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• W2072914101 abstract "To explore the scope and significance of alternate promoter usage and its putative inter-relationship to alternative splicing, we searched expression sequence tags for the 5′ region of acetylcholinesterase (ACHE) genes. Three and five novel first exons were identified in human and mouse ACHE genes, respectively. Reverse transcription-PCR and in situ hybridization validated most of the predicted transcripts, and sequence analyses of the corresponding genomic DNA regions suggest evolutionarily conserved promoters for each of the novel exons identified. Distinct tissue specificity and stress-related expression patterns of these exons predict combinatorial complexity with known 3′ alternative AChE mRNA transcripts. Unexpectedly one of the 5′ exons encodes an extended N terminus in-frame with the known AChE sequence, extending the increased complexity to the protein level. The resultant membrane variant(s), designated N-AChE, is developmentally regulated in human brain neurons and blood mononuclear cells. Alternative promoter usage combined with alternative splicing may thus lead to stress-dependent combinatorial complexity of AChE mRNA transcripts and their protein products. To explore the scope and significance of alternate promoter usage and its putative inter-relationship to alternative splicing, we searched expression sequence tags for the 5′ region of acetylcholinesterase (ACHE) genes. Three and five novel first exons were identified in human and mouse ACHE genes, respectively. Reverse transcription-PCR and in situ hybridization validated most of the predicted transcripts, and sequence analyses of the corresponding genomic DNA regions suggest evolutionarily conserved promoters for each of the novel exons identified. Distinct tissue specificity and stress-related expression patterns of these exons predict combinatorial complexity with known 3′ alternative AChE mRNA transcripts. Unexpectedly one of the 5′ exons encodes an extended N terminus in-frame with the known AChE sequence, extending the increased complexity to the protein level. The resultant membrane variant(s), designated N-AChE, is developmentally regulated in human brain neurons and blood mononuclear cells. Alternative promoter usage combined with alternative splicing may thus lead to stress-dependent combinatorial complexity of AChE mRNA transcripts and their protein products. Alternative splicing and alternate promoter usage both expand the complexity of gene products. While the massive contribution of alternative splicing to such expansion is widely recognized (1Graveley B.R. Trends Genet. 2001; 17: 100-107Abstract Full Text Full Text PDF PubMed Scopus (942) Google Scholar), less is known about the scope and significance of alternate promoter usage. Moreover the directionality of transcription processes raises the yet unresolved possibility that these two phenomena are inter-related, namely that the choice of the first exon determines downstream splice choices (2Cramer P. Pesce C.G. Baralle F.E. Kornblihtt A.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11456-11460Crossref PubMed Scopus (271) Google Scholar). The recent accumulation of genomic and gene expression data bases together with the development of sophisticated bioinformatic tools makes these questions amenable for experimental analysis as multiple gene products display both alternate promoter usage and alternative splicing variations. By alignment of expression sequence tags (ESTs) 1The abbreviations used are: EST, expression sequence tag; AChE, acetylcholinesterase; RT, reverse transcription; GR, glucocorticoid receptor; FISH, fluorescence in situ hybridization; PFC, prefrontal cortex; m, mouse; h, human; nt, nucleotide; ORF, open reading frame; GRE, glucocorticoid response element; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; AP, activating protein; EGR, early growth response; EKLF, erythroid Kruppel-like factor; HNF4, hepatocyte nuclear factor 4; MZF1, myeloid zinc finger 1; RREB1, Ras-responsive element binding 1; SP1, specificity protein 1; ZBP, zinc finger binding protein; ZF5F, zinc finger protein 5; TAL1, T cell acute lymphoblastic leukemia 1; ATF6, activating transcription factor 6; RFX1, regulatory factor X 1, AML1, acute myeloid leukemia 1; HSF1, heat-shock factor 1; PAX, paired homeobox; FKHD, forkhead. against genomic sequences, for example, it is possible to explore the different alternatively spliced products of a single gene (3Modrek B. Resch A. Grasso C. Lee C. Nucleic Acids Res. 2001; 29: 2850-2859Crossref PubMed Scopus (533) Google Scholar, 4Xie H. Zhu W.Y. Wasserman A. Grebinskiy V. Olson A. Mintz L. Genomics. 