The Former Annotated Human Pseudogene Dihydrofolate Reductase-Like 1 (DHFRL1) Is Expressed and Functional

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

Gráinne McEntee, Stefano Minguzzi, Kirsty O’Brien, Nadia Ben Larbi, Christine Loscher, Ciarán Ó’Fágáin, and Anne Parle-McDermott1

School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland

Edited by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved July 20, 2011 (received for review March 8, 2011)

Human dihydrofolate reductase (DHFR) was previously thought ability to reduce folic acid when compared to the rat version to be the only enzyme capable of the reduction of dihydrofolate of the enzyme (7). to tetrahydrofolate; an essential reaction necessary to ensure a Absence of DHFR activity leads to a rapid depletion of tetra- continuous supply of biologically active folate. DHFR has been hydrofolate and a consequent cessation in de novo DNA synthesis studied extensively from a number of perspectives because of and cell proliferation. This effect has led to the development of its role in health and disease. Although the presence of a number a range of antifolate drugs that target DHFR (and other folate of intronless DHFR pseudogenes has been known since the 1980s, it enzymes). Methotrexate is one such drug and has been in use in was assumed that none of these were expressed or functional. We chemotherapy for more than 50 years. Cells, however, can become show that humans do have a second dihydrofolate reductase drug resistant through mutation or amplification of the DHFR enzyme encoded by the former pseudogene DHFRP4, located on gene (4). Normally, however DHFR expression is tightly controlled chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate at the transcriptional, translational, and posttranslational level. reductase-like 1 (DHFRL1), gene is expressed and shares some com- Transcriptional control during the cell cycle is mediated by the monalities with DHFR. Recombinant DHFRL1 can complement a transcription factors Sp1 and E2F (8, 9) plus a noncoding RNA DHFR-negative phenotype in bacterial and mammalian cells but that is transcribed from the minor promoter (10). Regulation of BIOCHEMISTRY has a lower specific activity than DHFR. The Km for NADPH is similar DHFR at the translational level involves the binding of the DHFR for both enzymes but DHFRL1 has a higher Km for dihydrofolate protein to its own mRNA (11). The initial response of cells to when compared to DHFR. The need for a second reductase with methotrexate exposure is to upregulate DHFR protein level. This lowered affinity for its substrate may fulfill a specific cellular upregulation is thought to be mediated at the translational level requirement. The localization of DHFRL1 to the mitochondria, as (12–14); likely due to a conformational change of the DHFR demonstrated by confocal microscopy, indicates that mitochondrial mRNA complex (11, 15). At the posttranslational level recent dihydrofolate reductase activity may be optimal with a lowered evidence suggests that DHFR is subject to both monoubiquitina- affinity for dihydrofolate. We also found that DHFRL1 is capable of tion and sumoylation (16, 17). These posttranslational modifica- the same translational autoregulation as DHFR by binding to its tions are thought to be important for its localization at specific own mRNA; with each enzyme also capable of replacing the other. phases of the cell cycle. DHFR has also been reported to be regu- The identification of DHFRL1 will have implications for previous lated posttranslationally by p14ARF by an unknown indirect research involving DHFR. mechanism that affects protein stability (18). It is clear that DHFR has been extensively investigated, but ecent knowledge of the dihydrofolate reductase (DHFR)gene all the work to date has assumed that humans have just one Rfamily suggests one functional gene among several intronless expressed and functional DHFR. DHFR on chromosome 5 was pseudogenes (1, 2). The functional DHFR gene resides on chromo- thought to be the only human enzyme capable of carrying out some 5 (1) and encodes an enzyme that catalyzes the reduction of the reduction of dihydrofolate to tetrahydrofolate as the four re- dihydrofolate to the biologically active form, tetrahydrofolate. The ported pseudogenes were regarded as nonfunctional (Table S1). DHFR gene/enzyme has been studied extensively in relation to The intronless nature of the four DHFR pseudogenes indicates that health and disease given its crucial role in folate metabolism (3), they arose through reintegration of an mRNA intermediate (2). use as an antifolate drug target (4), and as a commonly used re- Although there are other dihydrofolate reductase-like sequences porter gene for molecular studies. in other species, this particular reintegration event may have been Folate mediated one-carbon metabolism is a cellular pathway a primate-specific event as similar intronless pseudogenes of where the essential B vitamin folate acts as a cofactor for a variety DHFR are not evident in nonprimate species (www.ensembl.org, of anabolic and catabolic reactions (5). This pathway is essential blast.ncbi.nlm.nih.gov/Blast.cgi). The DHFRP1 pseudogene located for the supply of cofactors for purine/pyrimidine synthesis, on chromosome 18 is polymorphic in the human population which cellular methylation reactions, and the supply of formylated is indicative of its recent evolutionary origins (19). The open read- methionine for protein synthesis in the mitochondria. The DHFR ing frame (ORF) of DHFRP1 is identical to the functional DHFR enzyme forms part of folate metabolism, ensuring there is a supply of the biologically active form of folate, i.e., tetrahydrofolate. Author contributions: A.P.