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• W2052632013 abstract "The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3′-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-basedin vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was specific for GAP-43 mRNA containing the HuD binding element in the 3′-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA. The neuronal ELAV-like RNA-binding protein HuD binds to a regulatory element in the 3′-untranslated region of the growth-associated protein-43 (GAP-43) mRNA. Here we report that overexpression of HuD protein in PC12 cells stabilizes the GAP-43 mRNA by delaying the onset of mRNA degradation and that this process depends on the size of the poly(A) tail. Using a polysome-basedin vitro mRNA decay assay, we found that addition of recombinant HuD protein to the system increased the half-life of full-length, capped, and polyadenylated GAP-43 mRNA and that this effect was caused in part by a decrease in the rate of deadenylation of the mRNA. This stabilization was specific for GAP-43 mRNA containing the HuD binding element in the 3′-untranslated region and a poly(A) tail of at least 150 A nucleotides. In correlation with the effect of HuD on GAP-43 mRNA stability, we found that HuD binds GAP-43 mRNAs with long tails (A150) with 10-fold higher affinity than to those with short tails (A30). We conclude that HuD stabilizes the GAP-43 mRNA through a mechanism that is dependent on the length of the poly(A) tail and involves changes in its affinity for the mRNA. untranslated region RNA recognition motif AU-rich element glutathione S-transferase granulocyte/macrophage colony-stimulating factor The growth-associated protein GAP-43 is a neuronal-specific phosphoprotein that is expressed primarily in neurons during both the initial axonal outgrowth and remodeling of synaptic connections for reviews (1Skene J.H.P. Annu. Rev. Neurosci. 1989; 12: 127-156Crossref PubMed Scopus (1012) Google Scholar, 2Benowitz L.I. Routtenberg A. Trends Neurosci. 1997; 20: 84-91Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 3Oestreicher A.B. DeGraan P.N.E. Gispen W.H. Verhagen J. Schrama L.H. Prog. Neurobiol. 1997; 53: 627-686Crossref PubMed Scopus (263) Google Scholar). The expression of GAP-43 is complex and features both transcriptional and post-transcriptional components (4Eggen B.J.L. Nielander H.B. Rensen-De Leeuw M.G.A. Schotman P. Gispen W.H. Schrama L.H. Brain Res. Mol. Brain Res. 1994; 23: 221-234Crossref PubMed Scopus (51) Google Scholar, 5Starr R.G., Lu, B. Federoff H.J. Brain Res. 1994; 638: 211-220Crossref PubMed Scopus (25) Google Scholar, 6Nedivi E. Basi G.S. Akey I.V. Skene J.H.P. J. Neurosci. 1992; 12: 691-704Crossref PubMed Google Scholar, 7Federoff H.J. Grabczyk E. Fishman M.C. J. Biol. Chem. 1988; 263: 19290-19295Abstract Full Text PDF PubMed Google Scholar, 8Perrone-Biozerozz N.I. Neve R.L. Irwin N. Lewis S. Fischer I. Benowitz L.I. Mol. Cell. Neurosci. 1991; 2: 402-409Crossref PubMed Scopus (62) Google Scholar, 9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar, 10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar, 11Namgung U. Routtenberg A. Eur. J. Neurosci. 2000; 12: 3124-3136Crossref PubMed Scopus (35) Google Scholar). Transcriptional factors of the basic helix-loop-helix family are known to control the neural specific expression of the GAP-43 gene (12Chiaramello A. Neuman T. Peavy D.R. Zuber M.X. J. Biol. Chem. 1996; 271: 22035-22043Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 13Kinney M. MacNamra R.K. Valcourt E. Routtenburg A. Brain Res. Mol. Brain Res. 1996; 38: 25-36Crossref PubMed Scopus (22) Google Scholar). Yet, and most surprisingly, in several instances, the levels of gene transcription do not correlate well with the accumulation of the mature GAP-43 mRNA (8Perrone-Biozerozz N.I. Neve R.L. Irwin N. Lewis S. Fischer I. Benowitz L.I. Mol. Cell. Neurosci. 1991; 2: 402-409Crossref PubMed Scopus (62) Google Scholar, 9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar, 11Namgung U. Routtenberg A. Eur. J. Neurosci. 