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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

Cellular proteins bind to the MYB SLR polyU RNA.(A) MYB SLR polyU structure analysis. The mfold predicted structure of the MYB SLR polyU is shown. (B) Electrophoretic mobility of radiolabeled MYB SLR polyU RNA transcripts (MYB SLR polyU; 310 bases), polyU tract-deleted (MYB SLR ΔpolyU; 291 bases), stem-loop deleted (MYB ΔSLR polyU; 249 bases) and 9 of the 19 polyU residues deleted (MYB SLR Δ9 polyU; 300 bases). RNAs were subjected to electrophoresis in a denaturing 4% acrylamide gel where probes migrate according to size and a 4% non-denaturing acrylamide gel where secondary structure is maintained. (C) LIM1215 whole cell extracts were incubated with radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract deleted (MYB SLR ΔpolyU) and stem-loop region deleted (MYB ΔSLR polyU) templates and subjected to UV-cross linking followed by SDS-PAGE. Two species (~50 kDa, black arrow and ~ 38 kDa, grey arrow) were observed with the MYB SLR polyU probe, and one species (~ 38 kDa, grey arrow) with the MYB SLR ΔpolyU probe. A ~20 kDa doublet is also evident with the MYB ΔSLR polyU and MYB SLR ΔpolyU probes, white arrow. (D) LIM1215 whole cell extract was depleted of NFκBp50 by incubation with NFκBp50 antibody. Depletion of NFκBp50 was confirmed by Western blot analysis. Depleted extracts were subsequently incubated with radiolabeled MYB SLR polyU RNA probe and subjected to UV-cross linking followed by SDS-PAGE. Two phosphorimaging exposures of the same SDS-PAGE gel are shown to highlight the reduction in the 50kDa signal as indicated by the asterisk. Phosphorimaging quantitation confirmed reduction of the 50kDa signal. Error bars represent mean ± SEM, ** P <0.01.
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pone.0122919.g001: Cellular proteins bind to the MYB SLR polyU RNA.(A) MYB SLR polyU structure analysis. The mfold predicted structure of the MYB SLR polyU is shown. (B) Electrophoretic mobility of radiolabeled MYB SLR polyU RNA transcripts (MYB SLR polyU; 310 bases), polyU tract-deleted (MYB SLR ΔpolyU; 291 bases), stem-loop deleted (MYB ΔSLR polyU; 249 bases) and 9 of the 19 polyU residues deleted (MYB SLR Δ9 polyU; 300 bases). RNAs were subjected to electrophoresis in a denaturing 4% acrylamide gel where probes migrate according to size and a 4% non-denaturing acrylamide gel where secondary structure is maintained. (C) LIM1215 whole cell extracts were incubated with radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract deleted (MYB SLR ΔpolyU) and stem-loop region deleted (MYB ΔSLR polyU) templates and subjected to UV-cross linking followed by SDS-PAGE. Two species (~50 kDa, black arrow and ~ 38 kDa, grey arrow) were observed with the MYB SLR polyU probe, and one species (~ 38 kDa, grey arrow) with the MYB SLR ΔpolyU probe. A ~20 kDa doublet is also evident with the MYB ΔSLR polyU and MYB SLR ΔpolyU probes, white arrow. (D) LIM1215 whole cell extract was depleted of NFκBp50 by incubation with NFκBp50 antibody. Depletion of NFκBp50 was confirmed by Western blot analysis. Depleted extracts were subsequently incubated with radiolabeled MYB SLR polyU RNA probe and subjected to UV-cross linking followed by SDS-PAGE. Two phosphorimaging exposures of the same SDS-PAGE gel are shown to highlight the reduction in the 50kDa signal as indicated by the asterisk. Phosphorimaging quantitation confirmed reduction of the 50kDa signal. Error bars represent mean ± SEM, ** P <0.01.

Mentions: Soluble protein was extracted from LIM1215 or 293 cells with NP40 lysis buffer (0.64% Nonidet P-40, 5 mM KCl, 2 mM MgCl2, 500 mM NaCl, 10 mM Tris, pH8). Soluble protein cell extracts or recombinant proteins were resolved on 10% SDS PAGE gels or 4–12% NuPAGE MOPs gels (Invitrogen) and transferred to PVDF membrane. Membranes were probed with anti-c-Myb1.1 [30], anti-V5 (V5-10; Sigma), anti-NFκBp50 (E10; Santa Cruz, SC), anti-CyclinT1 (T18; SC), anti-CDK9 (L19; SC), anti-HIV-1 Tat (ab42359), anti-NFκBp65 (A: SC), anti-NFκBp65 (C20; SC) or anti-pan-Actin (C4; MP Biomedicals). Membranes were probed with HRP secondary antibodies (Bio-Rad). For the NFκBp50 depletion experiment in Fig 1D, LIM1215 extracts were incubated overnight with anti-NFκBp50 (C19: SC) and NFκBp105 (C19: Cell Signaling). NFκBp50 immunoprecipitates were subsequently removed by incubating extracts with Protein A/G resin (Santa Cruz).


