Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA - PubMed (original) (raw)

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA

X Lin et al. Nucleic Acids Res. 2000.

Abstract

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5'-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434-634. In this paper, a 70 nt RNA sequence, corresponding to nt 480-550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434-634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5'-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434-634 or p644/TS480-550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT-PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480-550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434-634 and p644/TS480-550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(A) TS cDNA sequence. (B) Structure of heterologous luciferase plasmids. Details of construction of the luciferase heterologous plasmids are presented in Materials and Methods. The solid lines represent sequences of the pGL-2 basic plasmid. The solid box indicates the EGR-1 promoter and the hatched box represents the protein coding region of the luciferase gene. The open box represents the TS RNA sequence cloned into the _Hin_dIII restriction site of the p644 recombinant plasmid.

Figure 1

Figure 1

(A) TS cDNA sequence. (B) Structure of heterologous luciferase plasmids. Details of construction of the luciferase heterologous plasmids are presented in Materials and Methods. The solid lines represent sequences of the pGL-2 basic plasmid. The solid box indicates the EGR-1 promoter and the hatched box represents the protein coding region of the luciferase gene. The open box represents the TS RNA sequence cloned into the _Hin_dIII restriction site of the p644 recombinant plasmid.

Figure 2

Figure 2

Effects of mutation in the TS480–550 RNA sequence on translational inhibition of human TS mRNA by TS. (A) Translation reactions containing rabbit lysate were incubated with TS1–1524 mRNA (0.4 pmol) (lanes 1 and 2), TS94–1053 (0.4 pmol) (lanes 3 and 4) or TS94–1053m (0.4 pmol) (lanes 5 and 6). Human His-tagged TS protein (11 pmol) was included in the reaction mixtures where indicated (lanes 2, 4 and 6). As an internal control for translation, luciferase mRNA (0.4 pmol) was included in each reaction (lanes 1–6). Translation reactions were incubated at 30°C for 60 min and protein products were analyzed by SDS–PAGE and autoradiography as described in Materials and Methods. TS and luciferase protein products are indicated by the arrows. (B) Translational inhibition of TS mRNA sequences by TS. TS1–1524 (0.4 pmol) (triangle) and TS1–1524(m) RNA sequences (0.4 pmol) (cirlce) were incubated with human His-tagged TS protein (0–40 pmol) in the rabbit reticulocyte translation system as described in Materials and Methods. Proteins were resolved by SDS–PAGE and then subjected to autoradiography. The level of TS mRNA translation in the absence of TS protein represents 100%. Points represent the mean ± SE from three experiments.

Figure 3

Figure 3

Effect of TS RNA sequences on luciferase expression in vivo. Luciferase heterologous constructs were transiently expressed in H630 cells. Luciferase activity was measured 48 h post-transfection using the dual luciferase reporter assay system as described in Materials and Methods. The activity in cells transfected with the parent p644 plasmid was defined as 100%. Luciferase expression in cells transfected with p644/TS434–634, p644/TS480–580, p644/TS480–550 and p644/TS480–550(3′) was significantly less than in cells transfected with p644 (paired Student’s _t_-test, *P < 0.01). Each value represents the mean ± SE of three to five experiments.

Figure 4

Figure 4

Effect of TS RNA sequences on luciferase mRNA expression. (A) RNase protection assay; (B) RT–PCR analysis. Human colon cancer H630 cells were transiently transfected in the absence (lane 1) or presence of p644 (lane 2), p644/TS434–634 (lane 3), p644/TS480–550 (lane 4) or p644/TS1040–1240 (lane 5). Total cellular RNA was isolated and then subjected to either RNase protection or RT–PCR analysis as described in Materials and Methods. The arrowheads indicate the positions of the luciferase- and β-actin-associated bands.

Figure 5

Figure 5

Isolation of TS RNP complexes in human colon cancer cells transfected with luciferase heterologous constructs. (A) Ethidium bromide stain. An immunoprecipitation/RT–PCR method was used to isolate TS RNP complexes, as described in Materials and Methods. H630 cells were transiently transfected with p644/TS480–550 (lanes 1, 4–6), p644 (lane 2) or p644/TS1040–1240 (lane 3). Whole cell extracts were immunoprecipitated with an anti-TS monoclonal antibody (lanes 1–3 and 5) or with no antibody (lane 4) as outlined in Materials and Methods. The isolated nucleic acid fraction was reverse transcribed and PCR amplified using EGR-1- and luciferase-specific primers. In lane 5, the immunoprecipitated nucleic acid fraction was not subjected to reverse transcription. Lane 6 represents a control PCR reaction in which the p644/TS480–550 DNA template was subjected to PCR amplification using the same primer set as used in lanes 1–5. Lane M, size marker. (B) p644/TS480–550 RNA sequence. The 123 bp fragment predicted to be RT–PCR amplified is shown in relation to its position on the full-length p644 plasmid. The positions of the EGR-1 promoter and the luciferase gene are indicated.

Figure 6

Figure 6

Effect of 5-FU treatment on luciferase expression in vivo. Plasmids p644, p644/TS434–634, p644/TS480–550 and p644/TS1040–1240 were each transiently expressed in H630 cells. Twenty-four hours after transfection, cells were incubated in the absence (open bars) or presence (closed bars) of 1 µM 5-FU for an additional 24 h. Luciferase activity in cells transfected with p644 in the absence of 5-FU was defined as 100%. 5-FU treatment of cells transfected with either p644/TS434–634 or p644/TS480–550 resulted in a significant increase (P < 0.05) in luciferase activity, as determined by paired Student’s _t_-test. Each value represents the mean ± SE of three to five experiments.

Figure 7

Figure 7

Effect of TS480–550 on luciferase expression in HCT-C18 and HCT-C:His-TS(+) cells. Human colon cancer HCT-C18 and HCT-C:His-TS(+) cells were transiently transfected with either p644 (closed bars) or p644/TS480–550 (open bars). Luciferase activity was measured 48 h post-transfection using the dual luciferase assay system as described in Materials and Methods. The activity in cells transfected with the p644 plasmid was taken as 100%. Transfection with p644/TS480–550 resulted in a significant decrease in luciferase activity (P < 0.01), indicated by the asterisk, when compared to transfection with p644, as determined by paired Student’s _t_-test. Each value represents the mean ± SE of three to five experiments.

Similar articles

Cited by

References

    1. Friedkin M. and Kornberg A. (1957) Chemical Basis of Heredity. John Hopkins Press, Baltimore, MD, pp. 609–614.
    1. Danenberg P.V. (1977) Biochim. Biophys. Acta, 473, 73–79. - PubMed
    1. Carreras C. and Santi,D.V. (1995) Annu. Rev. Biochem., 64, 721–762. - PubMed
    1. Chu E., Koeller,D.M., Casey,J.L., Drake,J.C., Chabner,B.A., Elwood,P.C., Zinn,S. and Allegra,C.J. (1991) Proc. Natl Acad. Sci. USA, 88, 8977–8981. - PMC - PubMed
    1. Chu E., Voeller,D., Koeller,D.M., Drake,J.C., Takimoto,C.H., Maley,G.F., Maley,F. and Allegra,C.J. (1993) Proc. Natl Acad. Sci. USA, 90, 517–521. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources