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• W2035618537 abstract "Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. In this study, we observed that nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching ∼3.5-fold by 72 h. Given that the AChE 3′-untranslated region (UTR) contains an AU-rich element, we focused on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, our studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3′-UTR showed that this 3′-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots revealed an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrated that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a keytrans-acting factor in these events. Expression of acetylcholinesterase (AChE) is greatly enhanced during neuronal differentiation, but the nature of the molecular mechanisms remains to be fully defined. In this study, we observed that nerve growth factor treatment of PC12 cells leads to a progressive increase in the expression of AChE transcripts, reaching ∼3.5-fold by 72 h. Given that the AChE 3′-untranslated region (UTR) contains an AU-rich element, we focused on the potential role of the RNA-binding protein HuD in mediating the increase in AChE mRNA seen in differentiating neurons. Using PC12 cells engineered to stably express HuD or an antisense to HuD, our studies indicate that HuD can regulate the abundance of AChE transcripts in neuronal cells. Furthermore, transfection of a reporter construct containing the AChE 3′-UTR showed that this 3′-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the antisense. RNA gel shifts and Northwestern blots revealed an increase in the binding of several protein complexes in differentiated neurons. Immunoprecipitation experiments demonstrated that HuD can bind directly AChE transcripts. These results show the importance of post-transcriptional mechanisms in regulating AChE expression in differentiating neurons and implicate HuD as a keytrans-acting factor in these events. Acetylcholinesterase (AChE) 1The abbreviations used are: AChE, acetylcholinesterase; NGF, nerve growth factor; RT, reverse transcription; GRAP, giant rat acetylcholinesterase promoter; CAT, chloramphenicol acetyltransferase; 3′-UTR, 3′-untranslated region; REMSA, RNA-based electrophoretic mobility shift assay; DTT, dithiothreitol; ELAV, embryonic lethal abnormal vision; ARE, AU-rich element; PBS, phosphate-buffered saline; IP, immunoprecipitation is the enzyme responsible for the rapid hydrolysis of acetylcholine in the central and peripheral nervous systems (see, for review, Refs. 1Massoulie J. Pezzementi L. Bon S. Krejci E. Vallette F.M. Prog. Neurobiol. 1993; 41: 31-91Google Scholar, 2Legay C. Microsc. Res. Tech. 2000; 49: 56-72Google Scholar, 3Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Google Scholar, 4Soreq H. Seidman S. Nat. Rev. Neurosci. 2001; 2: 294-302Google Scholar). The enzyme exists in multiple molecular forms that differ in their C terminus and mode of anchoring to subcellular structures. The different C termini of the protein are generated through alternative splicing of a single gene, and three different mature mRNAs, referred to as the T (tail), H (hydrophobic), and R (readthrough) transcripts, can be produced. The pattern of expression of these transcripts is known to be tissue-specific because, for example, the T transcript is abundantly expressed in excitable cells such as skeletal muscle and neurons, whereas the H transcript is found predominantly in the hematopoietic lineage. Two polyadenylation signals can be used in the 3′-untranslated region (UTR) to produce a ∼2.4- or 3.2-kb transcripts. Choice of the polyadenylation signal is also tissue-specific because neurons appear to preferentially express the shorter form of the transcript whereas skeletal muscle express both species but in different amounts (5Legay C. Huchet M. Massoulie J. Changeux J.P. Eur. J. Neurosci. 1995; 7: 1803-1809Google Scholar, 6Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Google Scholar, 7Rachinsky T.L. Camp S. Li Y. Ekstrom T.J. Newton M. Taylor P. Neuron. 