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 35kb 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 453bp fragment, including 442 upstream of exon C and 11bp of exon C. A 346bp 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.
- alternative splicing
The nucleotide sequences reported in this study will appear in GenBank® and EMBL databases with accession nos. AY089985, AY089986, AY089987 and AY089988.
Abbreviations used: ALL, acute lymphoblastic leukaemia; CRE, cAMP-response element; MTE, multi-tissue; MTN, 12-lane human multi-tissue Northern blot; Mtx, methotrexate; poly(A)+, polyadenylated; RACE, rapid amplification of cDNA ends; RFC, reduced folate carrier; hRFC, human RFC; mRFC, murine RFC; RT, reverse transcriptase; Sp, stimulating protein; 5′-UTR, 5′-untranslated region.
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