Identification of an RNA binding site for human thymidylate synthase.

Abstract

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of "36 nt is protected from RNase Ti digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.Thymidylate synthase (TS; EC 2.1.1.45) catalyzes the conversion of deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate to thymidine monophosphate (dTMP) and dihydrofolate (1). Because this enzymatic reaction provides the sole de novo intracellular source of dTMP, TS is a critical therapeutic target enzyme in cancer chemotherapy (2, 3).In mammalian cells, the study of TS gene regulation has mainly focused on cell cycle-directed events. The increase in TS enzyme expression that accompanies entry into the S phase of the cell cycle appears to be regulated at both the transcriptional and the posttranscriptional level (4-9). More recently, Jolliff et al. (10) protein end product, TS, in an autoregulatory manner (13). Using an in vitro rabbit reticulocyte lysate system, we observed that recombinant human TS inhibited translation of TS mRNA. Preliminary studies using a gel mobility-shift assay confirmed binding of recombinant human TS to its corresponding mRNA.Here we provide evidence that TS protein binds specifically to TS RNA sequences in vitro. Two binding sites for TS have been identified, one located within the initial 188 nt of the TS RNA and the other between nt 434 and 634. Further studies reveal that a 35-nt RNA oligomer containing the TS translational initiation sequence retains the ability to specifically bind TS. MATERIALS AND METHODS Preparation of Plasmid Constructs and in Vitro mRNATranscripts. Full-length TS RNA transcript was synthesized with SP6 RNA polymerase (13). Full-length antisense TS RNA transcript was synthesized with T7 RNA polymerase after linearization of pcEHTS with Sst I. The pcEHTS-PST RNA (PST) transcript was synthesized after linearization of pcEHTS with Pst I. The 113-bp Pst I-EcoRI fragment from pcEHTS was cloned into the Pst I and EcoRI sites of pGEM-4Z (Promega), and pcEHTS-PST/EcoRI (PSTEcoRI) RNA was synthesized with SP6 RNA polymerase after linearization with HindIII. The 133-bp EcoRI-Ava I fragment from pcEHTS was cloned into the EcoRI...

Initiation of Protein Synthesis in Eukaryotic Cells

“…In the case of thymidylate synthetase mRNA, one of two elements recognizing the cellular enzyme includes the AUG initiation codon, the other being within the coding sequence [264]. As with the IRE/IRP interaction, binding may be regulated by a redox mechanism involving protein -SH groups [265].…”

Posttranscriptional Regulation of Mammalian Methionine Synthase by B12

“…Two other folatedependent enzymes, dihydrofolate reductase and thymidylate synthase (22)(23)(24), bind to their own mRNAs. This represses translation and represents a form of autoregulation.…”

Zebrafish p53 protein enhances the translation of its own mRNA in response to UV irradiation and CPT treatment

“…recombinant p53 protein was prepared as described in our previous report [17]. RNA-protein binding reaction mixtures (20 ll), containing 100 ng of biotin-labeled full-length p53, p53 5 0 UTR+ORF or p53 3 0 UTR mRNA and 1 lg of recombinant renatured p53 protein, were incubated at room temperature for 30 min and then cross-linked by UV irradiation for 15 min at 254 nm [18]. The mixtures were digested with RNaseA1 (1 mg/ml) at 37°C for 30 min and resolved on 12% SDS-PAGE.…”