1990; 5: 317-327Google Scholar). In addition to its role in cholinergic neurotransmission, converging lines of evidence indicate that it likely fulfills additional, non-catalytic functions within the nervous system (see, for review, Ref. 8Layer P.G. Willbold E. Prog. Histochem. Cytochem. 1995; 29: 1-94Google Scholar). For example, Northern blot analysis and in situhybridization experiments have revealed the presence of AChE mRNAs in non-cholinergic areas of the brain such as the cerebellum (9Bernard V. Legay C. Massoulie J. Bloch B. Neuroscience. 1995; 64: 995-1005Google Scholar, 10Hammond P. Rao R. Koenigsberger C. Brimijoin S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10933-10937Google Scholar, 11Landwehrmeyer B. Probst A. Palacios J.M. Mengod G. Neuroscience. 1993; 57: 615-634Google Scholar, 12Legay C. Bon S. Vernier P. Coussen F. Massoulie J. J. Neurochem. 1993; 60: 337-346Google Scholar). Moreover, AChE expression in the brain is known to precede the establishment of synaptic transmission and coincides with the period of neurite outgrowth (13Brimijoin S. Hammond P. Neuroscience. 1996; 71: 555-565Google Scholar, 14Robertson R.T. Yu J. News Physiol. Sci. 1993; 8: 266-272Google Scholar). Finally, several studies in which AChE levels have been experimentally manipulated directly support the notion that AChE is indeed involved in neurite outgrowth (see, for example, Refs. 13Brimijoin S. Hammond P. Neuroscience. 1996; 71: 555-565Google Scholar, 14Robertson R.T. Yu J. News Physiol. Sci. 1993; 8: 266-272Google Scholar, 15Blasina M.F. Faria A.C. Gardino P.F. Hokoc J.N. Almeida O.M. De Mello F.G. Arruti C. Dajas F. Cell Tissue Res. 2000; 299: 173-184Google Scholar and 17Dupree J.L. Bigbee J.W. J. Neurocytol. 1996; 25: 439-454Google Scholar, 18Jones S.A. Holmes C. Budd T.C. Greenfield S.A. Cell Tissue Res. 1995; 279: 323-330Google Scholar, 19Koenigsberger C. Chiappa S. Brimijoin S. J. Neurochem. 1997; 69: 1389-1397Google Scholar, 20Sharma K.V. Bigbee J.W. J. Neurosci. Res. 1998; 53: 454-464Google Scholar, 21Srivatsan M. Peretz B. Neuroscience. 1997; 77: 921-931Google Scholar, 22Sternfeld M. Ming G. Song H. Sela K. Timberg R. Poo M. Soreq H. J. Neurosci. 1998; 18: 1240-1249Google Scholar). In recent years, there have been several studies that have examined the basic molecular events that preside over AChE expression in developing and adult skeletal muscles (see, for example, Refs. 23Angus L.M. Chan R.Y. Jasmin B.J. J. Biol. Chem. 2001; 276: 17603-17609Google Scholar, 24Boudreau-Lariviere C. Chan R.Y. Wu J. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Google Scholar, 25Chan R.Y. Boudreau-Lariviere C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Google Scholar, 26Krejci E. Legay C. Thomine S. Sketelj J. Massoulie J. J. Neurosci. 1999; 19: 10672-10679Google Scholar, 27Luo Z.D. Wang Y. Werlen G. Camp S. Chien K.R. Taylor P. Mol. Pharmacol. 1999; 56: 886-894Google Scholar, 28Pregelj P. Sketelj J. J. Neurosci. Res. 2002; 67: 114-121Google Scholar, 29Rimer M. Randall W.R. Biochem. Biophys. Res. Commun. 1999; 260: 251-255Google Scholar, 30Siow N.L. Choi R.C. Cheng A.W. Jiang J.X. Wan D.C. Zhu S.Q. Tsim K.W. J. Biol. Chem. 2002; 277: 36129-36136Google Scholar). By contrast, there is relatively little information concerning the mechanisms regulating AChE expression in neurons. In addition, of the few available reports, there also appear to be some contradictory findings. In particular, Greene and Rukenstein (31Greene L.A. Rukenstein A. J. Biol. Chem. 1981; 256: 6363-6367Google Scholar) have provided evidence indicating that differentiation of PC12 cells, which leads to an increase in AChE expression, induces an increase in AChE gene transcription. Alternatively, embryonic P19 carcinoma cells induced to differentiate into neurons via retinoic acid treatment failed to increase the transcriptional activity of the AChE gene, thereby indicating that post-transcriptional mechanisms represent key events in regulating the abundance of AChE transcripts during neuronal development (32Coleman B.A. Taylor P. J. Biol. Chem. 1996; 271: 4410-4416Google Scholar). Given the diverse and key functions of AChE within the nervous system, it appears important to gain a more complete understanding of the molecular mechanisms that control AChE expression in neurons. Accordingly, we have initiated a series of experiments in attempts to characterize some of the molecular events involved in AChE expression during neuronal differentiation. Specifically, we have examined the importance of transcriptional and post-transcriptional mechanisms in the regulation of AChE during NGF-induced differentiation of PC12 cells. PC12 cells, a rat pheochromocytoma-derived cell line (33Greene L.A. Aletta J.M. Ruckenstein A. Greene S.H. Methods Enzymol. 