ALTERNATIVE SPLICING OF THE HUMAN SEROTONIN TRANSPORTER GENE

Citation
Cc. Bradley et Rd. Blakely, ALTERNATIVE SPLICING OF THE HUMAN SEROTONIN TRANSPORTER GENE, Journal of neurochemistry, 69(4), 1997, pp. 1356-1367
Citations number
68
Categorie Soggetti
Biology,Neurosciences
Journal title
ISSN journal
00223042
Volume
69
Issue
4
Year of publication
1997
Pages
1356 - 1367
Database
ISI
SICI code
0022-3042(1997)69:4<1356:ASOTHS>2.0.ZU;2-P
Abstract
To explore the structural basis for regulation of human serotonin tran sporter (hSERT) gene expression, we used primer extension and 5' rapid amplification of cDNA ends (5'RACE) techniques to estimate levels of and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Prim er extension on JAR cell mRNA suggested the presence of significant hS ERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Usi ng 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we clo ned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reporte d noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of JAR cell mRNA blotted with exon-specific oligonucleotides revealed bo th exons 1A and 1B to be regulated in a cholera toxin-dependent manner . To clarify the structure of the hSERT gene including exon 1B, we iso lated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing band s on genomic Southern blots and was found to utilize 13 exons to encod e the transporter's coding sequences along with the two noncoding 5' e xons. Exon 1B was identified similar to 14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation si te for translation is located. Exon 1B is surrounded by several elemen ts potentially suitable for regulating serotonin transporter gene expr ession in vivo, including consensus sites for transcription factors AP -1, AP-2, CREB/ATF, and NF-kappa B. These data reveal additional compl exity in hSERT gene structure and expression that may be relevant to r egulated and compromised transporter expression in vivo.