The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional

Abstract

Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K(m) for NADPH is similar for both enzymes but DHFRL1 has a higher K(m) for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.

Fig. 1.

Complementation of DHFR-negative phenotype in a bacterial system. E. coli D3-157 streptomycin-resistant cells were transformed with either DHFR or DHFRL1 and grown in media with and without supplements/antibiotics. Strep, streptomycin 100 μg/mL; Amp, ampicillin 100 μg/mL; IPTG = 0.2 mM. Growth was measured at various time points until stationary phase was reached. (A) The original strain grew only in media containing thymidine and without ampicillin. (B) Cells transformed with empty vector only grew in the presence of thymidine with or without ampicillin and/or IPTG. (C) Cells transformed with DHFR grew as expected in media both with and without supplements, i.e., could grow in the absence of thymidine once DHFR expression was induced. (D) Cells transformed with DHFRL1 also complemented the phenotype similarly to recombinant DHFR.

Fig. 2.

Complementation of DHFR-negative phenotype in a mammalian system. Cell counts of transfected CHO DG44 cells after switching cells to media without supplements. Cells were counted after 1, 5, and 12 d in complementation media. Cells transfected with either DHFR (A) or DHFRL1 (B) had some cell death on day 5; however, by day 12 both sets of cells had recovered and were growing well in the complementation media. The cells transfected with DHFR grew more quickly than those transfected with DHFRL1. (C) Cells transfected with the empty vector alone did not survive without supplements.

Fig. 3.

EMSA shows that DHFRL1 and DHFR can bind to their own and each other’s mRNA. All binding reactions were resolved on a 4% nondenaturing polyacrylamide gel, then transferred to a nylon membrane for detection using the LightShift® Chemiluminescent EMSA Kit (Thermo Scientific). Band shifts are indicated by the arrows. (A) EMSA involving DHFR mRNA. A clear band shift is only observed in the presence of recombinant DHFR or DHFRL1 (lanes 2 and 4). Lane order, 1: DHFR mRNA only; 2: DHFR mRNA + DHFR; 3: DHFR mRNA + DHFR + unlabeled DHFR mRNA; 4: DHFR mRNA + DHFRL1; 5: DHFR mRNA + DHFRL1 + unlabeled DHFRL1 mRNA. (B) EMSA involving DHFRL1 mRNA. A clear band shift is only observed in the presence of recombinant DHFR or DHFRL1 (lanes 7 and 9). Lane order, 6: DHFRL1 mRNA only; 7: DHFRL1 mRNA + DHFR; 8: DHFRL1 mRNA + DHFR + unlabeled DHFR mRNA; 9: DHFRL1 mRNA + DHFRL1; 10: DHFRL1 mRNA + DHFRL1 + unlabeled DHFRL1 mRNA.

Fig. 4.

Localization of GFP-DHFRL1 in mitochondria by immunofluorescence microscopy. HEK293 cells were transiently transfected with GFP-DHFRL1 and visualized by confocal microscopy. The top left image shows GFP-DHFRL1 (green). The bottom left image shows mitochondria stained with MitoTracker CMTMRos (red). The top right image is the merged image; arrows show localization of GFP-DHFRL1 in the mitochondria. The bottom right image is the differential interference contrast (DIC) of the cells.