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• W2080636764 abstract "The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies. The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies. MicroRNAs (miRNAs) 2The abbreviations used are: miRNA, microRNA; nt, nucleotide; miRISC, miRNA-induced silencing complexe; siRNA, small interfering RNA; UTR, untranslated region; HA, hemagglutinin; PBS, phosphate-buffered saline; RT, reverse transcriptase; FACS, fluorescence-activated cell sorter; PHA, phytohemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; HIV-1, human immunodeficiency virus type 1; P-bodies, processing bodies. 2The abbreviations used are: miRNA, microRNA; nt, nucleotide; miRISC, miRNA-induced silencing complexe; siRNA, small interfering RNA; UTR, untranslated region; HA, hemagglutinin; PBS, phosphate-buffered saline; RT, reverse transcriptase; FACS, fluorescence-activated cell sorter; PHA, phytohemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; HIV-1, human immunodeficiency virus type 1; P-bodies, processing bodies. are 20-22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29225) Google Scholar, 2Ambros V. Chen X. Development. 2007; 134: 1635-1641Crossref PubMed Scopus (244) Google Scholar, 3Rana T.M. Nat. Rev. Mol. Cell. Biol. 2007; 8: 23-36Crossref PubMed Scopus (844) Google Scholar). More than 474 miRNAs have been identified in humans so far, and ∼30% of the genes in the human genome are predicted to be subject to miRNA regulation (4Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9777) Google Scholar). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3Rana T.M. Nat. Rev. Mol. Cell. Biol. 2007; 8: 23-36Crossref PubMed Scopus (844) Google Scholar). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29225) Google Scholar, 5Hutvagner G. Zamore P.D. Science. 2002; 297: 2056-2060Crossref PubMed Scopus (1628) Google Scholar, 6Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1443) Google Scholar, 7Ambros V. Lee R.C. Lavanway A. Williams P.T. Jewell D. Curr. Biol. 2003; 13: 807-818Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, 8Pillai R.S. Bhattacharyya S.N. Filipowicz W. Trends Cell Biol. 2007; 17: 118-126Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3′-untranslated region (3′-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29225) Google Scholar). A 7-nucleotide “seed” sequence (at positions 2-8 from the 5′-end) in miRNAs seems to be essential for this action (4Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9777) Google Scholar). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29225) Google Scholar, 3Rana T.M. Nat. Rev. Mol. Cell. Biol. 2007; 8: 23-36Crossref PubMed Scopus (844) Google Scholar, 7Ambros V. Lee R.C. Lavanway A. Williams P.T. Jewell D. Curr. Biol. 2003; 13: 807-818Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3Rana T.M. Nat. Rev. Mol. Cell. Biol. 2007; 8: 23-36Crossref PubMed Scopus (844) Google Scholar, 8Pillai R.S. Bhattacharyya S.N. Filipowicz W. Trends Cell Biol. 2007; 17: 118-126Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophila helicase Armitage, Mov10, possibly in a cell type-specific manner (9Chendrimada T.P. Gregory R.I. Kumaraswamy E. Norman J. Cooch N. Nishikura K. Shiekhattar R. Nature. 2005; 436: 740-744Crossref PubMed Scopus (1576) Google Scholar, 10Gregory R.I. Chendrimada T.P. Cooch N. Shiekhattar R. Cell. 2005; 123: 631-640Abstract Full Text Full Text PDF PubMed Scopus (1205) Google Scholar, 11Caudy A.A. Myers M. Hannon G.J. Hammond S.M. Genes Dev. 2002; 16: 2491-2496Crossref PubMed Scopus (538) Google Scholar, 12Lee Y. Hur I. Park S.Y. Kim Y.K. Suh M.R. Kim V.N. EMBO J. 2006; 25: 522-532Crossref PubMed Scopus (520) Google Scholar, 13Meister G. Landthaler M. Peters L. Chen P.Y. Urlaub H. Luhrmann R. Tuschl T. Curr. Biol. 2005; 15: 2149-2155Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). The miRNA-mediated translational repression consistently correlates with an accumulation of miRNA-bound mRNAs at cytoplasmic foci known as processing bodies (P-bodies) (8Pillai R.S. Bhattacharyya S.N. Filipowicz W. Trends Cell Biol. 2007; 17: 118-126Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Several lines of evidence have indicated that P-bodies are actively involved in miRNA-mediated mRNA repression (14Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell. Biol. 2007; 8: 9-22Crossref PubMed Scopus (744) Google Scholar). The P-body-associated protein GW182 associates directly with Ago-1 (15Liu J. Rivas F.V. Wohlschlegel J. Yates 3rd, J.R. Parker R. Hannon G.J. Nat. Cell Biol. 2005; 7: 1261-1266Crossref PubMed Scopus (508) Google Scholar, 16Behm-Ansmant I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (725) Google Scholar). Depletion of P-body components such as GW182 and Rck/p54 prevents translational repression of target mRNAs (8Pillai R.S. Bhattacharyya S.N. Filipowicz W. Trends Cell Biol. 2007; 17: 118-126Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar, 14Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell. Biol. 2007; 8: 9-22Crossref PubMed Scopus (744) Google Scholar, 15Liu J. Rivas F.V. Wohlschlegel J. Yates 3rd, J.R. Parker R. Hannon G.J. Nat. Cell Biol. 2005; 7: 1261-1266Crossref PubMed Scopus (508) Google Scholar, 16Behm-Ansmant I. Rehwinkel J. Doerks T. Stark A. Bork P. Izaurralde E. Genes Dev. 2006; 20: 1885-1898Crossref PubMed Scopus (725) Google Scholar, 17Bruno I. Wilkinson M.F. Cell. 2006; 125: 1036-1038Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 18Chu C.Y. Rana T.M. PLoS Biol. 2006; 4: e210Crossref PubMed Scopus (410) Google Scholar, 19Jakymiw A. Lian S. Eystathioy T. Li S. Satoh M. Hamel J.C. Fritzler M.J. Chan E.K. Nat. Cell Biol. 2005; 7: 1267-1274Crossref PubMed Scopus (314) Google Scholar). Furthermore, several miRISC-related components, such as miRNAs, mRNAs repressed by miRNAs, Ago-1, Ago-2, and Mov10, are found in P-bodies (14Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell. Biol. 2007; 8: 9-22Crossref PubMed Scopus (744) Google Scholar). P-body formation is a dynamic process that requires continuous accumulation of repressed mRNAs (20Eulalio A. Behm-Ansmant I. Schweizer D. Izaurralde E. Mol. Cell. Biol. 2007; 27: 3970-3981Crossref PubMed Scopus (500) Google Scholar). However, P-bodies serve not only as sites for RNA degradation, but also for storage of repressed mRNAs (15Liu J. Rivas F.V. Wohlschlegel J. Yates 3rd, J.R. Parker R. Hannon G.J. Nat. Cell Biol. 2005; 7: 1261-1266Crossref PubMed Scopus (508) Google Scholar). These mRNAs may later return to polysomes to synthesize new proteins (14Eulalio A. Behm-Ansmant I. Izaurralde E. Nat. Rev. Mol. Cell. Biol. 2007; 8: 9-22Crossref PubMed Scopus (744) Google Scholar). In fact, some cellular proteins can facilitate the exit of miRNA-bound mRNAs from P-bodies. For example, a stress situation may induce the relocation of HuR, an AU-rich element-binding protein, from the nucleus to P-bodies in the cytoplasm where it binds to the 3′-UTR of its target mRNA encoding CAT-1 (21Bhattacharyya S.N. Habermacher R. Martine U. Closs E.I. Filipowicz W. Cell. 2006; 125: 1111-1124Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar). This binding increases the stability of the miR-122-bound mRNA by assisting it to egress from the P-body and return to polysomes. However, the mechanism underlying this reverse transport of miRNA-bound mRNA out of P-bodies remains to be further clarified. The cellular apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G protein (APOBEC3G or A3G) is a potent antiretroviral factor that belongs to the cytidine deaminase family (22Sheehy A.M. Gaddis N.C. Choi J.D. Malim M.H. Nature. 2002; 418: 646-650Crossref PubMed Scopus (1901) Google Scholar, 23Turelli P. Trono D. Science. 2005; 307: 1061-1065Crossref PubMed Scopus (79) Google Scholar). A3G can be incorporated into HIV-1 particles and cause extensive C to U conversion in the viral minus-stranded DNA during reverse transcription (24Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (915) Google Scholar, 25Mangeat B. Turelli P. Caron G. Friedli M. Perrin L. Trono D. Nature. 