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

Abstract

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.

Transmembrane Domains 4, 5, 7, 8, and 10 of the Human Reduced Folate Carrier Are Important Structural or Functional Components of the Transmembrane Channel for Folate Substrates

“…Moreover, our previous results imply that Lys-411 in TMD11 is important for binding certain transport substrates (28) and, most recently, that it is a likely target for covalent modification by the activated ester N-hydroxysuccinimide Mtx. 4 This suggests that Lys-411 participates in binding to the glutamate moiety in Mtx and reduced folates. Compelling evidence from mutant studies establishes that Ser-313 and Arg-373 are functionally important and contribute to substrate binding specificity (25,26).…”

The role of folate receptor alpha (FRα) in the response of malignant pleural mesothelioma to pemetrexed-containing chemotherapy

“…The reduced folate carrier (RFC) is a bidirectional anion exchanger that has a high affinity for reduced folate co-factors and antifolates, but a low affinity for folic acid. It has a neutral pH optimum and is ubiquitously expressed (Whetstine et al, 2002). The folate receptor alpha (FRa) is an energy-dependent high-affinity, low-capacity folate-binding protein that delivers folates and antifolates into cells.…”

The Region between Transmembrane Domains 1 and 2 of the Reduced Folate Carrier Forms Part of the Substrate-binding Pocket

“…It is expressed as a membrane protein in a wide variety of tissues and cell types (1,3,4); and although highly conserved at the amino acid level, it varies in predicted size from 58 to 85 kDa depending upon the species (5-7). Hydrophobicity analyses have indicated that the RFC protein has 12 transmembrane-spanning segments with the N and C termini located intracellularly and a large intracellular loop between transmembrane domain (TM) 6 and TM7 (8).…”