1986; 147: 207-216Google Scholar), were cultured on culture dishes coated with type I collagen (Sigma) in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% horse serum, 5% fetal bovine serum, and 100 units/ml penicillin-streptomycin, in a humidified chamber at 37 °C containing 5% CO2. Stably transfected PC12 cells were maintained as described elsewhere (34Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Bizzozero N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Google Scholar). These lines were transfected with the pcDNA3 vector alone (pcDNA) or with plasmids containing the human HuD sequence in sense (pcHuD) or antisense (pDuH) orientation. All cells were plated at a density of 1–2 × 105 cells/cm2 and induced to differentiate by adding 100 ng/ml 7S-NGF (Sigma) to the culture medium. Culture media were changed every 72 h. Cultures of undifferentiated and differentiated PC12 cells (three 35-mm wells) were washed with cold phosphate-buffered saline (PBS), scraped, and homogenized on ice with a glass Kontes homogenizer in 0.5 ml of a high salt detergent buffer containing anti-proteolytic agents (10 mm Tris-HCl, pH 7.0, 10 mm EDTA, 1 m NaCl, 1% Triton X-100, 1 mg/ml bacitracin (Sigma), and 25 units/ml aprotinin (Sigma)). Following centrifugation of the homogenates (20,000 × g for 15 min at 4 °C), the supernatant was removed and stored immediately at −80 °C. AChE activity was measured using a modified version of the spectrophotometric method of Ellman et al. (35Ellman G.L. Courtney K.D. Andres V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Google Scholar) as described previously (36Jasmin B.J. Gisiger V. J. Neurosci. 1990; 10: 1444-1454Google Scholar). The total amount of protein present in the extracts was determined by the bicinchoninic acid assay (BCA; Pierce). Total RNA was extracted from undifferentiated and differentiated PC12 cell cultures (three 35-mm wells) using 0.5–1 ml of TRIzol reagent (Invitrogen) according to the instructions from the manufacturer. Briefly, the cells were scraped from the plates and disrupted by vigorous pipetting, followed by addition of chloroform. The resulting solution was mixed vigorously and centrifuged (12,000 × g for 15 min at 4 °C). The aqueous layer was then transferred to a fresh tube and combined with an equal volume of isopropanol. The RNA was precipitated by centrifugation, and the resulting pellet was washed twice with 75% ice-cold ethanol and resuspended in RNase-free water. All samples were stored in −80 °C until used. RNA from each sample was quantified using the AmershamBiosciences Gene Quant II RNA/DNA spectrophotometer and adjusted to a final concentration of 80 ng/μl. Reverse transcription of RNA was performed using 2 μl of each RNA sample at 42 °C for 45 min, followed by 5 min at 99 °C, as previously described elsewhere (37Boudreau-Lariviere C. Sveistrup H. Parry D.J. Jasmin B.J. Neuroscience. 1996; 73: 613-622Google Scholar, 38Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Google Scholar, 39Jasmin B.J. Lee R.K. Rotundo R.L. Neuron. 1993; 11: 467-477Google Scholar). Negative controls consist of the same RT mixture in which the RNA was replaced with 2 μl of RNase-free water. PCR was used to amplify cDNAs corresponding to AChE and S12 rRNA as described in detail elsewhere (23Angus L.M. Chan R.Y. Jasmin B.J. J. Biol. Chem. 2001; 276: 17603-17609Google Scholar, 37Boudreau-Lariviere C. Sveistrup H. Parry D.J. Jasmin B.J. Neuroscience. 1996; 73: 613-622Google Scholar, 38Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Google Scholar, 39Jasmin B.J. Lee R.K. Rotundo R.L. Neuron. 1993; 11: 467-477Google Scholar). Primers for AChE (see Ref. 12Legay C. Bon S. Vernier P. Coussen F. Massoulie J. J. Neurochem. 