-through) was collected, and the column was washed in >25 ml CBB. Optional 5-ml salt washes were then applied (0.6 m and 1.2 m KCl in CBB), and finally the poly(A)+ fraction eluted in 5 ml of 10 mm Tris-HCl, pH 7.5, in warm DEPC-treated distilled water, with 0.001% (w/v) xylene cyanol as an elution marker. 300 μl of nuclear lysate containing 1.5 mg of total protein, prepared as above in Buffer C, was diluted with an equal volume of distilled water to halve the NaCl concentration to 210 mm and loaded onto a 10-ml Sephacryl S-500 (Amersham Pharmacia Biotech) column, previously equilibrated with a 2-fold dilution of the same Buffer C at room temperature. Up to 80 fractions of 250 μl were collected by gravity flow for subsequent analysis. Nuclear extracts were dialyzed into sucrose gradient buffer (10 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1.5 mm MgCl2, 0.2% (v/v) Nonidet P-40). 300-μl samples containing 1.5 mg of total protein were loaded onto pre-formed 5-ml sucrose gradients, 15–30% sucrose (w/v), and spun at 12,000 rpm or 18,000 rpm for 18 h at 0 °C in a Sorvall AH650 rotor. Samples were collected manually into 18 fractions of 250 μl. To load the samples directly onto SDS-PAGE, samples were kept hot after boiling to prevent precipitation of sucrose. RNA was extracted with the RNeasy kit (Qiagen) and rRNA visualized by running samples onto a standard denaturing agarose gel. 20% sucrose cushions were used to pellet poly(A)+ RNP by ultracentrifugation at 40,000 rpm for 3 h using the same rotor. Density equilibrium gradient centrifugation was performed as follows. Nuclear extracts, containing 1.5 mg of total protein in a volume of 300 μl, either treated or untreated with RNase (200 μg RNase A for 30 min at 37 °C), were dialyzed against Nycodenz gradient low salt buffer (20 mmTris-HCl, pH 7.5, 2 mm MgCl2, 1 mmEDTA) and loaded onto a pre-formed 5-ml gradient of 20–60% Nycodenz dissolved in the above buffer. Samples were spun at 36,000 rpm for 18 h at 0 °C in a Sorvall AH650 rotor. Gradients were manually fractionated into 18 samples of 250 μl. Proteins were analyzed by adding SDS-PAGE buffer directly; the presence of Nycodenz presented no hindrance to pipetting these samples onto SDS-PAGE gels. RNA was extracted using RNeasy kits (Qiagen); Nycodenz did not hinder RNA extraction. The density of the samples was determined by measuring the refractive index and applying the formula, density (ρ) in g/cm3 = 3.242η − 3.323, where η is the refractive index. RNA samples extracted from gradients were reverse-transcribed using a cDNA kit (Roche Molecular Biochemical). 5-μl RNA samples were heated to 95 °C for 5 min, cooled on ice, and then combined with 100 pmol of random hexamer, 1 mm each dNTP, 1 unit of ribonuclease inhibitor, and 20 units of M-MuLV reverse transcriptase in a total volume of 30 μl, and incubated for 10 min at room temperature, 60 min at 42 °C, and then 10 min at 95 °C. 1 μl of each sample was used in PCR. Each 50-μl PCR reaction contained 10 pmol of each primer and was run for 30 cycles (1 min 95 °C, 1 min 58 °C, and 3 min 72 °C). Product sizes were compared against DNA markers on 2% agarose gels. The following primers were obtained (Genosys): mouse U1 snRNA, based on data base entry X01623, forward 5′-GCATACTTACCTGGCAGGGGAG-3′, reverse 5′-CAGGGGAGAGCGCGAACGCAGTC-3′, yielding a 166-base pair product; mouse U2 snRNA, based on data base entry X07913, forward 5′-GGTATCGCTTCTCGGCCTTTTGGC-3′, reverse 5′-GGGGGTGCACCGTTCCTGGAGG-3′, yielding a 192-base pair product. Protein fractions in SDS-PAGE loading buffer (2% SDS, 10% glycerol, 60 mmTris-HCl, pH 6.9, 100 mm dithiothreitol, 0.001% bromphenol blue, pH 8.3) were boiled for 5 min, separated by SDS-PAGE (10% polyacrylamide, NBL Gene Sciences Ltd.), and blotted onto polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech). The filter was blocked for 60 min with 5% non-fat dry milk in Tris-buffered saline (TBST: 50 mm NaCl, 20 mm Tris-HCl, pH 7.6, containing 0.1% (v/v) Tween 20) and