-M. designed research; G.M., S.M., K.O., and N.B.L. performed Up to now, DHFR was thought to be the only enzyme that could research; C.L. supervised confocal microscopy; C.O. supervised enzyme kinetics analysis; not only recycle folate metabolites back to tetrahydrofolate, but G.M., S.M., K.O., N.B.L., and A.P.-M. analyzed data; and G.M., S.M., and A.P.-M. wrote also reduce the synthetic form of folate, folic acid. This enzyme the paper. activity is significant given the widespread worldwide mandatory The authors declare no conflict of interest. and voluntary folic acid fortification of foods that has occurred This article is a PNAS Direct Submission. in recent years as a preventative measure against the occurrence Freely available online through the PNAS open access option. of neural tube defects (6). Despite the importance of DHFR 1To whom correspondence should be addressed. E-mail: [email protected]. activity, recent work has demonstrated that human liver DHFR This article contains supporting information online at www.pnas.org/lookup/suppl/ activity was quite variable between individuals and had limited doi:10.1073/pnas.1103605108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103605108 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 24, 2021 but despite this similarity, it does not appear to have a functional Complementation of a DHFR-Negative Phenotype in a Bacterial Sys- promoter and there is no evidence to suggest it is expressed. In this tem. Escherichia coli D3-157 is a streptomycin-resistant bacterial study, we provide evidence that in fact the pseudogene, formerly strain that has been mutated so that it no longer has DHFR known as DHFRP4 (DHFRL1) on chromosome 3 is not only ex- activity and requires the addition of thymidine to the medium for pressed, but that the translated protein (i) harbors enzyme activity, growth (23). To determine if our recombinant DHFRL1 has any (ii) can complement a DHFR null phenotype, (iii) is likely to auto enzyme activity, we transformed our DHFRL1 recombinant regulate itself and DHFR, and (iv) localizes to the mitochondria. clone (in pCR2.1) into E. coli D3-157 strain and grew cultures in media both in the presence/absence of thymidine and/or isopro- Results pyl β-D-1-thiogalactopyranoside (IPTG) and ampicillin. The Confirmation of Expression of DHFRL1 by Quantitative Reverse Tran- presence of ampicillin selects for those bacteria that have been scribed PCR (RT-qPCR) and Sequencing. A large scale cDNA sequen- transformed with pCR2.1 vector whereas IPTG induces expres- cing project (20) was the first indication that the DHFRL1 (or sion of the recombinant protein. As a positive control the strain DHFRP4) pseudogene was actually expressed. Annotation of the was also transformed with a DHFR recombinant clone (also in DHFRL1 mRNA entry (NM_176815) suggests that there are two pCR2.1). Our other controls included untransformed cultures transcripts produced by the DHFRL1 gene that differ in their 5′ and cultures transformed with empty pCR2.1 vector. The results untranslated (UTR) regions. Both transcripts would produce the are shown in Fig. 1. As expected, both the original D3-157 strain same protein sequence. We designed a successful RT-qPCR assay and bacteria transformed with the empty pCR2.1 vector survived to specifically amplify transcript variant 2 without a possibility of only in media where thymidine was present. They quickly died erroneous amplification of genomic DNA or other DHFR homo- in the media without thymidine. Our positive control, D3-157 logous sequences. The RT-qPCR assay was optimized and a num- transformed with DHFR, also behaved as predicted and grew ber of human cell lines were screened for expression including in media containing IPTG (plus ampicillin) but without thymi- SW480, SKBR3, L428, DG75, BT474, National Cancer Institute dine. D3-157 cells transformed with DHFRL1 behaved similar H1299, and Coriell lymphoblast cell lines. The DHFRL1 tran- to the positive control and grew in media both with and without script was expressed in all the cell lines tested at either a similar thymidine. The conclusion drawn from this experiment is that re- or lower level (relative ratio ranged from 1.0 to 0.11) to the recombinant DHFRL1 has sufficient DHFR enzyme activity to latively abundant