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)

Cellular proteins bind to the MYB SLR polyU RNA.(A) MYB SLR polyU structure analysis. The mfold predicted structure of the MYB SLR polyU is shown. (B) Electrophoretic mobility of radiolabeled MYB SLR polyU RNA transcripts (MYB SLR polyU; 310 bases), polyU tract-deleted (MYB SLR ΔpolyU; 291 bases), stem-loop deleted (MYB ΔSLR polyU; 249 bases) and 9 of the 19 polyU residues deleted (MYB SLR Δ9 polyU; 300 bases). RNAs were subjected to electrophoresis in a denaturing 4% acrylamide gel where probes migrate according to size and a 4% non-denaturing acrylamide gel where secondary structure is maintained. (C) LIM1215 whole cell extracts were incubated with radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract deleted (MYB SLR ΔpolyU) and stem-loop region deleted (MYB ΔSLR polyU) templates and subjected to UV-cross linking followed by SDS-PAGE. Two species (~50 kDa, black arrow and ~ 38 kDa, grey arrow) were observed with the MYB SLR polyU probe, and one species (~ 38 kDa, grey arrow) with the MYB SLR ΔpolyU probe. A ~20 kDa doublet is also evident with the MYB ΔSLR polyU and MYB SLR ΔpolyU probes, white arrow. (D) LIM1215 whole cell extract was depleted of NFκBp50 by incubation with NFκBp50 antibody. Depletion of NFκBp50 was confirmed by Western blot analysis. Depleted extracts were subsequently incubated with radiolabeled MYB SLR polyU RNA probe and subjected to UV-cross linking followed by SDS-PAGE. Two phosphorimaging exposures of the same SDS-PAGE gel are shown to highlight the reduction in the 50kDa signal as indicated by the asterisk. Phosphorimaging quantitation confirmed reduction of the 50kDa signal. Error bars represent mean ± SEM, ** P <0.01.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4390348&req=5

pone.0122919.g001: Cellular proteins bind to the MYB SLR polyU RNA.(A) MYB SLR polyU structure analysis. The mfold predicted structure of the MYB SLR polyU is shown. (B) Electrophoretic mobility of radiolabeled MYB SLR polyU RNA transcripts (MYB SLR polyU; 310 bases), polyU tract-deleted (MYB SLR ΔpolyU; 291 bases), stem-loop deleted (MYB ΔSLR polyU; 249 bases) and 9 of the 19 polyU residues deleted (MYB SLR Δ9 polyU; 300 bases). RNAs were subjected to electrophoresis in a denaturing 4% acrylamide gel where probes migrate according to size and a 4% non-denaturing acrylamide gel where secondary structure is maintained. (C) LIM1215 whole cell extracts were incubated with radiolabeled RNA probes generated from pBluescript II KS MYB SLR polyU, polyU tract deleted (MYB SLR ΔpolyU) and stem-loop region deleted (MYB ΔSLR polyU) templates and subjected to UV-cross linking followed by SDS-PAGE. Two species (~50 kDa, black arrow and ~ 38 kDa, grey arrow) were observed with the MYB SLR polyU probe, and one species (~ 38 kDa, grey arrow) with the MYB SLR ΔpolyU probe. A ~20 kDa doublet is also evident with the MYB ΔSLR polyU and MYB SLR ΔpolyU probes, white arrow. (D) LIM1215 whole cell extract was depleted of NFκBp50 by incubation with NFκBp50 antibody. Depletion of NFκBp50 was confirmed by Western blot analysis. Depleted extracts were subsequently incubated with radiolabeled MYB SLR polyU RNA probe and subjected to UV-cross linking followed by SDS-PAGE. Two phosphorimaging exposures of the same SDS-PAGE gel are shown to highlight the reduction in the 50kDa signal as indicated by the asterisk. Phosphorimaging quantitation confirmed reduction of the 50kDa signal. Error bars represent mean ± SEM, ** P <0.01.
Mentions: Soluble protein was extracted from LIM1215 or 293 cells with NP40 lysis buffer (0.64% Nonidet P-40, 5 mM KCl, 2 mM MgCl2, 500 mM NaCl, 10 mM Tris, pH8). Soluble protein cell extracts or recombinant proteins were resolved on 10% SDS PAGE gels or 4–12% NuPAGE MOPs gels (Invitrogen) and transferred to PVDF membrane. Membranes were probed with anti-c-Myb1.1 [30], anti-V5 (V5-10; Sigma), anti-NFκBp50 (E10; Santa Cruz, SC), anti-CyclinT1 (T18; SC), anti-CDK9 (L19; SC), anti-HIV-1 Tat (ab42359), anti-NFκBp65 (A: SC), anti-NFκBp65 (C20; SC) or anti-pan-Actin (C4; MP Biomedicals). Membranes were probed with HRP secondary antibodies (Bio-Rad). For the NFκBp50 depletion experiment in Fig 1D, LIM1215 extracts were incubated overnight with anti-NFκBp50 (C19: SC) and NFκBp105 (C19: Cell Signaling). NFκBp50 immunoprecipitates were subsequently removed by incubating extracts with Protein A/G resin (Santa Cruz).

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