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MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

Pereira LA, Hugo HJ, Malaterre J, Huiling X, Sonza S, Cures A, Purcell DF, Ramsland PA, Gerondakis S, Gonda TJ, Ramsay RG - PLoS ONE (2015)

Bottom Line: We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation.Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB.Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

View Article: PubMed Central - PubMed

Affiliation: Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, Locked Bag #1, Melbourne, Victoria, 8006, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

No MeSH data available.


Related in: MedlinePlus

NFκBp50 binds directly to the MYB SLR polyU.(A) Radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract-deleted (MYB SLR ΔpolyU) or stem-loop region deleted (MYB ΔSLR polyU) templates were incubated with 50 ng of NFκBp50 or in the case of the MYB SLR ΔpolyU probe with 25 ng and 50 ng NFκB p50 and the reactions resolved on a 5% Tris-glycine gel. Coomassie gel and Western blot analysis of the NFκBp50 protein are shown. (B) Radiolabeled RNA probes generated from pGEM-MYB SLR polyU or MYB SLR ΔpolyU were incubated with 50 ng of NFκBp50 and the reactions resolved on a 5% Tris-glycine gel. (C) Left; an RNA probe generated from pGEM-MYB SLR polyU was incubated with 50 ng of NFκBp50 and NFκBp50-MYB SLR polyU RNA-protein complexes were supershifted by the addition of anti-NFκBp50 antibody. Anti-NFκBp65 antibody was used as a control. Right; RNA shifts were performed as above and the reactions subjected to UV-cross linking and SDS-PAGE. (D) NFκBp50 RHD analysed by Coomassie staining and Western blot analysis. (E) Left; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with increasing amounts of recombinant NFκBp50 RHD and the reactions resolved on a 5% Tris-glycine gel. Right; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with recombinant NFκBp50 RHD and NFκBp50-MYB SLR polyU RNA-protein complexes were super-shifted by the addition of anti-NFκBp50 antibody. Anti-rabbit IgG was used as a control. In (A-E) the black arrows indicate the position of the NFκBp50-MYB polyU SLR or NFκBp50-MYB SLR ΔpolyU RNA complexes; white arrows show the position of the complexes in the presence of the anti-p50 antibody; grey arrow indicates free MYB polyU SLR or MYB SLR ΔpolyU RNA probes.
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pone.0122919.g002: NFκBp50 binds directly to the MYB SLR polyU.(A) Radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract-deleted (MYB SLR ΔpolyU) or stem-loop region deleted (MYB ΔSLR polyU) templates were incubated with 50 ng of NFκBp50 or in the case of the MYB SLR ΔpolyU probe with 25 ng and 50 ng NFκB p50 and the reactions resolved on a 5% Tris-glycine gel. Coomassie gel and Western blot analysis of the NFκBp50 protein are shown. (B) Radiolabeled RNA probes generated from pGEM-MYB SLR polyU or MYB SLR ΔpolyU were incubated with 50 ng of NFκBp50 and the reactions resolved on a 5% Tris-glycine gel. (C) Left; an RNA probe generated from pGEM-MYB SLR polyU was incubated with 50 ng of NFκBp50 and NFκBp50-MYB SLR polyU RNA-protein complexes were supershifted by the addition of anti-NFκBp50 antibody. Anti-NFκBp65 antibody was used as a control. Right; RNA shifts were performed as above and the reactions subjected to UV-cross linking and SDS-PAGE. (D) NFκBp50 RHD analysed by Coomassie staining and Western blot analysis. (E) Left; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with increasing amounts of recombinant NFκBp50 RHD and the reactions resolved on a 5% Tris-glycine gel. Right; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with recombinant NFκBp50 RHD and NFκBp50-MYB SLR polyU RNA-protein complexes were super-shifted by the addition of anti-NFκBp50 antibody. Anti-rabbit IgG was used as a control. In (A-E) the black arrows indicate the position of the NFκBp50-MYB polyU SLR or NFκBp50-MYB SLR ΔpolyU RNA complexes; white arrows show the position of the complexes in the presence of the anti-p50 antibody; grey arrow indicates free MYB polyU SLR or MYB SLR ΔpolyU RNA probes.

Mentions: The above experiments suggested that NFκBp50 could bind to the MYB SLR RNA. To examine this possibility RNA EMSAs were performed with recombinant NFκBp50 and intronic MYB RNA probes MYB SLR polyU, MYB ΔSLR polyU or MYB SLR ΔpolyU. NFκBp50 formed a complex with MYB SLR polyU RNA but was unable to shift probe in which the SLR was deleted (MYB ΔSLR polyU). Removal of the polyU tract (MYB SLR ΔpolyU) substantially reduced NFκBp50 binding (Fig 2A).


MYB elongation is regulated by the nucleic acid binding of NFκB p50 to the intronic stem-loop region.

