The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter

• Published in: Biochemical journal Vol. 367; no. Pt 3; pp. 629 - 640
• Main Authors: Whetstine, Johnathan R, Flatley, Robin M, Matherly, Larry H
• Format: Journal Article
• Language: English
• Published: England 01.11.2002
• Subjects: 5' Untranslated Regions; Base Sequence; Blotting, Northern; Carrier Proteins - genetics; DNA; Exons; Humans; Membrane Transport Proteins; Molecular Sequence Data; Promoter Regions, Genetic; Reduced Folate Carrier Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - genetics; Tumor Cells, Cultured
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20020512

Our previous study identified two alternate non-coding upstream exons (A and B) in the human reduced folate carrier (hRFC) gene, each controlled by a separate promoter. Each minimal promoter was regulated by unique cis -elements and transcription factors, including stimulating protein (Sp) 1 and Sp3 and the basic leucine zipper family of proteins, suggesting opportunities for cell- and tissue-specific regulation. Studies were performed to explore the expression patterns of hRFC in human tissues and cell lines. Levels of hRFC transcripts were measured on a multi-tissue mRNA array from 76 human tissues and tumour cell lines and on a multi-tissue Northern blot of representative tissues, each probed with full-length hRFC cDNA. hRFC transcripts were ubiquitously expressed, with the highest level in placenta and the lowest level in skeletal muscle. By rapid amplification of cDNA 5'-ends assay from nine tissues and two cell lines, hRFC transcripts containing both A and B 5'-untranslated regions (UTRs) were identified. However, five additional 5'-UTRs (designated A1, A2, C, D and E) were detected, mapping over 35 kb upstream from the hRFC translation start site. The 5'-UTRs were characterized by multiple transcription start sites and/or alternative splice forms. At least 18 unique hRFC transcripts were detected. A novel promoter was localized to a 453 bp fragment, including 442 upstream of exon C and 11 bp of exon C. A 346 bp repressor flanked the 3'-end of this promoter. Our results suggest an intricate regulation of hRFC gene expression involving multiple promoters and non-coding exons. Moreover, they provide a transcriptional framework for understanding the role of hRFC in the pathophysiology of folate deficiency and antifolate drug selectivity.