2002; 80: 326-330Crossref PubMed Scopus (43) Google Scholar). However, EST data bases are biased toward the 3′ end of mRNAs and occasionally contain genomic contaminations that may cause misinterpretation of the genomic information (5Sorek R. Safer H.M. Nucleic Acids Res. 2003; 31: 1067-1074Crossref PubMed Scopus (77) Google Scholar). To correctly evaluate the inter-relationship between alternate promoter usage and alternative splicing, it is therefore necessary to characterize the identified variants using traditional molecular biology tools at the RNA level and, if applicable, at the protein level as well. The acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) provides an adequate example for such a study. AChE pre-mRNA is subject to stimulus-induced 3′ alternative splicing (6Meshorer E. Erb C. Gazit R. Pavlovsky L. Kaufer D. Friedman A. Glick D. Ben-Arie N. Soreq H. Science. 2002; 295: 508-512Crossref PubMed Scopus (205) Google Scholar), and previous evidence has suggested that it is also subject to alternate promoter usage (7Mutero A. Camp S. Taylor P. J. Biol. Chem. 1995; 270: 1866-1872Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Atanasova E. Chiappa S. Wieben E. Brimijoin S. J. Biol. Chem. 1999; 274: 21078-21084Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). We analyzed the genomic regions flanking the human and mouse ACHE genes and found an unexpected, evolutionarily conserved diversity of alternate exons at their 5′ end. The newly identified exons are stress- and glucocorticoid-regulated and show no apparent connection to the 3′ splice variations. One of these new exons was further found to initiate an N-terminal extension to the canonic AChE protein, enabling a combinatorial complexity of developmentally controlled human membrane AChE protein variant(s). Our findings highlight independent yet inter-related expansions of ACHE gene products by 5′ and 3′ variations in AChE mRNA. Human Tissues—The use of human embryos, cord blood, and adult tissues in this study was approved by the Tel-Aviv Sourasky Medical Center Ethics Committee according to the regulations of the Helsinki accords. Tissue samples from spontaneously aborted human fetuses at different ages (16, 25, and 34 gestational weeks) were transferred immediately to 4% paraformaldehyde, embedded in paraffin, and sliced (7 μm). Fresh samples of umbilical cord blood cells were obtained following normal deliveries. Adult human brain samples were collected within 4 h postmortem from a 70-year-old patient with cardiac arrhythmia. Tissues were frozen immediately in liquid nitrogen. Brain homogenates (in 0.1 m phosphate buffer, 1% Triton X-100) were immunoblotted using standard procedures. Animals—Central nervous system-specific glucocorticoid receptor (GR) mutants (GRNesCre), control littermates (GRLoxP/LoxP) (9Tronche F. Kellendonk C. Kretz O. Gass P. Anlag K. Orban P.C. Bock R. Klein R. Schutz G. Nat. Genet. 1999; 23: 99-103Crossref PubMed Scopus (1456) Google Scholar), and FVB/N male mice were kept under a 12-h dark/12-h light diurnal schedule with food and water ad libitum. Stress experiments included a 30-min immobilization in 50-ml conical tubes. Mice were sacrificed by decapitation 2 h after immobilization, and brains were dissected on ice, frozen in liquid nitrogen or fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, sliced to 5-7-μm sections, and collected by adhesion to Superfrost®-Plus slides (Menzel-Glaser, Braunschweig, Germany). For all experiments, naïve age-matched males served as controls. These experiments were approved by the animal committee in the Hebrew University. Computational Resources—The human (GenBank™ accession number AF002993) and mouse (GenBank™ accession number AF312033) ACHE loci were analyzed using the National Center for Biotechnology Information for access to the GenBank™ as well as to Blast, Entrez, Locus Link, Structure, Protein, and