Pereira LA, Hugo HJ, Malaterre J, Huiling X, Sonza S, Cures A, Purcell DF, Ramsland PA, Gerondakis S, Gonda TJ, Ramsay RG - PLoS ONE (2015)

NFκBp50 binds directly to the MYB SLR polyU.(A) Radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract-deleted (MYB SLR ΔpolyU) or stem-loop region deleted (MYB ΔSLR polyU) templates were incubated with 50 ng of NFκBp50 or in the case of the MYB SLR ΔpolyU probe with 25 ng and 50 ng NFκB p50 and the reactions resolved on a 5% Tris-glycine gel. Coomassie gel and Western blot analysis of the NFκBp50 protein are shown. (B) Radiolabeled RNA probes generated from pGEM-MYB SLR polyU or MYB SLR ΔpolyU were incubated with 50 ng of NFκBp50 and the reactions resolved on a 5% Tris-glycine gel. (C) Left; an RNA probe generated from pGEM-MYB SLR polyU was incubated with 50 ng of NFκBp50 and NFκBp50-MYB SLR polyU RNA-protein complexes were supershifted by the addition of anti-NFκBp50 antibody. Anti-NFκBp65 antibody was used as a control. Right; RNA shifts were performed as above and the reactions subjected to UV-cross linking and SDS-PAGE. (D) NFκBp50 RHD analysed by Coomassie staining and Western blot analysis. (E) Left; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with increasing amounts of recombinant NFκBp50 RHD and the reactions resolved on a 5% Tris-glycine gel. Right; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with recombinant NFκBp50 RHD and NFκBp50-MYB SLR polyU RNA-protein complexes were super-shifted by the addition of anti-NFκBp50 antibody. Anti-rabbit IgG was used as a control. In (A-E) the black arrows indicate the position of the NFκBp50-MYB polyU SLR or NFκBp50-MYB SLR ΔpolyU RNA complexes; white arrows show the position of the complexes in the presence of the anti-p50 antibody; grey arrow indicates free MYB polyU SLR or MYB SLR ΔpolyU RNA probes.
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Related In: Results  -  Collection

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pone.0122919.g002: NFκBp50 binds directly to the MYB SLR polyU.(A) Radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract-deleted (MYB SLR ΔpolyU) or stem-loop region deleted (MYB ΔSLR polyU) templates were incubated with 50 ng of NFκBp50 or in the case of the MYB SLR ΔpolyU probe with 25 ng and 50 ng NFκB p50 and the reactions resolved on a 5% Tris-glycine gel. Coomassie gel and Western blot analysis of the NFκBp50 protein are shown. (B) Radiolabeled RNA probes generated from pGEM-MYB SLR polyU or MYB SLR ΔpolyU were incubated with 50 ng of NFκBp50 and the reactions resolved on a 5% Tris-glycine gel. (C) Left; an RNA probe generated from pGEM-MYB SLR polyU was incubated with 50 ng of NFκBp50 and NFκBp50-MYB SLR polyU RNA-protein complexes were supershifted by the addition of anti-NFκBp50 antibody. Anti-NFκBp65 antibody was used as a control. Right; RNA shifts were performed as above and the reactions subjected to UV-cross linking and SDS-PAGE. (D) NFκBp50 RHD analysed by Coomassie staining and Western blot analysis. (E) Left; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with increasing amounts of recombinant NFκBp50 RHD and the reactions resolved on a 5% Tris-glycine gel. Right; an RNA probe generated from pBluescript II KS MYB SLR polyU was incubated with recombinant NFκBp50 RHD and NFκBp50-MYB SLR polyU RNA-protein complexes were super-shifted by the addition of anti-NFκBp50 antibody. Anti-rabbit IgG was used as a control. In (A-E) the black arrows indicate the position of the NFκBp50-MYB polyU SLR or NFκBp50-MYB SLR ΔpolyU RNA complexes; white arrows show the position of the complexes in the presence of the anti-p50 antibody; grey arrow indicates free MYB polyU SLR or MYB SLR ΔpolyU RNA probes.
Mentions: The above experiments suggested that NFκBp50 could bind to the MYB SLR RNA. To examine this possibility RNA EMSAs were performed with recombinant NFκBp50 and intronic MYB RNA probes MYB SLR polyU, MYB ΔSLR polyU or MYB SLR ΔpolyU. NFκBp50 formed a complex with MYB SLR polyU RNA but was unable to shift probe in which the SLR was deleted (MYB ΔSLR polyU). Removal of the polyU tract (MYB SLR ΔpolyU) substantially reduced NFκBp50 binding (Fig 2A).

Bottom Line: We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation.Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB.Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

View Article: PubMed Central - PubMed

Affiliation: Differentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, Locked Bag #1, Melbourne, Victoria, 8006, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, 3010, Australia.

ABSTRACT
MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFκBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFκBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFκBp50, mutation of which abrogated the interaction of NFκBp50 with the SLR polyU and impaired NFκBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFκBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFκBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFκBp50 required for MYB regulation.

No MeSH data available.


Related in: MedlinePlus