The Divergent 5‘ Termini of the α Human Folate Receptor (hFR) mRNAs Originate from Two Tissue-Specific Promoters and Alternative Splicing: Characterization of the α hFR Gene Structure

Abstract

The human KB cell or α folate receptor (α hFR) is a membrane glycoprotein of 42 kDa that participates in the internalization of folates and antifolates. Seven independent α hFR cDNA isoforms have been reported that contain unique 5‘ termini but share a common open reading frame (ORF). To investigate the molecular basis of these heterogeneous 5‘ sequences, we determined the sequence of the α hFR gene from two clones isolated from a human lymphocyte λ DASH genomic library. The gene is composed of seven exons that span 6.8 kb. The ORF is encoded by exons 4 through 7 while the reported 5‘ termini of the cDNA isoforms (including two novel cDNAs designated KB2 and KB4) are encoded by exons 1 through 4. Using RNase protection assays, we demonstrate that transcripts corresponding to the KB1 and KB4 cDNAs originate from promoters upstream from exon 1 and exon 4, designated P1 and P4, respectively, and that these mRNA isoforms are the most abundant transcripts expressed in KB cells and selected normal tissues (including kidney, lung, and cerebellum). We observed a heterogeneous start site within exon 1 from the P1 promoter while transcripts from the P4 promoter originate from a single site. In addition, we detected tissue specificity for the P1 and P4 promoter utilization. Transcripts originating from the P1 promoter are the most abundant transcripts expressed by human cerebellum and kidney. In contrast, transcripts from the P4 promoter are the most abundant transcripts expressed by human KB cells and lung. Total RNA from KB cells also protects a 66 bp fragment of an exon 3 riboprobe that is consistent with an alternatively spliced transcript. To examine the functional activity of the predicted P1 and P4 promoters, α hFR promoter-CAT chimeric plasmids were constructed using sequences flanking exon 1 and exon 4. We observed a 7.5- and 10-fold increase in CAT activity in HeLa cells transiently transfected with the P1 and P4 promoter constructs, respectively. These data demonstrate that a single gene encodes the divergent 5‘ termini of the α hFR cDNAs and that the α hFR transcripts are transcribed from two promoters that are activated in a tissue-specific manner.