control transcript glucuronidase beta (Fig. S1). complement a DHFR-negative phenotype in a bacterial system. The highest level of expression was observed in cell line BT474. Direct sequencing of the purified PCR product confirmed that Complementation of a DHFR-Negative Phenotype in a Mammalian DHFRL1 System. CHO DG44 cells are Chinese hamster ovary cell mutants the assay was specific and not amplifying the functional E. coli DHFR gene (Fig. S2). This data confirmed that the DHFRL1 lacking in dihydrofolate reductase (24). Similar to the gene was being transcribed. An examination of the DHFRL1 entry D3-157 bacterial cell line, these cells required the media to be supplemented with thymidine and hypoxanthine to grow. Having in the Unigene database (http://www.ncbi.nlm.nih.gov/UniGene/ shown complementation of the phenotype in a bacterial system ESTProfileViewer.cgi?uglist=Hs.718516) indicates that DHFRL1 we wanted to replicate those results in a mammalian system. The is expressed in a variety of normal human tissues and developmen- CHO DG44 cells were transfected with a mammalian expression tal states. vector with either a DHFR or a DHFRL1 insert. The cells transfected with DHFR were used as a positive control, normal un- Sequence Analysis of DHFRL1. Comparison of the amino acid transfected CHO DG44 cells were a negative control, and cells sequences of DHFR with DHFRL1 shows that they are 92% transfected with an empty vector acted as a quality control for the identical (Fig. S3). The four motifs required for dihydrofolate re- experiment. After transfection, cells were left in complete growth ductase activity are conserved except for three amino acid resi- medium for 48 h. Transfected cells were then positively selected dues. The most significant of these is a conserved tryptophan at by exploiting the neomycin resistance gene on the expression vec- position 24 (W24); DHFRL1 has an arginine (R) at this position. tor by adding 500 μg∕mL G418. At this stage the untransfected Previous site-directed mutagenesis experiments of the human CHO DG44 cells quickly died off. The cells were left in selective enzyme showed that replacement of this tryptophan with phenyl- media for a further 14 d at which point they were switched to alanine (F) resulted in a 50% decrease in stability and a drop in media containing G418 but without thymidine or hypoxanthine efficiency of 48% under intracellular conditions (21). This data supplements. The cells remained in the complementation media suggested that the W24 to arginine (R) change in DHFRL1 for 12 d. Cells were counted using trypan blue on days 1, 5, and would result in an enzyme capable of dihydrofolate reductase 12. Results are shown in Fig. 2. On day 1 both DHFR and activity but with altered catalytic characteristics versus wild type. DHFRL1 transfected cells had approximately 2 × 106 cells∕mL, The DHFRL1 sequence is preserved from primates to humans with cells transfected with empty vector having slightly less at (www.ensembl.org) with the same R24 change preserved in the 1.4 × 106 cells∕mL. Cells transfected with empty vector did not chimpanzee. This conservation may indicate that this amino acid survive and were completely dead by day 5. Cells transfected with change is significant for a functional role of DHFRL1 that may either DHFR or DHFRL1 did show significant cell death by day be distinct from DHFR, i.e., a type of subfunctionalization. The 5, however, they had recovered sufficiently by day 12. The cell amino acid sequences necessary for DHFR mRNA binding (15) death in transfected cells is likely to be related to how the plasmid and sumoylation (17) are all conserved in DHFRL1 indicating was incorporated into the genome. It is possible that for these that this new dihydrofolate reductase is subject to similar transla- cells the neomycin resistance gene could be active whereas the tional regulation and posttranslational modifications. Additional DHFR or DHFRL1 gene was silenced. If this type of gene activa- sequence analysis of DHFRL1 indicates that the translational tion was the case those cells would have survived in the selection signals required are present within the 5′ UTR of DHFRL1. media which had the required supplements but were unable to A comparison of the DHFR and DHFRL1 5′ UTR sequences sur- survive once the supplements were removed. At day 12, cells rounding the initiating ATG encoding methionine shows that transfected with DHFR had recovered their numbers to what DHFRL1 differs at just one base: ctgtcAUGt (DHFRL1) versus they were on day 1. Cells transfected with DHFRL1 were a ctgtcAUGg (DHFR). A reassessment of the translation initiation lot slower to recover. This data may indicate that although codons in vertebrates (22) found that approximately 50% of DHFRL1 does have enzyme activity it is not as active as DHFR. cytoplasmic translated transcripts do not contain a “g” at the +4 This theory was tested by harvesting protein from transfected position. cells at day 12 in complementation media and carrying out an