2003; 424: 99-103Crossref PubMed Scopus (1238) Google Scholar, 26Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (502) Google Scholar), which can trigger its degradation by virion-associated uracil DNA glycosylase-2 (UNG2) and apurinic/apyrimidinic endonucleases (APE) or lethal hypermutation in the HIV-1 genome (26Yu Q. Konig R. Pillai S. Chiles K. Kearney M. Palmer S. Richman D. Coffin J.M. Landau N.R. Nat. Struct. Mol. Biol. 2004; 11: 435-442Crossref PubMed Scopus (502) Google Scholar, 27Yang B. Chen K. Zhang C. Huang S. Zhang H. J. Biol. Chem. 2007; 282: 11667-11675Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). However, accumulating evidence indicates that A3G protein carrying mutations in the catalytic domain of the cytidine deaminase retains substantial anti-HIV-1 activity (24Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (915) Google Scholar, 28Newman E.N. Holmes R.K. Craig H.M. Klein K.C. Lingappa J.R. Malim M.H. Sheehy A.M. Curr. Biol. 2005; 15: 166-170Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 29Bogerd H.P. Wiegand H.L. Doehle B.P. Cullen B.R. Virology. 2007; 364: 486-493Crossref PubMed Scopus (59) Google Scholar, 30Holmes R.K. Koning F.A. Bishop K.N. Malim M.H. J. Biol. Chem. 2007; 282: 2587-2595Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 31Bishop K.N. Holmes R.K. Malim M.H. J. Virol. 2006; 80: 8450-8458Crossref PubMed Scopus (245) Google Scholar). Interestingly, A3G is found in P-bodies and stress granules (32Gallois-Montbrun S. Kramer B. Swanson C.M. Byers H. Lynham S. Ward M. Malim M.H. J. Virol. 2007; 81: 2165-2178Crossref PubMed Scopus (213) Google Scholar, 33Wichroski M.J. Robb G.B. Rana T.M. PLoS Pathog. 2006; 2: e41Crossref PubMed Scopus (159) Google Scholar). It is associated with a high molecular mass structure (>700 kDa) in replicating cells, and this interaction is RNase-sensitive (34Chiu Y.L. Soros V.B. Kreisberg J.F. Stopak K. Yonemoto W. Greene W.C. Nature. 2005; 435: 108-114Crossref PubMed Scopus (390) Google Scholar, 35Chiu Y.L. Witkowska H.E. Hall S.C. Santiago M. Soros V.B. Esnault C. Heidmann T. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15588-15593Crossref PubMed Scopus (208) Google Scholar). Further studies indicate that A3G interacts with many RNA-binding proteins, among which are several miRNA-related proteins, such as Ago1, Ago2, Mov10, and poly(A)-binding protein 1 (PABP1). These interactions are either partially or completely resistant to RNase A digestion (32Gallois-Montbrun S. Kramer B. Swanson C.M. Byers H. Lynham S. Ward M. Malim M.H. J. Virol. 2007; 81: 2165-2178Crossref PubMed Scopus (213) Google Scholar, 35Chiu Y.L. Witkowska H.E. Hall S.C. Santiago M. Soros V.B. Esnault C. Heidmann T. Greene W.C. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15588-15593Crossref PubMed Scopus (208) Google Scholar, 36Kozak S.L. Marin M. Rose K.M. Bystrom C. Kabat D. J. Biol. Chem. 2006; 281: 29105-29119Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). 3H. Zhang, unpublished data. 3H. Zhang, unpublished data. Aside from its inhibitory function in relation to endogenous retroviruses and other retrotransposons (37Esnault C. Heidmann O. Delebecque F. Dewannieux M. Ribet D. Hance A.J. Heidmann T. Schwartz O. Nature. 2005; 433: 430-433Crossref PubMed Scopus (278) Google Scholar, 38Bogerd H.P. Wiegand H.L. Doehle B.P. Lueders K.K. Cullen B.R. Nucleic Acids Res. 2006; 34: 89-95Crossref PubMed Scopus (218) Google Scholar, 39Hulme A.E. Bogerd H.P. Cullen B.R. Moran J.V. Gene (Amst.). 2007; 390: 199-205Crossref PubMed Scopus (126) Google Scholar, 40Dutko J.A. Schafer A. Kenny A.E. Cullen B.R. Curcio M.J. Curr. Biol. 2005; 15: 661-666Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 41Esnault C. Millet J. Schwartz O. Heidmann T. Nucleic Acids Res. 2006; 34: 1522-1531Crossref PubMed Scopus (97) Google Scholar), the major cellular function of A3G seems to be related to P-body-related RNA processing and metabolism. As recent development has indicated that the function of P-body is closely related to miRNA activity, we therefore investigated the possibility of a connection between A3G and miRNA function. Plasmid Constructions—The miRNA reporter constructs used in this study (pmir16-luc, pmir10b-luc, pmir25-luc, and pEGFP-c1-let-7a) were obtained by directly inserting the annealed corresponding miRNA-binding sites into the 3′-UTR region of the luciferase gene in the