OMIM (Online Mendelian Inheritance in Man) data base resources. Expert Protein Analysis System at the Swiss Institute of Bioinformatics was used for access to a variety of data manipulation programs and protein data bases. The Baylor College of Medicine Search Launcher 2See searchlauncher.bcm.tmc.edu. served for data manipulation and to derive display programs. The MatInspector program at Genomatix or the Cister software 3See zlab.bu.edu/~mfrith/cister.shtml. was used to find transcription factor binding sites. RNA Extraction and cDNA Preparation—Total RNA was extracted from animal and human tissues using the EZ-RNA total RNA isolation kit (Biological Industries, Beit Haemek, Israel) as instructed, diluted in diethyl pyrocarbonate-treated water to a concentration of 100 ng/μl, and stored at -70 °C until use. Human RNA from leukemic T-lymphocytes, liver, and testis was obtained from Ambion (Austin, TX). Super-Script reverse transcriptase (Invitrogen) was used with either poly(dT) or random hexamers. Gene-specific primers (see below) were used for one-step RT-PCR (Qiagen, Hilden, Germany). Fluorescence in Situ Hybridization (FISH)—Paraffin-embedded sections (mouse horizontal whole brain sections or human sagittal sections from whole embryos or human adult prefrontal cortex) were subjected to deparaffination with xylene (two 5-min washes) followed by decreasing ethanol washes (100, 75, 50, and 25%) and then a wash in phosphate-buffered saline with 0.5% Tween 20 and incubation with 10 mg/ml proteinase K (8 min at room temperature). Hybridization in a humidified chamber involved 10 mg/ml probe (in 50% formamide, 5× SSC, 10 mg/ml tRNA, 10 mg/ml heparin for 90 min at 52 °C). Sections were then washed twice at 60 °C with 50% formamide, 5× SSC, and 0.5% SDS; twice in 50% formamide, 2× SSC at 60 °C; and twice in Tris-buffered saline + 0.1% Tween 20 (TBST) at room temperature and then blocked in 1% skim milk (Bio-Rad) for 30 min. Biotin-labeled probes (see Supplemental Table I) were detected by incubating sections with streptavidin-Cy3 conjugates (CyDye™, Amersham Biosciences) for 30 min followed by three washes in TBST. Sections were mounted with IMMU-MOUNT (Shandon Inc., Pittsburgh, PA). Polymerase Chain Reaction—PCR was used for detecting different transcripts in various tissues and to confirm sequences. The PCR mixture contained 2 units of Taq DNA polymerase (Sigma), deoxynucleotide mixture (0.2 mm each) (Sigma), forward/reverse primers (see Supplemental Table II, 0.5 μm each), and 300 ng of template (cDNA or genomic DNA). Each of the 35 cycles included denaturation (1 min at 95 °C), annealing (1 min at 60 °C), and elongation (72 °C at 1 min). Antibodies—High affinity polyclonal rabbit IgG antibodies against the human hE1d-encoded N-terminal domain were custom-made (Eurogentec, Seraing, Belgium). Two 16-amino acid residue peptides from the coding sequence of human exon hE1d (hN-AChE) were synthesized (KVRSHPSGNQHRPTRG and GSRSFHCRRGVRPRPA), mixed, and injected together into two rabbits. Additional boost injections were given at 2, 4, and 8 weeks thereafter. Final bleeding was carried out after week 16. Enzyme-linked immunosorbent assay screening with the synthetic peptides served to identify successful antibody production. The synthetic peptides were further used for affinity purification of the antibodies. Dilutions of 1:500 of the affinity-purified antiserum were used for Western blotting. Flow Cytometry—Mononuclear fractions of cord blood cells were separated by FACS lysing solution (BD Biosciences, diluted 1:100 in double-distilled water, 12-min incubation). Cells were either stained directly with phycoerythrin-conjugated CD34 (BD Biosciences), allophycocyanine-conjugated CD3 (Caltag, Burlingame, CA), allophycocyanine-conjugated CD19 (BD Biosciences), peridinin chlorophyll protein-conjugated CD45 (BD Biosciences), and phycoerythrin-conjugated interleukin 7 (R&D Systems Inc., Minneapolis, MN) antibodies or first permeabilized and fixed (Fix and Perm kit, Caltag; 7 min) and then stained. Isotype controls served to distinguish unspecific labeling. Rabbit anti-hN-AChE antibodies were detected on these cells using fluorescein isothiocyanate-conjugated goat anti-rabbit Fab antibodies (Jackson Immunoresearch Laboratories, Inc., Westgrove, PA). Multiparameter flow cytometry was performed using the FACScalibur flow cytometry system (BD Biosciences) equipped with Cellquest software (BD Biosciences). hN-AChE expression was assessed in fresh CD45+ cells by analyzing 50,000 gated events. 