mRNA Instability in the Nucleus Due to a Novel Open Reading Frame Element Is a Major Determinant of the Narrow Tissue Specificity of Folate Receptor α

The folate receptor (FR) types α and β are glycosyl-phosphatidylinositol-anchored cell surface glycoproteins (11, 40). The expression of FR-α among normal tissues is restricted to the luminal surface of certain epithelial cells (37, 38, 39). FR-α is consistently and vastly overexpressed in major subtypes of ovarian and uterine carcinoma and testicular choriocarcinoma and less frequently in carcinomas of the breast, colon, and kidney (5, 27, 32, 39). Owing to its inaccessibility in normal tissues via the bloodstream and its high expression in malignant cells, FR-α offers a means of obtaining targeted delivery of diagnostic and therapeutic agents to the receptor-rich tumors. Accordingly, a wide variety of FR-α-mediated diagnostic and therapeutic approaches are currently in various stages of preclinical and clinical testing (8, 9a, 12, 15, 17, 21, 25, 30, 36). FR-β bears approximately 70% amino acid sequence identity to FR-α (23, 40) and is limited in its expression to cells in the myelomonocytic lineage (19, 28, 35). The entire FR-α gene (Fig. ​(Fig.1)1) consists of seven exons and six introns and two TATA-less promoters, namely, the P1 promoter, located upstream of the first exon, and the P4 promoter, located in the fourth exon (6). The P1 promoter gives rise to multiple, alternatively spliced transcripts, which appear to show some degree of selectivity among different tissues, whereas the P4 promoter generates a single mRNA species (6). P1 promoter transcripts, however, are translated much less efficiently than the P4 promoter transcript (24); moreover, the major FR-α mRNA in malignant cells appears to be P4 promoter driven (6). FIG. 1. FR-α gene organization. The 7.5-kb FR-α gene structure is shown with seven exons (labeled 1 to 7) and six introns (labeled A to F). The two promoters, P1 and P4, are shown in shaded boxes. The P1 promoter occurs upstream of exon 1, and ... Here, we report a systematic investigation of the molecular basis for the narrow tissue and tumor specificity of FR-α. For the detailed studies, we chose two model cell lines (HeLa cervical carcinoma and JAR choriocarcinoma) that are known to overexpress FR-α and three (HT3 cervical carcinoma, MG63 osteosarcoma, and Caki1 kidney carcinoma) that are FR negative, and we subsequently tested additional FR-α-positive and -negative cell lines. Our findings indicate that there is not a tight tissue-specific control of FR-α transcription, since a relatively low level of transcription occurs in FR-α-negative as well as FR-α-positive cells. The results point to differential transcript stability in the nucleus, directed by a novel instability determinant in the open reading frame (ORF) as the principal mechanism governing the tissue specificity of FR-α in the model cell lines. Constitutive gene transcription and protein synthesis throughout the cell cycle, combined with a slow turnover of the protein, appear to ensure a high level of FR-α in HeLa cells despite the low transcription rate. Cytosolic mRNA stability has emerged as a dominant principle in gene regulation (26), but although the metabolism of snRNAs and snoRNAs has been well studied, gene regulation at the level of mRNA stability in the nucleus has received relatively little attention. The singular role of nuclear mRNA stability in determining the tissue specificity of expression of the FR-α gene is discussed in this context.