1993; 60: 337-346Google Scholar) and S12 rRNA (used as an internal control; see Ref. 40Forster E. Otten U. Frotscher M. Neurosci. Lett. 1993; 155: 216-219Google Scholar) were synthesized based on available sequences that have been previously described, and they amplified products of 670 and 368 bp, respectively. PCR cycling parameters for AChE and S12 rRNA consisted of denaturation for 1 min at 94 °C, followed by primer annealing and extension for 3 min at 70 °C for AChE and primer annealing for 1 min at 54 °C and extension for 2 min at 72 °C for S12 rRNA, followed by a 10-min elongation step at 72 °C. PCR products were visualized and quantified on ethidium bromide-stained 1.5% agarose gels. Quantitation of the labeling intensity of the PCR products was performed using the Kodak Digital Science Image Station 440 CF and related Kodak Digital Science 1D Image Analysis Software (Eastman Kodak Co.). All values obtained for AChE were corrected according to the corresponding level of S12 rRNA present in the sample. All RT-PCR experiments aimed at determining the relative abundance of AChE transcripts were performed using cycle numbers that fell within the linear range of amplification (37Boudreau-Lariviere C. Sveistrup H. Parry D.J. Jasmin B.J. Neuroscience. 1996; 73: 613-622Google Scholar, 38Michel R.N. Vu C.Q. Tetzlaff W. Jasmin B.J. J. Cell Biol. 1994; 127: 1061-1069Google Scholar, 39Jasmin B.J. Lee R.K. Rotundo R.L. Neuron. 1993; 11: 467-477Google Scholar). The cycle numbers were between 24 and 27 for AChE and 22 for S12 rRNA. RT-PCR conditions (primer concentration, input RNA, choice of RT primer, cycling conditions) were initially optimized, and these were identical for all experiments. Appropriate precautions (use of sterile filtered tips and gloves) were taken to prevent contamination of the samples and degradation of the RNA. Samples, including the negative control, were always prepared using the same RT and PCR reagents and master mixes, and were run in parallel. In all experiments, PCR products were never detected in the negative controls. Nuclear run-on assays were performed as described in detail elsewhere (23Angus L.M. Chan R.Y. Jasmin B.J. J. Biol. Chem. 2001; 276: 17603-17609Google Scholar, 41Boudreau-Lariviere C. Jasmin B.J. FEBS Lett. 1999; 444: 22-26Google Scholar, 42Chan R.Y. Adatia F.A. Krupa A.M. Jasmin B.J. J. Biol. Chem. 1998; 273: 9727-9733Google Scholar). Briefly, nuclei were isolated from undifferentiated and differentiated PC12 cells (two 250-ml flasks) and resuspended in a transcription buffer containing GTP, ATP, CTP, and 25 μCi of [α-32P]UTP. RNA was transcribed for 60 min at 30 °C in the presence of an RNase inhibitor (Promega, Madison, WI). Following a 30-min RQ1 DNase I (Promega) treatment, the nascent radiolabeled RNA was extracted using TRIzol reagent (see above) and hybridized for 48 h to 10 μg of linearized AChE cDNA (2 kb) immobilized on a Protran pure nitrocellulose membrane (Schleicher & Schuell). After hybridization the membranes were washed thoroughly at 42 °C in a 1× saline-sodium citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) solution and subjected to autoradiography. The intensity of the resulting signals was quantified using a STORM PhosphorImager and the accompanying ImageQuant software (Amersham Biosciences). The signals corresponding to AChE were standardized relative to the signal obtained from genomic DNA. The recently described 5.3-kb AChE promoter fragment termed GRAP (25Chan R.Y. Boudreau-Lariviere C. Angus L.M. Mankal F.A. Jasmin B.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4627-4632Google Scholar) was subcloned into a LacZ reporter vector. In addition, the 3′-UTR from the mouse ∼2.4-kb AChE transcript, which contains the shorter 3′-UTR in comparison to the ∼3.2-kb AChE mRNA (see Introduction), was amplified by RT-PCR and first inserted into the pGL3 vector. For these experiments, we focused on the short 3′-UTR as opposed to the longer one because (i) previous studies showed that it appears to contain important cis-acting regulatory elements (24Boudreau-Lariviere C. Chan R.Y. Wu J. Jasmin B.J. J. Neurochem. 2000; 74: 2250-2258Google Scholar, 43Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Google Scholar, 44Sketelj J. Crne-Finderle N. Strukelj B. Trontelj J.V. Pette D. J. Neurosci. 