2000; 12: 3124-3136Crossref PubMed Scopus (35) Google Scholar, 14Cantallops I. Routtenberg A. J. Neurobiol. 1999; 41: 208-220Crossref PubMed Scopus (50) Google Scholar, 15Namgung U. Matsuyama S. Routtenburg A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11675-11680Crossref PubMed Scopus (50) Google Scholar). In PC12 cells induced to differentiate by nerve growth factor, GAP-43 mRNA levels are regulated primarily through selective changes in the rate of degradation of the mRNA (9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar). This process depends on the activation of protein kinase C and is mediated by the interaction of highly conserved sequences in the 3′-untranslated region (3′-UTR)1 of the mRNA with neuronal-specific RNA-binding proteins (16Kohn D.T. Tsai K.-C. Cansino V.V. Neve R.L. Perrone-Biozerozz N.I. Brain Res. Mol. Brain Res. 1996; 36: 240-250Crossref PubMed Scopus (41) Google Scholar). One of these proteins was identified as the ELAV-like protein HuD (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar, 17Chung S.M. Eckrich M. Perrone-Biozerozz N.I. Kohn D.T. Furneaux H. J. Biol. Chem. 1997; 272: 6593-6598Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Blocking endogenous HuD expression using antisense RNA was found to decrease the levels of the GAP-43 mRNA and protein in PC12 cells and to limit process outgrowth (18Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Biozerozz N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Crossref PubMed Scopus (113) Google Scholar). In contrast, overexpression of HuD in PC12 cells causes an increase in GAP-43 protein levels and cellular differentiation, even in the absence of nerve growth factor (19Anderson K.D. Morin M.A. Beckel-Mitchener A. Mobarak C.D. Neve R.L. Furneaux H.M. Burry R. Perrone-Biozerozz N.I. J. Neurochem. 2000; 75: 1103-1114Crossref PubMed Scopus (71) Google Scholar). In light of these observations, we proposed that HuD is the main regulatory protein involved in the post-transcriptional regulation of the GAP-43 gene. HuD belongs to the highly conserved elav(embryonic lethal abnormalvision) family of RNA-binding proteins (20Antic D. Keene J.D. Am. J. Hum. Genet. 1997; 61: 273-278Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 21Brennan C.M. Steitz J.A. Cell Mol Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (888) Google Scholar). HuD is one of four mammalian ELAV-like proteins that have been identified, three of which are exclusively expressed in the nervous system. These proteins contain three RNA recognition motifs (RRMs); RRMs I and II bind to AU-rich motifs (22Liu J. Dalmau J. Szabo A. Rosenfeld M. Huber J. Furneaux H.M. Neurology. 1995; 45: 544-550Crossref PubMed Scopus (126) Google Scholar), whereas RRM III binds to long stretches of poly(A) (23Ma W.-J. Chung S. Furneaux H. Nucleic Acids Res. 1997; 25: 3564-3569Crossref PubMed Scopus (186) Google Scholar, 24Abe R. Sakashita E. Yamamoto K. Sakamoto H. Nucleic Acids Res. 1996; 24: 4895-4901Crossref PubMed Scopus (91) Google Scholar). HuD has been shown to bind to AU-rich elements (AREs) found in the 3′-UTRs of several mRNAs including c-Fos (25Chung S. Jiang L. Cheng S. Furneaux H. J. Biol. Chem. 1996; 271: 11518-11524Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), tau (26Aranda-Abreu G.E. Behar L. Chung S. Furneaux H. Ginzburg I. J. Neurosci. 