incubated with the primary antibody, with dilutions ranging from 1:500 to 1:10,000 determined empirically for each antibody, in TBST/half-strength blocking buffer (2.5% non-fat dry milk) overnight at 4 °C. Membranes were then washed 4 times for 10 min, incubated with the secondary antibody (goat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated, Sigma) for 60 min at room temperature, and washed 4 times for 10 min with TBST. Detection was performed by enhanced chemiluminescence (ECL Plus, Amersham Pharmacia Biotech). Blots were exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) for up to 60 min. Each blot was checked for proper protein transfer by staining the polyvinylidene difluoride membrane with 1% india ink in PBS. Antibodies used were as follows: anti-WT1 (C19) polyclonal; anti-Sp1 (PEP 2) polyclonal; anti-PCNA (PC10) monoclonal; anti-TBP (58C9) monoclonal, all from Santa Cruz Biotechnology; anti-U2AF65 polyclonal, courtesy of Rachel Davies; anti-p116 polyclonal ("Stan"), courtesy of A. Lührmann and T. Achsel; anti-hnRNP A1 monoclonal (9H10), courtesy of G. Dreyfuss; anti-p17 TMV coat protein, courtesy of S. Santa Cruz. A cDNA encoding the +/+ isoform of WT1 (including exons 5 and 9) with a thymidine kinase 5′ leader sequence was subcloned into a pSTBlue-1 plasmid vector (Novagen), as per manufacturer's specifications, and verified by automated ABI sequencing (Perkin-Elmer Applied Biosystems). In vitro translated WT1 was prepared using the TNT-coupled transcription/translation system (Promega), as per manufacturer's specifications, and labeling with [35S]methionine (1000 Ci/mmol, Amersham Pharmacia Biotech). The translation product was checked by autoradiography and Western blotting. Full-length mouse WT1 cDNAs (+exon 5 and +KTS) and two deletion constructs (N terminus, amino acids 1–235; and C terminus, amino acids 233–449) were obtained by PCR, adding appropriate restriction sites, subcloned into the vector pCGT7 kindly provided by Javier Caceres, and verified by automated ABI sequencing (Perkin-Elmer Applied Biosystems). 10 μg of each plasmid was transfected into COS7 cells by electroporation (1.00 kV; 25 microfarads), and into AC29 and HeLa cells using LipofectAMINE (Life Technologies, Inc.) as per manufacturer's specifications. Transcription was driven by the cytomegalovirus promoter. Following the thymidine kinase 5′-untranslated region, translation starts with the 11 amino acid prokaryotic epitope tag "T7" (MASMTGGQQMG); and a rabbit β-globin 3′-untranslated region follows the stop codon. Expression was tested by Western blotting and immunofluorescence, using a mouse monoclonal antibody directed against the T7 epitope (Novagen). For immunofluorescence analysis, cells were fixed for 10 min in 1:1 acetone:methanol and blocked in 2% bovine serum albumin in PBS, 7% (v/v) glycerol, + 0.02% sodium azide. Primary antibody dilutions used were 1:1000 (anti-T7 and anti-p116) and secondary dilutions 1:100 (FITC-conjugated goat anti-mouse and Texas Red-conjugated goat anti-rabbit; Sigma). Immunofluorescence was observed and recorded using a Zeiss Axioplan 2 microscope, 63× objective, with a Micro Imager 1400. This technique is typically used to prepare nuclear or cytoplasmic poly(A)+ RNP from a variety of sources, both animal and plant (for examples see Refs. 28Darnborough C.H. Ford P.J. Eur. J. Biochem. 1981; 113: 415-424Crossref PubMed Scopus (82) Google Scholar, 29Dreyfuss G. Choi Y.D. Adam S.A. Mol. Cell. Biol. 1984; 4: 1104-1114Crossref PubMed Scopus (150) Google Scholar, 30Dreyfuss G. Adam S. Choi Y.D. Mol. Cell. Biol. 1984; 4: 415-423Crossref PubMed Scopus (198) Google Scholar, 31Jeffery W.R. J. Biol. 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We found WT1 to be highly enriched in total extract poly(A)+ fractions, as was U2AF65, a U2 snRNP-associated splicing factor that recognizes the polypyrimidine tract at the 3′ splice site (35Zamore P.D. Patton J.G. Green M.R. Nature. 