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• W1991423906 abstract "Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3′-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function. Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3′-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function. Cytokine mRNAs in mammalian cells are typically targeted for rapid degradation by an ARE 1The abbreviations used are: ARE, A+U-rich element; hnRNP, heterogeneous nuclear ribonucleoprotein; HuR, Hu protein R; TTP, tristetraprolin; AUF1, AU-rich binding factor-1; AUBP, AU-rich binding protein; TNF, tumor necrosis factor; KSRP, KH-type splicing regulatory protein; ES, embryonic stem; PcAb, polyclonal antibody. present in the 3′-untranslated region (reviewed in Ref. 1Guhaniyogi J. Brewer G. Gene (Amst.). 2001; 265: 11-23Crossref PubMed Scopus (554) Google Scholar). The mechanism and process by which the ARE promotes rapid degradation of mRNAs are not well understood. AREs generally consist of repeating pentamers of the sequence AUUUA, which promotes the rapid cytoplasmic degradation of cytokine and proto-oncogene mRNAs (1Guhaniyogi J. Brewer G. Gene (Amst.). 2001; 265: 11-23Crossref PubMed Scopus (554) Google Scholar, 2Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3123) Google Scholar, 3Stoecklin G. Ming X.F. Looser R. Moroni C. Mol. Cell. Biol. 2000; 20: 3753-3763Crossref PubMed Scopus (129) Google Scholar). The accelerated decay of ARE mRNAs likely proceeds by rapid deadenylation followed by rapid degradation of the mRNA body (4Brewer G. Ross J. Mol. Cell. Biol. 1988; 8: 1697-1708Crossref PubMed Scopus (215) Google Scholar, 5Wilson T. Treisman R. Nature. 1988; 336: 396-399Crossref PubMed Scopus (506) Google Scholar, 6Shyu A.B. Greenberg M.E. Belasco J.G. Genes Dev. 1989; 3: 60-72Crossref PubMed Scopus (452) Google Scholar, 7Shyu A.B. Belasco J.G. Greenberg M.E. Genes Dev. 1991; 5: 221-231Crossref PubMed Scopus (404) Google Scholar, 8Xu N. Chen C.Y. Shyu A.B. Mol. Cell. Biol. 1997; 17: 4611-4621Crossref PubMed Scopus (307) Google Scholar, 9Ford L.P. Watson J. Keene J.D. Wilusz J. Genes Dev. 1999; 13: 188-201Crossref PubMed Scopus (219) Google Scholar). Although a number of proteins can be cross-linked in vitro to the ARE, only several have been shown to have regulatory functions for ARE mRNAs. Two AUBPs (AUF1 and TTP) promote rapid decay of ARE mRNAs, one (HuR) inhibits mRNA turnover, and one (KH-type splicing-regulatory protein (KSRP)) is suspected in promoting ARE mRNA degradation. The protein TTP promotes rapid decay of tumor necrosis factor (TNF) and granulocyte macrophage colony-stimulating factor mRNAs (10Lai W.S. Carballo E. Strum J.R. Kennington E.A. Phillips R.S. Blackshear P.J. Mol. Cell. Biol. 1999; 19: 4311-4323Crossref PubMed Scopus (635) Google Scholar, 11Carballo E. Lai W.S. Blackshear P.J. Blood. 2000; 95: 1891-1899Crossref PubMed Google Scholar). TTP overexpression further facilitates the rapid decay of TNF and granulocyte macrophage colony-stimulating factor mRNAs (12Lai W.S. Carballo E. Thorn J.M. Kennington E.A. Blackshear P.J. J. Biol. Chem. 2000; 275: 17827-17837Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), whereas in TTP knock-out mice these mRNAs are stabilized (11Carballo E. Lai W.S. Blackshear P.J. Blood. 2000; 95: 1891-1899Crossref PubMed Google Scholar, 13Taylor G.A. Carballo E. Lee D.M. Lai W.S. Thompson M.J. Patel D.D. Schenkman D.I. Gilkeson G.S. Broxmeyer H.E. Haynes B.F. Blackshear P.J. Immunity. 1996; 4: 445-454Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). The protein family known as AUF1 or hnRNP D also binds the ARE and is associated with rapid decay of ARE mRNAs, as originally shown in an in vitro ARE mRNA decay system (14Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (404) Google Scholar). AUF1 comprises four isoforms produced by differential splicing of a single transcript (15Wagner B. DeMaria C.T. Sun Y. Wilson G.M. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (238) Google Scholar). The four isoforms consist of a 37-kDa core protein (p37), a 40-kDa protein (p40) with an N-terminal 19-amino acid insertion of exon 2, a 42-kDa protein (p42) with a C-terminal 49-amino acid insertion of exon 7, and a 45-kDa protein (p45) containing both exon 2 and 7 insertions (reviewed in Ref. 1Guhaniyogi J. Brewer G. Gene (Amst.). 2001; 265: 11-23Crossref PubMed Scopus (554) Google Scholar). The different protein isoforms possess different ARE-RNA binding characteristics (16Kajita Y. Nakayama J.-I. Aizawa M. Ishikawa F. J. Biol. Chem. 1995; 270: 22167-22175Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), ubiquitination, and stabilities (17Laroia G. Schneider R.J. Nucleic Acids Res. 2002; 30: 1-7Crossref PubMed Scopus (54) Google Scholar). The p37 followed by the p40 isoform is most closely associated with promotion of ARE mRNA degradation in a number of studies (18Pende A. Tremmel K.D. DeMaria C.T. Blaxall B.C. Minobe W.A. Sherman J.A. Bisognano J.D. Bristow M.R. Brewer G. Port J.D. J. Biol. Chem. 1996; 271: 8493-8501Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 20Sirenko O.I. Lofquist A.K. DeMaria C.T. Morris J.S. Brewer G. Haskill S. Mol. Cell. Biol. 1997; 17: 3898-3906Crossref PubMed Scopus (135) Google Scholar, 21Laroia G. Cuesta R. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar, 22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar, 23Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar, 24Sarkar B. Xi Q. Liu J.-Y. Schneider R.J. Mol. Cell. Biol. 2003; 23: 6685-6693Crossref PubMed Scopus (122) Google Scholar). The Hu family of proteins (HuA/R, HuB, HuC, HuD) are the only proteins shown to date to stabilize ARE mRNAs. HuR and HuB inhibit ARE mRNA turnover when ectopically overexpressed (25Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (747) Google Scholar, 26Peng S.S. Chen C.Y. Xu N. Shyu A.B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (656) Google Scholar, 27Jain R.G. Andrews L.G. McGowan K.M. Pekala P.H. Keene J.D. Mol. Cell. Biol. 1997; 17: 954-962Crossref PubMed Scopus (182) Google Scholar), and antisense RNA knockdown of endogenous HuR expression decreases the half-lives of certain ARE-containing mRNAs (28Levy N.S. Chung S. Furneaux H. Levy A.P. J. Biol. Chem. 1998; 273: 6417-6423Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 29Rodriguez-Pascual F. Hausding M. Ihrig-Biedert I. Furneaux H. Levy A.P. Forstermann U. Kleinert H. J. Biol. Chem. 2000; 275: 26040-26049Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 30Wang W. Furneaux H. Cheng H. Caldwell M.C. Hutter D. Liu Y. Holbrook N. Gorospe M. Mol. Cell. Biol. 2000; 20: 760-769Crossref PubMed Scopus (468) Google Scholar). Other studies (30Wang W. Furneaux H. Cheng H. Caldwell M.C. Hutter D. Liu Y. Holbrook N. Gorospe M. Mol. Cell. Biol. 2000; 20: 760-769Crossref PubMed Scopus (468) Google Scholar, 31Dixon D.A. Tolley N.D. King P.H. Nabors L.B. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Clin. Investig. 2001; 108: 1657-1665Crossref PubMed Scopus (376) Google Scholar, 32Dean J.L. Wait R. Mahtani K.R. Sully G. Clark A.R. Saklatvala J. Mol. Cell. Biol. 2001; 21: 721-730Crossref PubMed Scopus (250) Google Scholar) have identified HuR as an important stabilizer of short lived AU-rich mRNAs in a variety of settings and in different cell types in culture. The protein KSRP has been implicated in ARE mRNA decay, but it has not been shown to directly promote rapid mRNA turnover (33Chen C.Y. Gherzi R. Ong S.E. Chan E.L. Raijmakers R. Pruijn G.J. Stoecklin G. Moroni C. Mann M. Karin M. Cell. 2001; 107: 451-464Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). KSRP has been found to copurify with the mammalian exosome, a large multiprotein complex containing 3′-5′-exoribonucleases and other proteins involved in mRNA degradation (33Chen C.Y. Gherzi R. Ong S.E. Chan E.L. Raijmakers R. Pruijn G.J. Stoecklin G. Moroni C. Mann M. Karin M. Cell. 2001; 107: 451-464Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). In summary, two or three AUBPs promote ARE mRNA destabilization (TTP, AUF1, and possibly KSRP), yet only one family (Hu proteins) is known to promote ARE mRNA stabilization. In mammalian cell lines, HuR is widely expressed (34Good P.