1999; 19: 6907-6917Crossref PubMed Google Scholar), p21 waf1 (27Joseph B. Orlian M. Furneaux H. J. Biol. Chem. 1998; 273: 20511-20516Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and GAP-43 (17Chung S.M. Eckrich M. Perrone-Biozerozz N.I. Kohn D.T. Furneaux H. J. Biol. Chem. 1997; 272: 6593-6598Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). In this study, we examined the mechanism by which HuD stabilizes the GAP-43 mRNA using a combination of in vivo and cell-free mRNA decay assays. We found that HuD increases the stability of this mRNA in both systems. This process depends on the presence of the HuD binding element in the GAP-43 3′-UTR and on the size of the poly(A) tail. GAP-43 mRNAs with short tails (A30) were found not only to be less stable than their A150 counterparts, they were also unable to be stabilized by HuD. Consistent with these findings, we observed that the affinity of HuD for the GAP-43 mRNA increased ∼10-fold when the RNA contained long versus short tails. Altogether, our results suggest that HuD controls GAP-43 mRNA stability by binding to its recognition element in the 3′-UTR and decreasing the initial rate of deadenylation of mRNAs with long poly(A) tails. The construction of the inducible expression vectors used for in the in vivomRNA decay assays has been described previously (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar, 16Kohn D.T. Tsai K.-C. Cansino V.V. Neve R.L. Perrone-Biozerozz N.I. Brain Res. Mol. Brain Res. 1996; 36: 240-250Crossref PubMed Scopus (41) Google Scholar). Briefly, the rat GAP-43 cDNA was cloned into the pMEP4 vector (Invitrogen), containing the human metallothionein-IIA promoter. In the presence of cadmium, this vector increases the expression of the transgene by ∼8–10-fold (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar). To co-express HuD in PC12 cells, we used a pcDNA3-derived construct containing the human HuD cDNA (a gift from H. Furneaux) downstream of the CMV promoter (pcHuD). As shown by Mobarak et al. (18Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Biozerozz N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Crossref PubMed Scopus (113) Google Scholar), this construct directs the constitutive expression of HuD in PC12 cells increasing the levels of HuD protein in the cells 2–3-fold. In addition to the generation of mammalian expression vectors, additional constructs were generated for the synthesis of radiolabeled RNAs by in vitro transcription. A full-length rat GAP-43 cDNA was generated by RT-PCR using specific 5′- and 3′-UTR flanking primers (5′-GGAATAAGGATCCGAGGAGGAAAGGAG-3′ and 5′-GACGTCGACGCTAATTGGCACATTTGC-3′, respectively). This fragment was cloned into the BamHI site of the SP64-poly(A) (Promega, Madison, WI) to yield pSP64-GAP. When this plasmid is linearized withSmaI, the resulting transcript contains no poly(A) tail, but when it is digested with EcoRI, the RNA contains a tail of exactly 30 As. To delete the HuD binding site from the GAP-43 3′-UTR, we used a trans-PCR protocol described by Neve and Neve (28Neve R.L.N. Neve K.A. Methods Neurosci. 1995; 25: 163-174Crossref Scopus (6) Google Scholar). Briefly, PCR reactions used the full-length GAP-43 cDNA as template and the following primers, 5′-ACACACTTGGAACTCCCACAGGGCCACACGCACCAG-3′ and 5′-GCCCTGTGGGAGTTCCAAGTGTGTGTGTGCAATGTT-3′ to loop out the internal 27-nucleotide sequence. A second round of PCR used the flanking 5′- and 3′-UTR primers described above to yield the full-length product containing a single 3′-UTR deletion. This PCR product was then cloned into pCR-Script (Stratagene, La Jolla, CA), and the resulting plasmid was called pΔHuD. To verify the specificity of the in vitro decay assay, the entire GAP-43 3′-UTR was replaced by that of β-globin and cloned into pMEP4 as described by Tsai et al. (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar). This plasmid (called C2) was digested withHindIII and XhoI and the resulting insert, BG3′UTR, was cloned into pBlueScript II (Stratagene). GAP-43-deficient PC12 cells (PC12-N36 clone, a gift from Richard Burry) were maintained in RPMI media supplemented with 7.5% horse serum and 2.5% fetal calf serum. PC12 cells were transfected by electroporation with pMEP4-GAP-43, pcHuD, or empty vectors as previously described (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar, 16Kohn D.T. Tsai K.