1998; 18: 1944-1952Google Scholar), and (ii) the ∼2.4-kb AChE transcript is considerably more abundant in nervous tissues (12Legay C. Bon S. Vernier P. Coussen F. Massoulie J. J. Neurochem. 1993; 60: 337-346Google Scholar) including PC12 cells (45Li Y. Liu L. Kang J. Sheng J.G. Barger S.W. Mrak R.E. Griffin W.S. J. Neurosci. 2000; 20: 149-155Google Scholar, 46Meshorer E. Erb C. Gazit R. Pavlovsky L. Kaufer D. Friedman A. Glick D. Ben Arie N. Soreq H. Science. 2002; 295: 508-512Google Scholar). The 3′-UTR was subsequently cloned into a luciferase reporter construct driven by the thymidine kinase promoter (phRG-TK) (Promega). Plasmid DNA was prepared using the Mega-Prep procedure (Qiagen, Chatsworth, CA). DNA pellets were resuspended in 10 mmTris-HCl, pH 8.5. Transfections were performed using the LipofectAMINE reagent kit (Invitrogen) according to the instructions from the manufacturer. Undifferentiated cells (1–2 × 105cells/cm2) were transfected with 0.5 μg of the appropriate reporter gene construct and 0.5 μg of the constitutively expressed chloramphenicol acetyltransferase (CAT) plasmid driven by the SV40 promoter used, in this case, to control for transfection efficiency. Transfected cells were induced to differentiate 24 h later by the addition of NGF. To determine reporter gene activity, undifferentiated and differentiated cells were washed with cold PBS and lysed in Reporter-Lysis buffer (Promega) following two cycles of freezing and thawing. The extracts were then centrifuged (15,000 ×g for 2 min at 4 °C), and the resulting supernatants were assayed for β-galactosidase, luciferase, or CAT activities using available kits (Promega). The β-galactosidase and luciferase activities were normalized to CAT levels. Background values, obtained by transfecting promoterless LacZ and luciferase plasmids, were subtracted from the activities obtained with the reporter constructs. cDNAs encoding different lengths of the AChE 3′-UTR were obtained by PCR amplification of the plasmid template pGL3–3′-UTR (see above). The PCR primers employed to amplify: 1) the full-length AChE 3′-UTR, 2) a truncated fragment in which the AU-rich element was absent (−ARE), and 3) a small fragment encompassing the ARE (see Fig. 5A), were designed to include a T7 promoter. An in vitro T7 transcription system (Promega) was used to synthesize radiolabeled AChE 3′-UTR fragments. Briefly, the transcription reaction containing 0.5 μg of PCR fragment, 5 μCi of [α-32P]UTP, nucleotides, RNase inhibitor, and T7 polymerase, was carried out at 37 °C for 1 h. The template PCR fragments were digested with 1 unit of RQ1 DNase I (Promega) for 30 min at 37 °C. The resulting radiolabeled RNA was purified on an RNase-free G-25 RNA purification column (Roche Diagnostics Corp., Indianapolis, IN). The integrity of the RNA was confirmed by gel electrophoresis. Unlabeled RNA probes were generated by the same method and used in cold competition assays. RNA-based electrophoretic mobility shift assays (REMSAs) and Northwestern blots were performed using total protein extracts obtained from undifferentiated and differentiated cells (two 250-ml flasks). The cells were washed with cold PBS, scraped, and lysed in 300 μl of homogenization buffer (0.3 m sucrose, 60 mm NaCl, 15 mm Tris, pH 8.0, 10 mmEDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mmbenzamidine, 10 μg/μl leupeptin, 10 μg/μl pepstatin A, 1 μg/μl aprotinin, pH 7.4). The samples were centrifuged (15,000 × g for 15 min at 4 °C), and the resulting supernatant was stored at −80 °C until used. The total amount of protein present in the extracts was determined by the BCA method (see above). REMSAs were performed as described elsewhere in detail (47Alterio J. Mallet J. Biguet N.F. Mol. Cell Neurosci. 2001; 17: 179-189Google Scholar, 48Hew Y. Lau C. Grzelczak Z. Keeley F.W. J. Biol. Chem. 2000; 275: 24857-24864Google Scholar, 49Wilson G.M. Brewer G. Methods. 