Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Abstract

Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to generate thymidylate and dihydrofolate. This enzymatic reaction provides for the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis. As a result, TS remains a critical target enzyme in cancer chemotherapy (25, 60a).In addition to its role in enzyme catalysis, there is evidence that TS also functions as an RNA binding protein (5-7). Studies from this laboratory have demonstrated that the translation of human TS mRNA is regulated by its own protein product via a negative autoregulatory mechanism (5). The repression of TS mRNA translation by TS is mediated by specific binding of the protein to at least two distinct cis-acting sequences on its own mRNA (6). The first site corresponds to a 188-nucleotide (nt) sequence that includes the translational start site, while the second site is contained within a 100-nt sequence in the protein-coding region. However, in the presence of the nucleotide substrate dUMP or 5-fluoro-2Ј-deoxyuridine-5Ј-monophosphate (FdUMP), TS is unable to directly interact with its own mRNA, thus allowing for the synthesis of new protein to proceed (5, 7). Several in vitro studies have shown that shortterm exposure of human colon and breast cancer cells to TSinhibitory compounds, such as 5-fluorouracil (5-FU) or the antifolate analog ZD1694, is associated with an increased level of TS protein expression but no corresponding change in the levels of TS mRNA (8,9,35). These findings are consistent with earlier observations that both the RNA binding and the translational inhibition activities of TS are impaired in the presence of either nucleotide and/or folate substrates. Thus, the ability to regulate the expression of TS at the translational level in the setting of acute cytotoxic stress suggests that this regulatory event has biological relevance. Moreover, this process may represent an important mechanism by which normal cellular synthetic function can be controlled and a mechanism for the rapid development of cellular resistance in response to exposure to nucleotide inhibitors of TS, such as 5-FU, and antifolate inhibitors of TS, such as ZD1694, LY231514, and AG331.An immunoprecipitation-reverse transcription (RT)-PCR technique was recently developed to isolate from an intact cultured human colon cancer cell line a TS-ribonucleoprotein (RNP) complex made up of TS protein and TS mRNA (10). In addition to complexing with its own mRNA, TS formed an RNP complex with the mRNA of the c-myc transcription factor. Subsequent studies with RNA electrophoretic gel mobility shift assays (EMSAs) confirmed that the interaction between TS and c-myc mRNA was specific and identified the C-terminal coding region as being an important cis-acting regulatory element (11). Furthermore, in vitro translation experiments demonstrated that TS protein specifically repressed the translation of c-myc mRNA (11). Recent work has shown that TS is * Corresponding author. Mailing ad...

Nuclear thymidylate synthase expression, p53 expression and 5FU response in colorectal carcinoma

“…However, previous studies have demonstrated the presence of the enzyme in the nuclei of rat hepatoma cells using immunogold electron microscopy and autoradiography (Samsonoff et al, 1997), and along the nuclear periphery of yeasts using immunofluorescence analysis (Poon and Storms, 1994). The significance of this subcellular localisation is uncertain but, as previously mentioned, TS protein and p53 mRNA form a complex which is speculated to have a nucleolar location (Chu et al, 1999). TS protein is also known to bind to other mRNAs, e.g.…”

“…As previously mentioned, TS protein has been shown to bind to p53 mRNA, thereby reducing expression of p53 protein (Chu et al, 1999). Trying to predict, from these findings, the association between TS and p53 proteins in vivo is, however, complicated by observations that wild-type p53 protein can, in turn, inhibit TS promoter activity (Lee et al, 1997).…”

“…Despite the fact that nuclear staining has also been recorded (Johnston et al, 1991), we are unaware of any work into the clinicopathological significance of nuclear expression of TS in vivo. The presence of TS in the nucleus is of particular interest in light of the recent demonstration that TS protein and p53 mRNA form a biologically active complex and speculation that this complex may be localised within the nucleolus (Chu et al, 1999). There has also been interest in associations between TS and p53 on a more clinical level.…”

Clinical application of biological markers for treatments of resectable non-small-cell lung cancers

“…Its expression is associated with tumour cell proliferation (Navalgund et al, 1980), as demonstrated in the present study. Thymidylate synthase protein also downregulates p53 expression through TS protein -p53 mRNA interaction (Chu et al, 1999). In addition, TS can induce a transformed phenotype in mammalian cells (Rahman et al, 2004).…”

Binding of PurH to a Muscle-specific Splicing Enhancer Functionally Correlates with Exon Inclusion in Vivo

“…In the nucleus, PCD/DCoH is found in a 2:2 heterotetrameric complex with HNF1 (hepatocyte nuclear factor 1) where it is required to stabilize HNF1 binding to its target sequences for activated transcription (48 -50). Thymidylate synthetase forms a complex with p53 mRNA in vivo that is thought to regulate p53 translation (51). The best studied example of an enzyme having a role in RNA metabolism is the iron-binding protein 1.…”