-C. Cansino V.V. Neve R.L. Perrone-Biozerozz N.I. Brain Res. Mol. Brain Res. 1996; 36: 240-250Crossref PubMed Scopus (41) Google Scholar). Stable transfectants were maintained in the presence of 150 μg/ml hygromycin B (for pMEP-4 constructs) and/or 500 μg/ml G418 (for pcDNA3-derived constructs), and used for GAP-43 expression analysis. In vivo mRNA decay studies were performed in pMEP-4-GAP-43-transfected PC12 cells essentially as described by Tsai et al. (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar). Briefly, cells were pre-incubated with 5 μm CdCl2 for 16 h to induce GAP-43 transcription from the human metallothionein IIA promoter. To start the decay analysis, the medium was washed out and replaced with fresh medium without cadmium (time 0). Cultures were incubated for various time periods, cells were then harvested, and RNA isolated using Tri Reagent (Sigma). For H-mapping studies, the RNA was further purified using the RNeasy minikit (Qiagen, Valencia, CA). Fifteen μg of total RNA extracted from cells was run on 1.1% formaldehyde-agarose gel as previously described (9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar). Membranes were probed for GAP-43 mRNA or glyceraldehyde-3-phosphate dehydrogenase mRNA using32P-radiolabeled cDNA probes generated by random priming (Prime-a-Gene, Promega). Membranes were scanned on a PhosphorImager (Storm 860, Invitrogen) and analyzed using the ImageQuant software package. The density of the GAP-43 bands was normalized to that of glyceraldehyde-3-phosphate dehydrogenase mRNA, and results were expressed as percentage of the mRNA remaining relative to time 0 as previously described (9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar). H-mapping was performed essentially as described by Brewer and Ross (29Brewer G. Ross J. Methods Enzymol. 1990; 181: 203-209Google Scholar) with a few modifications. For in vivo decay assays, 30 μg of total RNA extracted from transfected cells were dissolved in 1 mm EDTA and annealed to an antisense oligonucleotide (5′-CTTAAAGTTCAGGCATGTTCTTGGT-3′) that is complementary to a sequence overlapping the beginning of the 3′-UTR. After digestion with RNase H (United States Biochemical Corp.) (0.75 units/reaction), the resulting fragments were run on a 1.35% NuSieve® 3:1 agarose gel (FMC, Rockland, ME) and probed as described above. For in vitro decay assays, radiolabeled RNAs were purified using RNeasy minicolumns. Purified RNAs were annealed to the antisense oligonucleotide and treated with RNase H as described above. Fragments were analyzed using 10% polyacrylamide-urea gels. Capped and polyadenylated RNAs were synthesized by a combination of in vitro transcription and subsequent polyadenylation. To radiolabel the body of the RNA, RNAs were transcribed in vitro in the presence of 1 mm cap analog 7mG(5′)ppp(5′)G (Invitrogen) and of [α-32P]UTP (PerkinElmer Life Sciences, 80 Ci/mmol). Wild type full-length GAP-43 RNA was synthesized in vitro using SP6 RNA polymerase (New England Biolabs, Beverly, MA) and pSP64 GAP. Constructs were linearized either with SmaI to prepare RNAs without tails or EcoRI to prepare RNAs with tails of 30 As. GAP-43 RNAs without the HuD binding site were synthesized using EcoRI-digested pΔHuD and T3 RNA polymerase. To prepare chimeric GAP-43 RNAs with the β-globin 3′-UTR, BG3′UTR was linearized with XhoI and plasmids transcribed with T3 RNA polymerase. For studies measuring the decay of the poly(A) tail, unlabeled RNAs were transcribed in the presence of cold nucleotides and poly(A) tails were labeled in vitro as described below. Radiolabeled RNAs were polyadenylated in vitro using 1.5–2 units/fmol RNA of yeast poly(A) polymerase (United States Biochemical Corp.) and 500 μm cold ATP according to the manufacturer's protocol. To prepare radioactive poly(A) tails, polyadenylation reactions were performed in the presence of 50 μm ATP plus 25 μCi of [α-32P]ATP. Comparison of the sizes of mRNA with and without tails against an RNA ladder (Invitrogen) revealed that under these conditions the length of the poly(A) tail was between 150 and 200 As (data not shown). In vitro decay reactions were performed as described by Brewer and Ross (29Brewer G. Ross J. Methods Enzymol. 