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103605108 McEntee et al. Downloaded by guest on September 24, 2021 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 ¼ 0.2 mM. BIOCHEMISTRY 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.

enzyme assay to test for activity (Fig. S4). As expected both Characterization of Recombinant DHFRL1 Protein. DHFR enzyme DHFR and DHFRL1 transfected cells showed enzyme activity, as acts by reducing dihydrofolate into tetrahydrofolate in the pre- measured by a decrease in absorbance at 340 nm over a 10 min sence of NADPH. To determine if DHFRL1 has similar enzyme time period. DHFRL1 activity begins to level off after 5 min activity we produced a purified recombinant DHFRL1 protein whereas DHFR still shows strong enzyme activity after 10 min. with a GST tag that we subsequently cleaved off. We then tested Measurement of specific activity showed that cells transfected this recombinant protein for enzyme activity using a standard, with DHFR (0.5832 μmol∕ min ∕mg) had approximately five and compared the results to a recombinant DHFR protein pro- times higher activity than that of DHFRL1 (0.154697 μmol∕ duced in the same manner. Initial results indicated that DHFRL1 min ∕mg). These results correlate with the specific activity mea- protein did have enzyme activity. However the specific activity of sured using recombinant purified protein (see below). DHFRL1 was roughly two-thirds (70%) that of DHFR (Table 1).

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.

McEntee et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 24, 2021 Table 1. Kinetic analysis of recombinant DHFRL1 versus DHFR Enzyme Dihydrofolic acid* NADPH† K ‡ μ V μ ∕ ∕ K § μ V μ ∕ ∕ μ ∕ ∕ m M max mol min mL m M max mol min mL Specific activity mol min mg DHFR 2.5–300 μM‡ 20.1–2.1¶ 0.0132–0.0004 3.6–0.4 0.021–0.0004 6.1–0.3 DHFRL1 2.5–700 μM‡ 209.3–18.5 0.0226–0.0004 3.4–0.7 0.038–0.001 4.3–0.3 *At constant 50 μM NADPH †At constant 60 μM dihydrofolic acid ‡Dihydrofolic acid concentration ranges ¶Standard error §NADPH concentration ranged from 2.5–150 μM