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4557-4561Crossref PubMed Scopus (273) Google Scholar) but at different levels (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar, 25Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (747) Google Scholar). It is possible that destabilization of ARE mRNAs involves multiple redundant protein functions, or it is restricted to different tissues by different proteins. Additionally, the fact that HuR, a major expressed member of the Hu protein family, stabilizes ARE mRNAs suggests that it is expressed in most tissues to provide for regulation in opposition to that of the destabilizing AUBPs, that in some tissues destabilizing AUBPs are likely unopposed in function, or that other inhibitors of ARE mRNA turnover remain to be identified. As might be expected for proteins that regulate proto-oncogene and cytokine mRNA stability, both HuR and AUF1 expression levels have been implicated in carcinogenesis. Overexpression of HuR has been associated with malignant brain and lung tumorigenesis (35Nabors L.B. Gillespie G.Y. Harkins L. King P.H. Cancer Res. 2001; 61: 2154-2161PubMed Google Scholar, 36Blaxall B.C. Dwyer-Nield L.D. Bauer A.K. Bohlmeyer T.J. Malkinson A.M. Port J.D. Mol. Carcinog. 2000; 28: 76-83Crossref PubMed Scopus (104) Google Scholar) and cellular proliferation (37Wang W. Yang X. Cristofalo V.J. Holbrook N.J. Gorospe M. Mol. Cell. Biol. 2001; 21: 5889-5898Crossref PubMed Scopus (144) Google Scholar). AUF1 levels have also been associated with tumorigenesis in a mouse model (38Gouble A. Grazide S. Meggetto F. Mercier P. Delsol G. Morello D. Cancer Res. 2002; 62: 1489-1495PubMed Google Scholar). There has been only a limited analysis of the adult mouse tissue distribution of the different regulators of ARE mRNA stability, and no study has examined the distribution of all AUBPs simultaneously in mice, in which much of the research on cytokine and proto-oncogene mRNA stability takes place. Here we report the tissue distribution of the major known regulators of ARE mRNA stability. These results suggest that HuR and AUF1 are found at the highest levels primarily in the same tissues (thymus and spleen, followed by intestine), whereas TTP is distributed in a largely non-overlapping manner with HuR. KSRP, which may play a role in ARE mRNA destabilization, was distributed widely. Antibodies—Anti-hnRNP D (5B9) monoclonal antibodies were kindly provided by Dr. G. Dreyfuss (University of Pennsylvania, Philadelphia, PA). Rabbit polyclonal antibodies to recombinant p37 AUF1 were developed in our laboratory to bacterially produced and purified p37 protein, affinity purified and concentrated (PcAb 995). Antibodies to HuR (3A2 monoclonal antibody, Santa Cruz), TTP (H-120 polyclonal antibody, Santa Cruz), and β-tubulin monoclonal antibody (Sigma) were purchased commercially. A polyclonal antibody against KSRP (C2742) was a gift from Dr. D. Black (Howard Hughes Medical Institute/University of California, Los Angeles, CA). Cell Culture and Northern Blot Analysis—Chinese hamster ovary and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum or bovine calf serum, respectively. Mouse embryonic stem (ES) cells were cultured on gelatin-coated plates and maintained in Dulbecco's modified Eagle's medium with high glucose supplemented with 15% fetal bovine serum, β-mercaptoethanol (100 μm), non-essential amino acids (100 μm), 1 mm sodium pyruvate, 2 mml-glutamine, and 1000 units/ml leukemia inhibitory factor. Total RNA was isolated from ES cells by using TRIzol reagent (Invitrogen). 