1992; 355: 609-614Crossref PubMed Scopus (457) Google Scholar); p116, a U5 snRNP-associated GTPase structurally related to the ribosomal translocase EF2 (36Fabrizio P. Laggerbauer B. Lauber J. Lane W.S. Lÿhrmann R. Nucleic Acids Res. 1997; 16: 4092-4106Google Scholar); and hnRNP A1, a core hnRNP particle component (Fig. 1 B). Similar WT1 enrichment was seen in nuclear poly(A)+ fractions (not shown). Based on Western blots, we estimate up to 100-fold enrichment of WT1 in poly(A)+fractions. In contrast, PCNA (proliferating cell nuclear antigen, involved in DNA replication), Sp1 (transcription factor, containing zinc fingers structurally related to WT1), and TBP (TATA-binding protein, involved in basal transcription) were not detected in the poly(A)+ fraction. Before elution in distilled water, it is possible to wash off bound proteins with increasing salt concentrations. For example, theXenopus oocyte mRNP-associated RNA helicase, Xp54, can be eluted in 0.6 m KCl, whereas the Y box "mRNA masking" proteins remain bound in high salt and are finally eluted, bound to mRNA, in distilled water (37Ladomery M. Wade E. Sommerville J. Nucleic Acids Res. 1997; 25: 965-973Crossref PubMed Scopus (129) Google Scholar). Similarly, whereas specific poly(A)+ proteins eluted in the 0.6 m KCl or 1.2 m KCl salt washes, others eluted in distilled water. In this experiment, we estimate that approximately 5% of total extract protein eluted in the 0.6 m KCl wash, 5% in the 1.2m KCl wash, and 4% in the final distilled water elution. The bulk of WT1, U2AF65, and p116 remained bound after the 1.2m salt wash (Fig. 1 C). In contrast, hnRNP A1 eluted mostly in the 1.2 m wash, consistent with the reported salt sensitivity of core hnRNP particles (38Beyer A.L. Christensen M.E. Walker B.W. Le Stourgeon W.M. Cell. 1977; 11: 127-138Abstract Full Text PDF PubMed Scopus (294) Google Scholar). Given the possibility that WT1 may have a direct affinity for oligo(dT), two controls were performed (Fig. 1 D). First, binding of WT1 in M15 extract to oligo(dT) did not occur in the absence of 0.3m KCl, salt being required for hybridization of poly(A) sequences to oligo(dT). Second, in vitro translated WT1, present in RNase-treated reticulocyte lysate diluted 20-fold with binding buffer (including 0.3 m KCl), also did not bind. To determine the size of macromolecular complexes containing WT1, nuclear extracts were applied to a 10-ml Sephacryl-500 column, and up to 80 fractions were collected by gravity flow. The elution profiles of WT1, U2AF65, and p116 were compared (Fig. 2). WT1 in AC29 extract eluted from fraction 20 onward and reached a peak in fractions 30–33. WT1 derived from fetal kidney (day E17) nuclear extract eluted in a similar fashion. Particles eluting in these fractions would be expected to include single, or multiple spliceosomes associated with nascent pre-mRNP. In contrast, U2AF65 eluted in a much broader range, up to fraction 48, approaching the elution peak for a monomer (bovine serum albumin, 66 kDa), whereas the U5 snRNP-associated protein p116 was mainly present in fractions 30–40. As a further marker, we used TMV (tobacco mosaic virus), a well characterized plant virus. TMV viruses consist of genomic RNA packaged by the coat protein p17 into large particles (>200S); as expected, TMV eluted in early fractions. AC29 nuclear extract was applied to a 15–30% sucrose gradient and initially spun at 12,000 rpm (Fig. 3 A). Cytoplasmic extract was run in parallel on a similar gradient as a marker. The bulk of WT1 overlapped in fractions 5–7 with p116 but not hnRNP A1 and U2AF65; however, a significant proportion of WT1 and hnRNP A1 also pelleted in fraction 18. When extracts were run at 24,000 rpm, all of the WT1 signal was now in the pellet (not shown). To investigate further the sedimentation of WT1 relative to p116, another sucrose gradient was run, this time at an intermediate speed of 18,000 rpm (Fig. 3 B). Cytoplasmic extract was again run in parallel to ac
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