Having shown that recombinant DHFRL1 did have enzyme Staining of the nuclei demonstrated that DHFRL1 does not activity we then went on to characterize the enzyme by calculating localize to the nucleus in these unsynchronized cells. However, Km values for both the cofactor NADPH and the substrate dihy- intense fluorescence was detected in the mitochondria (Fig. 4) drofolic acid (Table 1). The Km values of both enzymes for the demonstrating that DHFRL1 localizes to this organelle. cofactor NADPH were similar. For the substrate dihydrofolate, however, DHFRL1 displayed a Km value of 209.3 μMversusthe Discussion DHFR value of 20.1 μM (Table 1). The altered values of DHFRL1 We have demonstrated that the former human pseudogene DHFRP4 DHFRL1 compared to DHFR may be driven by the W24R change in or is not only expressed but is functional. DHFRL1, but this suggestion requires further investigation. Previously, DHFR was thought to be the only enzyme capable of dihydrofolate reductase activity in humans. The data that we have DHFRL1 has the Ability to Bind its own mRNA and that of DHFR. presented refutes this long held assumption and the sequence Previous studies have established that the DHFR protein can similarity between DHFR and DHFRL1 indicates that much of act as an RNA binding protein and bind to its own mRNA to the previous work on DHFR may well have been unable to dis- suppress translation (11). The amino acids essential for this pro- tinguish between the two forms. DHFRL1 is capable of comple- cess have also been identified in previous work (15, 25). Our menting DHFR knockout phenotypes in both bacterial and sequence analysis of DHFRL1 shows that those essential amino mammalian cells (Fig. 1, Fig. 2, and Fig. S4). However, our data acids are also present in the DHFRL1 protein (Fig. S3). More- also indicate that although both enzymes share commonalities over, the 27-nt mRNA sequence of DHFR, which was shown to they have distinct differences in their affinity for dihydrofolate. be strictly necessary for the binding with DHFR protein (25), Gene duplication is a major contribution to diversity of function differs in only one nucleotide from the DHFRL1 sequence. For (27). The neofunctionalization model allows for the gain of a new these reasons, we hypothesized that DHFRL1 protein may bind function in one of the duplicates, which is thought to occur to its own mRNA and also may have the ability to bind DHFR through an initial relaxation of selective constraints (28). There- mRNA. Initially we tested the ability of DHFRL1 protein to bind fore, new genes can arise through duplications that will drive to DHFR mRNA by electrophoretic mobility shift assay. We used the evolution and adaptation of a species. The duplication event DHFR binding to DHFR mRNA as a positive control. Results that gave rise to DHFRL1 may have happened after primates shown in Fig. 3A clearly show a mobility shift for both the positive control and for DHFRL1 protein. We expanded the experiment to include DHFRL1 mRNA and tested not only the ability of DHFRL1 protein to bind to its own mRNA but also explored the possibility that DHFR protein could bind to DHFRL1 mRNA (Fig. 3B). As expected, DHFRL1 protein did bind to DHFRL1 mRNA and a clear mobility shift can be seen for this sample. A clear shift can also be seen for the sample containing DHFRL1 mRNA and DHFR protein, indicating that DHFR protein can also bind to DHFRL1 mRNA as well as its own mRNA. We re- peated the EMSA experiment but included purified GST protein as an additional negative control to ensure that the binding of DHFR and DHFRL1 was not being mediated by the GST tag (Fig. S5). No mobility shift was detected for DHFR nor DHFRL1 mRNA when only GST protein was added. From these experiments we conclude that recombinant DHFRL1 protein acts as an RNA binding protein not only for its own mRNA but also for DHFR mRNA. We have also shown that, in turn, DHFR protein can also bind to DHFRL1 mRNA in addition to its own mRNA. Fig. 3. EMSA shows that DHFRL1 and DHFR can bind to their own and each These results indicate that DHFRL1 can not only regulate its own other’s mRNA. All binding reactions were resolved on a 4% nondenaturing translation but may also play a role in the regulation of DHFR polyacrylamide gel, then transferred to a nylon membrane for detection protein translation. using the LightShift® Chemiluminescent EMSA Kit (Thermo Scientific). Band shifts are indicated by the arrows. (A) EMSA involving DHFR mRNA. A clear Subcellular Localization of DHFRL1. Folate enzymes are known to band shift is only observed in the presence of recombinant DHFR or DHFRL1 reside in the cytoplasm, nucleus, and mitochondria (26). DHFR (lanes 2 and 4). Lane order, 1: DHFR mRNA only; 2: DHFR mRNA + DHFR; 3: has previously been reported to localize primarily in the cyto- DHFR mRNA + DHFR + unlabeled DHFR mRNA; 4: DHFR mRNA + DHFRL1; 5: plasm but a small percentage has been shown to go to the nucleus DHFR mRNA + DHFRL1 + unlabeled DHFRL1 mRNA. (B) EMSA involving DHFRL1 mRNA. A clear band shift is only observed in the presence of recom- at the synthesis phase of the cell cycle (17). We examined the sub- binant DHFR or DHFRL1 (lanes 7 and 9). Lane order, 6: DHFRL1 mRNA only; 7: cellular localization of DHFRL1 in unsynchronized HEK293 DHFRL1 mRNA + DHFR; 8: DHFRL1 mRNA + DHFR + unlabeled DHFR mRNA; 9: cells that were transiently transfected with GFP-DHFRL1 and DHFRL1 mRNA + DHFRL1; 10: DHFRL1 mRNA + DHFRL1 + unlabeled DHFRL1 examined using immunofluoresence and confocal microscopy. mRNA.