5′ Diversity of Murine AChE mRNAs—EST data base searches using the 5′ region of the mouse (m) ACHE gene revealed the existence of five putative alternative first exons (Table I and Fig. 1A). The most proximal exon was termed mE1a. The EST clone containing this sequence (GenBank™ accession number BB606349, mouse eyeball) extends from position -787 to -680 (relative to the translational ATG start present in the mouse exon 2) and continues with exon 2 (Fig. 1, A and B), skipping over a 657-nucleotide (nt) intron (termed mouse mI1a) that possesses consensus GT-AG splice sites. RT-PCR and sequencing confirmed the existence of this transcript in the mouse cortex (GenBank™ deposit, accession number AY389982).Table IAlternative 5′ exons of mouse acetylcholinesteraseExonNumber of ESTsRepresentative EST evidencePositionaFor convenience, the - in front of all position numbers was eliminated. (from ATG)Intron sizeSplice sitesORFPrevious referenceConfirmationntME1a1BB606349, eyeball, P0787 to 680657GT-AGNoYesME1b0945 to 733710GT-AGNoYesME1c-long1AK036443, bone, adult1762 to 22NoNoME1c18BB639234, thymus, P31762 to 16711648GT-AGNo10YesME1d02271 to 19801957GT-AGNo8YesME1d′02271 to 20081986GT-AGNoYesME1e02518 to 24032380GT-AGYesNoa For convenience, the - in front of all position numbers was eliminated. Open table in a new tab A second first exon, named mE1b, was found by RT-PCR using a forward primer located in the -945 to -923 region with a reverse primer on exon 2 (see Supplemental Table II). The resulting product extends from this primer to position -733 and skips over a 710-nt intron (mI1b), which includes consensus GT-AG splice sites (Fig. 1, A and B). This exon as well was confirmed in the mouse cortex by RT-PCR and sequencing (GenBank™ deposit, accession number AY389981). Upstream to mE1b, at -1762 to -1671, we found the “classical” exon 1 (10Li Y. Camp S. Rachinsky T.L. Getman D. Taylor P. J. Biol. Chem. 1991; 266: 23083-23090Abstract Full Text PDF PubMed Google Scholar), renamed here mE1c, in 18 different reported homologous EST clones (GenBank™ accession number BB639234, Table I). When this first exon is fused to exon 2, a 1648-nt intron (mI1c) that contains consensus GT-AG splice sites is spliced out. Sequencing of an RT-PCR-amplified DNA fragment confirmed the existence of mE1c in the mouse cortex. An additional mRNA transcript that contains mE1c but proceeds through the genomic sequence was named mE1c-long. Two longer ESTs indeed initiated at mE1c (GenBank™ accession numbers BB629342, adult bone, and CA327701, whole brain embryo) and extend through the entire genomic sequence to exon 2 (GenBank™ accession number AK036443, adult male bone). In these ESTs, exon 2 is fused to exon 3. Splicing of intron 2 rules out the possibility of genomic DNA contamination as the source of this mE1c-long variant. Further upstream, an alternative first exon (8Atanasova E. Chiappa S. Wieben E. Brimijoin S. J. Biol. Chem. 1999; 274: 21078-21084Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) had previously been found at position -2271 to -1980 followed by a 1957-nt intron (mI1d). This first exon was found to be fused with exon 2. We confirmed the expression of the corresponding transcript in the prefrontal cortex by RT-PCR and sequencing. This exon was named mE1d. Two alternative splice donors that differ by 29 nucleotides were observed in the mouse cortex. We named the shorter form mE1d′ (GenBank™ deposit, accession number AY389980). Upstream from mE1d, three different putative ORFs (positions -2518 to -2402, -2925 to -2522, and -3129 to -2933) were found in a continuous reading frame with that of the classical protein. These could add 46, 142, or 73 amino acids (respectively) to the common ORF beginning at exon 2. Of these, the mE1e ORF shares 79% sequence similarity with the corresponding region in the human ACHE gene and its translated sequence (see below) and was thus regarded as a candidate. Fig. 1, A and B, depicts the different mouse 5′ exons. 