1999; 17: 74-83Google Scholar). Forty μg of protein extract were incubated for 20 min at room temperature with 1 × 105 cpm of 32P-labeled AChE 3′-UTR fragments in 2× binding buffer (20 mm Hepes, pH 7.9, 3 mm MgCl2, 50 mm KCl, 1 mm DTT, 5% glycerol, 0.2 μg/μl yeast tRNA) in a total volume of 20 μl. The unbound RNA was digested with ribonuclease T1 (Calbiochem, San Diego, CA) for 20 min at 37 °C, and the samples were then incubated at room temperature for 10 min with heparin (2.5 mg/ml). This mixture was separated by 4 or 6% native polyacrylamide gel electrophoresis with 0.5× TBE (Tris borate-EDTA) running buffer. The gels were subsequently dried under vacuum at 80 °C for 1 h and exposed to x-ray film at −70 °C. Competition assays were performed by incubating a 25 m excess of cold probe with the protein extract for 10 min prior to the incubation with the radiolabeled probe. Northwestern analyses were performed according to the procedure described elsewhere (50Erondu N.E. Nwankwo J. Zhong Y. Boes M. Dake B. Bar R.S. Mol. Endocrinol. 1999; 13: 495-504Google Scholar, 51Sagesser R. Martinez E. Tsagris M. Tabler M. Nucleic Acids Res. 1997; 25: 3816-3822Google Scholar). Fifty μg of protein extract diluted in SDS buffer (50 mm Tris-HCl, pH 6.8, 100 mm DTT, 2% SDS, 0.1% bromphenol blue, 10% glycerol) were denatured at 100 °C for 3 min and separated by 8% SDS-PAGE. After separation, proteins were electroblotted onto a polyvinylidene difluoride membrane (Schleicher & Schuell). The membrane was then incubated in renaturation buffer (15 mm Hepes, pH 7.9, 50 mm KCl, 0.1 mm MnCl2, 0.1 mm ZnCl2, 0.1 mm EDTA, 0.5 μm DTT, 0.1% (w/v) Ficoll 400 d-L, 0.1% (w/v) polyvinylpyrrolidone, and 0.01% (v/v) Igepal CA-630 (a Nonidet P-40 substitute) in RNase-free water) at 4 °C overnight. Following pre-hybridization at room temperature for 1 h in renaturation buffer containing 0.2 mg/ml yeast tRNA, the membrane was incubated for 4 h with 1 × 106cpm/ml of a probe corresponding to the radiolabeled AChE 3′-UTR RNA dissolved in renaturation buffer containing 0.2 mg/ml yeast tRNA and 5 mg/ml heparin at room temperature. After several 5-min washes, the membrane was put to x-ray film at −70 °C. To ensure that equivalent amounts of proteins were loaded for each sample, membranes were also stained with Ponceau S (Sigma), following exposure to x-ray films. The RNA-binding protein HuD was immunoprecipitated from a total protein extract as described (52Chung S. Eckrich M. Perrone-Bizzozero N. Kohn D.T. Furneaux H. J. Biol. Chem. 1997; 272: 6593-6598Google Scholar). Total protein was extracted from PC12 cells stably transfected to express HuD (8 × 100-mm plate). To this end, the cells were pelleted, resuspended in 0.3 ml of immunoprecipitation (IP) buffer (1% Igepal CA-630, 10 mmTris-HCl, pH 7.5, 1% bovine serum albumin, 150 mm NaCl, 2 mm EDTA, 25 μg/μl pepstatin A, 2.5 μg/μl aprotinin, 10 units of RNase inhibitor), and sonicated (10-s pulse at 50% duty cycle and a power output of 1 using the Branson Sonifier 450). Protein samples (200 μg) were incubated for 1 h at 4 °C, with an affinity-purified antibody to HuD previously described (34Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Bizzozero N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Google Scholar) or with normal rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA) in IP buffer. This reaction mixture was subsequently added to 20 μl of pre-washed A/G chimera Sepharose beads (Pierce) and incubated by gentle shaking at 4 °C for 1 h. The mixture was centrifuged (10,000 × g for 20 s), and the supernatant was removed. After several washes with IP buffer, the RNA was extracted from the pellet using the TRIzol reagent and analyzed by RT-PCR as described above. Cultured cells were washed in 1× PBS; resuspended in a homogenization buffer containing 0.3 msucrose, 60 mm NaCl, 15 mm Tris-HCl, pH 8.0, 10 mm EDTA, 0.1 mm β-mercaptoethanol, 0.01 mm phenylmethylsulfonyl fluoride, 0.01 mmbenzamidine, 1 μg of leupeptin, 10 μg of pepstatin A, and 1 μg of aprotinin; and sonicated (see above). Following centrifugation, the supernatant was recovered, aliquoted, and stored at −80 °C. The concentration of proteins in each sample was determined using the BCA method (see above). For Western blotting, 50 μg of protein extracts were denaturated in SDS loading buffer and subjected to SDS-PAGE using a 10% gel. The proteins were then transferred onto a polyvinylidene difluoride membrane (Sigma). Following transfer, the membranes were