Dihydrofolate Reductase Protein Inhibits Its Own Translation by Binding to Dihydrofolate Reductase mRNA Sequences within the Coding Region

Abstract

Previous studies suggest that dihydrofolate reductase (DHFR) regulates its own translation. Moreover, intracellular levels of DHFR protein increase following exposure to the antifolate methotrexate (MTX), suggesting that MTX may release the translational inhibition mediated by DHFR [Chu et al. (1993) Biochemistry 32,4756-4760; Ercikan et al. (1993) Adv. Exp. Med. Biol. 338, 537-540]. To further investigate the role of DHFR in translational autoregulation, we have considered the possibility that DHFR directly contacts its cognate mRNA. Binding studies using a series of truncated DHFR mRNAs as probes localized the DHFR/RNA interaction to a 100-base-pair region containing two putative stem-loop structures; initial studies indicated that both of these loop structures are involved in protein binding. Moreover, the binding of MTX to DHFR prevents interaction of the protein with its cognate mRNA, thereby relieving translational autoregulation.

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation

“…Notably, we demonstrate that this mechanism can be utilized for regulation of transgene expression when transgenes are located in a 3Ј cis arrangement with DHFR FS . By codon-optimizing DHFR FS to remove the mRNA binding sequence previously identified (15,16), we find that MTX continues to induce an increase in DHFR FS expression independent of the mRNA sequence and that the addition of TYMS SS unexpectedly blunts this increase. This finding suggests a novel method of post-transcriptional DHFR expression that is regulated by the enzymatic action of TYMS SS .…”

“…This construct is referred to as FLAG-DHFR FS -2A-eGFP pSBSO (D FS G). The DHFR FS was also codon-optimized to remove the original codon sequence, with specific attention paid to the coding region where DHFR is known to bind its own mRNA (15,16 . A selection-free RFP plasmid NLS-mCherry pSBSO (RFP) was utilized as a control.…”

“…Furthermore, MTX was shown to prevent this binding (14) and lead to an increase in DHFR protein expression. The nucleotide sequence to which DHFR protein binds DHFR mRNA within the coding domain is known (15,16), and the specific amino acids that DHFR protein utilizes to bind DHFR mRNA have been elucidated (17). However, subsequent studies found that a mutation to mRNA binding amino acids of DHFR still resulted in increased DHFR protein expression in the presence of MTX (18).…”

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

“…At first glance, it appears unusual that the coding region of an mRNA is the target of a translational regulatory mechanism; however, genome-wide analyses of interactions of regulatory RNA-binding proteins with mRNAs have revealed surprisingly large numbers of interactions that take place in the coding regions of the cognate mRNAs (Hogan et al, 2008;Hafner et al, 2010). Previous studies have also identified specific regulatory proteins that bind to the coding regions of certain mRNAs, such as the thymidylate synthase and dihydrofolate reductase, both of which bind to the coding regions of their own mRNAs (Ercikan-Abali et al, 1997;Lin et al, 2000;Zhang et al, 2010) and the RNA-binding protein HuR, which interacts with the coding region of CD83 RNA (Prechtel et al, 2006). Target sites for microRNAs are also not restricted to the 3 0 -UTRs but can occur in the coding region of certain mRNAs.…”

Resistance to antifolates

“…This has been attributed to a phenomenon in which the DHFR message binds to DHFR, thus regulating its availability for translation. In the presence of an antifolate, the message is released, translation is enhanced, and synthesis of protein is increased (Chu et al, 1993;Ercikan et al, 1993;Ercikan-Abali et al, 1997;Schmitz et al, 2001). Regulation of DHFR expression by this mechanism was absent in the malaria parasite and suggested as a factor in the utility of antifolates in the treatment of this disease (Zhang and Rathod, 2002).…”