1990; 181: 203-209Google Scholar) with minor modifications. Radiolabeled RNAs (2 fmol/reaction) were preincubated in 25 μl of decay buffer (100 mm potassium acetate, 2 mm magnesium acetate, 2 mmdithiothreitol (DTE). 10 mm creatine phosphate, 1 μg of creatine phosphokinase, 1 mm ATP, 0.4 mm GTP, 0.1 mm spermine, 10 mm Tris-HCl (pH 7.6), 80 units/ml RNasin) in the absence (control) or presence of 1 μg of recombinant GST-HuD fusion protein. Recombinant proteins were prepared using pGEX-2T-HuD (a gift from Henry Furneaux) as described by Chunget al. (25Chung S. Jiang L. Cheng S. Furneaux H. J. Biol. Chem. 1996; 271: 11518-11524Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). To control for the specificity of GST-HuD, some control reactions used 1 μg of GST protein instead of the fusion protein. No differences were observed in the presence or absence of GST protein (data not shown). To begin the decay, isolated polysomes (0.1A 600 unit/fmol of RNA) were added and samples incubated for various times. Decay reactions were terminated by addition of an equal volume of 2× PK buffer (200 mmTris-HCl (pH 7.5), 440 mm NaCl, 2% SDS, and 25 mm EDTA). Following proteinase K digestion, RNAs were isolated by phenol/chloroform extraction and ethanol precipitation and samples were analyzed in denaturing 10% polyacrylamide TBE, 8.3m urea gels. All gels were dried and analyzed using a Personal Molecular Imager FX (Bio-Rad) and the accompanying Quantity One software. Filter binding studies were performed as described by Chung et al. (25Chung S. Jiang L. Cheng S. Furneaux H. J. Biol. Chem. 1996; 271: 11518-11524Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Briefly, 2 fmol of [α-32P]UTP-radiolabeled full-length GAP-43 RNAs, with tails of 30 or 150 As, were incubated in the presence of recombinant HuD protein in binding buffer (50 mm Tris (pH 7.4), 1 mm MgCl2, 1 mm EDTA, 150 mm NaCl, and 1 mm DTE) at 37 °C for 10 min. Protein concentrations for these studies ranged from of 30 to 600 nm. Samples were then transferred to nitrocellulose filters, and counts were analyzed by scintillation counting. We have previously shown that HuD stabilizes the GAP-43 mRNA in transfected PC12 cells and neurons, leading to increased protein expression and neuronal differentiation (18Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Biozerozz N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Crossref PubMed Scopus (113) Google Scholar, 19Anderson K.D. Morin M.A. Beckel-Mitchener A. Mobarak C.D. Neve R.L. Furneaux H.M. Burry R. Perrone-Biozerozz N.I. J. Neurochem. 2000; 75: 1103-1114Crossref PubMed Scopus (71) Google Scholar, 30Anderson K.D. Sengupta J. Morin M. Neve R.L. Valenzuela C.F. Perrone-Biozerozz N.I. Exp. Neurol. 2001; 168: 250-258Crossref PubMed Scopus (78) Google Scholar). In this study, we sought to characterize the mechanism by which HuD exerts these effects using a combination of in vivo and in vitromRNA decay assays. Initial studies examined the decay of the GAP-43 mRNA in transfected GAP-43-deficient PC12 cells using an inducible expression system. The pMEP4-GAP vector allows the transient expression of the GAP-43 mRNA by cadmium induction; its subsequent decay can then be measured by Northern blot analysis in the absence of any transcriptional inhibitors (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar, 16Kohn D.T. Tsai K.-C. Cansino V.V. Neve R.L. Perrone-Biozerozz N.I. Brain Res. Mol. Brain Res. 1996; 36: 240-250Crossref PubMed Scopus (41) Google Scholar). In addition, to assess the effect of HuD in GAP-43 mRNA stability, some cultures were co-transfected with a constitutive HuD expression vector (pcHuD) that increases HuD protein level in the cell by 2–3-fold (18Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Biozerozz N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Crossref PubMed Scopus (113) Google Scholar). As shown in Fig. 1, in the absence of exogenous HuD, the GAP-43 mRNA decayed with a half-life (t 12) of ∼5 h, similar to that previously calculated using