Combinatorial Complexity of 5′ Alternative Acetylcholinesterase Transcripts and Protein Products

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.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 (1), 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 (2). 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) 1 against genomic sequences, for example, it is possible to explore the different alternatively spliced products of a single gene (3, 4). However, EST data bases are biased toward the 3Ј end of mRNAs and occasionally contain genomic contaminations that may cause misinterpretation of the genomic information (5). 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 3Ј alternative splicing (6), and previous evidence has suggested that it is also subject to alternate promoter usage (7,8). We analyzed the genomic regions flanking the human and mouse ACHE genes and found an unexpected, evolutionarily conserved diversity of alternate exons at their 5Ј end. The newly id...

Differential localization of acetylcholinesterase in neuronal and non‐neuronal cells

“…Exon 1d forward (E1df): 5′-CCTGGTGACGAAAGTCCGA-3′ [Meshorer et al, 2004] Exon 1d reverse (E1dr): 5′-TCCTCCACCCAGGAGCCAGAG-3′ [Meshorer et al, 2004] Exon 3 forward (E3f): 5′-CGGGTCTACGCCTACGTCTTTGAACACCGTGCTTC-3′ [Karpel et al, 1994] Exon 5 reverse (E5r): 5′-TCACAGCCGCCGGAGGTGGGAG-3′ Exon 6 reverse (E6r): 5′-CACAGGTCTGAGCAGCGATCCTGCTTGCTG-3′ [Karpel et al, 1994].…”

“…pGS-AChE, a plasmid containing the cDNA encoding the human AChE cDNA E4/E6 splice variant was a gift from Dr. Oksana Lockridge (University of Nebraska Medical School, Omaha, NE). PCR was performed on 5 μl of cDNA and PCR conditions were as described previously [Karpel et al, 1994;Meshorer et al, 2004]. For E1d, denaturation 94°C 1min; annealing 60°C 1 min; synthesis 72°C 1 min × 39 cycles.…”

“…Anti-huAChE was diluted 1:1,500 in blocking buffer (5% non-fat dehydrated milk in PBS/0.3% Tween-20) and secondary anti-mouse IgG conjugated to HRP (Cell Signaling Technology, Beverly, MA) was diluted 1:1,000 in blocking buffer. Primary anti-AChE core polyclonal antibody (N19; Santa Cruz Biotechnologies, Inc., Santa Cruz, CA) has previously been used to detect AChE in immunoprecipitation and immunoblot assays [Birikh et al, 2003;Darreh-Shori et al, 2004;Meshorer et al, 2004]. N19 was diluted 1:500 in blocking buffer, and secondary anti-goat IgG conjugated to HRP (Vector Laboratories, Inc., Burlingame, CA) was diluted 1:2,000 in blocking buffer.…”

“…The variant that contains E6 is termed E4/E6, and is expressed in neuronal tissue. Recently, a novel alternative exon 1 (E1d) has been identified in the human AChE gene, and was shown to be expressed in neuronal tissue and leukocytes [Meshorer et al, 2004]. All three splice variants were detected in SH-SY5Y cells (Fig.…”

“…The different isoforms have been shown by reverse-transcriptase polymerase chain reaction (RT-PCR) to result from alternative splicing of AChE mRNA leading to isoforms of AChE protein differing in their 3′ exon composition [Karpel et al, 1994;Perry et al, 2002]. Although long thought that the transcriptional processing of AChE is complex and cell-regulated, additional isoforms continue to be demonstrated [Meshorer et al, 2004].…”