incubated with antibodies directed against HuD (34Mobarak C.D. Anderson K.D. Morin M. Beckel-Mitchener A. Rogers S.L. Furneaux H. King P. Perrone-Bizzozero N.I. Mol. Biol. Cell. 2000; 11: 3191-3203Google Scholar) and revealed using a commercially available ECL kit from Pierce. An analysis of variance was performed to evaluate the effects of NGF-induced neuronal differentiation on AChE expression. The Fisher's Least Square Difference test was used to determine whether the differences seen between group means were significant. The level of significance was set at p < 0.05. Data are expressed as mean ± S.E. throughout. In a first series of experiments, we examined the expression of AChE during the process of NGF-induced neuronal differentiation of PC12 cells. Initially, we compared the level of cell-associated AChE between undifferentiated and 24-, 48-, and 72-h differentiated PC12 cells. Previous studies have demonstrated that undifferentiated PC12 cells express a basal level of AChE activity that increases upon NGF stimulation (31Greene L.A. Rukenstein A. J. Biol. Chem. 1981; 256: 6363-6367Google Scholar, 53Inestrosa N.C. Reiness C.G. Reichardt L.F. Hall Z.W. J. Neurosci. 1981; 1: 1260-1267Google Scholar, 54Lucas C.A. Czlonkowska A. Kreutzberg G.W. Neurosci. Lett. 1980; 18: 333-337Google Scholar). In agreement with these earlier reports, Fig.1 A shows that AChE activity increases considerably during differentiation. In fact, the cell-associated activity increased significantly by ∼2.5-fold (p < 0.01) within the first 48 h, and reached a maximal 5-fold induction (p < 0.0001) following 72 h of NGF treatment. We next examined the impact of NGF on the relative abundance of AChE transcripts in PC12 cells. We focused on the level of the T transcript because this transcript is the predominant splice variant found in nervous tissues (12Legay C. Bon S. Vernier P. Coussen F. Massoulie J. J. Neurochem. 1993; 60: 337-346Google Scholar) and PC12 cells (45Li Y. Liu L. Kang J. Sheng J.G. Barger S.W. Mrak R.E. Griffin W.S. J. Neurosci. 2000; 20: 149-155Google Scholar, 46Meshorer E. Erb C. Gazit R. Pavlovsky L. Kaufer D. Friedman A. Glick D. Ben Arie N. Soreq H. Science. 2002; 295: 508-512Google Scholar). As illustrated in Fig.1 B, AChE mRNAs could be detected in undifferentiated cells. However, treatment of PC12 cells with NGF led to a pronounced increase in the levels of AChE mRNA. These increases were highly significant, reaching more than 2-fold (p < 0.002) and 3.5-fold (p < 0.0001) by 48 and 72 h, respectively (Fig. 1 C). The relative amount of S12 rRNA, which was used as an internal control for these assays, did not change during differentiation. The observed increase in transcript level was directly related to the NGF treatment and not to the length of time in culture or cell density because, in separate studies, both of these factors had no effect on AChE expression (data not shown). In addition, NGF removal from the culture medium 24 h after the initial treatment resulted in a gradual decrease in AChE mRNA levels (data not shown). Because recent studies demonstrated the importance of transcriptional events in regulating AChE expression at the early stages of muscle differentiation (23Angus L.M. Chan R.Y. Jasmin B.J. J. Biol. Chem. 2001; 276: 17603-17609Google Scholar, 30Siow N.L. Choi R.C. Cheng A.W. Jiang J.X. Wan D.C. Zhu S.Q. Tsim K.W. J. Biol. Chem. 2002; 277: 36129-36136Google Scholar), we determined whether the increase in the abundance of AChE transcripts seen in differentiating PC12 cells could be linked to an increase in transcription. To this end, we performed transfection studies using an AChE promoter-reporter gene construct that cont" @default.
• W2035618537 created "2016-06-24" @default.
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• W2035618537 date "2003-02-01" @default.
• W2035618537 modified "2023-10-16" @default.
• W2035618537 title "Post-transcriptional Regulation of Acetylcholinesterase mRNAs in Nerve Growth Factor-treated PC12 Cells by the RNA-binding Protein HuD" @default.
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• W2035618537 doi "https://doi.org/10.1074/jbc.m209383200" @default.
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