5′ Diversity of Human AChE mRNAs—EST data base searches using the 5′ region of the human (h) ACHE gene revealed the existence of at least four alternative first exons (Table II). The previously identified mouse EST clone (mE1a GenBank™ accession number BB606349, see above) suggests the existence of the alternative first exon named hE1a.Table IIAlternative 5′ exons of human acetylcholinesteraseExonNumber of ESTsRepresentative EST evidencePositionaFor convenience, the - in front of all position numbers was eliminated. (from ATG)Intron sizeSplice sitesORFExon 2 startPrevious referenceConfirmationnthE1a1BB606349, eyeball, P0768 to 732NoNohE1b23BG707892, hypothalamus1681 to 15761543GT-AGNoCAG11YeshE1c1BI667712, hypothalamus1859 to 18241803GT-AGNoACGNohE1d3BX420294, fetal brain2720 to 23182294GT-AGYesACGYesa For convenience, the - in front of all position numbers was eliminated. Open table in a new tab The known first exon at -1681 to -1576 (relative to the translational start site ATG present in the human exon 2) (11Ben Aziz-Aloya R. Sternfeld M. Soreq H. Prog. Brain Res. 1993; 98: 147-153Crossref PubMed Scopus (26) Google Scholar) is named here hE1b (represented by EST clone GenBank™ accession number BG707892, human brain hypothalamus). A 1543-nt intron (hI1b) separates hE1b from exon 2 (position -23). We confirmed the existence of hE1b by RT-PCR and sequencing. An additional EST clone contained the genomic sequence located at position -1859 to -1824 (GenBank™ accession number BI667712, human brain hypothalamus). This putative first exon, fused to exon 2 at position -20 (ACG), was named hE1c. The corresponding 1803-nt intron (hI1c) includes donor and acceptor splice sites (GT-AG). Exon 2 starts with two optional acceptor splice sites located 3 nucleotides apart (both AG dinucleotide; Fig. 1, C and D). Therefore, when fused to hE1c, exon 2 starts at a different position than the one described previously. Our attempts to confirm the existence of this transcript failed. An additional EST clone (GenBank™ accession number BX420294, human fetal brain) contained a putative first exon located further upstream at position -2720 to -2318 (exon hE1d) fused to exon 2 at position -20. This implies the existence of a 2294-nt intron (hI1d). The existence of this mRNA was confirmed by RT-PCR and sequencing (GenBank™ deposit, accession number AY389977; Fig. 1, C and D). Intriguingly hE1d harbors a translation start codon (ATG, position -2495) creating a continuous reading frame with that of the classical ATG in exon 2 (12Soreq H. Ben-Aziz R. Prody C.A. Seidman S. Gnatt A. Neville L. Lieman-Hurwitz J. Lev-Lehman E. Ginzberg D. Lipidot-Lifson Y. Zakut H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9688-9692Crossref PubMed Scopus (182) Google Scholar), thus potentially adding 66 amino acids to the AChE protein. An additional ATG (position -2478) in the same ORF may yield a shorter 61-amino acid domain. Sequence homology with mE1e, which lacks the first ATG, suggests that the second ATG is more likely to serve as the translational start site. Putative Promoters for the Novel Exons—Using luciferase assays, Atanasova et al. (8Atanasova E. Chiappa S. Wieben E. Brimijoin S. J. Biol. Chem. 1999; 274: 21078-21084Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) demonstrated the functionality of the promoter located upstream to mE1d (referred to in their work as exon E1a). In our study, the Cister and Chip2Promoter programs enabled promoter predictions, which are shown in Fig. 2A for the murine and human ACHE genes. These programs search for regions with motif conservation predicting high probability of being transcriptionally active promoters. These are based primarily on the density of putative transcription factor binding sites. Several regions with such high probability were revealed by this search. Promoter prediction analyses of the region containing the novel alternative first exons revealed a plausible promoter for each of the newly identified exons of both the mouse and human genes (Fig. 2, A and B). The probability score of the alternative promoters was found to be similar to that of the previously described promoter (upstream to mE1b in mouse and hE1b in human), supporting the notion that they might be functionally active. A particularly high probability to function as a promoter was observed for the mouse region upstream to exon mE1a. In