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 binding affinity for MDR-1 RNA. (A) An electromobility shift assay showing binding to [32P] MDR-1 RNA nts 746–962 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–4000 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 from three separate experiments was then used to plot the saturation binding curve as shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: CRD-BP binding affinity for MDR-1 RNA. (A) An electromobility shift assay showing binding to [32P] MDR-1 RNA nts 746–962 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–4000 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 from three separate experiments was then used to plot the saturation binding curve as shown.

Mentions: Keeping in mind the limitation of the EMSA, we also performed saturation-binding experiments to determine if CRD-BP could bind with other radiolabeled RNA substrates. When challenged with [32P] β-globin RNA, CRD-BP up to 3070 nM was unable to form an observable RNA–protein complex (Figure 4C). However, when CRD-BP was challenged with [32P] MDR-1, two RNA–protein complexes that exhibited a similar profile to that of the CRD-BP-c-myc CRD interaction were observed; Kd = 500 nM (Figure 5). The analysis of the combined binding data from three separate experiments revealed a level of cooperativity was present between the formation of the first and second binding complexes (Figure 5B). The cooperativity observed is also reflected in the Hill coefficient, which for CRD-BP versus c-myc CRD (Figure 4B) was 2.06, and 2.6 when challenged with MDR-1 RNA (Figure 5B). The Hill coefficients in both cases suggest that two molecules of CRD-BP bind with one molecule of c-myc CRD RNA or with one molecule of MDR-1 RNA at identical or non-identical sites. Evidence to support a two RNA-binding site model is provided by the kinetic analysis of the interactions between IMP-1 and IGF-II RNA (39). In this study, IMP-1 was shown to bind through a cooperative, sequential mechanism via two binding sites within the IGF-II transcript (39). Interestingly, dissociation constant of about 2 nM was reported for the interaction between IMP-1 and IGF-II RNA (39). The significantly higher dissociation constant determined in this study could reflect the inefficiency of renaturation of CRD-BP and c-myc CRD RNA as discussed above, or/and the use of His6-tagged CRD-BP since it has been previously reported that tagged-IMP-1 exhibited poor RNA binding (14).Figure 5.


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 MDR-1 RNA. (A) An electromobility shift assay showing binding to [32P] MDR-1 RNA nts 746–962 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–4000 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 from three separate experiments was then used to plot the saturation binding curve as shown.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 5: CRD-BP binding affinity for MDR-1 RNA. (A) An electromobility shift assay showing binding to [32P] MDR-1 RNA nts 746–962 by an increasing amount of purified recombinant His6-tagged CRD-BP (0–4000 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 from three separate experiments was then used to plot the saturation binding curve as shown.
Mentions: Keeping in mind the limitation of the EMSA, we also performed saturation-binding experiments to determine if CRD-BP could bind with other radiolabeled RNA substrates. When challenged with [32P] β-globin RNA, CRD-BP up to 3070 nM was unable to form an observable RNA–protein complex (Figure 4C). However, when CRD-BP was challenged with [32P] MDR-1, two RNA–protein complexes that exhibited a similar profile to that of the CRD-BP-c-myc CRD interaction were observed; Kd = 500 nM (Figure 5). The analysis of the combined binding data from three separate experiments revealed a level of cooperativity was present between the formation of the first and second binding complexes (Figure 5B). The cooperativity observed is also reflected in the Hill coefficient, which for CRD-BP versus c-myc CRD (Figure 4B) was 2.06, and 2.6 when challenged with MDR-1 RNA (Figure 5B). The Hill coefficients in both cases suggest that two molecules of CRD-BP bind with one molecule of c-myc CRD RNA or with one molecule of MDR-1 RNA at identical or non-identical sites. Evidence to support a two RNA-binding site model is provided by the kinetic analysis of the interactions between IMP-1 and IGF-II RNA (39). In this study, IMP-1 was shown to bind through a cooperative, sequential mechanism via two binding sites within the IGF-II transcript (39). Interestingly, dissociation constant of about 2 nM was reported for the interaction between IMP-1 and IGF-II RNA (39). The significantly higher dissociation constant determined in this study could reflect the inefficiency of renaturation of CRD-BP and c-myc CRD RNA as discussed above, or/and the use of His6-tagged CRD-BP since it has been previously reported that tagged-IMP-1 exhibited poor RNA binding (14).Figure 5.

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