radioisotopic mRNA decay methods (9Perrone-Biozerozz N.I. Cansino V.V. Kohn D.T. J. Cell Biol. 1993; 120: 1263-1270Crossref PubMed Scopus (148) Google Scholar). Overexpression of HuD decreased the overall rate of decay of the mRNA, and the resulting t 12 was calculated as 8.5 h. Regression analysis also indicated that the best fit for mRNA turnover in the presence of HuD was obtained using a two-rate exponential decay instead of a single rate as in the case of the GAP-43 mRNA alone. The biphasic decay profile consisted of an initial 3-h delay in the onset of degradation of the mRNA, followed a by similar second rate decay as in the absence of exogenous HuD. Delays in the onset of mRNA decay similar to this have been observed for mRNAs containing type I AREs in the 3′-UTR, such as c-Fos (31Chen C.Y., Xu, N. Shyu A.B. Mol. Cell. Biol. 1995; 15: 5777-5788Crossref PubMed Scopus (249) Google Scholar). In that case, the lag in decay reflected the time required to degrade the poly(A) tail. To test whether a similar mechanism was operating on the GAP-43 mRNA, subsequent experiments used H-mapping to monitor the decay of the 3′ end of the mRNA. After in vivo decay assays, RNAs were annealed to an antisense oligonucleotide against the 5′ end of the GAP-43 3′-UTR and these RNA-DNA hybrids were digested with RNase H. The resulting fragments contained either the 5′-UTR and coding region or the entire GAP-43 3′-UTR and the poly(A) tail. As shown in Fig.2, in the absence of excess HuD, both fragments decayed simultaneously with a rate similar to that of the entire mRNA. While the 5′ end consisted of a band of unique size, the 3′ end fragments showed greater heterogeneity, possibly because they contain poly(A) tails of varying sizes. This heterogeneity did not change during the decay, as 3′ fragments disappeared without an apparent change in the relative abundance of the different size species. Therefore, the broad 3′ end bands are more likely caused by the imprecise nature of the polyadenylation reaction than to a distributive deadenylation. The results in Fig. 2 also show that overexpression of HuD protein resulted in the stabilization of both fragments. The finding of similar decay rates for the 3′ and 5′ end fragments without the formation of intermediate size products could be the result of one of two alternative decay mechanisms. The first possibility is that the decay reaction is very processive. Supporting this possibility, processive deadenylation was shown to occur during the degradation of other ARE-containing mRNAs such as that for GM-CSF (31Chen C.Y., Xu, N. Shyu A.B. Mol. Cell. Biol. 1995; 15: 5777-5788Crossref PubMed Scopus (249) Google Scholar). Alternatively, the mRNA may be the target of endonucleolytic attack. To address these issues, subsequent studies utilized a cell-free mRNA decay system where individual components could be controlled more easily. We used a polysome-based mRNA decay system (29Brewer G. Ross J. Methods Enzymol. 1990; 181: 203-209Google Scholar) to examine the effect of HuD on the half-life of in vitrosynthesized GAP-43 mRNA. Isolated polysomes are known to contain all the enzymatic activities required for mRNA degradation. Although mRNAs decay at much faster rates in vitro thanin vivo, the system faithfully reproduces the effect ofcis- and trans-acting factors on mRNA half-life (32Brewer G. Ross J. Mol. Cell. Biol. 1988; 8: 1697-1708Crossref PubMed Scopus (215) Google Scholar). In vitro decay reactions used a radiolabeled capped and polyadenylated full-length GAP-43 mRNA as the substrate. Given that other RNA-binding proteins interact with the GAP-43 mRNA (16Kohn D.T. Tsai K.