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CRD-BP shields c-myc and MDR-1 RNA from endonucleolytic attack by a mammalian endoribonuclease.

Sparanese D, Lee CH - Nucleic Acids Res. (2007)

Bottom Line: In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage.Finally, we have identified RNA sequences required for CRD-BP binding.These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada.

ABSTRACT
The c-myc mRNA coding region determinant-binding protein (CRD-BP) has high affinity for the coding region determinant (CRD) of c-myc mRNA. Such affinity is believed to protect c-myc CRD from endonucleolytic attack. We have recently purified a mammalian endoribonuclease which can cleave within the c-myc CRD in vitro. The availability of this purified endonuclease has made it possible to directly test the interaction between CRD-BP and the endonuclease in regulating c-myc CRD RNA cleavage. In this study, we have identified the coding region of MDR-1 RNA as a new target for CRD-BP. CRD-BP has the same affinity for c-myc CRD nts 1705-1886 and MDR-1 RNA nts 746-962 with K(d) of 500 nM. The concentration-dependent affinity of CRD-BP to these transcripts correlated with the concentration-dependent blocking of endonuclease-mediated cleavage by CRD-BP. In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage. Finally, we have identified RNA sequences required for CRD-BP binding. These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.

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CRD-BP-binding affinity for c-myc CRD RNA and β-globin RNA. (A) An electromobility shift assay showing binding to [32P] c-myc CRD RNA nts 1705–1886 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–1470 nM). (B) Binding activity in (A) was quantified using the PhorphorImager to compare the relative amount of radiolabeled unbound RNA shifted into slower-migrating Complex I and Complex II. Data obtained was then used to plot the saturation binding curve as shown. (C) An electromobility shift assay assessing the ability to bind to [32P] β-globin RNA nts 1–145 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–3070 nM).
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Figure 4: CRD-BP-binding affinity for c-myc CRD RNA and β-globin RNA. (A) An electromobility shift assay showing binding to [32P] c-myc CRD RNA nts 1705–1886 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–1470 nM). (B) Binding activity in (A) was quantified using the PhorphorImager to compare the relative amount of radiolabeled unbound RNA shifted into slower-migrating Complex I and Complex II. Data obtained was then used to plot the saturation binding curve as shown. (C) An electromobility shift assay assessing the ability to bind to [32P] β-globin RNA nts 1–145 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–3070 nM).

Mentions: To further determine if the recombinant CRD-BP-c-myc CRD [32P] RNA nts 1705–1886 association was specific, we chalenged the interaction with unlabeled competitor RNA. Our EMSA competition assays revealed that up to 50X molar excess of unlabeled β-globin competitor RNA was unable to dilute the binding between CRD-BP and [32P] c-myc CRD (lanes 15–18, Figure 3A; lanes 7–8, Figure 3B). The inability of unlabeled β-globin competitor to dilute the CRD-BP-c-myc CRD interaction in conjunction with the saturation-binding experiment shown in Figure 4C support the observation that the 145-nt β-globin RNA fragment is not a bona fide CRD-BP target in vitro. The β-globin results from this investigation are also supported by the two earlier reports (12,13).Figure 3.


CRD-BP shields c-myc and MDR-1 RNA from endonucleolytic attack by a mammalian endoribonuclease.

Sparanese D, Lee CH - Nucleic Acids Res. (2007)

CRD-BP-binding affinity for c-myc CRD RNA and β-globin RNA. (A) An electromobility shift assay showing binding to [32P] c-myc CRD RNA nts 1705–1886 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–1470 nM). (B) Binding activity in (A) was quantified using the PhorphorImager to compare the relative amount of radiolabeled unbound RNA shifted into slower-migrating Complex I and Complex II. Data obtained was then used to plot the saturation binding curve as shown. (C) An electromobility shift assay assessing the ability to bind to [32P] β-globin RNA nts 1–145 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–3070 nM).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851641&req=5

Figure 4: CRD-BP-binding affinity for c-myc CRD RNA and β-globin RNA. (A) An electromobility shift assay showing binding to [32P] c-myc CRD RNA nts 1705–1886 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–1470 nM). (B) Binding activity in (A) was quantified using the PhorphorImager to compare the relative amount of radiolabeled unbound RNA shifted into slower-migrating Complex I and Complex II. Data obtained was then used to plot the saturation binding curve as shown. (C) An electromobility shift assay assessing the ability to bind to [32P] β-globin RNA nts 1–145 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–3070 nM).
Mentions: To further determine if the recombinant CRD-BP-c-myc CRD [32P] RNA nts 1705–1886 association was specific, we chalenged the interaction with unlabeled competitor RNA. Our EMSA competition assays revealed that up to 50X molar excess of unlabeled β-globin competitor RNA was unable to dilute the binding between CRD-BP and [32P] c-myc CRD (lanes 15–18, Figure 3A; lanes 7–8, Figure 3B). The inability of unlabeled β-globin competitor to dilute the CRD-BP-c-myc CRD interaction in conjunction with the saturation-binding experiment shown in Figure 4C support the observation that the 145-nt β-globin RNA fragment is not a bona fide CRD-BP target in vitro. The β-globin results from this investigation are also supported by the two earlier reports (12,13).Figure 3.

Bottom Line: In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage.Finally, we have identified RNA sequences required for CRD-BP binding.These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, BC V2N 4Z9, Canada.

ABSTRACT
The c-myc mRNA coding region determinant-binding protein (CRD-BP) has high affinity for the coding region determinant (CRD) of c-myc mRNA. Such affinity is believed to protect c-myc CRD from endonucleolytic attack. We have recently purified a mammalian endoribonuclease which can cleave within the c-myc CRD in vitro. The availability of this purified endonuclease has made it possible to directly test the interaction between CRD-BP and the endonuclease in regulating c-myc CRD RNA cleavage. In this study, we have identified the coding region of MDR-1 RNA as a new target for CRD-BP. CRD-BP has the same affinity for c-myc CRD nts 1705-1886 and MDR-1 RNA nts 746-962 with K(d) of 500 nM. The concentration-dependent affinity of CRD-BP to these transcripts correlated with the concentration-dependent blocking of endonuclease-mediated cleavage by CRD-BP. In contrast, three other recombinant proteins tested which had no affinity for c-myc CRD did not block endonuclease-mediated cleavage. Finally, we have identified RNA sequences required for CRD-BP binding. These results provide the first direct evidence that CRD-BP can indeed protect c-myc CRD cleavage initiated by an endoribonuclease, and the framework for further investigation into the interactions between CRD-BP, c-myc mRNA, MDR-1 mRNA and the endoribonuclease in cells.

Show MeSH
Related in: MedlinePlus