4of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1103605108 McEntee et al. Downloaded by guest on September 24, 2021 chondria or nucleus. We found no evidence that DHFRL1 localizes to the nucleus in unsynchronized cells, however, analysis of cells from the synthesis phase of the cell cycle would be required to definitely rule this localization out. We found clear evidence that DHFRL1 localizes to the mitochondria (Fig. 4). Although DHFRL1 does possess a potential sumoylation site, its localization to the mitochondria indicates that its primary role is to support mitochondrial DNA synthesis and replication. The relevance of DHFRL1 mRNA binding (discussed above) is not immediately apparent, given its mitochondrial localization. It is unlikely that the entire complement of DHFRL1-translated protein is present in the mitochondria at any one time and that a small percentage remains in the cytoplasm. This putative cytoplasmic percentage provides the opportunity for DHFRL1 to target DHFRL1 and DHFR mRNA. Moreover, the localization of DHFRL1 may also change at different phases of the cell cycle, in response to the extracellular environment or in a pathological si- tuation. The fact that DHFR can also bind DHFR and DHFRL1 mRNA has obvious biological relevance given that DHFR prin- cipally resides in the cytoplasm. The mechanism of mitochondrial localization is unclear given the lack of any obvious sequence that would indicate that DHFRL1 is targeted to the mitochondria. Mitochondria targeting sequences (MTS) do not appear to share a consensus primary sequence rather they share similar overall Fig. 4. Localization of GFP-DHFRL1 in mitochondria by immunofluorescence characteristics. Most MTS lack acidic residues and are rich in microscopy. HEK293 cells were transiently transfected with GFP-DHFRL1 and the positively charged amino acids arginine and lysine and the visualized by confocal microscopy. The top left image shows GFP-DHFRL1 hydroxylated amino acids serine and threonine (34). A secondary (green). The bottom left image shows mitochondria stained with MitoTracker feature common among MTS is the formation of an amphiphilic BIOCHEMISTRY CMTMRos (red). The top right image is the merged image; arrows show lo- α helices found on the surface of the protein (35). Hurt and calization of GFP-DHFRL1 in the mitochondria. The bottom right image is the – differential interference contrast (DIC) of the cells. Schatz found that amino acids 1 85 on mouse DHFR had the potential to be an MTS; however, it was inactive within the folded diverged from their most recent common ancestor. An mRNA protein (36). These amino acids are highly conserved within copy of DHFR reintegrated into the genome possibly before pri- DHFRL1 and, therefore, the localization of DHFRL1 in the mates diverged from humans, giving rise to DHFRL1 plus three mitochondria may be related to the folding of the enzyme follow- additional intronless pseudogenes (TableS1). A number of amino ing translation, possibly revealing the presence or absence of ami- acid substitutions accumulated in DHFRL1, the most significant no acids that facilitate its import (37). However, this suggestion that we noted appears to be replacement of the conserved tryp- requires further investigation. The identification and localization tophan in the catalytic site (Fig. S3). Under our conditions of of DHFRL1 emphasizes the importance of the mitochondria in measurement, the impact of the replacement of tryptophan with folate metabolism and will inform current research in this area. arginine (W24R) appears to result in a drop in the specific activity The conservation of DHFRL1 from primates to humans indi- of DHFRL1 by nearly 30% compared to DHFR (Table 1). The cates that this second human dihydrofolate reductase enzyme has Km for NADPH was not significantly different. DHFRL1, how- a specific role to play. The identification of DHFRL1 now means ever, consistently showed a notably higher Km value than DHFR that DHFR regulation, function, and antifolate drug responses for dihydrofolate. This difference possibly underlies the appar- will have to be reassessed in the context of its paralogue. A dif- ently lower specific activity of DHFRL1, but further investigation ferential response to antifolate drugs such as methotrexate may of this point would require a complete kinetic analysis. lead the way for more improved therapeutic treatments. Why do Apart from enzyme activity, the amino acid conservation we need a second DHFR enzyme? The answer to this question is between DHFR and DHFRL1 also indicated that DHFRL1 may likely to relate to its subcellular localization and possibly its tissue also be capable of binding its own mRNA in a similar fashion to specificity. A DHFR with reduced affinity for its substrate may be DHFR; resulting in suppression of translation (11). Our EMSA a specific requirement for one-carbon flux through the mitochon- analysis demonstrated that this proposal is in fact the case and dria. Its localization to the mitochondria adds to the recent list of that each enzyme can substitute for each other, i.e., DHFRL1 folate enzymes that have also been identified in this organelle and can bind DHFR mRNA and DHFR can bind DHFRL1 mRNA highlights the importance of mitochondrial folate metabolism. It (Fig. 3). This binding is significant, particularly if the enzymes has been assumed that the DHFR pseudogenes are nonfunc- have different affinities for antifolate drugs such as methotrexate. tional and thus irrelevant. Our data show that this assumption is The initial response of cells to methotrexate is to upregulate incorrect and that the human DHFR enzyme is not alone. protein levels through disruption of the DHFR∶mRNA complex (29). Our finding that DHFRL1 can also prevent DHFR mRNA Materials and Methods translation (and vice versa) indicates that this autoregulation Quantitative Reverse Transcribed PCR. Assay details and confirmation of mechanism and response to methotrexate needs to be reconsid- DHFRL1 mRNA expression are detailed in SI Materials and Methods. ered. We also examined the cellular localization of DHFRL1. The compartmentalization of folate metabolism has been pre- Sequence Analysis. Alignment of the amino acid sequences for DHFR (P00374.2) and DHFRL1 (AAH63379.1) were carried out using CLUSTAL viously established (26), with several mitochondrial-specific – 2.0.08. Relevant catalytic motifs were identified using PRINTS (www.bioinf. enzymes identified in recent years (30 33). Moreover, a small manchester.ac.uk/dbbrowser/PRINTS/). Other relevant amino acid residues fraction of DHFR has been reported to localize to the nucleus were identified from the literature (15, 17, 21, 38–40). The Ensembl genome during the synthesis phase of the cell cycle (17). In this context, browser (www.ensembl.org) and BLAST (blast.ncbi.nlm.gov/Blast.cgi) were we considered whether DHFRL1 can also localize to the mito- used to examine DHFRL1 sequences in other species.

