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. 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, as experiments were approved by the in the human and mouse ACHE were analyzed using the Center for for to the as as to and in data at the of was used for to a of data and protein data The of for data and to display The at or the was used to transcription factor binding RNA and RNA was from and human tissues using the RNA as in water to a of and at Human RNA from and was obtained from reverse was used with or were used for in brain or human from or human adult prefrontal were to with by and and a in with and with at in a (in for at were at with and in at and in at and in 1% for were by with for by in were with was used for different transcripts in tissues and to The 2 of DNA and of or genomic of the included at at and at against the human N-terminal were from the sequence of human exon were and and together two were at and was after with the to The were further used for of the of of the were used for of cord blood cells were by in were with and or first and fixed and and to were on these cells using was using the with expression was in cells by 5′ of AChE data using the 5′ region of the mouse ACHE gene the of five putative alternative first exons and The most exon was The EST this sequence mouse from to to the in the mouse exon and with exon 2 1, and a mouse that splice and the of this in the mouse 5′ exons of mouse of EST the in of was to to to to to to to the in of was in a new first was found by using a in the to region with a reverse on exon 2 The from this to and a splice 1, and exon as was in the mouse by and to at to we found the exon Camp S. D. Taylor P. J. Biol. Chem. Full Text PDF PubMed Google Scholar), in different EST this first exon is to exon a that splice is spliced of an DNA the of in the mouse mRNA that the genomic sequence was at adult and brain and the genomic sequence to exon 2 adult male these exon 2 is to exon of 2 the possibility of genomic DNA as the of this an alternative first exon E. Chiappa S. Wieben E. Brimijoin S. J. Biol. Chem. 1999; 274: 21078-21084Abstract Full Text Full Text PDF PubMed Scopus (28) Google found at to by a first exon was found to with exon We the expression of the corresponding in the prefrontal by and exon was alternative splice that by were in the mouse We the from different putative to to and to were found in a reading with that of the or to the at exon the sequence with the corresponding region in the human ACHE gene and its sequence and was thus as a 1, and the different mouse 5′ 5′ of Human AChE data using the 5′ region of the human ACHE gene the of at alternative first exons The identified mouse EST the of the alternative first exon 5′ exons of human of EST the in of was 2 to to to to the in of was in a new The known first exon at to to the in the human exon R. Soreq H. Brain Res. PubMed Scopus Google is by EST human brain from exon 2 We the of by and EST the genomic sequence at to human brain putative first to exon 2 at was The corresponding and splice 2 with two splice 1, and to exon 2 at a different the one Our to the of this EST human a putative first exon further at to to exon 2 at the of a The of this mRNA was by and 1, and a a reading with that of the in exon 2 H. R. S. A. L. J. E. D. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), thus to the AChE in the may a with the first that the is to as the for the E. Chiappa S. Wieben E. Brimijoin S. J. Biol. Chem. 1999; 274: 21078-21084Abstract Full Text Full Text PDF PubMed Scopus (28) Google the of the promoter to to in their as exon the and promoter are in for the and human ACHE genes. for regions with of are on the of putative transcription factor binding regions with such were by this analyses of the region the novel alternative first exons a promoter for each of the newly identified exons of both the mouse and human genes and The of the alternative promoters was found to to that of the promoter to in mouse and in the that to as a promoter was for the mouse region to exon the human we identified on to the mouse is a for a exon it splice and no were found in the region between exon 2 and exon in the human the region from the of as a promoter a in for this sequence The and the to sequences, for human at the of the transcription factor binding that most are to of the putative alternative promoters and evolutionarily conserved in both human and putative DNA were to one of the identified example, a conserved binding for the transcription factor development G. 2002; PubMed Google Scholar), was found in and and a putative binding for growth factor was found in and putative glucocorticoid response were identified in the region of the human ACHE gene in and one to and one was identified in the mouse gene was therefore to the glucocorticoid response of these newly identified transcripts. and 5′ Alternative from the Human ACHE one one and one for within the transcription Nat. Genet. 2002; PubMed Scopus Google Scholar). different for the ACHE of the and of to the of exon The and were analyzed using the and the of The was found to the for and the for and for DNA in this and were found within the human and mouse regions the alternative first exons were a for these DNA in human and The gene to ACHE is C. P. S. Nucleic Acids Res. 2001; 29: PubMed Scopus Google Scholar). Tissue of the in in mouse of the mRNAs the different alternative first exons was by was found to in brain PFC, and was also in the and in or was in most of the tissues with the of and was the most widely from was in the brain PFC, and and or While it is to the expression between different products to in and a tissue as was a of the expression of each in each We that is the most to its expression and to its by we in the tissues the expression of the different AChE 3′ was in tissues for and the expression was to the expression of was in of the brain regions and in was in and and in the and was in of the brain regions as as in and was from and the of the 5′ variants the expression with a single 3′ that 5′ splicing patterns 3′ splicing in the mRNA. The different 5′ and different 3′ splice may thus transcripts. Distinct of the 5′ for the expression patterns of the novel we for for for and exons to in cell and example, of the neurons in the and mRNA was in the of neurons for AChE mRNA transcripts in of these also highlight expression patterns for the alternative AChE neurons within the or of exons neurons expression of the 5′ exons in the of cell was in was in the and was in cells in it was in both cell and processes and was in neurons of the the cells interspersed in the molecular these it an by neurons were with the for Human AChE mRNA tissue of mRNA in was in from human 25, and 34 mRNA was in the nervous and was in the development expression with increased of cells and increased in both the nervous and the to of the neurons were as as 2 of the cells were at 25, by of the cells were a developmentally regulated expression we analysis on RNA from human cells G. A. G. J. B. O. N. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google and human cells were for were The developmentally regulated expression of mRNA in the and in for corresponding analysis of its putative were against two short from the for on sections, these human mononuclear cells in blood cell were according to their and their expression of the blood cell blood cell and and both of AChE C. D. S. V. Soreq H. J. (in Google Scholar), the most with and of the cells as with of the and 4% of the were and were To further the we used for cells early and of these may in one cell in and of the cells and early and increased to in To is as predicted from its the were following of the cells. and to and that is at the and of the D. Friedman A. S. Soreq H. PubMed Scopus Google yet long (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 expression of mRNA an AChE with a to the accumulation of AChE The ACHE gene a in a L. N. D. 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PubMed Scopus Google was found for the sequence in the data to the human a putative as as an to an predicted that of and of protein the an to the using the from different regions of the human brain in a for the protein in and the it was most The and were also expression was To we to mRNA in situ analysis of human mRNA with of the cells in the to of the cells mRNA in in of the neurons thus both in their cell and in The recent of human and mouse genomic together with EST data bases and the of novel alternative 5′ exons and corresponding promoters in the human and mouse ACHE genes. human L. N. D. Soreq H. Genet. PubMed Scopus Google and mouse (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. 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Genet. 1999; 23: 99-103Crossref PubMed Scopus (1456) Google different and were found to in response to these the of for its both and were under in One possible that following of different one the of the of the are no and of may the of novel 5′ alternative splicing patterns of AChE mRNA has and the complexity and of AChE mRNA variants to that were the 5′ splice variations extended membrane of AChE in brain neurons and cells. While the and membrane directionality of these variants further this long known in AChE and new The apparent of this extended in and its expression patterns and for its We for early to this and for and for human and Glick for this with