STRUCTURAL-ANALYSIS OF THE HUMAN RFC-1 GENE ENCODING A FOLATE TRANSPORTER REVEALS MULTIPLE PROMOTERS AND ALTERNATIVELY SPLICED TRANSCRIPTS WITH 5'-END HETEROGENEITY

The organization and structure of the human RFC-1 gene encoding a fola te transporter were determined. The RFC-1 gene spans 22.5 kb and was f ound to be distributed in eight exons, including five primary exons an d three alternatives of exon 1. Most splice junctions conform to conse nsus sequences for such junctions. The human RFC-1 gene differs from t he mouse and hamster genes both in terms of the total number of exons and in regard to alternatives of exon 1 which encode 5' end heterogene ity. Previously described cDNA variants (GenBank/EMBL accession no. U1 9720) are now shown to incorporate one of two alternatives (exons 1a a nd 1b) to exon 1 and exons 2-6 as a result of RNA splicing. Another va riant also described may not be full length in that it incorporates a probable alternative (exon 1c) to exon 1 along with exon 2 and a trunc ated exon 3. A relatively GC-rich region of the genome 5' of the alter natives to exon 1 appears to be distinctly promoter like and incorpora tes a number of putative cis-acting elements, including multiple SP1 s ites, involved in the regulation of transcription. Primer extension an alysis of this upstream region in two human cell types revealed a simi lar pattern of multiple transcription start sites (tsp) proximal to th e 5' end of exon 1. However, there was a greater number of potential t sp within the region immediately upstream of exon 1b than within the r egions upstream of exons 1a and 1c. The existence of true alternatives to exon 1 in this gene incorporating different 5' ends indicates that its transcription is under the control of multiple promoters. The ide ntity of two such promoters was obtained by functional deletion analys is, showing that expression of a luciferase reporter gene was directed separately by discrete stretches of nucleotide sequence proximal to e xon 1a (promoter 1) or exon 1b (promoter 2) in transient transfection experiments. Promoter 1 appeared to have a three-fold lower basal acti vity than promoter 2, but was enhanced up to nine-fold in fusion const ructs containing an SV40 enhancer element. Also, promoter 2 partly con sists of a highly GC-rich direct repeat element containing at least th ree putative SP-1 and 3 putative MZF1 sites. Finally, the activity of these promoters relative to each other was consistent with the results of primer extension analysis showing a greater multiple and usage of tsp within promoter 2 (exon 1b) than within promoter 1 (exons 1a and 1 c), suggesting that the variant incorporating exon 1b was the most abu ndant. (C) 1998 Elsevier Science B.V. All rights reserved.