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

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.