the human gene, we identified hE1a based on homology to the mouse mE1a. Exon hE1a is a weak candidate for being a true exon because it lacks consensus splice sites and because no ESTs were found in the entire region between exon 2 and exon hE1b in the human sequence. Intriguingly the region located upstream from hE1a displays the highest probability of functioning as a promoter (Fig. 2A), perhaps suggesting a change in function for this sequence during evolution. The Cister and the Chip2Promoter program, which does not apply to murine sequences, yielded similar predictions for human promoters. A closer look at the distribution of the transcription factor binding sites revealed that most sites are common to several of the putative alternative promoters and evolutionarily conserved in both human and mouse. Several putative DNA targets, however, were unique to one of the identified promoters. For example, a conserved binding site for the transcription factor Dlx, highly expressed during organ development (13Panganiban G. Rubenstein J.L. Development. 2002; 129: 4371-4386Crossref PubMed Google Scholar), was found in mP1 and hP1, and a putative binding site for transforming growth intestinal factor was found in mP2 and hP2. Of interest, three putative glucocorticoid response elements (GREs) were identified in the upstream region of the human ACHE gene (one in hP3 and one adjacent to hE1a, Fig. 2A), and one was identified in the mouse gene (mP2, Fig. 2A). It was therefore tempting to test the glucocorticoid and/or stress response of these newly identified transcripts. Short Interspersed Nuclear Elements (SINEs) and Long Interspersed Nuclear Elements (LINEs) Separate 5′ Alternative Exons from the Distal Human ACHE Promoter—On average, one might expect one SINE and one LINE for approximately every 2-3.5 kb except within the transcription unit itself (14Batzer M.A. Deininger P.L. Nat. Rev. Genet. 2002; 3: 370-379Crossref PubMed Scopus (1064) Google Scholar). A totally different outcome emerged for the currently available GenBank™ ACHE sequences (20 kb of the human, GenBank™ accession number AF002993, and 9.5 kb of mouse, GenBank™ accession number AF312033) upstream to the translation start sites of exon 2. The SINE and LINE distributions were analyzed using the Eldorado software (Genomatix) and the RepeatMasker algorithm (Baylor College of Medicine Search Launcher). The density was found to be 6-fold higher than the expected average for SINEs and almost 2-fold higher than the expected average for LINEs (Fig. 2, C and D). This leaves little room for any functional DNA in this area. In contrast, exceptionally few repeats (one and three, respectively) were found within the human and mouse 3.5-kb regions where the alternative first exons were identified, supporting a functional role for these DNA fragments in human and mouse. The closest gene upstream to ACHE is located ∼180 kb away (15Wilson M.D. Riemer C. Martindale D.W. Schnupf P. Boright A.P. Cheung T.L. Hardy D.M. Schwartz S. Scherer S.W. Tsui L.C. Miller W. Koop B.F. Nucleic Acids Res. 2001; 29: 1352-1365Crossref PubMed Scopus (45) Google Scholar). Tissue Distribution of the Novel Exons in Mouse—Tissue distribution in mouse of the mRNAs containing the different alternative first exons was studied by RT-PCR (Fig. 3A). Exon mE1a was found to be expressed in every examined brain region, including hippocampus, cortex, PFC, brainstem, and basal nuclei. Exon mE1a was also expressed in the thymus, heart, liver, intestine, and spleen but not in kidney, testis, muscle, or spinal cord. Exon mE1b was detected in most of the tissues examined with the exception of liver, intestine, and muscle. Exon mE1c was the most widely expressed. It was, however, absent from intestine. Exon mE1d was detected in the brain (hippocampus, PFC, brainstem, and basal nuclei) and heart but not spleen, thymus, intestine, or liver. While it is difficult to compare the expression levels between different RT-PCR products due to differences in primer and target efficiency, a tissue distribution analysis, as was conducted, gives a good hint of the relative expression levels of each variant in each tissue. We conclude that mE1c is the most abundant transcript due to its almost ubiquitous expression and due to its over-representation by ESTs (Table I). For comparison, we investigated in the same tissues the expression profiles of the different AChE 3′ variants. “Synaptic” AChE-S was strongly expressed in all tissues examined except for thymus, liver, and the small intestine where only weak expression was observed, similar to the expression pattern of mE1c. “Read-through” AChE-R was strongly expressed in all of the brain regions tested and in spleen. It was moderately expressed in heart, muscle, kidney, spinal cord, and liver and very poorly expressed in the testis, thymus, and intestine. “Erythrocytic” AChE-E was expressed in all of the examined brain regions as well as in heart, kidney, spinal cord, liver, spleen, and muscle. It was absent from testis, thymus, and the small intestine. Thus, none of the 5′ variants shared the same expression pattern with a single 3′ variant, suggesting that 5′ splicing patterns do not always dictate 3′ splicing in the mature mRNA. The four different 5′ and three different 3′ splice options may thus potentially yield 12 distinct transcripts. Distinct Neuronal Distributions of the 5′ Murine Exons—To achieve cellular resolution levels for the expression patterns of the novel exons, we designed 40-50-mer 5′-biotinylated fully 2′-O-methylated riboprobes for FISH (see “Experimental Procedures” for details). Fig. 3B presents representative FISH profiles for mE1a, mE1b, and mE1d. These three exons all appeared to be expressed in neurons. They displayed, however, distinct cell type specificities and subcellular distributions. For example, essentially all of the deep layer neurons in the PFC displayed pronounced mE1a levels and considerably lower mE1b labeling. Exon mE1d mRNA was particularly concentrated in the uppermost layer of PFC neurons (Fig. 3B, I), suggesting distinct levels for AChE mRNA transcripts in specific subsets of PFC neurons. Whereas these differences potentially reflect probe efficiencies, they also highlight distinct expression patterns for the various alternative AChE mRNAs. Thus, hippocampal CA2 neurons within the same or adjacent sections displayed consistently low levels of all three exons (Fig. 3B, II), whereas cerebellar neurons showed differential expression of the various 5′ exons (Fig. 3B, III). Exon mE1a accumulated in the cytoplasm of Purkinje cell perikarya but was only faintly detected in other cerebellar neurons. mE1b was poorly expressed in the cerebellum, and mE1d was strongly expressed in Purkinje cells in which it was labeled in both cell bodies and axonal processes (Fig. 3B, IV and V). In addition, mE1d was transcribed in other neurons of the cerebellum, including the cells interspersed in the molecular layer. In these neurons, it displayed an asymmetric labeling pattern accompanied by neurite labeling. Granular neurons were poorly labeled with the probe for mE1d. Human hE1d AChE mRNA Expression—The tissue distribution of transcribed hE1d mRNA in several developmental stages was explored in paraffin sections from human embryos aged 16, 25, and 34 weeks. At week 16, hE1d mRNA was only weakly detected in the nervous system and was absent in the thymus. As development proceeded, hE1d expression became more pronounced with increased density of positive cells and increased labeling intensity in both the nervous system and the thymus. At week 34, up to 50 ± 10% of the neurons were positive (Fig. 4A). In contrast, as low as 2 ± 1.5% of the thymus cells were hE1d mRNA-positive at week 25, but by week 34, over 8 ± 1.5% of the cells were positive (p < 0.0005, two-tailed Student's t test). Since hE1d demonstrated a developmentally regulated expression pattern, we performed RT-PCR analysis on RNA extracted from human embryonic stem cells (16Drukker M. Katz G. Urbach A. Schuldiner M. Markel G. Itskovitz-Eldor J. Reubinoff B. Mandelboim O. Benvenisty N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9864-9869Crossref PubMed Scopus (572) Google Scholar) and le" @default.
• W2072914101 created "2016-06-24" @default.
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• W2072914101 date "2004-07-01" @default.
• W2072914101 modified "2023-10-02" @default.
• W2072914101 title "Combinatorial Complexity of 5′ Alternative Acetylcholinesterase Transcripts and Protein Products" @default.
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