Expression Patterns of the Multiple Transcripts from the Folylpolyglutamate Synthetase Gene in Human Leukemias and Normal Differentiated Tissues

Abstract

Folylpoly-␥-glutamate synthetase (FPGS) catalyzes the activation of folate antimetabolites in mammalian tissues and tumors. We have determined the sequence, abundance, and function of human FPGS transcripts and found some striking differences to transcription of the mouse gene that allow production of FPGS isoforms in mouse liver and dividing tissues. Multiple human transcripts were identified, including the homolog of the mouse transcripts that initiate at two upstream exons. However, the human FPGS upstream promoter is infrequently used, and transcripts from this promoter include sequences homologous with only one of the upstream exons found in the mouse. The downstream promoter generates an array of transcripts, some of which do not produce active enzyme, a phenomenon not seen in the mouse. Hence, the dual promoter mechanism directing expression of FPGS isozymes in mouse tissues is not conserved in humans, and, unlike the mouse downstream promoter, the human downstream promoter is active in both dividing and differentiated tissues. This study raises questions about the differences in function served by the two mouse FPGS isozymes and how, or if, human tissues fulfill these functions. How humans and mice produce FPGS in only a subset of tissues using such different promoter structures also becomes a central issue.

Primary Acute Lymphoblastic Leukemia Cells Use a Novel Promoter and 5′Noncoding Exon for the Human Reduced Folate Carrier That Encodes a Modified Carrier Translated from an Upstream Translational Start

“…5Ј RACE is not sufficiently reliable for quantitating frequencies of transcript forms, because it is based on PCR and may be affected by template secondary structures (15). Accordingly, we used real-time RT-PCR, with primers to the individual 5ЈUTRs identified in ALL specimens by 5ЈRACE and downstream primers to hRFC coding sequence to quantitate levels of the individual 5ЈUTRs in 26 ALL specimens (includes 14 of 15 B-precursor ALL and 6 T-ALL samples used for 5ЈRACE and 6 additional T-ALL samples).…”

“…To date, promoter activity has been localized to the 5Ј regions proximal to exons A, B, and C (9 -11). Whereas the biological role of each 5ЈUTR has yet to be established, alternate noncoding exons can influence transcript stabilities, intracellular targeting, and/or translational efficiencies, or even encode modified proteins translated from upstream AUGs (12)(13)(14)(15)(16)(17)(18). For hRFC, cell-and tissuespecific usage of each promoter and noncoding exon can be envisaged to function in regulating relative levels of hRFC transcripts and protein in tissues and tumors in response to folate cofactor requirements or other cell-or tissue-specific signals.…”

Structure and Regulation of the Murine Reduced Folate Carrier Gene

“…has an inherent statistical bias, since 5Ј-RACE results can be significantly impacted by template secondary structures (29). Thus, to better quantitate mRFC transcript levels and the individual 5Ј-UTRs identified by 5Ј-RACE, we used real time RT-PCR.…”

Identification and Characterization of Genetic Variation in the Folylpolyglutamate Synthase Gene

“…The ability of the FPGS allozymes to complement the FPGS mutation in AuxB1 cells was measured as previously described (20). DNA (10 Ag) from the pSF-GFP FPGS allozyme expression constructs was used to electroporate 1 Â 10 6 AuxB1 cells for 15 ms at 370 V. The cells were then plated at a density of 5,000 cells/plate in 60-mm plates in either a(+)MEM or a(À)MEM (with 10% dialyzed fetal bovine serum) with G418 (1.2 mg/mL; three replicates each).…”