Limits...
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.

Show MeSH

Related in: MedlinePlus

CRD-BP is capable of shielding MDR-1 RNA from endonucleolytic attack by the mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] MDR-1 RNA nts 746–962 as described in the Materials and methods section, without (lane 2) or with the presence of 500 nM (lane 5), 750 nM (lane 4) or 1500 nM (lane 3) of purified recombinant His6-tagged CRD-BP. Lane 2 had the input RNA only. (B) As in (A), the purified endoribonuclease was incubated with [32P] β-globin RNA nts 1–145 without (lane 3) or with the presence of 750 nM (lane 4) or 1500 nM (lane 5) of purified recombinant His6-tagged CRD-BP. Lane 1 had the input RNA only.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851641&req=5

Figure 7: CRD-BP is capable of shielding MDR-1 RNA from endonucleolytic attack by the mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] MDR-1 RNA nts 746–962 as described in the Materials and methods section, without (lane 2) or with the presence of 500 nM (lane 5), 750 nM (lane 4) or 1500 nM (lane 3) of purified recombinant His6-tagged CRD-BP. Lane 2 had the input RNA only. (B) As in (A), the purified endoribonuclease was incubated with [32P] β-globin RNA nts 1–145 without (lane 3) or with the presence of 750 nM (lane 4) or 1500 nM (lane 5) of purified recombinant His6-tagged CRD-BP. Lane 1 had the input RNA only.

Mentions: CRD-BP was shown to bind with considerable affinity and specificity to MDR-1 RNA (Figure 5) and we have previously shown that the endonuclease is capable of cleaving MDR-1 RNA (31). It was therefore anticipated that CRD-BP would also be capable of blocking MDR-1 cleavage by the mammalian endoribonuclease. As predicted, CRD-BP was observed to shield MDR-1 RNA from endonucleolytic attack in a concentration-dependent manner (Figure 7A), and the most significant cleavage reductions were observed at 1500 nM (lane 3, Figure 7A). The consistent reduction observed in all regions suggests that the entire MDR-1 RNA was protected via CRD–BP interactions. We have also tested the ability of CRD-BP to block cleavage of β-globin RNA by the endonuclease. As expected, CRD-BP that had no affinity for β-globin RNA (Figure 4C) did not effectively block the endonuclease from cleaving this transcript (Figure 7B).Figure 7.


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 is capable of shielding MDR-1 RNA from endonucleolytic attack by the mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] MDR-1 RNA nts 746–962 as described in the Materials and methods section, without (lane 2) or with the presence of 500 nM (lane 5), 750 nM (lane 4) or 1500 nM (lane 3) of purified recombinant His6-tagged CRD-BP. Lane 2 had the input RNA only. (B) As in (A), the purified endoribonuclease was incubated with [32P] β-globin RNA nts 1–145 without (lane 3) or with the presence of 750 nM (lane 4) or 1500 nM (lane 5) of purified recombinant His6-tagged CRD-BP. Lane 1 had the input RNA only.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 7: CRD-BP is capable of shielding MDR-1 RNA from endonucleolytic attack by the mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] MDR-1 RNA nts 746–962 as described in the Materials and methods section, without (lane 2) or with the presence of 500 nM (lane 5), 750 nM (lane 4) or 1500 nM (lane 3) of purified recombinant His6-tagged CRD-BP. Lane 2 had the input RNA only. (B) As in (A), the purified endoribonuclease was incubated with [32P] β-globin RNA nts 1–145 without (lane 3) or with the presence of 750 nM (lane 4) or 1500 nM (lane 5) of purified recombinant His6-tagged CRD-BP. Lane 1 had the input RNA only.
Mentions: CRD-BP was shown to bind with considerable affinity and specificity to MDR-1 RNA (Figure 5) and we have previously shown that the endonuclease is capable of cleaving MDR-1 RNA (31). It was therefore anticipated that CRD-BP would also be capable of blocking MDR-1 cleavage by the mammalian endoribonuclease. As predicted, CRD-BP was observed to shield MDR-1 RNA from endonucleolytic attack in a concentration-dependent manner (Figure 7A), and the most significant cleavage reductions were observed at 1500 nM (lane 3, Figure 7A). The consistent reduction observed in all regions suggests that the entire MDR-1 RNA was protected via CRD–BP interactions. We have also tested the ability of CRD-BP to block cleavage of β-globin RNA by the endonuclease. As expected, CRD-BP that had no affinity for β-globin RNA (Figure 4C) did not effectively block the endonuclease from cleaving this transcript (Figure 7B).Figure 7.

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