RESUMO
Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.
Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Timidilato Sintase/metabolismo , Sequência de Bases , Sítios de Ligação , Neoplasias do Colo/metabolismo , Primers do DNA/química , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Ligação Proteica , Biossíntese de Proteínas , Ribonucleoproteínas/química , Células Tumorais CultivadasRESUMO
A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.
Assuntos
Genes p53 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Timidilato Sintase/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Regulação da Expressão Gênica , Humanos , Substâncias Macromoleculares , Polirribossomos/química , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , Ratos , Ribonucleoproteínas/química , Timidilato Sintase/química , TransfecçãoRESUMO
Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/genética , RNA/metabolismo , Timidilato Sintase/metabolismo , Sequência de Bases , Western Blotting , Neoplasias do Colo , Genes Reporter/genética , Humanos , Mutação/genética , Ensaios de Proteção de Nucleases , Conformação de Ácido Nucleico , Plasmídeos/genética , Testes de Precipitina , Ligação Proteica , RNA/química , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes de Fusão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Timidilato Sintase/genética , Timidilato Sintase/isolamento & purificação , Transfecção , Células Tumorais CultivadasAssuntos
Reação em Cadeia da Polimerase/métodos , Testes de Precipitina/métodos , Proteínas de Ligação a RNA/isolamento & purificação , Ribonucleoproteínas/isolamento & purificação , Extratos Celulares , Células Cultivadas , DNA Complementar , Biblioteca Gênica , RNA Mensageiro/análise , Transformação GenéticaRESUMO
We developed an immunoprecipitation-RNA-random PCR (rPCR) method to isolate cellular RNA sequences that bind to the folate-dependent enzyme thymidylate synthase (TS). Using this approach, nine different cellular RNAs that formed a ribonucleoprotein (RNP) complex with thymidylate synthase (TS) in human colon cancer cells were identified. RNA binding experiments revealed that seven of these RNAs bound TS with relatively high affinity (IC50 values ranging from 1.5 to 6 nM). One of the RNAs was shown to encode the interferon (IFN)-induced 15 kDa protein. Western immunoblot analyses demonstrated that the level of IFN-induced 15 kDa protein was significantly decreased in human colon cancer H630-R10 cells compared with parent H630 cells. While the level of IFN-induced 15 kDa mRNA expression was the same in parent and TS-overexpressing cell lines, the level of IFN-induced 15 kDa RNA bound to TS in the form of a RNP complex was markedly higher in H630-R10 cells relative to parent H630 cells. These studies begin to define a number of cellular target RNA sequences with which TS interacts and suggest that these TS protein-cellular RNA interactions may have a biological role.