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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 shields the c-myc CRD RNA from endonucleolytic attack by a mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] c-myc CRD RNA nts 1705–1886 as described in the Materials and methods section, with (lanes 4, 5, 9, 10, 14, 15) or without (lanes 3, 8 and 13) the presence of 500, 750 or 1500 nM of purified recombinant His6-tagged CRD-BP as shown. (B) As in (A), the purified endoribonuclease was incubated with the radiolabeled c-myc CRD RNA in the absence (lane 2) or presence of 1500 nM of Rpp20 (lane 4), Rpp21 (lane 6) and Rpp40 (lane 8). Lanes 3, 5 and 7 had the purified recombinant proteins only. Numbers on the right indicate the cleavage sites generated by the mammalian endoribonuclease on the CRD RNA substrates.
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Figure 6: CRD-BP shields the c-myc CRD RNA from endonucleolytic attack by a mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] c-myc CRD RNA nts 1705–1886 as described in the Materials and methods section, with (lanes 4, 5, 9, 10, 14, 15) or without (lanes 3, 8 and 13) the presence of 500, 750 or 1500 nM of purified recombinant His6-tagged CRD-BP as shown. (B) As in (A), the purified endoribonuclease was incubated with the radiolabeled c-myc CRD RNA in the absence (lane 2) or presence of 1500 nM of Rpp20 (lane 4), Rpp21 (lane 6) and Rpp40 (lane 8). Lanes 3, 5 and 7 had the purified recombinant proteins only. Numbers on the right indicate the cleavage sites generated by the mammalian endoribonuclease on the CRD RNA substrates.

Mentions: 5′-End labeled c-myc CRD nts 1705–1886 was incubated with one unit of the purified endonuclease in the presence or absence of recombinant CRD-BP. As shown in Figure 6A, the typical ten distinct cleavage sites at nts 1742, 1747, 1751, 1757, 1768, 1771, 1773, 1775, 1845 and 1855 were generated upon incubation with the purified endonuclease (lanes 3, 8 and 13) (31). For ease in quantification and categorization, we have named the separate cleavage sites as Region I (1845, 1855), Region II (1768, 1771, 1773, 1775) and Region III (1742, 1747, 1751, 1757) (Figure 6). Incubation of the transcript with recombinant CRD-BP at 500 nM (lane 2), 750 nM (lane 7) and 1500 nM (lane 12) alone had no effect, indicating that CRD-BP lacks RNase activity. It was anticipated that c-myc cleavage would be reduced using CRD-BP concentrations equal to the measured dissociation constant because 50% of c-myc CRD would be bound at 500 nM (Figure 4A and B). This prediction was confirmed by the reduction in cleavage fragments observed in all three regions indicated in Figure 6A (compare lanes 4–5 with lane 3). At 500 nM, CRD-BP was responsible for a 43% reduction in Region I, a 72% reduction in Region II and a 77% reduction in Region III.Figure 6.


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 shields the c-myc CRD RNA from endonucleolytic attack by a mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] c-myc CRD RNA nts 1705–1886 as described in the Materials and methods section, with (lanes 4, 5, 9, 10, 14, 15) or without (lanes 3, 8 and 13) the presence of 500, 750 or 1500 nM of purified recombinant His6-tagged CRD-BP as shown. (B) As in (A), the purified endoribonuclease was incubated with the radiolabeled c-myc CRD RNA in the absence (lane 2) or presence of 1500 nM of Rpp20 (lane 4), Rpp21 (lane 6) and Rpp40 (lane 8). Lanes 3, 5 and 7 had the purified recombinant proteins only. Numbers on the right indicate the cleavage sites generated by the mammalian endoribonuclease on the CRD RNA substrates.
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Related In: Results  -  Collection

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Figure 6: CRD-BP shields the c-myc CRD RNA from endonucleolytic attack by a mammalian endoribonuclease. (A) One unit of the purified mammalian endoribonuclease was incubated with [32P] c-myc CRD RNA nts 1705–1886 as described in the Materials and methods section, with (lanes 4, 5, 9, 10, 14, 15) or without (lanes 3, 8 and 13) the presence of 500, 750 or 1500 nM of purified recombinant His6-tagged CRD-BP as shown. (B) As in (A), the purified endoribonuclease was incubated with the radiolabeled c-myc CRD RNA in the absence (lane 2) or presence of 1500 nM of Rpp20 (lane 4), Rpp21 (lane 6) and Rpp40 (lane 8). Lanes 3, 5 and 7 had the purified recombinant proteins only. Numbers on the right indicate the cleavage sites generated by the mammalian endoribonuclease on the CRD RNA substrates.
Mentions: 5′-End labeled c-myc CRD nts 1705–1886 was incubated with one unit of the purified endonuclease in the presence or absence of recombinant CRD-BP. As shown in Figure 6A, the typical ten distinct cleavage sites at nts 1742, 1747, 1751, 1757, 1768, 1771, 1773, 1775, 1845 and 1855 were generated upon incubation with the purified endonuclease (lanes 3, 8 and 13) (31). For ease in quantification and categorization, we have named the separate cleavage sites as Region I (1845, 1855), Region II (1768, 1771, 1773, 1775) and Region III (1742, 1747, 1751, 1757) (Figure 6). Incubation of the transcript with recombinant CRD-BP at 500 nM (lane 2), 750 nM (lane 7) and 1500 nM (lane 12) alone had no effect, indicating that CRD-BP lacks RNase activity. It was anticipated that c-myc cleavage would be reduced using CRD-BP concentrations equal to the measured dissociation constant because 50% of c-myc CRD would be bound at 500 nM (Figure 4A and B). This prediction was confirmed by the reduction in cleavage fragments observed in all three regions indicated in Figure 6A (compare lanes 4–5 with lane 3). At 500 nM, CRD-BP was responsible for a 43% reduction in Region I, a 72% reduction in Region II and a 77% reduction in Region III.Figure 6.

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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Related in: MedlinePlus