McEntee et al. PNAS Early Edition ∣ 5of6 Downloaded by guest on September 24, 2021 Expression and Purification of Recombinant DHFR and DHFRL1. The expression EMSA. EMSA analysis was carried using a Pierce ®RNA 3′ End Biotinylation vectors were constructed similar to the approach described by Wang et al. Kit (Thermo Scientific cat. no. 20160) plus purified recombinant DHFRL1 or (41) and are described in SI Materials and Methods. The induced GST-DHFRL1 DHFR. Details are provided in SI Materials and Methods. or -DHFR fusion proteins were purified using glutathione agarose [Invitrogen catalog (cat.) no. G2879] and further purified as described in SI Materials and Localization of DHFRL1 Protein. The Invitrogen DHFRL1 ORF clone (cat. no. Methods. IOH26763) was inserted into pcDNA6.2/N-EmGFP-DEST (Invitrogen, cat. no. V356-20) expression vector using the Gateway Cloning System. Plasmid DNA Enzyme Activity Assay and Determination of Km Values. Enzyme activity and was isolated using a Qiagen Mini Prep Kit (cat. no. 12123). HEK293 cells, K 5 m values were tested using a Dihydrofolate Assay Kit (Sigma cat. no. grown on cover slips (1 × 10 cells∕mL), were transfected with 5 μg of plasmid CS03040-1KT) as described in SI Materials and Methods. DNA using Lipofectamine 2000 reagent (Invitrogen, cat. no. 11668500). Six hours after transfection, the transfection medium was removed and Complementation of DHFR-Negative Phenotype in a Bacterial System. Recom- replaced with complete growth medium. The cells were retransfected again binant clones for DHFRL1 and DHFR were constructed as detailed in SI 24 h after the initial transfection using the same conditions. A further 48 h Materials and Methods. Recombinant plasmid DNA was transformed into later, the cells were incubated for 20 min at 37 °C with MitoTraker CMTMROS a DHFR-negative E. coli cell line D3-157 (American Type Culture Collection (Molecular Probes Invitrogen, cat. no. M7512) at 200 nM. The cells were then cat. no. 47050). Cultures were then grown for 72 h in media containing strep- 3 × 5 tomycin (100 μg∕mL) and various combinations of Ampicillin (100 μg∕mL), fixed in paraformaldehyde on ice for 30 min. Following rinsing min in IPTG (0.2 mM), and Thymidine (50 μg∕mL). Samples were taken at 0, 2, 4, PBS baths, the cover slips were mounted on slides with antifade medium 6, 22, 30, 48, and 72 h and growth was measured in a spectrophotometer (Dako). Slide preparations were observed using a Zeiss Axio Observer. at 600 nm. Z1 equipped with a Zeiss 710 and ConfoCor3 laser scanning confocal head (Carl Zeiss, Inc.). Images were analyzed using Zen 2008 software. Complementation of DHFR-Negative Phenotype in a Mammalian System. Mammalian expression vectors of DHFRL1 and DHFR were constructed ACKNOWLEDGMENTS. This work was funded by the Health Research Board of and transfected as detailed in SI Materials and Methods. Ireland, HRB/2009/54.

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