20 μg of total RNA was resolved in agarose/formaldehyde gels and transferred to nylon membrane. The blot was hybridized to 32P-labeled probes prepared against the 220-bp murine AUF1 exon 1 and exposed to film. Animal Studies—Experiments were carried out in accordance with National Institutes of Health guidelines for animal treatment, housing, and euthanasia. Young adult C57BL/6 male and female mice (6 weeks old) were sacrificed by CO2 asphyxiation. The organs were removed rapidly by dissection, cut into thin slices, homogenized, and snap-frozen in liquid nitrogen for later use or fixed in neutral-buffered formaldehyde. Homogenization was carried out using 5 volumes of homogenization buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 1× complete protease inhibitor mixture (Roche)). After incubation on ice for 15 min, samples were clarified by centrifugation at 12,000 rpm at 4 °C for 10 min. Then the soluble supernatant was reserved. Protein concentrations were determined by Bradford assay (Bio-Rad). Protein Analysis—For Western immunoblot analysis, equal amounts of protein extracts from murine tissues or cells were denatured by the addition of 2× SDS-PAGE buffer (100 mm Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 4% SDS, bromphenol blue, 20% glycerol) and heating to 100 °C for 5 min. Equal amounts of cell lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane, and immunoblotting was performed using primary antibody as indicated above. Immunoblots were developed using the ECL system. Immunohistochemistry Analysis—For immunohistochemistry, surgically excised organs were fixed in 10% buffered formalin, dehydrated through a series of ethanol followed by xylene clearing, and embedded in paraffin. Sections were cut to a 5-μm thickness and heated overnight in at 56 °C. Sections were deparaffinized in xylene and rehydrated through a series of alcohol. Immunohistochemical staining was performed using a Vectastain ABC kit (Vector Laboratories) The sections were subjected to microwave antigen retrieval using a sodium citrate buffer. Endogenous peroxidase activity was blocked by a 10-min incubation in 3% H2O2 in phosphate-buffered saline followed by rinsing in phosphate-buffered saline. Nonspecific binding of the secondary antibody was blocked by incubating in 15% normal goat serum for 1 h at room temperature. Sections were subsequently incubated overnight at 4 °C in anti-AUF1 polyclonal antibody (PcAb 995), which was diluted at 1:200 with phosphate-buffered saline containing 5% normal goat serum. Following incubation with biotinylated secondary antibody and ABC reagent, staining was visualized using a 3,3′-diaminobenzidine substrate kit (Vector Laboratories). The sections were then counterstained with hematoxylin, cleared, and mounted with Permount medium (Sigma). Validation of AUF1 Antibodies—Previous studies have not collectively compared the major AUBP regulators of ARE mRNA stability in mice in a variety of tissues and organs. In addition, our immunoblot studies indicate that the antibody (5B9) often used to detect hnRNP D/AUF1 strongly cross-reacts with another protein of similar molecular mass to the p45 AUF1 isoform (Fig. 1B), confounding previous immunohistochemical and immunoblot studies. We therefore first developed and affinity purified a highly specific polyclonal rabbit antibody (PcAb 995) to AUF1 that does not cross-react with other polypeptides within the range of 35-50 kDa. To demonstrate the specificity of this antibody, protein extracts from wild type and AUF1 knock-out ES cells were used. The details of the development of the AUF1 gene knock-out cells will be reported elsewhere. Northern mRNA analysis confirmed that the homozygous AUF1 knock-out ES cells (-/-) do not express AUF1 mRNA (Fig. 1A) compared with wild type (+/+) and heterozygous (+/-) ES cells. Using the same ES cells, anti-hnRNP D/AUF1 monoclonal antibody (5B9), which has been widely used in the literature, was found to cross-react with a slightly larger polypeptide. In 12% SDS-PAGE, an unidentified polypeptide was recognized by the 5B9 but not the PcAb 995 antibodies; this polypeptide migrates very closely with, but is clearly distinct from, the largest isoform of AUF1, as it remained present even in homozygous AUF1 knock-out ES cells (Fig. 1B, compare left with middle panel). This cross-reactivity could lead to misinterpretation of the unknown protein as the p45 AUF1 isoform and, accordingly, the actual p45 AUF1 protein as the p42 AUF1 isoform. The high affinity