-C. Cansino V.V. Neve R.L. Perrone-Biozerozz N.I. Brain Res. Mol. Brain Res. 1996; 36: 240-250Crossref PubMed Scopus (41) Google Scholar), it was important to preserve the structure of the mRNA as closely as that in the cells. Radiolabeled mRNAs were added to decay reactions containing purified polysomes from adult rat brain, in the presence or absence of recombinant HuD protein. Control reactions used recombinant GST at the same molar concentration, whereas others omitted recombinant protein altogether. Both controls yielded identical results (data not shown). Initial studies evaluated the specificity of the in vitrodecay system by examining the structural requirements for GAP-43 mRNA decay. As shown in Fig. 3, the same structural elements that are necessary for effective translation, the 5′ cap and poly(A) tail, were also required for effective mRNA degradation. In addition, swapping the GAP-43 3′-UTR for that of β-globin dramatically increased the stability of the mRNA. This is consistent with the known stabilizing effect of the β-globin 3′-UTR when attached downstream of any unstable mRNA, including that of GAP-43, and is comparable with previous observations in cultured PC12 cells (10Tsai K. Cansino V.V. Kohn D.T. Neve R.L. Perrone-Biozerozz N.I. J. Neurosci. 1997; 17: 1950-1958Crossref PubMed Google Scholar). Once the specificity of the in vitro system was established, we examined the effect of HuD on the half-life of full-length GAP-43 mRNAs. Similar to the results obtained in PC12 cells (Fig. 1), addition of recombinant HuD protein increased the half-life of the GAP-43 mRNA by ∼2–3-fold (Fig. 4). However, we found virtually no delay in the onset of mRNA decayin vitro, and the GAP-43 mRNA began to decay 2 min after starting the reaction. The lack of an apparent delay in the cell-free system may be caused by the increased speed of mRNA degradation, which would make it difficult to detect small delays in the decay. Alternatively, additional factors not present in the in vitro system may contribute to the delay. In any case, it is clear that the presence of excess HuD protein increases GAP-43 mRNA stability both in vivo and in vitro. There are two possible explanations for this stabilization. First, HuD could be affecting the turnover of the mRNA by binding to both the 3′-UTR and the poly(A) tail. Alternatively, the third RNA recognition motif (RRM III) could be binding to the poly(A) tail nonspecifically, preventing degradation of the messenger RNA. To test the requirement of the interaction of HuD with the GAP-43 3′-UTR in mRNA stabilization, we synthesized a mRNA in which the single 27-nucleotide HuD binding site was deleted. This mRNA (ΔHuD) was then used in decay experiments in the presence or absence of GST-HuD protein. As shown in Fig. 5, HuD did not affect the decay of GAP-43 mRNAs that lack the HuD binding site, indicating that this protein must specifically bind to its recognition site in the 3′-UTR to stabilize the GAP-43 mRNA. This is in agreement with a recent study demonstrating that mutations of the ARE of tumor necrosis factor-α abolished the capacity of HuR to stabilize the mRNA (33Di Marco S. Hel Z. Lachance C. Furneaux H. Radzioch D. Nucleic Acids Res. 2001; 29: 863-871Crossref PubMed Google Scholar).Figure 5HuD does not stabilize ΔHuD, a mRNA with a deletion in the HuD binding site in the 3′-UTR. A, pΔHuD was constructed and used for in vitro transcription as indicated under “Experimental Procedures.” A, the ΔHuD mRNA was analyzed by in vitro decay assays as described in Fig. 4.B, graph showing that the rate of degradation of the GAP-43 mRNA lacking the HuD binding site did not change in presence or absence of recombinant HuD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A separate question about the in vitro reaction was whether the decay of GAP-43 mRNA proceeded in a processive manner as was found to occur in vivo. To address this question, we used H-mapping to measure the decay of the 3′ and 5′ ends of the mRNA. As shown in Fig. 6, the fragments corresponding" @default.
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• W2052632013 date "2002-08-01" @default.
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• W2052632013 title "Poly(A) Tail Length-dependent Stabilization of GAP-43 mRNA by the RNA-binding Protein HuD" @default.
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