The Former Annotated Human Pseudogene Dihydrofolate Reductase-Like 1 (DHFRL1) Is Expressed and Functional
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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 sup- ply 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 re- combinant DHFRL1 has sufficient DHFR enzyme activity to latively abundant control transcript glucuronidase beta (Fig.