polyclonal anti-AUF1 antibody recognized four bands in wild type ES cells and in Chinese hamster ovary cells (the p40 and p42 AUF1 bands co-migrate) but not the cross-reactive polypeptide detected by the 5B9 monoclonal AUF1 antibody. Higher resolution of AUF1 proteins in SDS-10% PAGE suggests that the unknown protein that cross-reacts with the 5B9 antibody has a molecular mass of 46-47 kDa (Fig. 1B, right panel). These data therefore validate the use of the PcAb 995 antibody for this study. Antibodies were validated previously to KSRP (39Min H. Turck C.W. Nikolic J.M. Black D.L. Genes Dev. 1997; 11: 1023-1036Crossref PubMed Scopus (279) Google Scholar) and HuR, which has shown some cross-reactivity to HuD and HelN1 (HuB) but not HuC (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar). Tissue Distribution of AUBP Proteins—Six-week-old male and female C57BL/6 mice (young adults) were sacrificed, and organs and tissues were rapidly dissected and either snap-frozen in liquid nitrogen, fixed in formalin, and embedded in paraffin or immediately processed for SDS-PAGE. For electrophoresis, whole cell extracts were prepared by mechanical disruption in homogenization buffer and clarified by centrifugation, and equal amounts of protein were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose membrane, and immunoblot analysis was performed using primary antibodies as indicated and ECL for detection. Tissue Expression of HuR Protein—There are four members of the Hu family of RNA-binding proteins that participate in RNA processing events and ARE mRNA stability (reviewed in Ref. 40Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5-7Crossref PubMed Scopus (257) Google Scholar). HuB (HelN1), HuC, and HuD are developmentally regulated and tissue-restricted in expression, predominantly in the brain (40Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5-7Crossref PubMed Scopus (257) Google Scholar). HuR (also known as HuA) is expressed widely but at different levels in mammalian cell lines (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar, 25Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (747) Google Scholar, 40Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5-7Crossref PubMed Scopus (257) Google Scholar). However, a limited number of human and mouse tissues have been studied for HuR protein expression (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar, 25Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (747) Google Scholar, 41King P.H. Levine T.D. Fremeau Jr., R.T. Keene J.D. J. Neurosci. 1994; 14: 1943-1952Crossref PubMed Google Scholar, 42Ma W.-J. Cheng S. Campbell C. Wright A. Furneaux H. J. Biol. 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Studies have not investigated fully HuR protein expression levels in a variety of tissues from the adult mouse. Soluble whole cell protein extracts were prepared from male and female mouse tissues; organs were denatured and resolved by SDS-PAGE, and HuR was identified by immunoblot analysis using a monoclonal antibody to HuR (3A2), which most strongly recognizes the HuR member of the Hu family but also cross-reacts with HuB and HuD (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar). Normalization to equal protein levels and the lack of protein degradation were reflected in similar total polypeptide levels (within 2-fold) by Ponceau S staining, permitting direct comparisons between tissues (Fig. 2C). Immunoblot staining of poly(A)-binding protein in tissues (Fig. 2D) demonstrated more specifically the lack of degradation of proteins and that similar (within 2-fold) levels were present in equal amounts of tissue protein extracts. Similar results were found for immunoblot analysis of tubulin proteins as well (data not shown). Thus, it is highly unlikely that differential protein degradation could occur in these rapidly frozen tissues. In both male and female young adult mice HuR was predominantly expressed in the intestine, thymus, and spleen, followed by the liver (Fig. 2, A and B). The significant expression of HuR in proliferating lymphocytic cells (43Atasoy U. Watson J. Patel D. Keene J.D. J. Cell Sci. 1998; 111: 3145-3156Crossref PubMed Google Scholar) could play a role in the high levels observed here. HuR was almost undetectable in the brain, skeletal muscle, kidney, lung, and heart (Fig. 2, A and B), arguing against strong cross-reactivity of the 3A2 antibody with HuB, -C, or -D. In female mice, a low level of HuR expression was detected in the uterus and little in the ovary, whereas male mice expressed HuR strongly in the testes. These results are consistent with several reports demonstrating strong HuR expression in malignant murine lung and brain carcinomas but not in normal tissue (35Nabors L.B. Gillespie G.Y. Harkins L. King P.H. Cancer Res. 2001; 61: 2154-2161PubMed Google Scholar, 36Blaxall B.C. Dwyer-Nield L.D. Bauer A.K. Bohlmeyer T.J. Malkinson A.M. Port J.D. Mol. Carcinog. 2000; 28: 76-83Crossref PubMed Scopus (104) Google Scholar). A previous study (47Gouble A. Morello D. Oncogene. 2000; 19: 5377-5384Crossref PubMed Scopus (41) Google Scholar) of HuR protein tissue expression in the adult mouse reported strong expression in the spleen, thymus, and testes as found here but additionally showed strong expression in the brain, lung, and intestine. Thus, our studies concur with those of Gouble and Morello (47Gouble A. Morello D. Oncogene. 2000; 19: 5377-5384Crossref PubMed Scopus (41) Google Scholar), apart from the abundance of HuR in the brain. In this regard, a previous study (35Nabors L.B. Gillespie G.Y. Harkins L. King P.H. Cancer Res. 2001; 61: 2154-2161PubMed Google Scholar) reported strong HuR expression in brain tumors but not normal brain using a validated antibody to HuR, similar to our results. Because studies demonstrated that the adult mouse brain predominantly expresses HuC and -D forms of the Hu protein (48Okano H.J. Darnell R.B. J. Neurosci. 1997; 17: 3024-3037Crossref PubMed Google Scholar), we suspect that reports of high levels of HuR protein in the brain more likely reflect cross-reactivity to HuC and -D neuronal forms. The analysis of HuR in a limited number of human tissues, using a commercial source of tissue, showed strong expression in the spleen and testes as found here in mouse tissue (the thymus was not investigated) but differed in finding strong expression in the brain, kidney, and lung (22Gallouzi I.E. Brennan C.M. Stenberg M.G. Swanson M.S. Eversole A. Maizels N. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3073-3078Crossref PubMed Scopus (275) Google Scholar), which might reflect species differences or differences in the amount of protein analyzed. It is also interesting to note that a survey of reported HuR protein and mRNA expression levels in tissues suggests a possible disparity, potentially implicating translational control or differential mRNA versus protein stabilities in HuR regulation. Tissue and Isoform Expression of AUF1 Proteins—AUF1 consists of four isoforms (p37, p40, p42, and p45) derived from the same mRNA by differential splicing (1Guhaniyogi J. Brewer G. Gene (Amst.). 2001; 265: 11-23Crossref PubMed Scopus (554) Google Scholar). The p37 and p40 isoforms are most closely associated with the promotion of ARE mRNA decay (14Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (404) Google Scholar, 19DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Laroia G. Cuesta R. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar, 23Loflin P. Chen C.Y. Shyu A.B. Genes Dev. 1999; 13: 1884-1897Crossref PubMed Scopus (263) Google Scholar, 24Sarkar B. Xi Q. Liu J.-Y. Schneider R.J. Mol. Cell. Biol. 2003; 23: 6685-6693Crossref PubMed Scopus (122) Google Scholar, 49Laroia G. Sarkar B. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1842-1846Crossref PubMed Scopus (94) Google Scholar). The studies carried out demonstrating tissue- and isoform-specific distribution of AUF1" @default.
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• W1991423906 title "Tissue Distribution of AU-rich mRNA-binding Proteins Involved in Regulation of mRNA Decay" @default.
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