pMIR-REPORT vector (Ambion Inc., Austin, TX) between the SpeI and HindIII sites or into the 3′-UTR region of the green fluorescent protein gene, gfp, in the pEGFP-C1 vector (BD, Mountain View, CA) between the EcoRI and XhoI sites. A3B gene with a V5 epitope tag sequence at its 3′ terminus was amplified from the mRNA of H9 cells through RT-PCR, and the sequence was confirmed. Then the modified A3B was inserted into the pcDNA3 vector. Wild-type and mutant A3G-expressing plasmids were amplified by PCR using constructs described previously as templates and cloned into the pcDNA3 vector (24Zhang H. Yang B. Pomerantz R.J. Zhang C. Arunachalam S.C. Gao L. Nature. 2003; 424: 94-98Crossref PubMed Scopus (915) Google Scholar). Detailed information about the oligonucleotides used for cloning or PCR is provided in supplemental Table S1. Cell Isolation, Culture, and Transfection—Primary CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells using the CD4+ T cell isolation kit II (Miltenyi, Auburn, CA) and subsequently activated by treating with phytohemagglutinin (PHA) (42Chen K. Huang J. Zhang C. Huang S. Nunnari G. Wang F.X. Tong X. Gao L. Nikisher K. Zhang H. J. Virol. 2006; 80: 7645-7657Crossref PubMed Scopus (127) Google Scholar). The purity of CD4 T-cells can reach 98%. For isolation of monocytes, CD14+ cells were purified from PBMCs by positive selection with CD14+ micromeads (Miltenyi, Auburn, CA) using auto MACS according to the manufacturerʼns instructions. The purity of the CD14+ cells is large than 98% as determined by FACS staining with CD14 antibody. Monocytes were further cultured in complete RPMI in the presence of 0.5 ng/ml recombinant human macrophage colony stimulating factor (M-CSF, R&D Systems) and 0.5 ng/ml granulocyte colony-stimulating factor (G-CSF, R&D Systems) for 7 days. The activated CD4+ T cells and H9 T cells were transfected using an Amaxa nucleofector apparatus (Amaxa Biosystems, Gaithersburg, MD), as described (42Chen K. Huang J. Zhang C. Huang S. Nunnari G. Wang F.X. Tong X. Gao L. Nikisher K. Zhang H. J. Virol. 2006; 80: 7645-7657Crossref PubMed Scopus (127) Google Scholar). 293T or HeLa cells were transfected using FuGENE 6 (Roche Applied Science, Indianapolis, IN) for plasmids or HiPerfect (Qiagen, Valencia, CA) for siRNAs or antisense miRNA inhibitors. Macrophages were transfected using jetPEI (Polyplus-Transfection Inc. New York, NY). Synthesis of siRNAs and Antisense miRNA Inhibitors—The miRNA gene sequences were selected from the Sanger Center miRNA Registry. The siRNAs and synthetic antisense miRNA inhibitors (2′-O-methyl-oligoribonucleotides) against mir-16 and mir-28 were chemically synthesized by Integrated DNA Technologies (Coralville, IA). miRNA Array Analysis—Total RNA (10 μg) from 293T cells transfected with pcDNA-A3G-HA or the pcDNA3 parent vector was isolated with TRIzol reagent (Invitrogen). The following RNA processing, microarray fabrication, array hybridization, and data acquisition were performed at LC Sciences (Houston, TX). Briefly, 2-5 μg of total RNA sample, which was size-fractionated using a YM-100 Microcon centrifugal filter (Millipore, Billerica, MA) and the small RNAs (<300 nt) isolated were 3′-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Two different tags were used for the two RNA samples in dual-sample experiments. Hybridization was performed overnight on a Paraflo microfluidic chip using a microcirculation pump (Atactic Technologies, Houston, TX). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. Each region in the chip comprises a miRNA probe region, which detects miRNA transcripts listed in Sanger miRBase Release 9.0. Total 469 human miRNAs were tested. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. Hybridization used 100 μl of 6× SSPE buffer (0.90 m NaCl, 60 mm Na2HPO4, 6 mm EDTA, pH 6.8) containing 25% formamide at 34 °C. After hybridization detection used fluorescence labeling using tag-specific Cy3 and Cy5 dyes. Hybridization images were collected using a GenePix 4000B laser scanner (Molecular Device, Sunnyvale, CA) and digitized using Array-Pro image analysis software (Media Cybernetics, Bethesda, MD). Data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). The ratio of the two sets of detected signals (log2 transformed, balanced) and p values of the t test were calculated; differentially detected signals were those with less than 0.01 p values. Real-time RT-PCR Detection—To confirm the miRNA array results, “stem-loop” real-time reverse transcription (RT)-PCR was used to detect cellular miRNAs, as described, but with minor modifications (43Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (3998) Google Scholar). The primers for RT-PCR to detect miRNA were designed based on the miRNA sequences provided by the Sanger Center miRNA Registry (supplemental Table S1). The miRNAs were isolated from 293T cells with the mirVana miRNA isolation kit (Ambion). RT reactions were performed by means of the iScript cDNA synthesis kit (Bio-Rad). Real-time PCR was performed on the 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). U6 RNA was used as an endogenous control for miRNA detection, while β-actin or β-tubulin mRNA was measured as an endogenous control for luciferase gene expression detection. The cycle number at which the product level exceeded an arbitrarily chosen threshold (CT) was determined for each target sequence, and the amount of each miRNA relative to U6 RNA (or luciferase to β-actin mRNA) was described using the formula 2-ΔCT, where ΔCT = CT(miRNA (or luciferase) - CT U6 RNA (or β-actin). Flow Cytometric Analysis—Primary CD4+ T cells and H9 cells transfected with gfp-containing plasmids were subjected to flow cytometric analysis on a Beckman Coulter cytometer (Fullerton, CA) at 48 h post-nucleofection. The mean fluorescence intensity (MFI) and positive percentage rate (%) of green fluorescing cells was determined. Luciferase Assay—A luciferase assay was performed as described (42Chen K. Huang J. Zhang C. Huang S. Nunnari G. Wang F.X. Tong X. Gao L. Nikisher K. Zhang H. J. Virol. 2006; 80: 7645-7657Crossref PubMed Scopus (127) Google Scholar). Immunoprecipitation and Western Blotting—The co-immunoprecipitation analysis and Western blotting assays were performed as described (44Yang S. Sun Y. Zhang H. J. Biol. Chem. 2001; 276: 4889-4893Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Rabbit polyclonal anti-A3G and anti-A3F antibodies were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. Polysome Profile Analysis—Polysome profiles were determined as described with modifications (45Nottrott S. Simard M.J. Richter J.D. Nat. Struct. Mol. Biol. 2006; 13: 1108-1114Crossref PubMed Scopus (315) Google Scholar). Briefly, 293T cells were cultured in Dulbeccoʼns modified Eagleʼns medium, and treated with various reagents as indicated in the figure legend, and harvested at 48-h post-transfection at 70-80% confluency by replacing the culture media with fresh media containing cycloheximide (100 μg/ml; Sigma) for 30 min. Cells were washed with ice-cold phosphate-buffered saline (PBS) containing cycloheximide (50 μg/ml), followed by resuspension in an ice-cold lysis buffer (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 2 units/μl RNasin, and 0.5% (v/v) Triton X-100 for 10 min. Nuclei and other cellular debris were then removed by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatants were subsequently layered on top of 15-50% sucrose gradients. Centrifugation proceeded at 36,000 rpm for 90 min at 4 °C in a Beckmann SW41Ti rotor. The location of polysomes in the gradient was determined by measuring the absorbance at 254 nm using a spectrophotometer. RNAs in each fraction were extracted using TRIzol reagent and subjected to RT-PCR detection using primer pairs for the luciferase and β-tubulin genes. Immunofluorescence and in Situ Hybridization Analysis—For immunofluorescence analysis, HeLa cells were seeded onto coverslips in 6-well plates and transfected with 0.5 μg of pGFP-GW182delta1 plasmid (Addgene Inc., Cambridge, MA) (19Jakymiw A. Lian S. Eystathioy T. Li S. Satoh M. Hamel J.C. Fritzler M.J. Chan E.K. Nat. Cell Biol. 2005; 7: 1267-1274Crossref PubMed Scopus (314) Google Scholar) and 1 μg of pcDNA-A3G-HA using FuGENE 6. Cells were fixed at 36-h post-transfection with 4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton X-100 for 5 min, followed by blocking in a buffer containing 0.2% Triton X-100, 100 mm Tris-HCl (pH 7.5), 0.9% NaCl, and 2% bovine serum albumin. Monoclonal anti-HA (Sigma) was used at 1:500 dilution, and a secondary goat anti-mouse antibody conjugated to Texas Red (Abcam, Cambridge, MA) was used at a dilution of 1:1000 for detection. The coverslips were analyzed with a Zeiss LSM 510 META Confocal Laser Scanning Microscope System (Thornwood, NY). For in situ hybridization analysis, HeLa cells growing on gelatin-coated coverslips were transfected with the pGFP-GW182delta1 plasmid and pmir-16-luc together with pcDNA-A3G-HA, pcDNA3, or various antisense anti-mir inhibitors, as indicated in the figure legend. At 48-h post-transfection, cells were fixed for 10 min at room temperature in 4% formaldehyde, 10% acetic acid, and PBS. After washing twice in PBS, cells were permeabilized by treatment with 70% ethanol overnight. Probes and coverslip were denatured at 80 °C for 75 s. After rehydration in 2× SSC, (300 mm NaCl, 30 mm sodium citrate, pH 7.0), 50% formamide, the fixed and permeabilized cells (∼105) were hybridized for 1 h in a moist chamber at 37 °C in 40 μl of a mixture containing 10% dextran sulfate, 2 mm vanadyl-ribonucleoside complex, 0.02% RNase-free bovine serum albumin, 40 μg of Escherichia coli tRNA, 2× SSC, 50% formamide, and 10 μl of Cy3-labeled locked nucleic acid probes (Integrated DNA Technologies, Coralville, IA; for sequence of the probes, see supplemental Table S1), complementary to the luciferase coding regions diluted with 50% deionized formamide, 2× SSC, 10% dextran sulfate, 50 mm sodium phosphate (pH 7) to a final concentration of 10 nm. After washing three times for 5 min in a washing buffer containing 0.1× SSC and 50% formamide at 65 °C, followed by washing with PBS, the coverslips were mounted in antifade medium containing 0.1 μg/ml DAPI. Subsequently, the coverslips were analyzed with the Zeiss LSM 510 microscope. A3G Counteracts miRNA-mediated Repression of Protein Translation—We first examined the effect of A3G on the expression of miRNAs. Using a miRNA microarray method, we did not find that A3G significantly changed the miRNA expression in 293T cells (supplemental Figs. S1 and S2). A3G also did not significantly change the expression of miRNA processors such as Drosha and Dicer1 or RISC components such as Ago2 and Mov10 (supplemental Fig. S3). Further, A3G also did not change the level of expression of P-body components such as GW182, Xrn1 and Lsm1 (supplemental Fig. S3). Nevertheless, the microarray data did indicate that several miRNAs, such as mir-16, mir-10b, mir-25, and let-7a, are abundant in 293T cells. To study whether A3G affects the efficiency of miRNA-mediated translational repression, various 293T cell-enriched miRNA-binding sites with perfect or partial complementarity to their corresponding miRNAs were inserted into the 3′-UTR of luciferase (luc) or gfp (Fig. 1a). These plasmids were transfected into 293T cells, which naturally do not express A3G (22Sheehy A.M. Gaddis N.C. Choi J.D. Malim M.H. Nature. 2002; 418: 646-650Crossref PubMed Scopus (1901) Google Scholar, 27Yang B. Chen K. Zhang C. Huang S. Zhang H. J. Biol. Chem. 2007; 282: 11667-11675Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), with or without an A3G-HA-expressing plasmid. Fig. 1b shows that the presence of mir-16, mir-10b, or mir-25 miRNA-binding sites in the 3′-UTR of luc gene remarkably inhibited the expression of luciferase. Interestingly, A3G significantly counteracted this inhibition. Similar phenomenon can be observed in HeLa cells (Fig. 1c). To verif" @default.
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• W2080636764 date "2007-11-01" @default.
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• W2080636764 title "Derepression of MicroRNA-mediated Protein Translation Inhibition by Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G (APOBEC3G) and Its Family Members" @default.
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• W2080636764 doi "https://doi.org/10.1074/jbc.m705116200" @default.
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